Isolation and structural characterization of eight new resin glycosides, calyhedins XVI-XXIII, from the rhizomes of Calystegia hederacea.

Biological effects attributed to resin glycosides, including cytotoxicity against cancer cells and antibacterial, multidrug resistance-modulating, and antiviral activities have been documented. Penta-glycosides composed of calysolic acid A or calyhedic acid A, which are glycosidic acid components of the crude resin glycoside fraction of Calystegia hederacea, have not yet been isolated from this plant. In this study, eight new resin glycosides, termed calyhedins XVI (1)-XXIII (8), were isolated from the rhizomes of C. hederacea. Compounds 1-8 are penta- or hexa-glycosides with macrolactone structures, and their sugar moieties are partially acylated by five organic acids, including 2S-methylbutyric, (E)-2-methylbut-2-enoic, and 2R-methyl-3R-hydroxybutyric acids. Compounds 1-5 are the first identified macrocyclic resin glycosides with five monosaccharides obtained from this plant, and 2 and 4 are the first to be characterized as containing calyhedic acid A as the glycosidic acid component. Compounds 1-8 were of the four following macrolactone types: one with a 22-membered ring (5), another with a 23-membered ring (6-8), the third with a 27-membered ring (1, 3), and the fourth with a 28-membered ring (2, 4). Compounds 2-8 exhibited cytotoxic activity against HL-60 human promyelocytic leukemia cells comparable to that of the positive control, cisplatin.

Five new resin glycosides, calyhedins XI-XV, from Calystegia hederacea.

Calystegia hederacea Wall. (Convolvulaceae) is a perennial herbaceous vine that grows widely in India and East Asia. All parts of this plant are used to treat various disorders such as menoxenia and gonorrhea. Four new resin glycosides, calyhedins XI (1)-XIV (4), were isolated from the rhizomes of C. hederacea. A new glycoside, calyhedin XV (5), was isolated from its leaves and stems. Alkaline hydrolysis of 1 and 2 furnished a new glycosidic acid, calyhedic acid G (1a), from 1 and a new acid, calyhedic acid H (2a), from 2 along with 2S-methylbutyric acid and 2R-methyl-3R-hydroxybutyric (2R,3R-nilic) acid. The structures of 1-5, 1a, and 2a were determined using MS and NMR spectral analyses. Compounds 1a and 2a had the same sugar moiety, ß-D-glucopyranosyl-(1 → 6)-O-ß-D-glucopyranosyl-(1 → 6)-O-ß-D-glucopyranosyl-(1 → 3)-[O-ß-D-glucopyranosyl-(1 → 3)-O-α-L-rhamnopyranosyl-(1 → 2)]-O-ß-D-glucopyranosyl-(1 → 2)-ß-D-fucopyranose, while their aglycones were 11S-dihydroxyhexadecanoic acid and 12S-dihydroxyhexadecanoic acid, respectively. These compounds are the first glycosidic acids, with fucose as the monosaccharide component obtained from the resin glycosides of C. hederacea. Compounds 1-5, comprising either 1a or 2a, were heptaglycosides with macrolactone structures, and their sugar moieties were partially acylated with 5 mol of organic acids comprising 2S-methylbutyric, (E)-2-methylbut-2-enoic, and 2R,3R-nilic acids. Compounds 1 and 5 had 22-membered rings, while 2-4 had 28-membered rings. In addition, 1 and 5 exhibited cytotoxic activity against HL-60 human promyelocytic leukemia cells, comparable to that of the positive control cisplatin.

Four new resin glycosides, calyhedins VII-X, from the rhizomes of Calystegia hederacea.

Four new resin glycosides with macrolactone structures (jalapins), namely, calyhedins VII (1)-X (4), were isolated from the rhizomes of Calystegia hederacea Wall. (Convolvulaceae). The structures of 1-4 were determined based on spectroscopic data. They were classified into three ring types: a 27-membered ring (1), a 22-membered ring (2, 3), and a 23-membered ring (4). Their sugar moieties were partially acylated using five organic acids, including (E)-2-methylbut-2-enoic acid, 2S-methylbutyric acid, and 2 R-methyl-3R-hydroxybutyric acid. Compound 4 was the first genuine resin glycoside with calyhedic acid F as the glycosidic acid component. Additionally, the cytotoxic activities of 1, 2, and 4 towards HL-60 human promyelocytic leukaemia cells were evaluated. All compounds demonstrated almost the same activity as the positive control, cisplatin.

