Enzymatic synthesis of ß-d-fructofuranosyl α-d-glucopyranosyl-(1→2)-α-d-glucopyranoside using Escherichia coli glycoside phosphorylase YcjT.

YcjT is a kojibiose phosphorylase found in Escherichia coli. We found that sucrose was a good acceptor of YcjT in reverse phosphorolysis using ß-d-glucose 1-phosphate as a donor. The product was identified as ß-d-fructofuranosyl α-d-glucopyranosyl-(1→2)-α-d-glucopyranoside. This sugar was also synthesized from sucrose and maltose using YcjT and maltose phosphorylase and promoted the growth of the probiotic Bifidobacterium breve.

Functional characterization of a novel GH94 glycoside phosphorylase, 3-O-ß-d-glucopyranosyl ß-d-glucuronide phosphorylase, and implication of the metabolic pathway of acidic carbohydrates in Paenibacillus borealis.

Glycoside hydrolase family 94 (GH94) contains enzymes that reversibly catalyze the phosphorolysis of ß-glycosides. We conducted this study to investigate a GH94 protein (PBOR_13355) encoded in the genome of Paenibacillus borealis DSM 13188 with low sequence identity to known phosphorylases. Screening of acceptor substrates for reverse phosphorolysis in the presence of α-d-glucose 1-phosphate as a donor substrate showed that PBOR_13355 utilized d-glucuronic acid and p-nitrophenyl ß-d-glucuronide as acceptors. In the reaction with d-glucuronic acid, 3-O-ß-d-glucopyranosyl-d-glucuronic acid was synthesized. PBOR_13355 showed a higher apparent catalytic efficiency to p-nitrophenyl ß-d-glucuronide than to d-glucuronic acid, and thus, PBOR_13355 was concluded to be a novel glycoside phosphorylase, 3-O-ß-d-glucopyranosyl ß-d-glucuronide phosphorylase. PBOR_13360, encoded by the gene immediately downstream of the PBOR_13355 gene, was shown to be ß-glucuronidase. Collectively, PBOR_13355 and PBOR_13360 are predicted to work together in the cytosol to metabolize oligosaccharides containing the 3-O-ß-d-glucopyranosyl ß-d-glucuronide structure released from bacterial and plant acidic carbohydrates.

The Immunomodulating Effect of Phlorotannins from a Brown Alga, Eisenia nipponica, on Mice Stimulated with Ovalbumin through T Cell Regulation.

The immunomodulating effect of phlorotannin was investigated in mice stimulated by ovalbumin. When analyzing the main components of phlorotannin concentrate (PTC) from Eisenia nipponica, seven phlorotannins [eckol, 6,6'-bieckol, 6,8'-bieckol, 8,8'-bieckol, dieckol, phlorofucofuroeckol (PFF)-A, and PFF-B] were detected. These phlorotannins accounted for approximately 80% of PTC. Oral administration of PTC to mice daily for 21 days reduced serum immunoglobulin E (IgE) and total IgG1 levels attributable to Th2 cells. The production of splenic cytokines [interleukin (IL)-10 and transforming growth factor-ß1] and Treg cell-mediated expression of forkhead box protein P3 mRNA were significantly increased whereas the production of inflammatory cytokines (interferon-γ, IL-4, IL-5, and IL-17) by Th1, Th2, and Th17 cells was markedly suppressed. IL-21 production and basic leucine zipper ATF-like transcription factor mRNA expression attributable to follicular helper T (Tfh) cells were also suppressed. Flow cytometric analyses demonstrated increased number of Treg cells despite a decrease in the total T cell population. An increase in total B cells was also observed by the flow cytometric analyses in addition to increases in IL-10 production, which activates B cells. In contrast, the significantly suppressed production of inflammatory cytokines and moderate increase in Treg cell subpopulation indicated a direct impact of PTC on inflammatory lymphocytes (Th1, Th2, Th17, and Tfh). Thus, PTC may exert antiallergic effects by immunomodulation of T cells and inactivation of inflammatory lymphocyte.

Zebrafish obesogenic test identifies anti-adipogenic fraction in Moringa oreifera leaf extracts.

