Andrey Marchev

Bioprocessing of differentiated plant in vitro systems

Plant cells contain a wide range of interesting secondary metabolites, which are used as natural pigments and flavoring agents in foods and cosmetics as well as phyto‐pharmaceutical products. However, conventional industrial extraction from whole plants or parts of them is limited due to environmental and geographical issues. The production of secondary metabolites from in vitro cultures can be considered as alternative to classical technologies and allows a year‐round cultivation in the bioreactor under optimal conditions with constant high‐level quality and quantity. Compared to plant cell suspensions, differentiated plant in vitro systems offer the advantage that they are genetically stable. Moreover, the separation of the biomass from culture medium after fermentation is much easier. Nevertheless, several investigations in the literature described that differentiated plant in vitro systems are instable concerning the yield of the target metabolites, especially in submerged cultivations. Other major problems are associated with the challenges of cultivation conditions and bioreactor design as well as upscaling of the process. This article reviews bioreactor designs for cultivation of differentiated plant in vitro systems, secondary metabolite production in different bioreactor systems as well as aspects of process control, management, and modeling and gives perspectives for future cultivation methods.

Sage in vitro cultures: a promising tool for the production of bioactive terpenes and phenolic substances

Extracts of Salvia species are used in traditional medicine to treat various diseases. The economic importance of this genus has increased in recent years due to evidence that some of its secondary metabolites have valuable pharmaceutical and nutraceutical properties.The bioactivity of sage extracts is mainly due to their content of terpenes and polyphenols. The increasing demand for sage products combined with environmental, ecological and climatic limitations on the production of sage metabolites from field-grown plants have led to extensive investigations into biotechnological approaches for the production of Salvia phytochemicals. The purpose of this review is to evaluate recent progress in investigations of sage in vitro systems as tools for producing important terpenoids and polyphenols and in development of methods for manipulating regulatory processes to enhance secondary metabolite production in such systems.

Two-phase temporary immersion system for Agrobacterium rhizogenes genetic transformation of sage (Salvia tomentosa Mill.)

Hairy root cultures of Salvia tomentosa were initiated by transformation with Agrobacterium rhizogenes. To prevent necrosis in the explants and to protect young hairy roots, Amberlite XAD-4 resin, in combination with a temporary immersion cultivation system, was applied. HPLC analyzes showed that the resin adsorbed more than 93% of the released phenolic acids and 100% of the released flavonoids. The decreased content of the released phenolics significantly reduced their destructive effects on the plant tissues, prevented, and speeded up the appearance of hairy roots.

Oxidative stress and chronic inflammation in osteoarthritis: can NRF2 counteract these partners in crime?

Osteoarthritis (OA) is an age‐related joint degenerative disease associated with pain, joint deformity, and disability. The disease starts with cartilage damage but then progressively involves subchondral bone, causing an imbalance between osteoclast‐driven bone resorption and osteoblast‐driven remodeling. Here, we summarize the data for the role of oxidative stress and inflammation in OA pathology and discuss how these two processes are integrated during OA progression, as well as their contribution to abnormalities in cartilage/bone metabolism and integrity. At the cellular level, oxidative stress and inflammation are counteracted by transcription factor nuclear factor erythroid p45–related factor 2 (NRF2), and we describe the regulation of NRF2, highlighting its role in OA pathology. We also discuss the beneficial effect of some phytonutrients, including the therapeutic potential of NRF2 activation, in OA.

Metabolic alterations of Verbascum nigrum L. plants and SAArT transformed roots as revealed by NMR-based metabolomics

Verbascum nigrum L. (dark mullein) is known to be a rich source of bioactive secondary metabolites, including high value iridoid and phenylethanoid glycosides, hence the development of alternative approaches for their sustainable supply is highly relevant. Here we report the induction of a transformed root culture of V. nigrum by applying sonication assisted Agrobacterium rhizogenes-mediated genetic transformation. Further, nuclear magnetic resonance (NMR)-based metabolomics approach has been successfully applied to study metabolic differences of the V. nigrum mother plant and related transformed roots. 1H NMR fingerprinting in combination with 2D NMR and multivariate data analysis revealed that the most abundant molecule in the hairy root culture is glutamine, which is not found in the mother plant tissue. These findings provide important insights on the metabolic alterations as a result of the transformation of the host plant genome and development of so-called hairy roots.

Production of Oleanolic and Ursolic Acids by Callus Cultures of Salvia Tomentosa Mill.