Identification and characterization of organic and glycosidic acids in the crude resin glycoside fraction from the leaves and stems of Calystegia japonica.

The alkaline hydrolysis of the crude resin glycoside fraction from the leaves and stems of the plant Calystegia japonica Choisy (Convolvulaceae) yielded organic acid and glycosidic acid fractions. The organic acid fraction was esterified with p-bromophenacyl bromide to obtain p-bromophenacyl 2R-methyl-3R-hydroxybutyrate (1) and p-bromophenacyl (E)-2-methylbut-2-enoate (2). By treating the glycosidic acid fraction with trimethylsilyldiazomethane-hexane, seven new methyl esters of glycosidic acids, namely calyjaponic acid A methyl ester (3) calyjaponic acid B methyl ester (5), calyjaponic acid C methyl ester (6), calyjaponic acid D methyl ester (7), calyjaponic acid E methyl ester (8), calyjaponic acid F methyl ester (9), and calyjaponic acid G methyl ester (10), were isolated along with one known ester (4). Their structures were characterized based on spectroscopic and chemical analyses. Compounds 3-8 had the same sugar moiety, α-L-rhamnopyranosyl-(1 → 2)-O-ß-D-glucopyranosyl-(1 → 2)-[O-α-L-rhamnopyranosyl-(1 → 6)]-O-ß-D-glucopyranose, and the aglycones of 3-8 were methyl 3S,11S-dihydroxyhexadecanoate, methyl 3S,12S-dihydroxyhexadecanoate, methyl 11S-hydroxyhexadecanoate, methyl 11S-hydroxypentadecanoate, methyl 3S,11S-dihydroxypentadecanoate, and methyl 3S,12S-dihydroxypentadecanoate, respectively. Compounds 9 and 10 were derivatives of 3 and 4, respectively, in which the C-6 of the second glucosyl residue was methylated. Compounds 6-8 contained methyl esters of unusual odd-carbon fatty acids as aglycones. The cytotoxicity of the crude resin glycoside fraction and 3 against HL-60 human promyelocytic leukemia cells was evaluated further; both were either weakly active or inactive compared to the positive control, cisplatin.

Calysepins I-VII, Hexasaccharide Resin Glycosides from Calystegia sepium and Their Cytotoxic Evaluation.

Seven new hexasaccharide resin glycosides, named calysepins I-VII (1-7), with 27-membered rings, were obtained from the aerial parts of Calystegia sepium. Their structures with absolute configuration were established on the basis of spectroscopic data interpretation analysis and the use of chemical methods. They were defined as hexasaccharides composed of one d-quinovose, four d-glucose, and one l-rhamnose unit, and their sugar moieties were partially acylated by (2S)-methylbutanoic acid in 1-7 and (2R,3R)-nilic acid in 1-5 and 7, which mainly differed at the positions of acylation. Additionally, calysepin IV (4) exhibited cytotoxicity against A549 cells with an IC50 value of 5.2 µM.

Dichloromethane fractions of Calystegia soldanella induce S­phase arrest and apoptosis in HT­29 human colorectal cancer cells.

Calystegia soldanella is a halophyte and a perennial herb that grows on coastal sand dunes worldwide. Extracts from this plant have been previously revealed to have a variety of bioactive properties in humans. However, their effects on colorectal cancer cells remain poorly understood. In the present study, the potential biological activity of C. soldanella extracts in the colorectal cancer cell line HT­29 was examined. First, five solvent fractions [n­hexane, dichloromethane (DCM), ethyl acetate, n­butanol and water] were obtained from the crude extracts of C. soldanella through an organic solvent extraction method. In particular, the DCM fraction was demonstrated to exert marked dose­ and time­dependent inhibitory effects according to results from the cell viability assay. Data obtained from the apoptosis assay suggested that the inhibition of HT­29 cell viability induced by DCM treatment was attributed to increased apoptosis. The apoptotic rate was markedly increased in a dose­dependent manner, which was associated with the protein expression levels of apoptosis­related proteins, including increased Fas, Bad and Bax, and decreased pro­caspase­8, Bcl­2, Bcl­xL, pro­caspase­9, pro­caspase­7 and pro­caspase­3. A mitochondrial membrane potential assay demonstrated that more cells became depolarized and the extent of cytochrome c release was markedly increased in a dose­dependent manner in HT­29 cells treated with DCM. In addition, cell cycle analysis confirmed S­phase arrest following DCM fraction treatment, which was associated with decreased protein expression levels of cell cycle­related proteins, such as cyclin A, CDK2, cell division cycle 25 A and cyclin dependent kinase inhibitor 1. Based on these results, the present study suggested that the DCM fraction of the C. soldanella extract can inhibit HT­29 cell viability whilst inducing apoptosis through mitochondrial membrane potential regulation and S­phase arrest. These results also suggested that the DCM fraction has potential anticancer activity in HT­29 colorectal cells. Further research on the composition of the DCM fraction is warranted.