The zebrafish obesogenic test (ZOT) is a powerful tool for identifying anti-adipogenic compounds for in vivo screening. In our previous study, we found that Moringa oleifera (MO) leaf powder suppressed the accumulation of visceral adipose tissue (VAT) in ZOT. MO demonstrates a wide range of pharmacological effects; however, little is known about its functional constituents. To identify the anti-adipogenic components of MO leaves, we prepared extracts using different extraction methods and tested the obtained extracts and fractions using ZOT. We found that the dichloromethane extract and its hexane:EtOAc = 8:2 fraction reduced VAT accumulation in young zebrafish fed a high-fat diet. We also performed gene expression analysis in the zebrafish VAT and found that CCAAT/enhancer-binding protein beta and CCAAT/enhancer-binding protein delta (associated with early stages of adipogenesis) gene expression was downregulated after fraction 2 administration. We identified a new MO fraction that suppressed VAT accumulation by inhibiting early adipogenesis using the ZOT. Phenotype-driven zebrafish screening is a reasonable strategy for identifying bioactive components in natural products.

Preventive Effects of Green Tea Extract against Obesity Development in Zebrafish.

Various natural products (NPs) have been used to treat obesity and related diseases. However, the best way to fight obesity is preventive, with accurate body weight management through exercise, diet, or bioactive NPs to avoid obesity development. We demonstrated that green tea extract (GTE) is an anti-obesity NP using a zebrafish obesity model. Based on a hypothesis that GTE can prevent obesity, the objective of this study was to assess GTE's ability to attenuate obesity development. Juvenile zebrafish were pretreated with GTE for seven days before obesity induction via a high-fat diet; adult zebrafish were pretreated with GTE for two weeks before obesity induction by overfeeding. As a preventive intervention, GTE significantly decreased visceral adipose tissue accumulation in juveniles and ameliorated visceral adiposity and plasma triglyceride levels in adult zebrafish obesity models. RNA sequencing analysis was performed using liver tissues from adult obese zebrafish, with or without GTE administration, to investigate the underlying molecular mechanism. Transcriptome analysis revealed that preventive GTE treatment affects several pathways associated with anti-obesity regulation, including activation of STAT and downregulation of CEBP signaling pathways. In conclusion, GTE could be used as a preventive agent against obesity.

10-Gingerol Suppresses Osteoclastogenesis in RAW264.7 Cells and Zebrafish Osteoporotic Scales.

Osteoporosis is the most common aging-associated bone disease and is caused by hyperactivation of osteoclastic activity. We previously reported that the hexane extract of ginger rhizome [ginger hexane extract (GHE)] could suppress receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis in RAW264.7 cells. However, the anti-osteoclastic components in GHE have not yet been identified. In this study, we separated GHE into several fractions using silica gel column chromatography and evaluated their effects on osteoclastogenesis using a RAW264.7 cell osteoclast differentiation assay (in vitro) and the zebrafish scale model of osteoporosis (in vivo). We identified that the fractions containing 10-gingerol suppressed osteoclastogenesis in RAW264.7 cells detected by tartrate-resistant acid phosphatase (TRAP) staining. In zebrafish, GHE and 10-gingerol suppressed osteoclastogenesis in prednisolone-induced osteoporosis regenerated scales to promote normal regeneration. Gene expression analysis revealed that 10-gingerol suppressed osteoclast markers in RAW264.7 cells [osteoclast-associated immunoglobulin-like receptor, dendrocyte-expressed seven transmembrane protein, and matrix metallopeptidase-9 (Mmp9)] and zebrafish scales [osteoclast-specific cathepsin K (CTSK), mmp2, and mmp9]. Interestingly, nuclear factor of activated T-cells cytoplasmic 1, a master transcription regulator of osteoclast differentiation upstream of the osteoclastic activators, was downregulated in zebrafish scales but showed no alteration in RAW264.7 cells. In addition, 10-gingerol inhibited CTSK activity under cell-free conditions. This is the first study, to our knowledge, that has found that 10-gingerol in GHE could suppress osteoclastic activity in both in vitro and in vivo conditions.

Dieckol isolated from a brown alga, Eisenia nipponica, suppresses ear swelling from allergic inflammation in mouse.

