Rosmarinic Acid Production Research Articles

Bioproduction and Optimization of Rosmarinic Acid Production in Solenostemon Scutellarioides through Media Manipulation and Conservation of High Yielding Clone via Encapsulation

The present study describes the role of different exogenous phytohormones, polyamines and sucrose on growth and rosmarinic acid (RA) production in whole plant culture of Solenostemon scutellarioides. It was further aimed to conserve elite clones via synthetic seed technology. S. scutellarioides was treated either singly or in combination with different phytohormones. Cultures incubated with NAA (0.5 mg L(-1)) yielded the highest RA accumulation (g(-1FW)), but negatively affected the growth. So, overall RA content was insignificant. Cultures incubated with IBA, BAP and GA3 at low concentration significantly improved growth and RA bioaccumulation. In the combinatorial study, IBA+BAP+GA3 (0.5 mg L(-1) each) was found optimum for plant biomass and RA production (65.2% improvement of total RA). Amongst polyamines, putrescine (1 mg L(-1)) exhibited 20.4% improvement of total RA content. The intracellular RA accumulation (g(-1FW)) was significantly higher between 5 and 7% of sucrose concentrations. However, the total increase in RA content was inhibited due to deterioration of the culture with increasing sucrose concentration. Based on the effect of different treatments on growth and RA accumulation, a high yielding and stable plant line was selected for conservation via alginate encapsulation. Uniform shaped alginate coated synthetic seeds conserved up to 6 months exhibited high regeneration potential and RA content.

The effect of tea fermentation on rosmarinic acid and antioxidant properties using selected in vitro sprout culture of Orthosiphon aristatus as a model study

Orthosiphon aristatus, an Indonesian medicinal plant, is normally used as a traditional herbal tea. Recently, this plant has begun to attract attention due to its antioxidant properties. However, little is known about tea fermentation effect on antioxidant properties of this plant. Thus, to extend the tea fermentation study, in vitro sprout culture of this plant was established as a new feature model. This model plant was selected based on three reasons. Firstly, as a native tropical plant, to grow this plant in sub-tropic area is considered difficult. Secondly, the in vitro sprout culture is more genetically stable compared to other types of in vitro cultures. Thirdly, results showed that this in vitro sprout culture grew faster and produced higher biomass than in vitro tissue culture. Both characteristics are important in producing tea leaves. Accordingly, the aim of the current study was twofold. First was to establish high rosmarinic acid line of in vitro sprout culture of Orthosiphon aristatus by elicitation. Second was to evaluate the effect of tea fermentation on antioxidant properties of this plant. Results showed that yeast extract (5 g/L) elicitation resulted in the highest production of rosmarinic acid. This elicited plant was subjected to partial and full tea fermentation. Results revealed that both tea fermentations decreased the level of rosmarinic acid, total phenolic compounds, flavonoids, and flavonols. These decreases were concomitant with reduced antioxidant activities as measured by 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity, Trolox equivalent antioxidant capacity (TEAC), and Superoxide dismutase (SOD)-like activity assays. HPLC results showed that the longer the tea fermentation was, the greater reduction rosmarinic acid was found. High correlation value of 0.922 between rosmarinic acid and antioxidant activities was also observed. These results indicated that rosmarinic acid is the major contributor to the antioxidant activities of Orthosiphon aristatus. These results may provide useful information, in particular, for the food and pharmaceutical industries in the development of functional foods desiring maximum potential health benefits from Orthosiphon aristatus.

Molecular cloning of SoHPPR encoding a hydroxyphenylpyruvate reductase, and its expression in cell suspension cultures of Salvia officinalis

Rosmarinic acid (RA) is a naturally-derived compound present in medicinal plants, herbs and spices, and is well-known for its antioxidant, antibacterial and medicinal properties. It has been recently considered for use as a food preservative, nutraceutical, and for medicinal purposes. Undifferentiated cells (callus and cell suspensions) cultured in vitro are often used for the production of secondary metabolites, and for identifying biosynthetic pathways. In this work Salvia officinalis L. (common sage) cell suspension cultures were established, and a specific cell line was selected for the high antioxidant capacity of its methanolic extract, which was characterized by a high content of RA. Scavenger activity (DPPH test) and total RA content were evaluated during cell growth. Gene coding for Hydroxyphenylpyruvate reductase, the key-enzyme responsible for the RA metabolic biosynthesis, was cloned from common sage (SoHPPR). Its transcript expression level was monitored during cell suspension cultures, and showed a relationship with scavenger activity and RA yield. Our results suggest the potential use of this gene as a marker and target for the modulation of RA production in controlled conditions.