Altered glycosylation associated with dedifferentiation of hepatocellular carcinoma: a lectin microarray-based study.

BACKGROUND: Altered glycosylation associated with hepatocellular carcinoma (HCC) is well documented. However, few reports have investigated the association between dedifferentiation and glycosylation. Therefore, the aim of this study was to analyze glycosylation associated with dedifferentiation of HCC within the same nodule and to investigate glycosyltransferase related to the glycosylation. METHODS: We analyzed resected HCC specimens (n = 50) using lectin microarray to comprehensively and sensitively analyze glycan profiles, and identify changes to glycosylation between well- and moderately-differentiated components within the same nodule. Moreover, we performed immunohistochemical staining of mannosyl(α-1,3-)-glycoprotein ß-1,2-N-acetylglucosaminyltransferase (MGAT1), which is an essential glycosyltransferase that converts high-mannose glycans to complex- or hybrid-type N-glycans. RESULTS: Four lectins from Narcissus pseudonarcissus agglutinin (NPA), Concanavalin A, Galanthus nivalis agglutinin, and Calystegia sepium agglutinin were significantly elevated in moderately-differentiated components of HCC compared with well-differentiated components, and all lectins showed binding specificity to high-mannose glycans. Therefore, these structures were represented to a greater extent in moderately-differentiated components than in well-differentiated ones. Immunohistochemical staining revealed significantly increased NPA expression and decreased MGAT1 expression in moderately-differentiated components. Low MGAT1 expression in moderately-differentiated components of tumors was associated with intrahepatic metastasis and had tendency for poor prognosis. CONCLUSION: Dedifferentiation of well-differentiated HCC is associated with an increase in high-mannose glycans. MGAT1 may play a role in the dedifferentiation of HCC.

Identification and characterization of organic and glycosidic acids in crude resin glycoside fraction from Calystegia hederacea.

Resin glycosides are well known as the purgative ingredients, which are characteristic of convolvulaceous plants. Calystegia hederacea Wall. is a perennial herbaceous vine that is widespread throughout India and East Asia. All parts of this plant are used for the treatment of menoxenia, gonorrhea, etc. Alkaline hydrolysis of the crude resin glycoside fraction of the whole plants of C. hederacea yielded four new glycosidic acids, calyhedic acids A, B, C, and D, along with two known glycosidic acids, calysolic acids A and C, and three known organic acids, 2S-methylbutyric, tiglic, and 2R,3R-nilic acids. Their structures were characterized on the basis of spectroscopic data and chemical evidence. Calyhedic acids A, B, and D were penta-, hexa-, and hepta-glycosides of 12S-hydroxyhexadecanoic acid, respectively, and cayhedic acid C was an isomer of calyhedic acid D, in which the 12S-hydroxyhexadecanoyl residue of calyhedic acid D was replaced by a 11S-hydroxyhexadecanoyl residue. Additionally, cytotoxic activity toward HL-60 human promyelocytic leukemia cells of the crude resin glycoside fraction, the glycosidic acid fraction, calyhedic acid A, and calysolic acid A from C. hederacea was evaluated. Furthermore, to clarify the structure-activity relationship of resin glycosides, the activities of six genuine resin glycosides with calysolic acid A or calysolic acid C as the glycosidic acid, which were isolated from C. soldanella, were examined. Among them, the crude resin glycoside fraction and five genuine resin glycosides with macrolactone structures demonstrated clear cytotoxic activities, while the glycosidic acid fraction, calyhedric acid A, calysolic acid A, and a genuine non-macrolactone-type resin glycoside were either inactive or exhibited weaker activity than the tested macrolactone-type resin glycosides.

