A Compact Differential Dynamic Microscopy-based Device (cDDM): An Approach Tool for Early Detection of Hypercoagulable State in Transfusion-Dependent-ß-Thalassemia Patients.

ß-Thalassemia especially transfusion-dependent thalassemia (TDT) associates with a hypercoagulable state, which is the main cause of thromboembolic events (TEE). Plasma viscosity and rheological parameters could be essential markers for determining hypercoagulable state in ß-thalassemia patients. The traditional methods for measuring viscosity are often limited by large sample volumes and are impractical for routine clinical monitoring. The compact differential dynamic microscopy-based device (cDDM), an optical microscopy for quantitative rheological assessment, was developed and applied for prognosis of the hypercoagulable state in ß-TDT with and without splenectomy. The device was performed plasma viscosity measurement using low plasma volume (8 µL) and revealed a value as modulus of complex viscosity |η(ω)| in 7 min. We also parallelly demonstrated the correlation of the viscosity and related-coagulable parameters: complete blood count, prothrombin time (PT), activated partial thromboplastin time (APTT), protein C (PC), protein S (PS), CD62P and CD63 expression, and platelet aggregation test. The thalassemia plasma exhibited a higher value of |η(ω)| than healthy plasma, which can represent a different viscoelastic property among the groups. Even all related-coagulable parameters indicated hypercoagulable state in both nonsplenectomies and splenectomies ß-TDT patients when compared to control, only high platelet numbers significantly correlated to high plasma viscosity in the splenectomy group. However, the other coagulable parameters have shown a trend of positive relationship with high plasma viscosity in all ß-1thalassemia TDT patients. The relative results suggested that our device would be an approach tool for early detection of hypercoagulable state in transfusion-dependent-ß-thalassemia patients, which can help to prevent TEE and the critical consequent-complications.

Deferiprone-resveratrol hybrid attenuates iron accumulation, oxidative stress, and antioxidant defenses in iron-loaded human Huh7 hepatic cells.

Chronic liver diseases are complications of thalassemia with iron overload. Iron chelators are required to remove excessive iron, and antioxidants are supplemented to diminish harmful reactive oxygen species (ROS), purposing to ameliorate oxidative liver damage and dysfunctions. The deferiprone-resveratrol hybrid (DFP-RVT) is a synthetic iron chelator possessing anti-ß-amyloid peptide aggregation, anti-malarial activity, and hepatoprotection in plasmodium-infected mice. The study focuses on investigating the antioxidant, cytotoxicity, iron-chelating, anti-lipid peroxidation, and antioxidant defense properties of DFP-RVT in iron-loaded human hepatocellular carcinoma (Huh7) cells. In the findings, DFP-RVT dose dependently bound Fe(II) and Fe(III) and exerted stronger ABTS•- and DPPH•-scavenging (IC50 = 8.0 and 164 µM, respectively) and anti-RBC hemolytic activities (IC50 = 640 µM) than DFP but weaker than RVT (p < 0.01). DFP-RVT was neither toxic to Huh7 cells nor PBMCs. In addition, DFP-RVT diminished the level of redox-active iron (p < 0.01) and decreased the non-heme iron content (p < 0.01) in iron-loaded Huh7 cells effectively when compared without treatment in the order of DFP-RVT > RVT ∼ DFP treatments (50 µM each). Moreover, the compound decreased levels of hepatic ROS in a dose-dependent manner and the level of malondialdehyde, which was stronger than DFP but weaker than RVT. Furthermore, DFP-RVT restored the decrease in the GSH content and GPX and SOD activities (p < 0.01) in iron-loaded Huh7 cells in the dose-dependent manner, consistently in the order of RVT > DFP-RVT > DFP. Thus, the DFP-RVT hybrid possesses potent iron chelation, antioxidation, anti-lipid peroxidation, and antioxidant defense against oxidative liver damage under iron overload.

Iron Chelation Property, Antioxidant Activity, and Hepatoprotective Effect of 6-Gingerol-Rich Ginger (Zingiber officinale) Extract in Iron-Loaded Huh7 Cells.

