Rosmarinic acid production by Lavandula vera MM cell suspension: the effect of temperature

Lavandula vera MM cell suspension, grown at 28 degrees C in a 3-l bioreactor, produced rosmarinic acid maximally at 3 g l(-1)) though most biomass (33.2 g dry wt l(-1)) was at 30 degrees C.     

The influence of phenylalanine on accumulation of rosmarinic and caffeic acids by Lavandula vera MM cell culture

Lavandula vera MM cell suspension culture was grown in Linsmayer-Skoog medium with different concentrations of phenylalanine. Adding phenylalanine to the medium (0.1–0.5 g/l) enhanced accumulation of caffeic acid in parallel with rosmarinic acid. When 0.3 g phenylalanine/l was added, the yield of rosmarinic and caffeic acids reached 87 mg/l and 60 mg/l respectively, compared with 68 mg/l and 4 mg/l in controls (without phenylalanine).     

Release of rosmarinic acid by Lavandula vera MM cell suspension in two-phase culture systems

Studies were conducted on the cultivation of Lavandula vera MM cell suspensions in different culture systems for the release of extracellular rosmarinic acid (RA). It was established that during cultivation with Amberlite XAD-4 as a second phase, 6.4% of the total content of RA was adsorbed. When L. vera MM cell suspension was cultivated in an aqueous two-phase system formed by adding 4% polyethylene glycol (MW 20,000) and 7.5% dextran (MW 70,000), 11.8% of the total RA content was released into the top polyethylene glycol phase.     

Rosmarinic acid by Lavandula vera MM cell suspension: Phosphorus effect

Summary The growth of Lavandula vera MM cell suspension and biosynthesis of rosmarinic acid were followed during its cultivation in Linsmayer - Skoog (LS) medium (containing 170 mg/L KH₂PO₄, control cultivation) and in modified LS media, containing different concentration of KH₂PO₄. Doubled concentration of KH₂PO₄ (340 mg/L) caused an increase in the amount of biosynthesised cell biomass (17 g/L vs 13 g/L for control cultivation) and rosmarinic acid (140 mg/L vs 68 mg/L for control cultivation).     

Rosmarinic acid production by Lavandula vera MM cell-suspension culture

The time courses of growth and rosmarinic acid production by Lavandula vera MM cell suspension were investigated. The uptake of the main nutrients (sucrose, nitrogen, phosphorus, K, Ca, Mg) was followed during cultivation and the data on the physiology of the L. vera MM cell culture are presented. It was established that the cell culture synthesizes rosmarinic acid during the linear phase of growth for a relatively short period (between the 4th and 8th days of cultivation). The influence of sucrose concentration in the nutrient medium on cell growth and accumulation of rosmarinic acid by L. vera MM cell culture was investigated. The results showed that 7% sucrose in the nutrient medium ensured a steady growth of the cell suspension and increased the yield of rosmarinic acid (29.2 g/l dry biomass and 507.5 mg/l rosmarinic acid compared to 13.0 g/l dry biomass and 68.6 mg/l rosmarinic acid for the control cultivation with 3% sucrose). Received: 17 September 1996 / Received revision: 31 January 1997 / Accepted: 1 February 1997     

Production of rosmarinic acid by<Emphasis Type="Italic">Lavandula vera</Emphasis> MM cell suspension in bioreactor: effect of dissolved oxygen concentration and agitation

The relationship between dissolved oxygen (DO) concentration, agitation rate and growth of Lavandula vera MM and rosmarinic acid biosynthesis was investigated in 3 l laboratory bioreactor. Lavandula vera MM cell suspension accumulated the highest amounts of biomass (34.8 g/l) and rosmarinic acid (1870.6 mg/l) on day 12 of cultivation at 50% dissolved oxygen and agitation speed 100 rpm and at 30% dissolved oxygen and agitation speed 300 rpm, respectively.     

