Covalent anthocyanin–flavonol complexes from the violet-blue flowers of Allium ‘Blue Perfume’

Three covalent anthocyanin–flavonol complexes (pigments 1–3) were extracted from the violet-blue flower of Allium ‘Blue Perfume’ with 5% acetic acid-MeOH solution, in which pigment 1 was the dominant pigment. These three pigments are based on delphinidin 3-glucoside as their deacylanthocyanin and were acylated with malonyl kaempferol 3-sophoroside-7-glucosiduronic acid or malonyl-kaempferol 3-p-coumaroyl-tetraglycoside-7-glucosiduronic acid in addition to acylation with acetic acid.By spectroscopic and chemical methods, the structures of these three pigments 1–3 were determined to be: pigment 1, (6^I-O-(delphinidin 3-O-(3^I-O-(acetyl)-β-glucopyranoside^I)))(2^VI-O-(kaempferol 3-O-(2^II-O-(3^III-O-(β-glucopyranosyl^V)-β-glucopyranosyl^III)-4^II-O-(trans-p-coumaroyl)-6^II-O-(β-glucopyranosyl^IV)-β-glucopyranoside^II)-7-O-(β-glucosiduronic acid^VI))) malonate; pigment 2, (6^I-O-(delphinidin 3-O-(3^I-O-(acetyl)-β-glucopyranoside^I)))(2^VI-O-(kaempferol 3-O-(2^II-O-β-glucopyranosyl^III)-β-glucopyranoside^II)-7-O-(β-glucosiduronic acid^VI))); and pigment 3, (6^I-O-(delphinidin 3-O-(3^I-O-(acetyl)-β-glucopyranoside^I)))(2^VI-O-(kaempferol 3-O-(2^II-O-(3^III-O-(β-glucopyranosyl^V)-β-glucopyranosyl^III)-4^II-O-(cis-p-coumaroyl)-6^II-O-(β-glucopyranosyl^IV)-β-glucopyranoside^II)-7-O-(β-glucosiduronic acid^VI))) malonate.The structure of pigment 2 was analogous to that of a covalent anthocyanin–flavonol complex isolated from Allium schoenoprasum where delphinidin was observed in place of cyanidin. The three covalent anthocyanin–flavonol complexes (pigment 1–3) had a stable violet-blue color with three characteristic absorption maxima at 540, 547 and 618 nm in pH 5–6 buffer solution. From circular dichroism measurement of pigment 1 in the pH 6.0 buffer solution, cotton effects were observed at 533 (+), 604 (−) and 638 (−) nm. Based on these results, these covalent anthocyanin–flavonol complexes were presumed to maintain a stable intramolecular association between delphinidin and kaempferol units closely related to that observed between anthocyanin and hydroxycinnamic acid residues in polyacylated anthocyanins. Additionally, an acylated kaempferol glycoside (pigment 4) was isolated from the same flower extract, and its structure was determined to be kaempferol 3-O-sophoroside-7-O-(3-O-(malonyl)-β-glucopyranosiduronic acid).     

In vitro culture and the incidence of somaclonal variation in regenerated plants of Trifolium pratense L.

Red clover (Trifolium pratense L.) cvs ‘Altaswede’ (2n = 2x = 14) and ‘Norseman’ (2n = 4x = 28) have been used to investigate tissue culture initiation, plant regeneration and the occurrence of somaclonal variation. After callus induction shoots were induced both when calli on L2 medium containing 2 mg l⁻¹ 2,4-dichlorophenoxy acetic acid (2,4-D), 2 mg l⁻¹ 6-benzylaminopurine (BA) and 2 mg l⁻¹ 6-amino-purine (AP) were subcultured on media containing naphthalene acetic acid (NAA) (0.05 mg l⁻¹) and kinetin (KIN) (0.05 or 0.5 mg l⁻¹) and when embryogenic calli were cultured and subcultured on L2 medium containing 0.002 mg l⁻¹ 4-amino-3,5,6-trichloropicolinic acid (PIC) and 0.2 mg l⁻¹ BA. Shoot tip cultures were also established to induce multiple shoots for regeneration of plants via organogenesis.Regenerants from different regeneration pathways were evaluated for chromosome number stability, morphology and several biochemical traits. Regenerated plants showed stable isozyme banding patterns for malate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucose isomerase, phosphoglucomutase and shikimate dehydrogenase, as well as their nodule leghemoglobin profiles. Variations were detected in the chromosome number of some regenerants as well as in leaflet length-to-width ratio and leaflet number. Factors related to the incidence of somaclonal variation are discussed.     