We previously found a lipophilic fraction of the methanol/chloroform extract of a brown alga, Eisenia nipponica, that had an antiallergic effect in a murine ear swelling test. In this study, we purified the active component from the lipophilic fraction using high performance liquid chromatography and analyzed the mass and nuclear magnetic resonance spectra. This uncovered the phlorotannin dieckol, which exhibited antiallergic effects in an ear swelling test using mice sensitized by arachidonic acid, 12-O-tetradecanoylphorbol-13-acetate, and oxazolone. Mechanistic investigations indicated that dieckol suppressed degranulation, chemical mediator release, and the expression of mRNA such as cyclooxygenase-2, interleukin-6, and tumor necrosis factor-α in rat basophilic leukemia-2H3 cells. In summary, we isolated dieckol from E. nipponica and demonstrated its antiallergic mechanisms. PRACTICAL APPLICATIONS: As the incidence of allergies increases worldwide, so too does the demand for food components with antiallergic and anti-inflammatory properties. Given this trend, we focused on a brown alga that displays a variety of bioactivities. Here, we have isolated dieckol from the antiallergic lipophilic fraction of E. nipponica and found that it possesses diverse physiological activities that may prevent lifestyle-related diseases. Consequently, dieckol or the alga containing this phlorotannin could be used as a health food ingredient to combat not only allergies, but also variety of disorders including the undesirable effects of aging.

Significance of a family-6 carbohydrate-binding module in a modular feruloyl esterase for removing ferulic acid from insoluble wheat arabinoxylan.

Ruminiclostridium josui Fae1A is a modular enzyme consisting of an N-terminal signal peptide, family-1 carbohydrate esterase module (CE1), family-6 carbohydrate-binding module (CBM6), and dockerin module in that order. Recombinant CE1 and CBM6 polypeptides were collectively and separately produced as RjFae1A, RjCE1, and RjCBM6. RjFae1A showed higher feruloyl esterase activity than RjCE1 towards insoluble wheat arabinoxylan, but the latter was more active towards small synthetic substrates than the former. This suggests that CBM6 in RjFae1A plays an important role in releasing ferulic acid from the native substrate. RjCBM6 showed a higher affinity for soluble wheat arabinoxylan than for rye arabinoxylan and beechwood xylan in native affinity polyacrylamide gel electrophoresis. Isothermal titration calorimetry analysis demonstrated that RjCBM6 recognized a xylopyranosyl residue at the nonreducing ends of xylooligosaccharides. Moreover, it showed exceptional affinity for 23-α-l-arabinofuranosyl-xylotriose (A2XX) among the tested branched arabinoxylooligosaccharides. Fluorometric titration analysis demonstrated that xylobiose and A2XX competitively bound to RjCBM6, and both bound to the same site in RjCBM6. RjCBM6's preference for the xylopyranosyl residue at the nonreducing end of xylan chains explains why the positive effect of CBM6 on RjFae1A activity was observed only during short incubation but not after extended incubation.

Determination of Anthocyanins and Antioxidants in 'Titanbicus' Edible Flowers In Vitro and In Vivo.

Titanbicus (TB), a hybrid of Hibiscus moscheutos × H. coccineus (Medic.) Walt., has potential to be used as an edible flower. In this study, proximate nutritional content, anthocyanin content, total polyphenol content (TPC), and antioxidant activities in vitro and in vivo were investigated. Three cultivars of TB, namely Artemis (AR), Rhea (R), and Adonis (AD), were used as materials. Protein and carbohydrates were the primary macronutrients, while crude fat and ash were detected in trace amounts. Cyanidin 3-glucoside (Cy3-G) and cyanidin 3-sambubioside (Cy3-Sam), were identified in all TBs. The highest anthocyanin content was observed in AD (47.09 ± 1.45 mg/g extract), followed by R and AR (6.04 ± 0.20 and 2.72 ± 0.11 mg/g extract, respectively). The TPC of AD (225.01 ± 1.97 mg/g extract) was greater than that of AR and R (185.41 ± 3.24 and 144.10 ± 1.71 mg/g extract, respectively). AD exhibited the strongest in vitro antioxidant activity in hydrophilic oxygen radical absorbance capacity, compared to the other two TBs. In addition, AD extract suppressed the generation of reactive oxygen species in caudal fin of wounded zebrafish. Antioxidant activities of AD appeared to be related to its total anthocyanin content, Cy3-G, Cy3-Sam, and TPC. Our findings indicate that TB, particularly the AD cultivar, would be an attractive source of bioactive compounds with antioxidant activities, and can improve both nutritional value and appearance of food.