Enhanced rosmarinic acid production by Lavandula vera MM cell suspension culture through elicitation with vanadyl sulfate

The influence of elicitation on rosmarinic acid biosynthesis by Lavandula vera MM cell suspension culture was investigated using vanadyl sulfate as an abiotic elicitor. It was established that 12 h after treatment with 25 mg/l vanadyl sulfate the rosmarinic acid production was increased up to 3.92 g/l (2.8 times higher compared to the control cultivation). No significant amounts of rosmarinic acid were detected in the culture medium in comparison with its intracellular content. However, it was observed that the extracellular content of rosmarinic acid is 3.3 times higher compared to the control variant (4 h after treatment at elicitor concentration 25 mg/l).     

Elicitation of rosmarinic acid by <Emphasis Type="Italic">Lavandula vera</Emphasis> MM cell suspension culture with abiotic elicitors

The growth of Lavandula vera MM plant cell suspension culture and rosmarinic acid biosynthesis under elicitation with benzothiadiazole and methyl jasmonate were investigated. Upon elicitation with 50 μM methyl jasmonate, the production of rosmarinic acid was enhanced 2.4-fold (3348 mg/l) compared to the non-elicited cells. The influence of benzothiadiazole on rosmarinic acid biosynthesis was weaker and 12 h after its addition the achieved yields were 20–30% higher compared to the control variant at this time. The influence of both elicitors on rosmarinic acid secretion into the culture medium was also discussed.     

Radical quenching by rosmarinic acid from Lavandula vera MM cell culture

This study was conducted to evaluate the radical scavenging capacities of extracts and preparations from a Lavandula vera MM plant cell culture with different rosmarinic acid content and to compare them with pure rosmarinic and caffeic acids as well. The methods, which were used are superoxide anion and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radicals scavenging assays. Results showed that extracts and preparations from Lavandula vera MM possess strong radical scavengers, as the best both radical scavengers appeared to be the fractions with enriched rosmarinic acid content, obtained after ethylacetate fractioning (47.7% inhibition of superoxide radicals and 14.2 microM 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid equivalents, respectively). These data reveal the possibilities for application of these preparations as antioxidants.     

Rosmarinic acid production in Coleus cell cultures

Cell suspension cultures of Coleus blumei Benth. have been found to accumulate 8–11% of their dry weight as rosmarinic acid (-O-caffeoyl-3,4-dihydroxyphenyl-lactic acid). Actively-growing tissue converts >20% of exogenously supplied phenylalanine and tyrosine to the caffeoyl ester and this high rate of synthesis coincides with an increase in phenylalanine ammonia-lyase specific activity. Administration to the cultures of known phenylpropanoid precursors of rosmarinic acid failed to enhance the latter's production and in some cases inhibited it.Abbreviations RA rosmarinic acid (-O-caffeoyl-3,4-dihydroxyphenyllactic acid - DOPA dihydroxyphenylalanine - PAL phenylalanine ammonialyase - DOPL dihydroxyphenyl-lactic acid     

Rosmarinic acid and its derivatives: biotechnology and applications

Rosmarinic acid (RA) is one of the first secondary metabolites produced in plant cell cultures in extremely high yields, up to 19% of the cell dry weight. More complex derivatives of RA, such as rabdosiin and lithospermic acid B, later were also obtained in cell cultures at high yields. RA and its derivatives possess promising biological activities, such as improvement of cognitive performance, prevention of the development of Alzheimer’s disease, cardioprotective effects, reduction of the severity of kidney diseases and cancer chemoprevention. The TNF-α-induced NF-κB signaling pathway has emerged as a central target for RA. Despite these impressive activities and high yields, the biotechnological production of these metabolites on an industrial scale has not progressed. We summarized data suggesting that external stimuli, the Ca²⁺-dependent NADPH oxidase pathway and processes of protein phosphorylation/dephosphorylation are involved in the regulation of biosynthesis of these substances in cultured plant cells. In spite of growing information about pathways regulating biosynthesis of RA and its derivatives in cultured plant cells, the exact mechanism of regulation remains unknown. We suggest that further progress in the biotechnology of RA and its derivatives can be achieved by using new high-throughput techniques.     