Acetylated allose-containing flavonoid glucosides from Stachys anisochila

In continuation of our chemosystematic study of Stachys (Labiatae) we have isolated the previously reported isoscutellarein 7-O-[6″'-O-acetyl-β-D-allopyranosyl-(1 → 2)-β-D-glucopyranoside] (1) and 3′-hydroxy-4′-O-methylisoscutellarein 7-O-[6″'-O-acetyl-β-D-allopyranosyl-(1 → 2)-β-D-glucopyranoside] (4) and four new allose-containing flavonoid glycosides from S. anisochila. The new glycosides are hypolaetin 7-O-[6″'-O-acetyl-β-D-allopyranosyl-(1 → 2)-β-D-glucopyranside] (6) as well as the three corresponding diacetyl analogues of 1, 4 and 6, isoscutellarein 7-O-[6″'-O-acetyl-β-D-allopyranosyl-(1 → 2)-6″-O-acetyl-β-D-glucopyranoside], 3′-hydroxy-4′-O-methylisoscutellarein 7-O-[6″'-O-acetyl-β-D-allopyranosyl-(1 → 2)-6″-O-acetyl-β-D-glucopyranoside] and hypolaetin 7-O-[6″'-O-acetyl-β-D-allopyranosyl-(1 → 2)-6″-O-acetyl-β-D-glucopyranoside]. Extensive two-dimensional NMR studies (proton-carbon correlations, COSY experiments) allowed assignment of all ¹H NMR sugar signals and a correction of the ¹³C NMR signal assignments for C-2 and C-3 of the allose.     

Prodigiosin formation by Serratia marcescens in a chemostat

In a study of the control of metabolite formation, prodigiosin production by Serratia marcescens was used as a model. Specific production rates of prodigiosin formation were determined using batch culture technique. Sucrose as carbon source and NH₄NO₃ as nitrogen source resulted in a specific production rate of 0.476 mg prodigiosin (g cell dry weight)⁻¹ h⁻¹. Prodigiosin formation and productivity was inversely correlated to growth rate when the bacterium was grown under carbon limitation on a defined medium in a chemostat culture. The maximum specific growth rate (μ_max) was 0.54 h⁻¹ and prodigiosin was formed in amounts over 1 mg l⁻¹ up to a growth rate (μ) of 0.3 h⁻¹ at steady state conditions. At a dilution rate of 0.1 h⁻¹ growth at steady state with carbon and phosphate limitation supported prodigiosin formation giving a similar specific yield [1.17 mg prodigiosin (g cell dry weight)⁻¹ and 0.94 mg g⁻¹, respectively], however, cells grown with nitrogen limitation [(NH₄)₂SO₄] did not form prodigiosin. Productivity in batch culture was 1.33 mg l⁻¹ h⁻¹ as compared to 0.57 mg l⁻¹ h⁻¹ in the chemostat.     

Isoflavone production in Cyclopia subternata Vogel (honeybush) suspension cultures grown in shake flasks and stirred-tank bioreactor

Suspension cultures of the endemic South-African plant Cyclopia subternata were established for the first time and evaluated for the presence of isoflavones. The influence of light, as well as medium supplementation strategies with phenylalanine, casein hydrolysate and coconut water on biomass growth and isoflavone production were examined. The highest levels of 7-O-β-glucosides of calycosin, pseudobaptigenin and formononetin (275.57, 125.37 and 147.28 mg/100 g DW, respectively) were recorded for cultures grown in the absence of light, whereas coconut water substantially promoted biomass growth. Cell suspensions were subsequently grown in the 2-l stirred-tank bioreactor. Maximum productivity of 7-O-β-glucosides of calycosin, pseudobaptigenin and formononetin (0.96, 0.44 and 0.22 mg l^?1 day^?1, respectively) in bioreactor-cultivated cells was obtained for biomass grown in the dark and supplemented with coconut water. The results indicate that C. subternata suspension cultures can be utilised for the production of the specified isoflavone derivatives absent in the intact plant.     