Isolation and Characterization of Antihyperglycemic Compounds from Vigna angularis Extracts.

Functional foods that inhibit α-amylase and α-glucosidase activity are effective for regulating the blood glucose level and preventing hyperglycemia. Extracts of adzuki beans (ABs, Vigna angularis), widely eaten in East Asia, can inhibit α-amylase and α-glucosidase activity. In this study, we identified and evaluated the components in an AB water extract (ABWE) after boiling, which is an essential process for cooking ABs. The ABWE before boiling inhibited α-amylase and α-glucosidase activity and the boiled ABWE showed slightly stronger inhibitory effects. High-performance liquid chromatography, liquid chromatography-mass spectrometry, and nuclear magnetic resonance analyses identified (+)-catechin 7-O-ß-d-glucopyranoside (C7G), (+)-epicatechin 7-O-ß-d-glucopyranoside (E7G), and (+)-catechin as the bioactive components in boiled ABWE. Interestingly, the quantity of E7G significantly increased after boiling (from 0% to 17.1 ± 1.3%). E7G showed stronger inhibition of α-amylase and α-glucosidase than C7G; the IC50 values for α-amylase were 0.74 ± 0.04 mg/mL (C7G) and 0.40 ± 0.09 mg/mL (E7G), and for α-glucosidase the IC50 values were 0.085 ± 0.032 mg/mL (C7G) and 0.051 ± 0.007 mg/mL (E7G). Our findings suggest that C7G and E7G are the main active components in ABWE as they inhibit α-amylase and α-glucosidase and their inhibitory effect is not lost after boiling. These results support the effectiveness of boiled ABs in the promotion of health. PRACTICAL APPLICATION: We identified (+)-catechin 7-O-ß-d-glucopyranoside (C7G), (+)-epicatechin 7-O-ß-d-glucopyranoside (E7G), and (+)-catechin in adzuki bean extracts and commercially available boiled adzuki bean products. Interestingly, the E7G content was increased by boiling, and this compound showed strong inhibitory activity toward α-amylase and α-glucosidase. These results support the consumption of boiled adzuki beans to prevent acute rises in blood glucose level.

Antiosteoporotic Effects of Acer palmatum Extract on Osteoclastogenesis and Osteoblastogenesis.

Osteoporosis is a systemic skeletal disease that causes bone weakness and fragility. Consuming bone-beneficial nutrients through diet can prevent and treat osteoporosis. Acer palmatum (Japanese maple) leaves are used to make tea, but there have been few reports of their health benefits, especially regarding bone homeostasis. In this study, we evaluated the effects of A. palmatum hot water extract (APE) on osteoclastogenesis and osteoblastogenesis in cultured cells. APE suppressed the number of tartrate-resistant acid phosphatase-positive multinucleated osteoclasts in RANKL induced RAW264.7 cells. Furthermore, APE facilitated Alkaline phosphatase activity and calcium deposition during osteoblast differentiation in MC3T3-E1 cells. High-performance liquid chromatography analysis was performed to investigate the effective components of APE, and four flavonoids orientin, isoorientin, vitexin, and isovitexin were identified with the LC-MS analysis. Treatment with fractionated APE suppressed osteoclastogenesis and facilitated osteoblastogenesis in cultured cells. These findings suggest that APE contains antiosteoporotic compounds; thus, APE might have health promoting effects that help prevent osteoporosis by inhibiting osteoclastogenesis and facilitating osteoblastogenesis.

Orally Administered Phlorotannins from Eisenia arborea Suppress Chemical Mediator Release and Cyclooxygenase-2 Signaling to Alleviate Mouse Ear Swelling.