Distinct roles for each N-glycan branch interacting with mannose-binding type Jacalin-related lectins Orysata and Calsepa.

Mannose-binding type Jacalin-related lectins (mJRLs) bind to branched N-glycans via conserved sugar-binding sites. Despite, significant 3D structural similarities, each mJRL is known to have a unique binding preference toward various N-glycans. However, the molecular basis of varying binding preference is substantially unknown. Here, we report a detailed comparison of N-glycan-binding preference for two mJRLs, Orysata and Calsepa using frontal affinity chromatography (FAC), X-ray and molecular modeling. The FAC analysis using a panel of N-glycans shows difference in N-glycan-binding preference between the lectins. Orysata shows broader specificity toward most high-mannose-type glycans as well as biantennary complex-type glycans bearing an extension on the Manα1-6 branch. Whereas, Calsepa shows narrow specificity to complex-type glycans with bisecting GlcNAc. The X-ray crystallographic structure reveals that two Orysata lectins bind to one biantennary N-glycan (2:1 binding) where one lectin binds to mannose of the α1-3 branch, while the other interacts with an N-acetylglucosamine of the α1-6 branch. In contrast, Calsepa shows 1:1 binding where α1-3 branch and core chitobiose region N-glycan interacts with lectin, while α1-6 branch is flipped-back to the chitobiose core. Molecular dynamics study of Orysata bound to N-glycans substantiate possibility of two-binding modes for each N-glycan. Binding free energies calculated separately for α1-3 and α1-6 branches of each N-glycan suggest both branches can bind to Orysata. Overall these results suggest that each branch of N-glycan has a distinct role in binding to mJRLs and the nonbinding branch can contribute significantly to the binding affinity and hence to the specificity.

A new resin glycoside from Calystegia soldanella and its antiviral activity towards herpes.

A new resin glycoside, named calysolin XVIII (1), was isolated from the leaves, stems and roots of Calystegia soldanella Roem. et Schult. (Convolvulaceae). The structure of 1 was defined as 11S-jalapinolic acid 11-O-ß-d-glucopyranosyl-(1 → 3)-O-(2-O-2S-methylbutyryl,4-O-3-hydroxy-2-methylenebutyryl)-α-l-rhamnopyranosyl-(1 → 2)-[O-ß-d-glucopyranosyl-(1 → 6)-O-(34-di-O-2S-methylbutyryl)-ß-d-glucopyranosyl-(1 → 3)]-O-ß-d-glucopyranosyl-(1 → 2)-ß-d-quinovopyranoside, intramolecular 1,2″'″'-ester on the basis of spectroscopic data. Compound 1 is the first known resin glycoside to feature 3-hydroxy-2-methylenebutyric acid as a component organic acid. In addition, 1 demonstrated an antiviral activity against herpes simplex virus type 1, with an IC50 value 2.3 µM.

Antiviral Activity of Four New Resin Glycosides Calysolins XIV-XVII from Calystegia soldanella against Herpes Simplex Virus.

Four new resin glycosides, named calysolins XIV (1), XV (2), XVI (3), and XVII (4) were isolated from the leaves, stems, and roots of Calystegia soldanella ROEM.. et SCHULT. (Convolvulaceae). Their structures were determined based on spectroscopic and chemical evidence, and consisted of two different types: those (1) with a macrolactone structure and those (2-4) with a non-macrolactone structure. Their sugar moieties were partially acylated by specific organic acids, including tiglic, 2S-methylbutyric, and 2S,3S-nilic acids. Additionally, evaluation of the antiviral activity of 1-4 revealed effects against the herpes simplex virus type 1.

Flavonoids and their qualitative variation in Calystegia soldanella and related species (Convolvulaceae).