A new complex triterpenoid saponin from Samanea saman with haemolytic activity and adjuvant effect

A new complex triterpenoid saponin was isolated from the stem bark of Samanea saman by using chromatographic methods. Its structure was established as 3-[(2-O-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy]-2,23-dihydroxy-(2β,3β,4α)-olean-12-en-28-oic acid O-β-d-glucopyranosyl-(1 → 3)-O-[O-β-d-glucopyranosyl-(1 → 4)]-O-6-deoxy-α-l-mannopyranosyl-(1 → 2)-6-O-[4-O-[(2E,6S)-2,6-dimethyl-1-oxo-2,7-octadienyl]-6-deoxy-α-l-mannopyranosyl)oxy]-β-d-glucopyranosyl ester (1). Structural elucidation was performed using detailed analyses of ¹H and ¹³C NMR spectra including 2D NMR spectroscopic techniques and chemical conversions. The haemolytic activity of the saponin was evaluated using in vitro assays, and its adjuvant potential on the cellular immune response against ovalbumin antigen was investigated using in vivo models.     

Synthesis of 2-(p-trifluoroacetamidophenyl)ethylO-α-l-fucopyranosyl-(1–3)-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl) (1–3)-O-β-d-galactopyranosyl-(1–4)-β-d-glucopyranoside,a tetrasaccharide fragment of a tumour-associated glycosphingolipid

The tetrasaccharide 2-(p-trifluoroacetamidophenyl)ethylO-α-l-fucopyranosyl-(1–3)-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-(1–3)-O-β-d-galactopyranosyl-(1–4)-β-d-glucopyranoside was synthesized from thioglycoside intermediates. The key step was a methyl triflate promoted glycosidation of a lactose-derived 3′,4′-diol with a disaccharide thioglycoside to give a β(1–3)-linked tetrasaccharide derivative in 67% yield.     

Enzymatically derived aldouronic acids from Cryptomeria japonica arabinoglucuronoxylan

An arabinoglucuronoxylan was extracted from the holocellulose of sugi (Cryptomeria japonica) wood with 10% KOH and subjected to hydrolysis by partially purified xylanase fraction from a commercial cellulase preparation “Meicelase”. Neutral sugars liberated were analyzed by size exclusion chromatography showing the presence of xylooligosaccharides up to xylohexaose. Aldouronic acids liberated were purified by preparative anion exchange chromatography. Their structures were identified by monosaccharide analysis, comparison of their volume distribution coefficients (Dvs) with those of the authentic samples in anion exchange chromatography and ¹H and ¹³C NMR spectroscopy, resulting in the characterization of eight aldouronic acids including acids consisting of two 4-O-Me-α-D-GlcAp residues and 3-5 D-Xyl residues.

1.

Fr. 1-S1: (aldohexaouronic acid, MeGlcA³Xyl₅), O-β-Xylp-(1 → 4)-O-β-D-Xylp-(1 → 4)-[O-(4-O-Me-α-D-GlcAp)-(1 → 2)]-O-β-Xylp-(1 → 4)-O-β-D-Xylp-(1 → 4)-D-Xyl

2.

Fr. 1-S2: (aldopentaouronic acid, MeGlcA³Xyl₄), O-β-Xylp-(1 → 4)-[O-(4-O-Me-α-D-GlcAp)-(1 → 2)]-O-β-D-Xylp-(1 → 4)-O-β-Xylp-(1 → 4)-D-Xyl

3.

