Kinetic Study of Subcritical Water Extraction of Scopoletin, Alizarin, and Rutin from Morinda citrifolia.

Noni fruits (Morinda citrifolia) are a source of phenolic bioactive compounds (scopoletin, alizarin, and rutin), which have antioxidant, antimicrobial, anticancer, and anti-inflammatory activities. In this study, subcritical water was applied to determine the extraction yields and kinetics of phenolic compounds from noni fruits. The scopoletin and alizarin yields increased with the increase in temperature from 100 to 140 °C, while that of rutin increased up to 120 °C and then decreased at 140 °C. The yields of all the compounds rapidly increased from 1 to 2 mL/min and then slightly up to 3 mL/min of water flow rate. The extraction kinetics were assessed using two mathematical models. The two-site kinetic desorption model had a better fit for all experimental conditions throughout the extraction cycle and best described the extraction kinetics of phenolic compounds from noni fruits. The diffusion coefficients of scopoletin and alizarin at 140 °C and 3 mL/min were 3.7- and 16.2-fold higher than those at 100 °C and 1 mL/min, respectively. The activation energies of alizarin were 2.9- to 8.5-fold higher than those of scopoletin at various flow rates. Thus, subcritical water could be an excellent solvent with higher extraction yields and shorter extraction times using an environmentally friendly solvent.

Organic Acid-Catalyzed Subcritical Water Hydrolysis of Immature Citrus unshiu Pomace.

Immature Citrus unshiu pomace (ICUP) was hydrolyzed under organic acid-catalyzed, subcritical water (SW) conditions to produce flavonoid monoglucosides (hesperetin-7-O-glycoside and prunin) and aglycons (hesperetin and naringenin) with high biological activities. The results of single-factor experiments showed that with 8 h of hydrolysis and an increasing citric acid concentration, the yield of flavonoid monoglucosides (hesperetin-7-O-glycoside and prunin) increased from 0 to 7% citric acid. Afterward, the hesperetin-7-O-glycoside yield remained constant (from 7 to 19% citric acid) while the pruning yield decreased with 19% of citric acid, whereas the aglycon yield increased continuously. In response surface methodology analysis, a citric acid concentration and hydrolysis duration of 13.34% and 7.94 h were predicted to produce the highest monoglucoside yield of 15.41 mg/g, while 18.48% citric acid and a 9.65 h hydrolysis duration produced the highest aglycon yield of 10.00 mg/g. The inhibitory activities of the SW hydrolysates against pancreatic lipase (PL) and xanthine oxidase (XO) were greatly affected by citric acid concentration and hydrolysis duration, respectively. PL and α-glucosidase inhibition rates of 88.2% and 62.7%, respectively, were achieved with 18.48% citric acid and an 8 h hydrolysis duration, compared to 72.8% for XO with 16% citric acid and 12 h of hydrolysis. This study confirms the potential of citric acid-catalyzed SW hydrolysis of ICUP for producing flavonoid monoglucosides and aglycons with enhanced enzyme inhibitory activities.

Extraction of flavanones from immature Citrus unshiu pomace: process optimization and antioxidant evaluation.

Dietary guidelines recommend the consumption of flavonoid-rich extracts for several health benefits. Although immature Citrus unshiu pomace (ICUP) contains high levels of flavanone glycosides, many studies have concentrated on the optimization of flavonoid extraction from mature citrus peels. Therefore, we developed an optimized extraction method for hesperidin and narirutin from ICUP, and evaluated their antioxidant activities using ten different assay methods. The extraction conditions for the highest flavonoid yields based on a response surface methodology were 80.3 °C, 58.4% (ethanol concentration), 40 mL/g (solvent/feed), and 30 min, where the hesperidin and narirutin yields were 66.6% and 82.3%, respectively. The number of extractions was also optimized as two extraction steps, where the hesperidin and narirutin yields were 92.1% and 97.2%, respectively. Ethanol was more effective than methanol and acetone. The ethanol extract showed high scavenging activities against reactive oxygen species but relatively low scavenging activities for nitrogen radicals and reactive nitrogen species. The antioxidant activities showed a higher correlation with hesperidin content than narirutin content in the extracts. This study confirms the potential of an optimized method for producing antioxidant-rich extracts for the functional food and nutraceutical industries.

Semi-Continuous Subcritical Water Extraction of Flavonoids from Citrus unshiu Peel: Their Antioxidant and Enzyme Inhibitory Activities.