Coastal species are exposed to severe environmental stresses, e.g. salt and UV-B. The plants adapt themselves to such harsh environment by controlling morphological features and chemical defense systems. Flavonoids are known as efficient anti-stress polyphenols produced by plants. Most flavonoids show antioxidant activity, and their properties are important for plants to survive under high-stress conditions such as those in a coastal area. Among the compounds, ortho-dihydroxylated flavonoids act as strong antioxidants. In this survey, we elucidated the flavonoid composition of a seashore species Calystegia soldanella, which is distributed not only on the seashore, but also by the inland freshwater lake, Lake Biwa. Seven flavonol glycosides, i.e. quercetin 3-0- rutinoside, 3-O-glucoside, 3-O-rhamnoside and 3-O-apiosyl-(1-->2)-[rhamnosyl-(1-->6)-glucoside], and kaempferol 3-O-rutinoside, 3-O-glucoside and 3-0- rhamnoside were isolated from the leaves of C. soldanella. In addition, it was shown that the quercetin (Qu) to kaempferol (Km) ratio of coastal populations was higher than that of lakeshore populations. In general, these differences of Qu/Km ratio depend on flavonoid 3'-hydroxylase (F3'H) transcription. RT-PCR analysis suggested that F3'H of C. soldanella is regulated translationally or post-translationally, but not transcriptionally. Furthermore, quantitative and qualitative differences in flavonoid composition occurred among three Calystegia species, C. soldanella, C. japonica and C. hederacea.

PTP1B inhibitory effect of alkyl p-coumarates from Calystegia soldanella.

In this report, the PTP1B inhibitory effect of Calystegia soldanella was investigated. Bioassay-guided fractionation of the crude extracts revealed that the n-hexane fraction had the strongest PTP1B inhibitory effect. Nine known alkyl p-coumarates were isolated from the n-hexane fraction, and each compound was evaluated for its effect on PTP1B. All compounds effectively inhibited PTP1B activity. The IC50 values of the compounds were 3 (10.8 µg/mL) > 2 (15.5 µg/mL) > 7 (26.6 µg/mL) > 1 (37.0 µg/mL) > 8 (41.2 µg/mL) > 9 (43.4 µg/mL) > 5 (44.7 µg/mL) > 4 (> 50 µg/mL) > 6 (> 50 µg/mL).

Calysolins X-XIII, resin glycosides from Calystegia soldanella, and their antiviral activity toward herpes simplex virus.

Four new resin glycosides having macrolactone structures (jalapins), named calysolins X (1)-XIII (4), were isolated from the leaves, stems, and roots of Calystegia soldanella ROEM. et SCHULT. (Convolvulaceae). Their structures were determined on the basis of spectroscopic data as well as chemical evidence. The sugar moieties of 1-4 were partially acylated by some organic acids, including tiglic acid, 2S-methylbutyric acid, and 2S,3S-nilic acid. Additionally, the antiviral activity of 1-4 toward herpes simplex virus type 1 was evaluated. All the compounds showed antiviral activity.

Calysolins V-IX, resin glycosides from Calystegia soldanella and their antiviral activity toward herpes.

Five new resin glycosides having macrolactone structures (jalapins), named calysolins V-IX (1-5), were isolated from the leaves, stems, and roots of Calystegia soldanella ROEM. et SCHULT. (Convolvulaceae). Their structures were determined on the basis of spectroscopic data as well as chemical evidence. The isolated compounds could be classified into two macrolactone types-one having a 22-membered ring (1-4) and the other with a 27-membered ring (5). The sugar moieties of 1-5 were found to exist in partially acylated forms comprising 2S-methylbutyric acid and tiglic acid. Compounds 4 and 5 are the first representatives of the calysolic acid C as the component glycosidic acid. Additionally, the antiviral activity of 1-5, together with calysolins I-IV and soldanelline B, which are previously isolated jalapins from this plant, toward herpes simplex virus type 1 was evaluated. All the compounds showed antiviral activity.

Perception of host plant volatiles in Hyalesthes obsoletus: behavior, morphology, and electrophysiology.

The Palearctic planthopper Hyalesthes obsoletus is the natural vector of the grapevine yellow disease Bois noir. Grapevine is an occasional host plant of this polyphagous planthopper. To deepen our knowledge of the role of plant volatile organic compounds for H. obsoletus host plant searching, we carried out behavioral, morphological, and electrophysiological studies. We tested the attraction of H. obsoletus to nettle, field bindweed, hedge bindweed, chaste tree, and grapevine by using a Y-shaped olfactometer. The results showed a significant attraction of male H. obsoletus to chaste tree, and of the females to nettle. Male H. obsoletus were repelled by odor from hedge bindweed. Ultrastructural studies of the antennae showed at least two types of olfactory sensilla at the antennal pedicel: plaque organs and trichoid sensilla. Volatile organic compounds from nettle and chaste tree were collected, and the extracts were analyzed by coupling gas-chromatography to both mass-spectrometry and electroantennography. The volatile organic compounds that elicited electrophysiological responses in male and female antennae were identified. These findings are discussed with respect to behavior of H. obsoletus males and females in the field.