Iron is essential for numerous biological processes; however, an iron imbalance can contribute to a number of diseases. An excess of iron can accumulate in the body and subsequently induce the production of reactive oxygen species (ROS), leading to oxidative tissue damage and organ dysfunction. The liver, a major iron storage site, is vulnerable to this iron-induced oxidative damage; however, this issue can be overcome by the chelation of excess iron. This study aimed to investigate the effect of 6-gingerol-rich ginger (Zingiber officinale) extract on iron chelation, antioxidation, and hepatoprotective function in protecting against iron-induced oxidative liver cell injury. In experiments, 6-gingerol was confirmed to be a main bioactive component of the ginger extract and possessed free radical scavenging activity, decreasing ABTS•+ and DPPH• radical levels, and inhibiting AAPH-induced red blood cell hemolysis. Interestingly, the extract significantly reduced the levels of labile cellular iron (LCI), intracellular ROS, and lipid peroxidation products (TBARS) in iron-loaded human hepatoma (Huh7) cells. In conclusion, this work highlights the iron chelation property of 6-gingerol-rich ginger extract and its antioxidant activity, which could potentially protect the liver from iron-induced oxidative tissue damage.

Testing the Feasibility and Dietary Impact of Macaroni Fortified with Green Tea and Turmeric Curcumin Extract in Diabetic Rats.

Macaroni is a commercially available Italian food product that is popular among consumers around the world. The supplementation of green tea extract (GTE) and turmeric curcumin extract (TCE) in macaroni may serve as promising and beneficial bioactive ingredients. We aimed to produce functional macaroni, assess the degree of consumer satisfaction and study the antidiabetic activity in diabetic rats. In this study, macaroni was fortified with GTE, TCE and a mixture of GTE and TCE ratio of 1:1, w/w (GTE/TCE). The resulting products were then analyzed in terms of their chemical compositions, while the degree of consumer satisfaction was monitored and the hypoglycemic and hypolipidemic effects in streptozotocin (STZ)-rats were investigated. GTE/TCE-M exhibited the strongest antioxidant activity (p < 0.05), while phenolics were most abundant in GTE-M. The overall preference for GTE-M, TCE-M and GTE/TCE-M were within ranges of 4.7-5.1, 5.9-6.7 and 6.2-8.2, respectively, in the nine-point hedonic scale. Consumption of these three preparations of macaroni (30 and 300 mg/kg each) neither decreased nor exacerbated increasing blood glucose levels in diabetic rats, while GTE-M (30 mg/kg) tended to lower increased serum triglyceride and cholesterol levels. In conclusion, GTE/TCE-M containing high amounts of bioactive EGCG and curcumin exerted the strongest degree of antioxidant activity and received the highest level of acceptance. Importantly, consumption of GTE-M tentatively ameliorated serum lipid abnormalities in diabetic STZ-induced rats by inhibiting lipase digestion and lipid absorption. Herein, we are proposing that GTE-fortified macaroni is a functional food that can mitigate certain metabolic syndromes.

A randomized placebo-controlled clinical trial of oral green tea epigallocatechin 3-gallate on erythropoiesis and oxidative stress in transfusion-dependent ß-thalassemia patients.

ß-Thalassemia patients suffer from ineffective erythropoiesis and increased red blood cell (RBC) hemolysis. Blood transfusion, erythropoietic enhancement, and antioxidant supplementation can ameliorate chronic anemia. Green tea extract (GTE) is comprised of catechin derivatives, of which epigallocatechin-3-gallate (EGCG) is the most abundant, presenting free-radical scavenging, iron-chelating, and erythropoiesis-protective effects. The present study aimed to evaluate the effects of GTE tablets on the primary outcome of erythropoiesis and oxidative stress parameters in transfusion-dependent ß-thalassemia (TDT) patients. Twenty-seven TDT patients were randomly divided into placebo and GTE tablet (50 and 100 mg EGCG equivalent) groups and assigned to consume the product once daily for 60 days. Blood was collected for analysis of hematological, biochemical, and oxidative stress parameters. Accordingly, consumption of GTE tablets improved blood hemoglobin levels when compared with the placebo; however, there were more responders to the GTE tablets. Interestingly, amounts of nonheme iron in RBC membranes tended to decrease in both GTE tablet groups when compared with the placebo. Importantly, consumption of GTE tablets lowered plasma levels of erythroferrone (p < 0.05) and reduced bilirubin non-significantly and dose-independently. Thus, GTE tablets could improve RBC hemolysis and modulate erythropoiesis regulators in transfusion-dependent thalassemia patients.

Perilla Fruit Oil-Fortified Soybean Milk Intake Alters Levels of Serum Triglycerides and Antioxidant Status, and Influences Phagocytotic Activity among Healthy Subjects: A Randomized Placebo-Controlled Trial.