Fr. 2-S1: (aldotetraouronic acid, MeGlcA³Xyl₃), O-(4-O-Me-α-D-GlcAp)-(1 → 2)-O-β-D-Xylp-(1 → 4)-O-β-D-Xylp-(1 → 4)-D-Xyl

4.

Fr. 3-S1: (aldotetraouronic acid, GlcA³Xyl₃), O-(α-D-GlcAp)-(1 → 2)-O-β-D-Xylp-(1 → 4)-O-β-Xylp-(1 → 4)-D-Xyl,

5.

Fr. 4-S1: (aldotriouronic acid, GlcA²Xyl₂), O-(4-O-Me-α-D-GlcAp)-(1 → 2)-O-β-D-Xylp-(1 → 4)-D-Xyl

6.

Fr. 4-S2: (MeGlc⁴MeGlcA³Xyl₅), O-β-D-Xylp-(1 → 4)-[O-(4-O-Me-α-D-GlcAp)]-(1 → 2)-O-β-D-Xylp-(1 → 4)-[O-(4-O-Me-α-D-GlcAp)]-(1 → 2)-O-β-D-Xylp-(1 → 4)-O-β-D-Xylp-(1 → 4)-D-Xyl

7.

Fr. 6-S1: (MeGlcA⁴MeGlcA³Xyl₄), O-(4-O-Me-α-D-GlcAp)-(1 → 2)-O-β-D-Xylp-(1 → 4)-O-[(4-O-Me-α-D-GlcAp)]-(1 → 2)-O-β-D-Xylp-(1 → 4)-O-β-D-Xylp-(1 → 4)-D-Xyl

8.

Fr. 7-S1: (MeGlcA³MeGlc²Xyl₃), O-(4-O-Me-α-D-GlcAp)-(1 → 2)-O-β-D-Xylp-(1 → 4)-O-[(4-O-Me-α-D-GlcAp)]-(1 → 2)-O-β-D-Xylp-(1 → 4)-D-Xyl

Fr. 4-S2 was a new acidic oligosaccharide. The distribution pattern of these vicinal uronic acid units along the D-xylan chain was discussed.     

Somatic embryogenesis and plant regeneration from callus cultures of Aconitum heterophyllum Wall

Plants were obtained via somatic embryogenesis in callus derived from in vitro raised leaf and petiole explants of Aconitum heterophyllum Wall. Callus was induced on a Murashige-Skoog medium supplemented with either 2,4-dichlorophenoxy acetic acid (2,4-d 1 mg l^-1) and kinetin (KN 0.5 mg l^-1) with coconut water (CW 10% v/v) or naphthalene acetic acid (NAA 5 mg l^-1) and benzylaminopurine (BAP 1 mg l^-1). Somatic embryos appeared after 2–3 months or 2 subculture passages when 2,4-d or NAA induced source of the callus was transferred to a MS medium containing BAP (1 mg l^-1) and NAA (0.1 mg l^-1). For successful plantlet formation, the somatic embryos were transferred to a medium containing 1/4 strength MS nutrient with indole-3-butyric acid (IBA 1 mg l^-1). Alternatively, the somatic embryos were dipped in a concentrated solution of IBA for 5 min and placed on a hormone free medium. Complete plantlets were formed after 4 weeks and were transferred successfully to soil.CIMAP Publication No. 1020.     

Phenolic glycosides from Cucumis melo var. inodorus seeds

A new phenolic glycoside (E)-4-hydroxycinnamyl alcohol 4-O-(2′-O-β-d-apiofuranosyl)(1″ → 2′)-β-d-glucopyranoside (1) was isolated and identified from Cucumis melo seeds together with benzyl O-β-d-glucopyranoside (2), 3,29-O-dibenzoylmultiflor-8-en-3α,7β,29-triol (3) and 3-O-p-amino-benzoyl-29-O-benzoylmultiflor-8-en-3α,7β,29-triol (4). Their structures were elucidated by extensive NMR experiments including ¹H–¹H (COSY, TOCSY, ROESY) and ¹H–¹³C (HSQC and HMBC) spectroscopy and chemical evidence. The multiflorane triterpene esters were identified as new melon constituents.     