ABSTRACT Callus cultures of Salvia tomentosa Mill. were induced and analyzed for their capacity to produce oleanolic and ursolic acids. The obtained callus lines showed high variability in their biosynthetic potentials. Flow cytometric investigations showed that this variability was due to polyploidization of plant cells in calli. Polyploidization of callus cell seems to be promoted by the growth regulators used for callus induction. One octaploid line (consisting of 8C, 16C and 32C cells) was selected as prospective producer of oleanolic (991.57 μg/g DW) and ursolic (641.85 μg/g DW) acids. To our knowledge, this is the first report for obtaining of triterpenes producing callus culture of S. tomentosa Mill.

Rhodiola rosea L.: from golden root to green cell factories

Rhodiola rosea L. is a worldwide popular plant with adaptogenic activities that have been and currently are exploited in the traditional medicine of many countries, as well as, examined in a number of clinical trials. More than 140 chemical structures have been identified which belong to several natural product classes, including phenylpropanoid glycosides, phenylethanoids, flavonoids and essential oils, and are mainly stored in the rhizomes and the roots of the plant. A number of mechanisms contribute to the adaptogenic activities of R. rosea preparations and its phytochemical constituents. Among them, the intrinsic inducible mammalian stress responses and their effector proteins, such as heat shock protein 70 (Hsp70), are the most prominent. Due to its popular medicinal use, which has led to depletion of its natural habitats, R. rosea is now considered as endangered in most parts of the world. Conservation, cultivation and micropropagation are all implemented as potential preservation strategies. A number of in vitro systems of R. rosea are being developed as sources of pharmaceutically valuable secondary metabolites. These are greatly facilitated by advances in elucidation of the biosynthetic pathways and the enzymes, which catalyse the production of these secondary metabolites in the plant. In addition, biotechnological approaches show promise towards achieving sustainable production of R. rosea secondary metabolites.

Chemical Compositions of Essential Oils from Leaves and Flowers of Salvia ringens Sibth. et Sm. Growing Wild in Bulgaria

Abstract The constituents of essential oils, obtained from different aerial parts (leaves and flowers) of Salvia ringens Sibth. et Sm. growing wild in Bulgaria, have been analyzed by GC-MS. The yields of leaves and flowers oils were similar (0.03 % v/w and 0.04 % v/w). 60 compounds were identified in both oils, which correspond to 97.7 % and 97.3 % of the total oils constituents for leaves and flower oils, respectively. Both leaves and flowers oils showed similar chemical compositions, with predominance of camphor (17.2 % and 18.8 %) and borneol (7.2 % and 8.7 %). Further analyses of minor component distributions proves that the both leaves and flowers could be equally used as a raw material for obtaining of camphor reach essential oil from Salvia ringens Sibth. et Sm.

TRITERPENES PRODUCTION BY RHIZOGENIC CALLUS OF SALVIA SCABIOSIFOLIA LAM. OBTAINED VIA AGROBACTERIUM RHIZOGENES MEDIATED GENETIC TRANSFORMATION

ABSTRACT Rhizogenic callus of Salvia scabiosifolia Lam. formed after transformation with Agrobacterium rhizogenes ATCC 15834 was isolated and investigated for triterpenes production. The rhizogenic callus was cultivated on MS medium free of phytohormones and showed stable morphological and growth characteristic. The genetic transformation was confirmed by PCR amplification of rolA gene, found in the genome of transformed rhizogenic callus cells. Cell suspension was successfully initiated by transferring rhizogenic callus on LS medium supplied with 0.2 mg/L 2,4-D. The suspension culture was stable, fast growing (accumulated 1.709 g dry biomass/L) and produced 829.14 7mu;g/g dry biomass oleanolic acid. This is the first report for initiation of rhizogenic cell suspension culture from the rare plant Salvia scabiosifolia Lam.

Chemical Composition of Essential Oil of Salvia scabiosifolia Lam. from Bulgaria

Abstract The chemical composition of the essential oils of Salvia scabiosifolia Lam. from Bulgaria, has been determined by GC and GC-MS. The yield of oil obtained by water distillation was 0.14 %. The basic components, among the identified 60, representing 97.5 % of the total oil, were camphor (19.2 %), borneol (7.8 %) and camphene (7.0 %). The other components (concentrations higher than 3.0 %) were α-caryophyllene (4.2 %), β-pinene (4.0 %), γ-terpinene (3.5 %), p-cymene (3.4 %), eucalyptol (3.3 %), limonene (3.2 %) and α-pinene (3.0 %).