This study aimed to develop perilla fruit oil (PFO)-fortified soybean milk (PFO-SM), identify its sensory acceptability, and evaluate its health outcomes. Our PFO-SM product was pasteurized, analyzed for its nutritional value, and had its acceptability assessed by an experienced and trained descriptive panel (n = 100) based on a relevant set of sensory attributes. A randomized clinical trial was conducted involving healthy subjects who were assigned to consume deionized water (DI), SM, PFO-SM, or black sesame-soybean milk (BS-SM) (n = 48 each, 180 mL/serving) daily for 30 d. Accordingly, health indices and analyzed blood biomarkers were recorded. Consequently, 1% PFO-SM (1.26 mg ALA rich) was generally associated with very high scores for overall acceptance, color, flavor, odor, taste, texture, and sweetness. We observed that PFO-SM lowered levels of serum triglycerides and erythrocyte reactive oxygen species, but increased phagocytosis and serum antioxidant activity (p < 0.05) when compared to SM and BS-SM. These findings indicate that PFO supplementation in soybean milk could enhance radical-scavenging and phagocytotic abilities in the blood of healthy persons. In this regard, it was determined to be more efficient than black sesame supplementation. We are now better positioned to recommend the consumption of PFO-SM drink for the reduction of many chronic diseases. Randomized clinical trial registration (Reference number 41389) by IRSCTN Registry.

Antioxidant Potential and Cytotoxic Effect of Isoflavones Extract from Thai Fermented Soybean (Thua-Nao).

Thua-nao, or Thai fermented soybeans, is a traditional Lanna fermented food in Northern Thailand. It is produced by using a specific bacterial species called Bacillus subtilis var. Thua-nao. We investigated the antioxidant activity and cytotoxic effect of isoflavones from Thua-nao. The phenolic compound contents and total flavonoid contents were determined by spectrophotometry. The antioxidant activity was examined using the ABTS, FRAP, and DPPH assays. The isoflavone contents and phenolic compositions were examined by the high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) techniques. The ability of isoflavones to inhibit human cancer cell growth was assessed by the MTT assay. The total phenolic content, total flavonoid content, and antioxidant activities of the isoflavones were 49.00 ± 0.51 mg GAE/g of dry extract (DE), 10.76 ± 0.82 mg QE/g of DE, 61.03 ± 0.97 µmol Trolox/g of DE, 66.54 ± 3.97 µM FeSO4/g of DE, and 22.47 ± 1.92% of DPPH inhibition, respectively. Additionally, the isoflavone extracts from Thua-nao had high isoflavone contents and polyphenolic compound compositions, especially daidzein and genistein. The isoflavone demonstrated a weak inhibition of MCF-7 and HEK293 cancer cell growth. It has a high antioxidant component, which is beneficial and can be developed for new therapeutic uses. However, further studies on the benefits of Thua-nao should be performed for realizing better and more effective uses soon.

Protection of Iron-Induced Oxidative Damage in Neuroblastoma (SH-SY5Y) Cells by Combination of 1-(N-Acetyl-6-aminohexyl)-3-hydroxy-2-methylpyridin-4-one and Green Tea Extract.

Iron is a crucial trace element and essential for many cellular processes; however, excessive iron accumulation can induce oxidative stress and cell damage. Neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease, have been associated with altered iron homoeostasis causing altered iron distribution and accumulation in brain tissue. This study aims to investigate the protective effect of 1-(N-acetyl-6-aminohexyl)-3-hydroxy-2-methylpyridin-4-one (CM1) in combination with green tea extract (GTE) on iron-induced oxidative stress in neuroblastoma (SH-SY5Y) cells. Cells were cultured in medium with or without ferric chloride loading. Their viability and mitochondrial activity were assessed using MTT and JC-1 staining methods. Levels of the cellular labile iron pool (LIP), reactive oxygen species (ROS), and lipid-peroxidation products were determined using calcein acetoxymethyl ester, 2',7'-dichlorohydrofluorescein diacetate, and TBARS-based assays, respectively. The viability of iron-loaded cells was found to be significantly increased after treatment with CM1 (10 µM) for 24 h. CM1 co-treatment with GTE resulted in a greater protective effect than their monotherapy. Combination of CM1 and GTE also reduced mitochondrial disruption and LIP content and ROS and TBARS production. In conclusion, the combination of CM1 and GTE exhibits protection against iron-induced oxidative stress in neuroblastoma cells.

Analgesic, anti-inflammatory and anti-ulcer properties of Thai Perilla frutescence fruit oil in animals.

