M. V. Veselova

Inhibitory effect of the Agrobacterium rhizogenes rolC gene on rabdosiin and rosmarinic acid production in Eritrichium sericeum and Lithospermum erythrorhizon transformed cell cultures

Rabdosiin and related caffeic acid metabolites have been proposed as active pharmacological agents demonstrating potent anti-HIV and antiallergic activities. We transformed Eritrichium sericeum and Lithospermum erythrorhizon seedlings by the rolC gene, which has been recently described as an activator of plant secondary metabolism. Surprisingly, the rolC-transformed cell cultures of both plants yielded two- to threefold less levels of rabdosiin and rosmarinic acid (RA) than respective control cultures. This result establishes an interesting precedent when the secondary metabolites are differently regulated by a single gene. We show that the rolC gene affects production of rabdosiin and RA irrespective of the methyl jasmonate (MeJA)-mediated and the Ca2+-dependent NADPH oxidase pathways. Cantharidin, an inhibitor of serine/threonine phosphatases, partly diminishes the rolC-gene inhibitory effect that indicates involvement of the rolC-gene-mediated signal in plant regulatory controls, mediated by protein phosphatases. We also show that the control MeJA-stimulated E. sericeum root culture produces (−)-rabdosiin up to 3.41% dry weight, representing the highest level of this substance for plant cell cultures reported so far.

Enhanced resveratrol accumulation in rolB transgenic cultures of Vitis amurensis correlates with unusual changes in CDPK gene expression.

It has been established that transformation of Vitis amurensis callus culture with the plant oncogene rolB of Agrobacterium rhizogenes results in a high level of resveratrol production in the transformed culture. In the present report, we investigated two rolB transgenic V. amurensis cell cultures with different levels of rolB expression and resveratrol production. We examined whether the calcium ion flux and later steps of the calcium-mediated signal transduction pathway play a role in resveratrol biosynthesis in the rolB transgenic cultures. It has been shown that the calcium channel blockers, LaCl(3), verapamil, and niflumic acid, significantly reduced the accumulation of resveratrol in the rolB transgenic cultures. The number of the calcium-dependent protein kinase (CDPK) transcript variants and abundance of some of the transcripts were considerably altered in the rolB transgenic cell cultures, as revealed by frequency analysis of RT-PCR products and real-time PCR. Some unusual CDPK transcripts with deletions and insertions in the kinase domain were isolated from cDNA probes of rolB-transformed cells. These results suggest that active resveratrol biosynthesis in rolB transgenic cultures of V. amurensis is Ca2+ dependent. We propose that the rolB gene has an important role in regulation of calcium-dependent transduction pathways in transformed cells.

High Rabdosiin and Rosmarinic Acid Production in Eritrichium sericeum Callus Cultures and the Effect of the Calli on Masugi-Nephritis in Rats

During an investigation of plant cell cultures that might be useful in the treatment of renal disorders, we established a vigorously-growing E-4 callus culture of Eritrichium sericeum that produced large amounts of caffeic acid metabolites, (−)-rabdosiin (1.8% dry wt) and rosmarinic acid (4.6% dry wt). Elicitation of the calli by methyl jasmonate induced a 38% increase in total polyphenol production. The most efficient method of eliciting (−)-rabdosiin biosynthesis was through the treatment of E-4 calli with cuprum glycerate, which induced an increase in (−)-rabdosiin production of as much as 4.1% dry wt. Oral administration of E-4 callus biomass (100 mg/kg/d for 30 d) to rats with induced Masugi-nephritis caused an increase in diuresis and lowered creatinine excretion and proteinuria levels as compared with Masugi-nephritis untreated rats. While all of the Masugi-nephritis untreated rats began to suffer, near a quarter of the E-4 treated rats remained in good health. This result indicates that the E-4 culture has the potential to alleviate the symptoms associated with nephritis.

Minor polyphenols from Maackia amurensis wood

The rare α,4,2′,4′-tetrahydroxydihydrochalcone, flavanones liquiritigenin and naringenin, isoflavones calycosin and 5-methoxydaidzein, and reduced stilbene dihydroresveratrol were isolated for the first time from Maackia amurensis wood. The structures of the pure compounds were established using 2D NMR COSY, NOE, HMBC, and HSQC experiments.

Catechin production in cultured cells of Taxus cuspidata and Taxus baccata

The main polyphenols in callus and cell suspension cultures of Taxus cuspidata and T. baccata were (+)-catechin and (−)-epicatechin, while lignans, such as (+)-taxiresinol, (+)-isotaxiresinol, (+)-isolariciresinol and (−)-secoisolariciresinol, were present in trace amounts. T. cuspidata cells contained 1.7% (+)-catechin and 2.4% (−)-epicatechin on dry wt basis but when stimulated with methyl jasmonate produced 3.4% catechin and 5.2% epicatechin. These are the highest levels of these metabolites obtained in plant cell cultures.