Phlorotannin is the collective term for polyphenols derived from brown algae belonging to the genera Ascopyllum, Ecklonia, Eisenia, Fucus and Sargassum etc. Since the incidence of allergies is currently increasing in the world, there is a focus on phlorotannins having anti-allergic and anti-inflammatory effects. In this study, six purified phlorotannins (eckol; 6,6'-bieckol; 6,8'-bieckol; 8,8'-bieckol; phlorofucofuroeckol (PFF)-A and PFF-B) from Eisenia arborea, orally administered to mice, were examined for their suppression effects on ear swelling. In considering the suppression, we also examined whether the phlorotannins suppressed release of chemical mediators (histamine, leukotriene B4 and prostaglandin E2), and mRNA expression and/or the activity of cyclooxygenase-2 (COX-2), using RBL-2H3 cells, a cultured mast cell model. Results showed that the phlorotnannins exhibited suppression effects in all experiments, with 6,8'-bieckol, 8,8'-bieckol and PFF-A showing the strongest of these effects. In conclusion, orally administered phlorotannins suppress mouse ear swelling, and this mechanism apparently involves suppression of chemical mediator release and COX-2 mRNA expression or activity. This is the first report of the anti-allergic effects of the orally administered purified phlorotannins in vivo. Phlorotannins show potential for use in functional foods or drugs.

Ruminiclostridium josui Abf62A-Axe6A: A tri-functional xylanolytic enzyme exhibiting α-l-arabinofuranosidase, endoxylanase, and acetylxylan esterase activities.

Ruminiclostridium josui Abf62A-Axe6A is a modular enzyme comprising (in order from the N-terminus): an N-terminal signal peptide, a glycoside hydrolase family 62 (GH62) catalytic module, a family 6 carbohydrate binding module (CBM6), a dockerin module and an additional carbohydrate esterase family 6 catalytic module (CE6). In this study, three Abf62A-Axe6A derivatives were constructed, overexpressed in Escherichia coli, purified, and biochemically characterized: RjAbf62A-Axe6A, containing all four modules but lacking the signal peptide; RjAbf62A-CBM6, containing the GH62 and CBM6 modules; and RjAxe6A, containing only CE6. RjAbf62A-Axe6A was highly active toward arabinoxylan and moderately active toward sugar beet arabinan, and released mainly arabinose. Analysis of the arabinoxylooligosaccharide hydrolysis products revealed that RjAbf62A-Axe6A released α-1,2- and α-1,3-linked arabinofuranose from both singly and doubly substituted xylosyl residues. Furthermore, RjAbf62A-Axe6A exhibited a weak activity toward linear 1,5-α-l arabinan and arabinooligosaccharides, indicating that it is capable of cleaving α-1,5-linkage. Surprisingly, RjAbf62A-Axe6A also demonstrated an endoxylanase activity toward birchwood and beechwood xylans and xylooligosaccharides. Although RjAbf62A-CBM6 exhibited a similar substrate specificity to RjAbf62A-Axe6A, RjAbf62A-CBM6 showed lower activities toward soluble arabinoxylans, birchwood and beechwood xylans and arabinoxylooligosaccharides but not toward insoluble arabinoxylan. RjAbf62A-Axe6A is the first reported GH62 enzyme with α-l-arabinofuranosidase and endoxylanase activities. Although both RjAbf62A-Axe6A and RjAxe6A had acetylxylan esterase activities, RjAbf62A-Axe6 exhibited a higher activity toward insoluble wheat arabinoxylan compared with RjAxe6.

Paenibacillus curdlanolyticus B-6 xylanase Xyn10C capable of producing a doubly arabinose-substituted xylose, α-L-Araf-(1→2)-[α-L-Araf-(1→3)]-D-Xylp, from rye arabinoxylan.