Influence of the carbon source on growth and rosmarinic acid production in suspension cultures of Coleus blumei

Suspension cultures of Coleus blumei were characterized with respect to growth and rosmarinic acid formation in media with different sugars and various sugar concentrations. Sucrose is the sugar with the highest stimulating effect on growth and rosmarinic acid accumulation, followed by glucose and fructose. The sugar alcohol mannitol cannot be metabolized by the plant cells. Sucrose is cleaved into glucose and fructose by the Coleus cells. Sucrose concentrations from 1 to 5% have an increasing positive effect on growth and rosmarinic acid synthesis in the cell cultures with a maximum rosmarinic acid content of 12% of the dry weight in medium with 5% sucrose; in medium with 6% sucrose rosmarinic acid accumulation obviously did not reach its highest level in the culture period of 14 days. A very high yield of rosmarinic acid (2 mg ml^-1 suspension) could also be achieved by maintaining a sucrose concentration of 2% during the whole culture period. The start of rosmarinic acid synthesis by the cell cultures seems to be regulated by the growth limitation when a nutrient, e.g. phosphate is depleted from the medium. The rate of rosmarinic acid accumulation is related to the amount of carbon left in the medium when growth ceases.Abbreviations RA rosmarinic acid     

Perfusion strategy for rosmarinic acid production by Anchusa officinalis

The production of an intracellular secondary metabolite rosmarinic acid (RA) by plant cell suspensions of Anchusa officinalis cultivated with intermittent medium exchange is investigated. Initially, a two-stage perfusion culture method was employed. After being cultured in the batch mode for ca. 6 days in B5 medium plus 3% sucrose, 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.1 mg/L kinetin (2,4-D B5 medium), Anchusa culture was cultivated to high cell density by perfusion during the growth stage using a hormone-free Gamborg B5 medium supplemented with 6% sucrose. This was followed by a production stage, in which a complete medium exchange into B5 medium plus 3% sucrose and 0.25 mg/L naphthleneacetic acid (NAA) was conducted. The two-stage perfusion culture had a higher maximum culture RA concentration but a lower RA content per cell than the batch stock culture maintained in the 2,4-D B5 medium. Higher culture RA concentration was due primarily to high cell density. The high packed cell volume, however, seemed to reduce the synergistic effect of NAA on RA synthesis. Subsequently, a single-stage perfusion culture method was investigated. The best result was obtained by growing the culture in the batch mode for ca. 10 days using B5 medium supplemented with 3% sucrose and 0.25 mg/L NAA, followed by perfusing the culture with B5 medium plus 6% sucrose and 0.25 mg/L NAA at a constant perfusion rate of 0.1/day. A maximum cell dry weight of 35 g/L and a RA concentration of almost 4 g/L were achieved. This is the highest RA concentration ever reported in the Anchusa culture. (c) 1993 John Wiley & Sons, Inc.     

In vitro production of gymnemic acid from cell suspension cultures of Gymnema sylvestre R. Br

Gymnema sylvestre is an important medicinal plant that bears bioactive compound namely gymnemic acids. The present work deals with the optimization of a cell suspension culture system of Gymnema sylvestre for the production of biomass and gymnemic acid, which has anti‐diabetic properties. We investigated the effect of inoculum densities (2.5–20.0 g/L), the strength of the Murashige and Skoog (MS) medium (0.25–2.0), carbon source (sucrose, glucose, fructose, maltose), and the concentration of the sucrose (1–8% w/v) to determine their effects on biomass accumulation and production of gymnemic acid. Overall, 10 g/L of inoculum density, full‐strength MS medium supplemented with 2,4‐dichlorophenoxy acetic acid (2.0 mg/L) and Kinetin (0.1 mg/L), and 3% w/v sucrose was found best for the accumulation of biomass and gymnemic acid content (9.95 mg/g dry weight). The results of the current study will be useful for bioprocess and biochemical engineers for large‐scale production of gymnemic acid in cell culture.     