Calysolins I-IV, resin glycosides from Calystegia soldanella.

Four new resin glycosides having intramolecular cyclic ester structures (jalapins), named calysolins I-IV (1-4), were isolated from the methanol extract of leaves, stems, and roots of Calystegia soldanella , along with one known jalapin (5) derivative. The structures of 1-4 were determined on the basis of spectroscopic data and chemical evidence. They fall into two types, one having a 22-membered ring (1 and 4) and the other with a 27-membered ring (2 and 3). The sugar moieties of 1-4 were partially acylated by some organic acids. Compound 4 is the first example of a hexaglycoside of jalapin.

Identification and characterization of component organic and glycosidic acids of crude resin glycoside fraction from Calystegia soldanella.

Alkaline hydrolysis of the crude resin glycoside fraction of the leaves, stems, and roots of Calystegia soldanella ROEM. et SCHULT. (Convolvulaceae) gave four new glycosidic acids, named calysolic acids A, B, C, and D, along with one known glycosidic acid, soldanellic acid B, and three organic acids, 2S-methylbutyric, tiglic, and 2S,3S-nilic acids. The structures of the new glycosidic acids were characterized on the basis of spectroscopic data and chemical evidence.

Suppressive effects of nitric oxide production and inducible nitric oxide synthase (iNOS) gene expression by Calystegia soldanella methanol extract on lipopolysaccharide-activated RAW 264.7 cells.

Since nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) has been found to be involved in various pathophysiological processes, including inflammation and carcinogenesis, the modulators of NO synthesis or expression have been considered as potential anti-inflammatory and cancer chemopreventive agents. In this study, to procure the iNOS inhibitors from natural products, we evaluated 57 methanol extracts of natural products including Korean indigenous plants for the inhibition of NO formation on lipopolysaccharide (LPS)-activated mouse macrophage-like RAW 264.7 cells. As a result, several extracts including those from Actinodaphne lancifolia, Calystegia soldanella, Caryratia japonica, Citrus dachibana, Dystaenia takeshimana, Erysimum aurantiacum, Hovenia undulata, Stewartia koreana and Viburnum awabuki showed potent inhibitory activities of NO production (>70% inhibition at the test concentration of 40 microg/ml). In particular, the extract of Calystegia soldanella showed a potential inhibition of NO production in a dose-dependent manner (IC50=4.3 microg/ml). Subsequent study also exhibited that the extract of Calystegia soldanella significantly suppressed iNOS protein and gene expression in a dose-dependent manner. These results suggest that Calystegia soldanella might be a new potential candidate for developing an iNOS inhibitor from natural products and also could be warranted for further elucidation of active principles for the development of new anti-inflammatory and/or cancer chemopreventive agents.

The crystal structure of the Calystegia sepium agglutinin reveals a novel quaternary arrangement of lectin subunits with a beta-prism fold.

The high number of quaternary structures observed for lectins highlights the important role of these oligomeric assemblies during carbohydrate recognition events. Although a large diversity in the mode of association of lectin subunits is frequently observed, the oligomeric assemblies of plant lectins display small variations within a single family. The crystal structure of the mannose-binding jacalin-related lectin from Calystegia sepium (Calsepa) has been determined at 1.37-A resolution. Calsepa exhibits the same beta-prism fold as identified previously for other members of the family, but the shape and the hydrophobic character of its carbohydrate-binding site is unlike that of other members, consistent with surface plasmon resonance analysis showing a preference for methylated sugars. Calsepa reveals a novel dimeric assembly markedly dissimilar to those described earlier for Heltuba and jacalin but mimics the canonical 12-stranded beta-sandwich dimer found in legume lectins. The present structure exemplifies the adaptability of the beta-prism building block in the evolution of plant lectins and highlights the biological role of these quaternary structures for carbohydrate recognition.