Paenibacillus curdlanolyticus B-6 Xyn10C is a single module xylanase consisting of a glycoside hydrolase family-10 catalytic module. The recombinant enzyme, rXyn10C, was produced by Escherichia coli and characterized. rXyn10C was highly active toward soluble xylans derived from rye, birchwood, and oat spelt, and slightly active toward insoluble wheat arabinoxylan. It hydrolyzed xylooligosaccharides larger than xylotetraose to produce xylotriose, xylobiose, and xylose. When rye arabinoxylan and oat spelt xylan were treated with the enzyme and the hydrolysis products were analyzed by thin layer chromatography (TLC), two unknown hydrolysis products, U1 and U2, were detected in the upper position of xylose on a TLC plate. Electrospray ionization mass spectrometry and enzymatic analysis using Bacillus licheniformis α-L-arabinofuranosidase Axh43A indicated that U1 was α-L-Araf-(1→2)-[α-L-Araf-(1→3)]-D-Xylp and U2 was α-L-Araf-(1→2)-D-Xylp, suggesting that rXyn10C had strong activity toward a xylosidic linkage before and after a doubly arabinose-substituted xylose residue and was able to accommodate an α-1,2- and α-1,3-linked arabinose-substituted xylose unit in both the -1 and +1 subsites. A molecular docking study suggested that rXyn10C could accommodate a doubly arabinose-substituted xylose residue in its catalytic site, at subsite -1. This is the first report of a xylanase capable of producing α-L-Araf-(1→2)-[α-L-Araf-(1→3)]-D-Xylp from highly arabinosylated xylan.

Characterization of Xyn30A and Axh43A of Bacillus licheniformis SVD1 identified by its genomic analysis.

The genome sequence of Bacillus licheniformis SVD1, that produces a cellulolytic and hemi-cellulolytic multienzyme complex, was partially determined, indicating that the glycoside hydrolase system of this strain is highly similar to that of B. licheniformis ATCC14580. All of the fifty-six genes encoding glycoside hydrolases identified in B. licheniformis ATCC14580 were conserved in strain SVD1. In addition, two new genes, xyn30A and axh43A, were identified in the B. licheniformis SVD1 genome. The xyn30A gene was highly similar to Bacillus subtilis subsp. subtilis 168 xynC encoding for a glucuronoarabinoxylan endo-1,4-ß-xylanase. Xyn30A, produced by a recombinant Escherichia coli, had high activity toward 4-O-methyl-D-glucurono-D-xylan but showed definite activity toward oat-spelt xylan and unsubstituted xylooligosaccharides. Recombinant Axh43A, consisting of a family-43 catalytic module of the glycoside hydrolases and a family-6 carbohydrate-binding module (CBM), was an arabinoxylan arabinofuranohydrolase (α-L-arabinofuranosidase) classified as AXH-m23 and capable of releasing arabinosyl residues, which are linked to the C-2 or C-3 position of singly substituted xylose residues in arabinoxylan or arabinoxylan oligomers. The isolated CBM polypeptide had an affinity for soluble and insoluble xylans and removal of the CBM from Axh43A abolished the catalytic activity of the enzyme, indicating that the CBM plays an essential role in hydrolysis of arabinoxylan.

Isolation of a new anti-allergic phlorotannin, phlorofucofuroeckol-B, from an edible brown alga, Eisenia arborea.

Eisenia arborea is an edible brown alga occasionally used as a folk medicine in gynecopathy in Japan. A new phlorotannin was isolated from the alga during our search for naturally occurring anti-allergic compounds from edible algae guided by the inhibitory effect on histamine release from rat basophile leukemia (RBL)-2H3 cells. The phlorotannin was called "phlorofucofuroeckol-B." Its structure was determined by spectral analysis and chemical conversion. This paper describes the isolation, structure elucidation, and inhibitory effect of phlorofucofuroeckol-B on histamine release.

Isolation of five types of flavonol from seabuckthorn (Hippophae rhamnoides) and induction of apoptosis by some of the flavonols in human promyelotic leukemia HL-60 cells.