Perilla frutescens fruit oil (PFO) is rich in α-linolenic acid (ALA) and exhibits biological activities. We aimed to investigate analgesic, anti-inflammatory and anti-ulcer activities of PFO and PFO-supplemented soybean milk (PFO-SM) in animal models. Analgesic activity was assessed in acetic acid-induced writhing in mice, while anti-inflammatory activity was performed in ethyl phenylpropiolate (EPP)-induced ear edema and carrageenan-induced hind paw edema in rats. Anti-ulcer effects were conducted in water immersion stress, HCl/ethanol and indomethacin-induced gastric ulcer in rats. Distinctly, PFO, containing 6.96 mg ALA and 2.61 mg LA equivalence/g, did not induce acute toxicity (LD50 > 10 mL/kg) in mice. PFO (2.5 and 5 mL/kg) and PFO-SM (0.05 mL PFO equivalence/kg) inhibited incidences of writhing (16.8, 18.0 and 32.3%, respectively) in acetic acid-induced mice. In addition, topical applications of PFO (0.1 and 1 mL/ear) significantly inhibited EPP-induced ear edema (59.3 and 65.7%, respectively) in rats, while PFO-SM slightly inhibited ear edema (25.9%). However, PFO and PFO-SM did not inhibit carrageenan-induced hind paw edema in rats. Indeed, PFO (2.5 and 5 mL/kg) significantly inhibited gastric ulcers in rats that induced by water immersion stress (92.4 and 96.6%, respectively), HCl/ethanol (74.8 and 73.3%, respectively) and indomethacin (68.8 and 88.9%, respectively), while PFO-SM did not. PFO displayed potent analgesic, anti-inflammatory and anti-ulcer properties, while PFO-SM exerted only analgesic properties. Thus, Thai PFO and its functional drink offer potential benefits in treatment of analgesic, inflammatory diseases and gastric ulcer.

Extracts of Thai Perilla frutescens nutlets attenuate tumour necrosis factor-α-activated generation of microparticles, ICAM-1 and IL-6 in human endothelial cells.

Elevation of endothelial microparticles (EMPs) play an important role in the progression of inflammation-related vascular diseases such as cardiovascular diseases (CVDs). Thai perilla (Perilla frutescens) nutlets are rich in phenolic compounds and flavonoids that exert potent antioxidant and anti-inflammatory effects. We found that the ethyl acetate (EA) and ethanol (Eth) extracts of Thai perilla nutlets contain phenolic compounds such as luteolin, apigenin, chryseoriol and their glycosides, which exhibit antioxidant activity. The goal of the present study was to investigate the effects of the extracts on endothelial activation and EMPs generation in tumour necrosis factor-α (TNF-α)-induced EA.hy926 cells. We found that TNF-α (10 ng/ml) activated EA.hy926 cells and subsequently generated EMPs. Pre-treatment with the extracts significantly attenuated endothelial activation by decreasing the expression of the intracellular adhesion molecule-1 (ICAM-1) in a dose-dependent manner. Only the Eth extract showed protective effects against overproduction of interleukin-6 (IL-6) in the activated cells. Furthermore, the extracts significantly reduced TNF-α-enhanced EMPs generation in a dose-dependent manner. In conclusion, Thai perilla nutlet extracts, especially the Eth extract, may have potential to protect endothelium against vascular inflammation through the inhibition of endothelial activation and the generation of endothelial microparticles (EMPs).

Thai Perilla frutescens fruit oil alleviates carbon tetrachloride-induced hepatotoxicities in rats

Objective: To study the effect of perilla fruit oil against carbon tetrachloride (CCl4)-induced liver damage in rats. Methods: Perilla fruit oil was analyzed in terms of fatty acids, tocopherols and tocotrienols using chromatography. Sub-chronic toxicity of perilla fruit oil was investigated in rats for 90 d followed by a 28 d recovery period. Hematological, biochemical and pathological parameters were determined. To evaluate hepatoprotection, rats were divided into five groups and orally administered with Tween 80 for 10 d; Tween 80, silymarin, perilla fruit oil (0.1 mL/200 g) and perilla fruit oil (1 mL/200 g) for 10 d together with subcutaneous injection of CCl4 (2 mL/200 g) on days 9 and 10. Liver enzymes and pathological parameters were determined. Results: Perilla fruit oil contained α-linolenic acid (56.55％ of total fatty acid), β-tocopherol (49.50 mg/kg) and γ-tocotrienol (43.65 mg/kg). Rats showed significant changes in the percentage of monocytes and platelet indices following perilla fruit oil consumption for 90 d; in the percentage of neutrophils and lymphocytes, and RBC indices in the recovery period when compared with the deionized water group. Total protein and creatinine levels were increased while alkaline phosphatase and aspartate aminotransferase levels were decreased (P < 0.05). Organ weight index and pathological indicators did not change significantly. The liver of CCl4-induced rats showed remarkable centrilobular fatty changes, which was ameliorated by perilla fruit oil pretreatment. Aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase levels were decreased (P < 0.05) in rats given perilla fruit oil. Conclusions: Perilla fruit oil is rich in α-linolenic acid, β-tocopherol and γ-tocotrienol and improves blood biomarker levels and protects against CCl4-induced hepatotoxicity. Further studies are required before supporting its use for the treatment of hepatitis.