We extracted and hydrolyzed bioactive flavonoids from C. unshiu peel using subcritical water (SW) in a semi-continuous mode. The individual flavonoid yields, antioxidant and enzyme inhibitory activities of the SW extracts were analyzed. The extraction yields of hesperidin and narirutin increased with increasing temperature from 145 °C to 165 °C. Hydrothermal hydrolysis products (HHP), such as monoglucosides (hesperetin-7-O-glucoside and prunin) and aglycones (hesperetin and naringenin) were obtained in the SW extracts at temperatures above 160 °C. The sum of hesperidin and its HHP in the SW extracts was strongly correlated with antioxidant activities, whereas the contents of hesperetin and naringenin were strongly correlated with enzyme inhibitory activities. Hesperetin exhibited the highest antioxidant activities (2,2-diphenyl-1-picrylhydrazyl radical scavenging activity, ferric-reducing antioxidant power, and oxygen radical absorbance capacity), whereas hesperetin-7-O-glucoside exhibited the highest enzyme inhibitory activities (angiotensin-І converting enzyme (ACE) and pancreatic lipase (PL)). Naringenin exhibited the highest enzyme inhibitory activities (xanthine oxidase and α-glucosidase). PMFs (sinensetin, nobiletin, and tangeretin) also exhibited relatively high inhibitory activities against ACE and PL. This study confirms the potential of SW for extracting and hydrolyzing bioactive flavonoids from C. unshiu peel using an environmentally friendly solvent (water) and a shorter extraction time.

Ishophloroglucin A, a Novel Phlorotannin for Standardizing the Anti-α-Glucosidase Activity of Ishige okamurae.

Nutraceutical use of algae requires understanding of the diversity and significance of their active compositions for intended activities. Ishige okamurae (I. okamurae) extract is well-known to possess α-glucosidase inhibitory activity; however, studies are needed to investigate its active composition in order to standardize its α-glucosidase inhibitory activity. In this study, we observed the intensity of the dominant compounds of each I. okamurae extract harvested between 2016 and 2017, and the different potency of each I. okamurae extract against α-glucosidase. By comparing the anti-α-glucosidase ability of the dominant compounds, a novel Ishophloroglucin A with highest α-glucosidase inhibitory activity was identified and suggested for standardization of anti-α-glucosidase activity in I. okamurae extract. Additionally, a validated analytical method for measurement of Ishophloroglucin A for future standardization of I. okamurae extract was established in this study. We suggest using Ishophloroglucin A to standardize anti-α-glucosidase potency of I. okamurae and propose the significance of standardization based on their composition for effective use of algae as marine-derived nutraceuticals.

Optimization of Subcritical Water Hydrolysis of Rutin into Isoquercetin and Quercetin.

Maximum production of isoquercetin and quercetin simultaneously from rutin by subcritical water hydrolysis (SWH) was optimized using the response surface methodology. Hydrolysis parameters such as temperature, time, and CO2 pressure were selected as independent variables, and isoquercetin and quercetin yields were selected as dependent variables. The regression models of the yield of isoquercetin and quercetin were valid due to the high F-value and low P-value. Furthermore, the high regression coefficient indicated that the polynomial model equation provides a good approximation of experimental results. In maximum production of isoquercetin from rutin, the hydrolysis temperature was the major factor, and the temperature or time can be lower if the CO2 pressure was increased high enough, thereby preventing the degradation of isoquercetin into quercetin. The yield of quercetin was considerably influenced by temperature instead of time and CO2 pressure. The optimal condition for maximum production of isoquercetin and quercetin simultaneously was temperature of 171.4°C, time of 10.0 min, and CO2 pressure of 11.0 MPa, where the predicted maximum yields of isoquercetin and quercetin were 13.7% and 53.3%, respectively. Hydrolysis temperature, time, and CO2 pressure for maximum production of isoquercetin were lower than those of quercetin. Thermal degradation products such as protocatechuic acid and 2,5-dihydroxyacetophenone were observed due to pyrolysis at high temperature. It was concluded that rutin can be easily converted into isoquercetin and quercetin by SWH under CO2 pressure, and this result can be applied for SWH of rutin-rich foodstuffs.

Immunostimulatory Activity of Opuntia ficus-indica var. Saboten Cladodes Fermented by Lactobacillus plantarum and Bacillus subtilis in RAW 264.7 Macrophages.