Five types of flavonol were isolated from seabuckthorn (Hippophae rhamnoides) and identified by mass, 1H- and 13C-NMR. The proliferations of human promyelotic leukemia HL-60 cells were inhibited as the concentrations of these flavonols were increased. The order of the extent of growth inhibition by the flavonols at a concentration of 20 microM is as follows: pentamethylquercetin > syringetin > isorhamnetin > quercetin > kaempherol > myricetin. Apoptotic morphological changes of the nucleus, including chromatin condensation were induced in the HL-60 cells treated with quercetin, kaempherol and myricetin, respectively, but not in the cells treated with the other flavonols. The fragmentations of DNA by quercetin, kaempherol and myricetin, respectively, to oligonucleosomal-sized fragments, a characteristic of apoptosis, were observed to be dose-dependent in the HL-60 cells. These findings suggest that growth inhibition by quercetin, kaempherol and myricetin, respectively, results from the induction of apoptosis by these flavonols. The other flavonols (pentamethylquercetin, syringetin and isorhamnetin) having methoxy (-OCH3) group inhibited more strongly than the above 3 flavonols without induction of apoptosis in the HL-60 cells. These findings suggest that mechanisms of growth inhibition by pentamethylquercetin, syringetin and isorhamnetin are different from the apoptosis caused by quercetin, kaempherol and myricetin.

2-O-methylisohemigossylic acid lactone, a sesquiterpene, isolated from roots of mokumen (Gossampinus malabarica) induces cell death and morphological change indicative of apoptotic chromatin condensation in human promyelotic leukemia HL-60 cells.

2-O-methylisohemigossylic acid lactone, a sesquiterpene, was purified from roots of mokumen (Gossampinus malabarica) and identified by Mass, and (1)H- and (13)-NMR. This sesquiterpene displayed strong growth inhibitory effect against human promyelotic leukemia HL-60 cells. Apoptotic morphological change of the nucleus, including chromatin condensation was induced in the HL-60 cells treated with the sesquiterpene. The fragmentation of DNA by the sesquiterpene to oligonucleosomal-sized fragments, a characteristic of apoptosis, was observed to be dose- and time-dependent in the HL-60 cells. Inhibitors of caspases suppressed the DNA fragmentation induced by the sesquiterpene. These findings suggest that growth inhibition by the sesquiterpene of HL-60 cells results from the induction of apoptosis by the sesqui-terpene, and that caspase cascade is involved in the induction of apoptosis by the compound in the HL-60 cells.

Induction of apoptosis by maackiain and trifolirhizin (maackiain glycoside) isolated from sanzukon (Sophora Subprostrate Chen et T. Chen) in human promyelotic leukemia HL-60 cells.

We have investigated the effects of maackiain and trifolirhizin (maackiain glycoside) isolated from sanzukon (Sophora Subprostrate Chen et T. Chen) on DNA of human promyelotic HL-60 leukemia cells. It was found that extent of induction of apoptosis by maackiain was larger than that by trifolirhizin in human leukemia HL-60 cells. Morphological changes showing apoptotic bodies were observed in the HL-60 cells treated with maackiain and trifolirhizin. The fragmentations of DNA by maackiain and trifolirhizin to oligonucleosomal-sized fragments that is a characteristic of apoptosis was observed to be concentration- and time-dependent in the HL-60 cells. The data of the present study show that the suppressions by maackiain and trifolrhizin of growth of the HL-60 cells result from the induction of apoptosis by these compounds, and that the extent of growth suppression and induction of apoptosis by maackiain was greater than that by the glycoside (trifolirhizin).

Induction of apoptosis by lupeol isolated from mokumen (Gossampinus malabarica L. Merr) in human promyelotic leukemia HL-60 cells.

We have investigated the effect of lupeol (lup-20(29)-ene-3beta-ene-3-ol) isolated from mokumen (Gossampinus malabarica L. Merr) on DNA of human promyelotic HL-60 leukemia cells. Induction of apoptosis by lupeol was observed in human leukemia HL-60 cells. Morphological change showing apoptotic bodies was observed in the HL-60 cells treated with lupeol. The fragmentation of DNA by lupeol to oligonucleosomal-sized fragments that is a characteristic of apoptosis was observed to be concentration- and time-dependent in the HL-60 cells. Flow cytometric analysis showed that the hypodiploid nuclei of HL-60 cells were increased to 28.5, 42.0 and 70.9% after a 3-day treatment with 75, 100 and 150 micro M lupeol, respectively. The data of the present study show that the suppression by lupeol of growth of the HL-60 cells results from the induction of apoptosis by this compound.