A chemically characterized ethanolic extract of Thai Perilla frutescens (L.) Britton fruits (nutlets) reduces oxidative stress and lipid peroxidation in human hepatoma (HuH7) cells.

Perilla frutescens is cultivated in East Asian countries including Thailand, and the nutlets (single-seeded fruits) are used as traditional and medicinal food. Perilla nutlets extracted by ethyl acetate (EA), 80% ethanol (Eth), and hot water (HW) sequentially were chemically characterized using high-resolution accurate liquid chromatography-mass spectrometry with the main compounds detected assigned as rosmarinic acid and derivatives of the flavones apigenin and luteolin, with the more diverse chemical composition observed with the Eth extract. All extracts showed dose-dependent free-radical scavenging activity, with the Eth extract the most potent (IC50  = 3.43 mg/ml for ABTS• scavenging and 0.27 mg/ml for DPPH• scavenging). The Eth extract also inhibited AAPH-induced hemolysis (IC50  = 0.07 mg/ml) more potently than did the HW (IC50  = 0.38 mg/ml) and EA extracts (IC50  = 1.63 mg/ml). An MTT test revealed all the extracts were noncytotoxic at concentrations up to 200 µg/ml. Only the Eth and EA extracts showed protective effects against the generation of reactive oxygen species and lipid peroxidation in FeCl3 -induced HuH7 cells in a dose-dependent manner. Our findings suggest the Eth extract of Thai perilla nutlets, containing rosmarinic acid and flavones and their derivatives, may have potential to provide protection against oxidative stress in hepatic disorders.

Anti-hemolytic, antibacterial and anti-cancer activities of methanolic extracts from leaves and stems of Polygonum odoratum

To investigate anti-hemolytic, antibacterial and anti-cancer activities of leaf and stem extracts fromPolygonum odoratum.Methods: Leaves and stems ofPolygonum odoratum were extracted using methanol and their anti-hemolytic activity was assessed using 2, 2′-Azobis (2-methylpropionamidine) dihydrochloride which is known to generate free radical damage on cell membranes of red blood cells. This damage, represented by hemolysis, was measured using spectrophotometry. Antibacterial activity was tested by using a broth microdilution method to find minimal inhibitory concentrations against eight bacterial strains. Anti-cancer activity of the extracts was evaluated against a human promyelocytic leukemic cell line (HL-60) by using MTT assay for cell viability and flow cytometry for apoptosis induction and cell cycle analysis.Results: Both leaf and stem extracts have anti-hemolytic activity. The results showed a significantly increased percentage of inhibition in a concentration-dependent manner. Interestingly, the leaf extract showed anti-hemolytic activity to a greater extent than the stem extract. Antibacterial activity of the extracts, as indicated by their minimal inhibitory concentration, using 12.5, 50, 25, 25 μg/mL, was measured againstStaphylococcus epidermidis, Enterococcus faecium,Enterococcus faecalisand Staphylococcus aureus. The leaf extracts also exhibited anti-cancer activity, demonstrated by significantly decreased cell viability of human promyelocytic cells (HL-60), with an IC50 of (350.00±1.85) μg/mL for 48 h and (38.00±0.92) μg/mL for 72 h. Additionally, HL-60 became apoptotic and accumulated in G1-phase after 48 hours of treatment.Conclusions: The extracts ofPolygonum odoratum exhibit potential anti-hemolytic activity. They also have antibacterial activity by inhibiting growth of Gram-positive bacteria. The leaf extract shows anti-cancer activity against HL-60 to a greater extent than the stem extract, causing decreased viability, increased G1-phase accumulation and apoptosis induction.