Antioxidant activity of polyphenols from the far-east plant Taxus cuspidata

Phenolic components of the wood and roots of far-east yew (Taxus cuspidata) have been isolated and investigated. Four lignans [(+)-taxiresinol, (+)-isotaxiresinol, (+)-isolariciresinol, (−)-secoisolariciresinol] and two catechins [(+)-catechin, (−)-epicatechin] were identified using spectroscopic techniques. HPLC data showed that wood, bark, roots, stems, and needles of the plant contain different amounts of lignans and catechins. The antioxidant and radical-scavenging activities of the polyphenols were evaluated on two in vitro model systems.

Deglycosylation of isoflavonoid glycosides from Maackia amurensis cell culture by β−D-glucosidase from Littorina sitkana hepatopancrease

Maakia amurensis (strain A-18) cell culture synthesizes a significant quantity of isoflavonoids, a large part of which consists of isoflavone glucosides and malonylglucosides. β−D-Hydrolase enzyme complexes from the marine mollusk Littorina sitkana and the marine mycelial fungus P. canescens were used to obtain isoflavones from their conjugated forms. The specificity of β−D-glucanases from L. sitkana for various glycosides was studied. The deglycosylation efficiency depended on the aglycon structure. The deglycosylated fraction of isoflavonoids obtained from M. amurensis cell culture exhibited antitumor activity.

AN ERITRICHIUM SERICEUM LEHM. (BORAGINACEAE) CELL CULTURE - A SOURCE OF POLYPHENOL COMPOUNDS WITH PHARMACOLOGICAL ACTIVITY

Polyphenol compounds consisting of caffeic acid oligomers were detected in cell cultures from Eritrichium sericeum Lehm.: (−)-rabdosiin, (+)-rosmarinic acid, and eritrichin; the quantitative contents of individual polyphenols were determined. The total content of polyphenol compounds in biotechnological raw material was 6.9% of dry tissue weight, which was 26.5 times greater than the content in the roots of the native plant. Studies of the pharmacological activity of the experimental Eritrichium sericeum Lehm. preparation, containing a complex of polyphenol compounds, demonstrated increases in renal excretory function and suppression of the exudative stage of inflammation as a result of prolonged use of the cell culture phytocomplex at a dose of 100 mg/kg.

Polyphenolic Metabolites from Iris pseudacorus

Plants of the genus Iris contain several polyphenolic compounds possessing antioxidant, antitumor, and antimicrobial activity [1–3]. Leaves of I. pseudacorus L. biosynthesize isoflavonoids and flavonoids under the influence of copper-chlorideinduced stress [4, 5]. The goal of the present work was to isolate and characterize structurally polyphenolic metabolites from the aerial part of I. pseudacorus without the influence of stress factors. The plant was collected on Sakhalin Island in Doltinsky District in August 2012. The Eurasian species I. pseudacorus was introduced to Sakhalin Island and propagated along the shores of reservoirs in the southern part of the island. Herbarium specimen No. 103568 is preserved in the herbarium of the Laboratory of Chemotaxonomy, PIBOC, FEB, RAS. The aerial part of the plant (450 g) was extracted successively with hexane and CHCl3. The hexane (5.8 g) and CHCl3 (3.3 g) extracts were chromatographed sequentially over columns of silica gel (100–150 m and 40–60 m) using hexane:EtOAc and then CHCl3:MeOH with increasing contents of EtOAc and MeOH, respectively. After additional purification, the obtained fractions underwent gel-filtration over a column of Sephadex LH-20 using CHCl3–MeOH (8:1) to afford compounds (mg) 1 (5.7), 2 (2.1), 3 (1.8), 4 (2.4), 5 (2.1), 6 (3.6), 7 (5.9), 8 (2.3), 9 (3.4), and 10 (1.6). PMR and 13C NMR spectra of 1–10 were recorded on a Bruker Avance III 700 instrument at 700 and176 MHz, respectively. Resonances in NMR spectra were assigned using homoand heteronuclear COSY, HMBC, HSQC, and NOESY experiments. Mass spectra (ESI) were obtained on a Shimadzu LCMS-2020 LC-MS (3.5 kV). 13C NMR spectra of 1, 2, 5, 8, and 9 were taken for the first time. The assignments of resonances for C-7, C-9, and C-2 in NMR spectra of 4 were refined [6].

Polyphenolic Metabolites from Iris pseudacorus Roots

Five known isoflavonoids (6–8, 11, 16), one flavonoid (14), four hydroxybenzoic acids (1–4), and a new 7-O-β-D-glucopyranoside of 5,7-dihydroxy-2′,6-dimethoxyisoflavone (5) were isolated from Iris pseudacorus roots and identified. The polyphenol contents in stems and roots of this plant were determined. The effect of 6, 8, 9, 11, 15, and 16 on the growth of colonies of HT-29 human colon carcinoma cells was studied. It was shown that trans-3-hydroxy-5,7-dimethoxyflavanone (9) exhibited pronounced inhibition of tumor-cell colony formation at a dose of 25 μM.