To increase the functionality of Opuntia ficus-indica var. saboten cladodes, it was fermented by Lactobacillus plantarum and Bacillus subtilis. Eighty percent methanol extracts were investigated for their effects on nitric oxide (NO) production, cytokine secretion, nuclear factor-κB (NF-κB) activity, and mitogen-activated protein kinase (MAPK) phosphorylation in RAW 264.7 cells. Methanol extracts of L. plantarum culture medium (LPCME) and B. subtilis culture medium (BSCME) did not affect lipopolysaccharide (LPS)-induced NO production but, at 500 µg/mL, increased interferon (IFN)-γ-induced NO production by 55.2 and 66.5 µM, respectively, in RAW 264.7 cells. In RAW 264.7 cells not treated with LPS and IFN-γ, LPCME did not affect NO production, but BSCME increased NO production significantly in a dose-dependent manner. In addition, BSCME induced the expression of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) in RAW 264.7 cells in a dose-dependent manner. BSCME at 500 µg/mL increased TNF-α and IL-1ß mRNA levels by 83.8% and 82.2%, respectively. BSCME increased NF-κB-dependent luciferase activity in a dose-dependent manner; 500 µg/mL BSCME increased activity 9.1-fold compared with the control. BSCME induced the phosphorylation of p38, c-JUN NH2-terminal protein kinase (JNK), and extracellular signal-regulated kinase (ERK) in a dose-dependent manner, but did not affect total ERK levels. In conclusion, BSCME exerted immunostimulatory effects, which were mediated by MAPK phosphorylation and NF-κB activation, resulting in increased TNF-α and IL-1ß gene expression in RAW 264.7 macrophages. Therefore, BSCM shows promise for use as an immunostimulatory therapeutic.

Enhancement of Phenolic Production and Antioxidant Activity from Buckwheat Leaves by Subcritical Water Extraction.

To enhance the production of phenolic compounds with high antioxidant activity and reduce the level of phototoxic fagopyrin, buckwheat leaves were extracted with subcritical water (SW) at 100~220°C for 10~50 min. The major phenolic compounds were quercetin, gallic acid, and protocatechuic acid. The cumulative amount of individual phenolic compounds increased with increasing extraction temperature from 100°C to 180°C and did not change significantly at 200°C and 220°C. The highest yield of individual phenolic compounds was 1,632.2 µg/g dry sample at 180°C, which was 4.7-fold higher than that (348.4 µg/g dry sample) at 100°C. Total phenolic content and total flavonoid content increased with increasing extraction temperature and decreased with increasing extraction time, and peaked at 41.1 mg gallic acid equivalents/g and 26.9 mg quercetin equivalents/g at 180°C/10 min, respectively. 2,2-Diphenyl-1-picrylhydrazyl free radical scavenging activity and ferric reducing ability of plasma reached 46.4 mg ascorbic acid equivalents/g and 72.3 mmol Fe2+/100 g at 180°C/10 min, respectively. The fagopyrin contents were reduced by 92.5~95.7%. Color values L* and b* decreased, and a* increased with increasing extraction temperature. SW extraction enhanced the yield of phenolic compounds with high antioxidant activity and reduced the fagopyrin content from buckwheat leaves.

Relationship between Chemical Structure and Antimicrobial Activities of Isothiocyanates from Cruciferous Vegetables against Oral Pathogens.

We evaluated the potentials of 10 isothiocyanates (ITCs) from cruciferous vegetables and radish root hydrolysate for inhibiting the growth of oral pathogens, with an emphasis on assessing any structure-function relationship. Structural differences in ITCs impacted their antimicrobial activities against oral pathogens differently. The indolyl ITC (indol-3-carbinol) was the most potent inhibitor of the growth of oral pathogens, followed by aromatic ITCs (benzyl ITC (BITC) and phenylethyl ITC (PEITC)) and aliphatic ITCs (erucin, iberin, and sulforaphene). Sulforaphene, which is similar in structure, but has one double bond, showed higher antimicrobial activity than sulforaphane. Erucin, which has a thiol group, showed higher antimicrobial activity than sulforaphane, which has a sulfinyl group. BITC and iberin with a short chain exhibited higher antimicrobial potential than PEITC and sulforaphane with a longer chain, respectively. ITCs have strong antimicrobial activities and may be useful in the prevention and management of dental caries.

Enhanced Production of Phenolic Compounds from Pumpkin Leaves by Subcritical Water Hydrolysis.