The synthesis of P1-2-acetamido-4-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-2-deoxy-α-d-glucopyranosyl P2-dolichyl pyrophosphate, (P1-di-N-acetyl-α-chitobiosyl P2-dolichyl pyrophosphate)

2-Acetamido-4-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-2-deoxy-α-d-glucopyranosyl phosphate, pure according to thin-layer and gas—liquid chromatography, optical rotation, and treatment with alkaline phosphatase and 2-acetamido-2-deoxy-β-d-glucosidase, was prepared by treatment of 2-methyl-[4-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-d-glucopyranosyl)-3,6-di-O-acetyl-1,2-dideoxy-α-d-glucopyrano]-[2,1-d]-2-oxazoline with dibenzyl phosphate, followed by the removal of the benzyl groups by catalytic hydrogenolysis, and O-deacetylation. In contrast, a sample prepared by the phosphoric acid procedure was shown to consist mainly of the β anomer. 2-Acetamido-4-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-d-glucopyranosyl)-3,6-di-O-acetyl-2-deoxy-α-d-glucopyranosyl phosphate was treated wit P¹-diphenyl P²-dolichyl pyrophosphate to give a fully acetylated pyrophosphoric diester, which was O-deacetylated to give P¹-2-acetamido-4-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-2-deoxy-α-d-glucopyranosyl P²-dolichyl pyrophosphate. This compound could be separated from the β anomer by t.l.c., and its behavior under dilute acid and alkaline conditions was investigated.     

Steroidal saponins from the leaves of Cordyline fruticosa (L.) A. Chev. and their cytotoxic and antimicrobial activity

Three new steroidal saponins, spirosta-5,25(27)-diene-1β,3β-diol-1-O-α-l-rhamnopyranosyl-(1→2)-β-d-fucopyranoside (fruticoside H) 1, 5α-spirost-25(27)-ene-1β,3β-diol-1-O-α-l-rhamnopyranosyl-(1→2)-(4-O-sulfo)-β-d-fucopyranoside (fruticoside I) 2, and (22S)-cholest-5-ene-1β,3β,16β,22-tetrol 1-O-β-galactopyranosyl-16-O-α-l-rhamnopyranoside (fruticoside J) 3, together with the known quercetin 3-O-β-d-glucopyranoside, quercetin 3-O-[6-trans-p-coumaroyl]-β-d-glucopyranoside, quercetin 3-rutinoside, apigenin 8-C-β-d-glucopyranoside and farrerol, were isolated from the leaves of Cordyline fruticosa. Their structures were elucidated by spectroscopic techniques (¹H NMR, ¹³C NMR, HSQC, ¹H–¹H COSY, HMBC, TOCSY, NOESY), mass spectrometry (HRESIMS, Tandem MS–MS), chemical methods and by comparison with published data. Compounds 1 and 2 showed moderate cytotoxic activity against MDA-MB 231 human breast adenocarcinoma cell line, HCT 116 human colon carcinoma cell line, and A375 human malignant melanoma cell line, while compound 3 was not active. Compound 2 also showed a moderate antibacterial activity against the Gram-positive Enterococcus faecalis.     

Dilignol glycosides from needles of Picea abies

(2R,3R)-2 3-Dihydro-2-(4′-hydroxy-3′-methoxyphenyl)-3-(hydroxymethyl)-7-methoxy-5-benzofuranpropanol 4′-O-β-d-glucopyranoside [dihydrodehydrodiconiferyl alcohol glucoside], (2R,3R)-2 3-dihydro-7-hydroxy-2-(4′-hydroxy-3′-methoxyphenyl)-3-(hydroxymethyl)-5-benzofuranpropanol 4′-O-β-d-glucopyranoside and 4′-O-α-l-rhamnopyranoside, 1-(4′-hydroxy-3′-methoxyphenyl)-2- [2″-hydroxy-4″-(3-hydroxypropyl)phenoxy]-1, 3-propanediol 1-O-β-d-glucopyranoside and 4′-O-β-d-xylopyranoside, 2,3-bis[(4′-hydroxy-3′-methoxyphenyl)-methyl]-1,4-butanediol 1-O-β-d-glucopyranoside [(?)-seco-isolariciresinol glucoside] and (1R,2S,3S)-1,2,3,4-tetrahydro-7-hydroxy-1-(4′-hydroxy-3′-methoxyphenyl)-6-methoxy-2 3-naphthalenedimethanol α²-O-β-d-xylopyranoside [(?)-isolariciresinol xyloside] have been isolated from needles of Picea abies and identified.     