Selection of cell lines for the production of rosmarinic acid from cell suspension cultures of Orthosihon stamineus benth

Summary Cell suspension cultures of Orthosiphon stamineus were established from friable calluses produced from leaf pieces of in vitro plantlets that were derived from nodal segments of the mother plants collected from three different geographical locations. Eight lines were eventually selected after seven subculture cycles based on the growth characteristic (plant height) of the plantlets from the three locations: two fast-growing lines (>5.1 cm tall), three intermediate-growing lines (3.1–5.0 cm tall), and three slow-growing lines (<3.0 cm tall). All eight lines grew well in liquid Murashige and Skoog medium supplemented with 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.4 μM 1-naphthaleneacetic acid (NAA). All cell lines exhibited the same growth pattern but produced different maximum cell biomass when cultured in this medium. The time of harvesting the plant cells from the culture medium and the geographical source of the original plant material were both found to affect the production of rosmarinic acid (RA) in cell cultures. Two cell lines were successfully selected and identified to produce high amounts of RA. These cell lines were a fast-growing cell line from Air Itam, Penang and an intermediate-growing cell line from Relau Agriculture Research Centre, Penang which could produce 5% [(w/w) dry weight] and 4.5% [(w/w) dry weight] of RA, respectively.     

The effect of calcium on flavanol production in cell suspension cultures of Polygonum hydropiper

Cultured Polygonum hydropiper cells maintained in Murashige and Skoog (MS) medium supplemented with 10^–6 m 2,4-D, 10^–6 m kinetin, 0.1% casamino acids and 3% sucrose were transferred to medium containing a higher concentration of calcium chloride (15 mm). The content of flavanols in the cells on the 6th day was approximately twice that of the control culture (31.9–60.7 mg/g dry wt). However, the contents of other secondary metabolites such as chlorogenic acid and gallic acid were not changed. The levels of flavanols in the culture medium remained unchanged throughout the 21-day culture period. Of the the inorganic components supplemented to the culture medium , only elevated levels of calcium chloride induced an increase in flavanol contents of the cells. The results indicated that the elevated concentration of calcium in the culture medium played an important role in activating the accumulation of flavanols. Received: 4 June 1998 / Revision received: 30 October 1998 / Accepted: 29 November 1998     

Significant enhancement of oleanolic acid accumulation by biotic elicitors in cell suspension cultures of Calendula officinalis L.

The effects of elicitors on cell growth and oleanolic acid (OA) accumulation in shaken cell suspension cultures of Calendula officinalis were investigated. Elicitors were added individually at various concentrations to 5-day-old cell cultures and their effects monitored at 24 h intervals for 4 days. Different effects on OA accumulation were observed depending on the day of treatment. Jasmonic acid was the most efficient elicitor. After 72 h of treatment with 100 μM JA, the intracellular content of OA reached its maximum value (0.84 mg g⁻¹ DW), which was 9.4-fold greater than that recorded in an untreated control cultures. The addition of chitosan at 50 mg l⁻¹ produced a 5-fold enhancement of OA accumulation (0.37 mg g⁻¹ DW) after 48 h of treatment. Treatment with yeast extract at 200 mg l⁻¹ for 96 h or with pectin at 2 mg l⁻¹ for 48 h produced identical cellular levels of OA (0.22 mg g⁻¹ DW). Lastly, 48 h elicitation with homogenate of the fungus Trichoderma viride produced a 1.8-fold increase in oleanolic acid content (0.12 mg g⁻¹ DW). In addition to significantly stimulating OA accumulation and its secretion into the culture medium, the elicitors also caused slight inhibition of cell growth.