Enhanced production of individual phenolic compounds by subcritical water hydrolysis (SWH) of pumpkin leaves was investigated at various temperatures ranging from 100 to 220°C at 20 min and at various reaction times ranging from 10 to 50 min at 160°C. Caffeic acid, p-coumaric acid, ferulic acid, and gentisic acid were the major phenolic compounds in the hydrolysate of pumpkin leaves. All phenolic compounds except gentisic acid showed the highest yield at 160°C, but gentisic acid showed the highest yield at 180°C. The cumulative amount of individual phenolic compounds gradually increased by 48.1, 52.2, and 78.4 µg/g dry matter at 100°C, 120°C, and 140°C, respectively, and then greatly increased by 1,477.1 µg/g dry matter at 160°C. The yields of caffeic acid and ferulic acid showed peaks at 20 min, while those of cinnamic acid, p-coumaric acid, p-hydroxybenzoic acid, and procatechuic acid showed peaks at 30 min. Antioxidant activities such as 2,2-diphenyl-1-picrylhydrazyl and ferric reducing antioxidant power values gradually increased with hydrolysis temperature and ranged from 6.77 to 12.42 mg ascorbic acid equivalents/g dry matter and from 4.25 to 8.92 mmol Fe(2+)/100 g dry matter, respectively. Color L* and b* values gradually decreased as hydrolysis temperature increased from 100°C to 140°C. At high temperatures (160°C to 220°C), L* and b* values decreased suddenly. The a* value peaked at 160°C and then decreased as temperature increased from 160°C to 220°C. These results suggest that SWH of pumpkin leaves was strongly influenced by hydrolysis temperature and may enhanced the production of phenolic compounds and antioxidant activities.

Formation and Stabilization of Raphasatin and Sulforaphene from Radish Roots by Endogenous Enzymolysis.

The biologically active compounds raphasatin and sulforaphene are formed during the hydrolysis of radishes by an endogenous myrosinase. Raphasatin is very unstable, and it is generated and simultaneously degraded to less active compounds during hydrolysis in aqueous media. This study determined the hydrolysis conditions to maximize the formation of raphasatin and sulforaphene by an endogenous myrosinase and minimize their degradation during the hydrolysis of radish roots. The reaction parameters, such as the reaction medium, reaction time, type of mixing, and reaction temperature were optimized. A stability test for raphasatin and sulforaphene was also performed during storage of the hydrolyzed products at 25°C for 10 days. The formation and breakdown of raphasatin and sulforaphene in radish roots by endogenous enzymolysis was strongly influenced by the reaction medium, reaction time, and type of mixing. The production and stabilization of raphasatin in radishes was efficient in water and dichloromethane with shaking for 15 min at 25°C. For sulforaphene, the favorable condition was water as the reaction medium without shaking for 10 min at 25°C. The maximum yields of raphasatin and sulforaphene were achieved in a concurrent hydrolysis reaction without shaking in water for 10 min and then with shaking in dichloromethane for 15 min at 25°C. Under these conditions, the yields of raphasatin and sulforaphene were maximized at 12.89 and 1.93 µmol/g of dry radish, respectively. The stabilities of raphasatin and sulforaphene in the hydrolyzed products were 56.4% and 86.5% after 10 days of storage in water and dichloromethane at 25°C.

Antioxidant and anticancer activities of broccoli by-products from different cultivars and maturity stages at harvest.

Many studies on broccoli have analyzed the functional components and their functionality in terms of antioxidant and anticancer activities; however, these studies have focused on the florets of different varieties. Investigation of the functionality of broccoli by-products such as leaves, stems, and leaf stems from different cultivars and harvest dates might be valuable for utilizing waste materials as useful food components. Total phenolics and sulforaphane contents, and antioxidant and anticancer activities were measured in the leaves, leaf stems, and stems of early-maturing (Kyoyoshi), middle-maturing (Myeongil 96), and late-maturing broccoli (SK3-085) at different harvest dates. Total phenolics in the leaves of Kyoyoshi were about 1.8-fold to 12.1-fold higher than those in all of the other cultivars and parts. The sulforaphane content of Kyoyoshi was 2.8-fold higher in the stems than in the florets. Antioxidant activities using 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity and oxygen radical absorbance capacity were highest in Kyoyoshi, followed by Myeongil 96 and SK3-085, most notably in the leaves harvested at the immature stage. Inhibition activity of cell growth against the NCI-H1299 cell lines was highest in the leaves of all cultivars in decreasing order of florets, leaf stems, and stems. The leaves harvested in October (nonflowering stage) had the highest inhibition activity, while those harvested in January (mature broccoli) showed the lowest. The results of this study demonstrate that broccoli leaves and stems contain high levels of total phenolics, and high antioxidant and anticancer activities and can provide opportunities for early-maturing broccoli as functional fresh raw vegetables.

Antioxidant and Anti-inflammatory Activities of Broccoli Florets in LPS-stimulated RAW 264.7 Cells.