Anthocyanins from red flowers of Camellia cultivar 'Dalicha'

Five anthocyanins, cyanidin 3-O-(2-O-β-xylopyranosyl-6-O-(Z)-p-coumaroyl)-β-galactopyranoside (2), cyanidin 3-O-(2-O-β-xylopyranosyl-6-O-(E)-p-coumaroyl)-β-galactopyranoside (3), cyanidin 3-O-(2-O-β-xylopyranosyl-6-O-(E)-caffeoyl)-β-galactopyranoside (4), cyanidin 3-O-(2-O-β-xylopyranosyl-6-O-acetyl)-β-galactopyranoside (5), and cyanidin 3-O-(2-O-β-xylopyranosyl-6-O-acetyl)-β-glucopyranoside (6), together with the known cyanidin 3-O-(2-O-β-xylopyranosyl)-β-galactopyranoside (1), were isolated from red flowers of Camellia cultivar ‘Dalicha’ (Camellia reticulata) by chromatography using open columns. Their structures were subsequently determined on the basis of spectroscopic analyses, i.e., ¹H NMR, ¹³C NMR, HMQC, HMBC, HR ESI-MS and UV-vis.     

Isolation and purification of flavonoid glucosides from Radix Astragali by high-speed counter-current chromatography

High-speed counter-current chromatography methods, combined with resin chromatography were applied to the separation and purification of flavonoid glycosides from the Chinese medicinal herb, Radix Astragali. Five flavonoid glycosides, namely calycosin-7-O-β-d-glucoside, ononin, (6aR, 11aR)-9,10-dimethoxypterocarpan-3-O-β-d-glucoside, (3R)-2′-hydroxy-3′,4′-dimethoxyisoflavan-7-O-β-d-glucoside and calycosin-7-O-β-d-glucoside-6′′-O-acetate, were obtained. Among them, calycosin-7-O-β-d-glucoside-6′′-O-acetate was preparatively separated from Radix Astragali for the first time. Their structures were identified by ESI–MS, ¹H NMR, ¹³C NMR, and 2D NMR.     

Methylated anthocyanidin glycosides from flowers of Canna indica

Methylated anthocyanin glycosides were isolated from red Canna indica flower and identified as malvidin 3-O-(6-O-acetyl-β-d-glucopyranoside)-5-O-β-d-glucopyranoside (1), malvidin 3,5-O-β-d-diglucopyranoside (2), cyanidin-3-O-(6″-O-α-rhamnopyranosyl-β-glucopyranoside (3), cyanidin-3-O-(6″-O-α-rhamnopyranosyl)-β-galactopyranoside (4), cyanidin-3-O-β-glucopyranoside (5) and cyanidin-O-β-galactopyranoside (6) by HPLC-PDA. Their structures were subsequently determined on the basis of spectroscopic analyses, that is, ¹H NMR, ¹³C NMR, HMQC, HMBC, ESI-MS, and UV-vis. Compounds (1-4) were found to be in major quantity while compounds (5-6) were in minor quantity.     

Synthesis of methyl O-(3-deoxy-3-fluoro-β-d-galactopyranosyl)-(1→6)-β-d-galactopyranoside and methyl O-(3-deoxy-3-fluoro-β-d-galactopyranosyl)-(1→6)-O-β-d-galactopyranosyl-(1→6)-β-d-galactopyranoside

Condensation of 2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-α-d-galactopyranosyl bromide (3) with methyl 2,3,4-tri-O-acetyl-β-d-galactopyranoside (4) gave a fully acetylated (1→6)-β-d-galactobiose fluorinated at the 3′-position which was deacetylated to give the title disaccharide. The corresponding trisaccharide was obtained by reaction of 4 with 2,3,4-tri-O-acetyl-6-O-chloroacetyl-α-d-galactopyranosyl bromide (5), dechloroacetylation of the formed methyl O-(2,3,4-tri-O-acetyl-6-O-chloroacetyl-β-d-galactopyranosyl)-(1→6)- 2,3,4-tri-O-acetyl-β-d-galactopyranoside to give methyl O-(2,3,4-tri-O-acetyl-β-d-galactopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-β-d-galactopyranoside (14), condensation with 3, and deacetylation. Dechloroacetylation of methyl O-(2,3,4-tri-O-acetyl-6-O-chloroacetyl-β-d-galactopyranosyl)-(1→6)-O-(2,3,4-tri-O-acetyl- β-d-galactopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-β-d-galactopyranoside, obtained by condensation of disaccharide 14 with bromide 5, was accompanied by extensive acetyl migration giving a mixture of products. These were deacetylated to give, crystalline for the first time, the methyl β-glycoside of (1→6)-β-d-galactotriose in high yield. The structures of the target compounds were confirmed by 500-MHz, 2D, ¹H- and conventional ¹³C- and ¹⁹F-n.m.r. spectroscopy.     

Structures of verbascoside and orobanchoside,caffeic acid sugar esters from Orobanche rapum-genistae

The complete structural elucidation of the two caffeic acid sugar esters verbascoside and orobanchoside, has been realized by ¹H and ¹³C NMR studies. It has been demonstrated that verbascoside is β-(3′,4′-dihydroxyphenyl)ethyl-O-α-L-rhamnopyranosyl(1→3)-β-D-(4-O-caffeoyl)-glucopyranoside, and orobanchoside is β-hydroxy-β-(3′,4′-dihydroxyphenyl)-ethyl-O-α-L-rhamnopyranosyl(1→2)-β-D-(4-O-caffeoyl)-glucopyranoside.     

Embryogenic protoplast cultures of orange jessamine (Murraya paniculata) and their regeneration into plants flowering in vitro

Embryogenic callus was induced from the hypocotyl region of seedlings germinated from immature embryos of orange jessamine (Murraya paniculata (L.) Jack) on Murashige & Tucker (1969) medium containing 50 g l^-1 sucrose, 5.0 mg l^-1 benzyladenine, 2.5 mg l^-1 2,4-dichlorophenoxyacetic acid and 600 mg l^-1 malt extract. Isolated protoplasts divided to produce callus on Murashige & Tucker (1969) medium containing 50 g l^-1 sucrose, 0.01 mg l^-1 gibberellin A₄₊₇ and 600 mg l^-1 malt extract. Callus developed to plantlets via somatic embryogenesis on Murashige & Tucker (1969) medium with 50 g l^-1 lactose but no plant growth regulators. These plantlets flowered in vitro on half strength Murashige & Tucker (1969) medium containing 50 g l^-1 sucrose after 2 months culture.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - FM full strength MT medium - FMG full strength MT medium +1 mg l^-1 GA₃ - GA₃ gibberellin A₃ - GA₄₊₇ gibberellin A₄₊₇ - HM half strength MT medium - HMG half strength MT medium +1 mg l^-1 GA₃ - MT Murashige & Tucker (1969)     

Three new flavonol glycosides from Nervilia fordii

Three new flavonol glycosides, nervilifordizins A–C (1–3), were isolated from the whole plant of Nervilia fordii. Their structures were elucidated as rhamnazin 3-O-β-d-xylopyranosyl-(1→4)-β-d-glucopyranoside (1), rhamnazin 3-O-β-d-glucopyranosyl-(1→4)-β-d-glucopyranoside (2) and rhamnazin 3-O-β-d-xylopyranosyl-(1→4)-β-d-glucopyranoside-4′-O-β-d-glucopyranoside (3) on the basis of extensive spectroscopic analysis, including HSQC, HMBC, ¹H–¹H COSY, and chemical evidences.