Broccoli (Brassica oleracea var. italia) florets were extracted with 80% methanol and the extract was sequentially fractionated with n-hexane, ethyl acetate, n-butanol, and distilled water. The extract and the fractions were evaluated for total phenolic content, sulforaphane content, antioxidant activity, and anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. The total phenolic content and sulforaphane content of the ethyl acetate fraction (EF) were 35.5 mg gallic acid equivalents/g and 620.2 µg/g, respectively. These values were higher than those of the 80% methanol extract and organic solvent fractions. The oxygen radical absorbance capacity of the EF [1,588.7 µM Trolox equivalents (TE)/mg] was 11-fold higher than that of the distilled water fraction (143.7 µM TE/mg). The EF inhibited nitric oxide release from LPS-stimulated RAW 264.7 cells in a dose-dependent manner and inhibited IκB-α degradation and nuclear factor-κB activation in LPS-stimulated RAW 264.7 cells. In conclusion, the EF of broccoli florets exerted potent antioxidant and anti-inflammatory effects.

Effects of selected plant extracts on anti-oxidative enzyme activities in rats.

The effects of plant extracts on the activity of anti-oxidative enzymes in rats were investigated. Sprague-Dawley rats were fed with the extracts and fractions from Rhus javanica, Malus sieboldii, and Ostrya japonica, and the activities of anti-oxidative enzymes, such as superoxide dismutase (SOD) and catalase (CAT) were investigated. SOD activities in blood serum and liver increased following an injection of 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH), and these increases were not cancelled out by feeding plant extracts. CAT activities in blood serum and liver increased following the AAPH injection, but these increases were reversed by feeding the extracts of R. javanica and O. japonica. This finding was similar to the result that the CAT activity increase induced by feeding alcohol was cancelled out by feeding ascorbic acid. These results suggest that anti-oxidative polyphenolics from these plants may act through the same mechanism as that of the well-known antioxidant ascorbic acid towards hydrogen peroxide.

Aquimarina litoralis sp. nov., isolated from a coastal seawater.

A strictly aerobic, red-pigmented, non-motile, catalase- and oxidase-positive, Gram-staining-negative bacterium, designated strain CNURIC011(T), was isolated from seawater off the coast of Jeju Island in Korea. A phylogenetic analysis based on 16S rRNA gene sequences showed that strain CNURIC011(T) belongs to the genus Aquimarina in the family Flavobacteriaceae. 16S rRNA gene sequence analysis revealed that the close relatives of the novel strain are Aquimarina latercula ATCC 23177(T), Aquimarina marcrocephali JAMB N27(T), Aquimarina intermedia KMM 6258(T), Aquimarina muelleri KMM 6020(T), and Aquimarina brevivitae SMK-19(T), with sequence similarities of 97.6, 96.6, 96.0, 95.6, and 94.2%, respectively. DNA-DNA hybridization revealed that the level of relatedness between strain CNURIC011(T) and Aquimarina latercula ATCC 23177(T) (=KCTC 2912(T)) was 4.9%. The DNA G+C content was 35.8 mol% and the major respiratory quinone was MK-6. The major fatty acids were iso-C(15:0) (14.9%), C(15:0) (13.9%), iso-C(17:0) 3-OH (12.6%), iso-C(15:1) G (7.3%), and iso-C(17:1) omega9c (7.2%). On the basis of phenotypic, phylogenetic, and genotypic data, strain CNURIC011(T) represents a novel species within the genus Aquimarina, for which the name Aquimarina litoralis sp. nov. is proposed. The type strain is CNURIC011(T) (=KCTC 22614(T) =JCM 15974(T)).

Gaetbulibacter jejuensis sp. nov., isolated from seawater.

A novel marine bacterium, designated strain CNURIC014(T) was isolated from coastal seawater of Jeju Island in Korea. Strain CNURIC014(T) formed yellow colonies on marine agar 2216 and the cells were Gram-negative, non-motile, strictly aerobic, rod-shaped. The temperature, pH and NaCl ranges for growth were 15-37 degrees C, pH 6.0-9.0 and 1.0-7.0% NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain CNURIC014(T) was most closely related to Gaetbulibacter marinus and Gaetbulibacter saemankumensis, with a sequence similarity of 95.1% and 94.6%, respectively. The DNA G+C content of the strain was 33.1 mol% and the major respiratory quinone was menaquinone-6. The major cellular fatty acids were iso-C(15:1) (22.8%), iso-C(15:0) (18.8%), summed feature 3 (iso-C(15:0) 2-OH/C(16:1) omega 7c, 12.9%) and iso-C(17:0) 3-OH (11.5%). On the basis of phenotypic, phylogenetic, and genotypic data, strain CNURIC014(T) represents a novel species within the genus Geatbulibacter, for which the name Gaetbulibacter jejuensis sp. nov. is proposed. The type strain is CNURIC014(T)(=KCTC 22615(T) =JCM 15976(T)).