Elicitor-induced rosmarinic acid accumulation and secondary metabolism enzyme activities in Solenostemon scutellarioides

This study aimed to improve rosmarinic acid (RA) production in the whole plant culture of Solenostemon scutellarioides through elicitation. Amongst selected elicitors methyl jasmonate (MJ), salicylic acid (SA), and yeast extract (YE) caused significant elevation in RA accumulation. Elicitation with MJ (50 μM) and SA (50 μM) caused almost 1.7 and 1.4-fold increase in RA accumulation, respectively, within day 1. While YE (100 μg ml−1) elicitation showed highest RA content (~1.5-fold) in day 3. Preceding the elicitor-induced RA accumulation, there was a notable alteration in the specific activities of RA biosynthetic enzymes viz. phenylalanine ammonia lyase, tyrosine aminotransferase, hydroxyl-phenylpyruvate reductase and rosmarinic acid synthase up on MJ (50 μM), SA (50 μM) and YE (100 mg ml−1) elicitation. Based on differential responses of aforementioned enzymes, RA synthesis was further scaled up through combination of elicitors in pre-optimized doses. In synergy study, at a time exposure with MJ + SA + YE and MJ + SA followed by YE after 24 h has been found to produce significant elevation of RA (2.0 and 1.9-fold, respectively) within 24 h while later maintained a steady state increased level (~1.7 ± 0.2-fold) over control up to day 7.

Accumulation of Tilianin and Rosmarinic Acid and Expression of Phenylpropanoid Biosynthetic Genes in Agastache rugosa

Korean mint (Agastache rugosa), a perennial, medicinal plant of the Labiatae family, has many useful constituents, including monoterpenes and phenylpropanoids. Among these, tilianin and rosmarinic acid, 2 well-known natural products, have many pharmacologically useful properties. Chalcone synthase (CHS) and chalcone isomerase (CHI) catalyze the first and second committed steps in the phenylpropanoid pathway of plants, leading to the production of tilianin. In this study, cDNAs encoding CHS (ArCHS) and CHI (ArCHI) were isolated from A. rugosa using rapid amplification of cDNA ends (RACE)-PCR. Amino acid sequence alignments showed that ArCHS and ArCHI shared high sequence identity and active sites with their respective orthologous genes. Quantitative real-time PCR analysis was used to determine the expression levels of genes involved in tilianin and rosmarinic acid biosyntheses in the flowers, leaves, stems, and roots of A. rugosa. High-performance liquid chromatography (HPLC) revealed that the accumulation pattern of tilianin matched the expression patterns of ArCHS and ArCHI in different organs of A. rugosa. Moreover, acacetin, the precursor of tilianin, also demonstrated an accumulation pattern congruent with the expression of these 2 genes. The transcription levels of ArPAL, ArC4H, and Ar4CL were the highest in the leaves or flowers of the plant, which also contained a relatively high amount of rosmarinic acid. However, the roots showed a significant content of rosmarinic acid, although the transcription of ArPAL, ArC4H, and Ar4CL were low. The findings of our study support the medicinal usefulness of A. rugosa and indicate targets for increasing tilianin and rosmarinic acid production in this plant.

The c4h, tat, hppr and hppd genes prompted engineering of rosmarinic acid biosynthetic pathway in Salvia miltiorrhiza hairy root cultures.

Rational engineering to produce biologically active plant compounds has been greatly impeded by our poor understanding of the regulatory and metabolic pathways underlying the biosynthesis of these compounds. Here we capitalized on our previously described gene-to-metabolite network in order to engineer rosmarinic acid (RA) biosynthesis pathway for the production of beneficial RA and lithospermic acid B (LAB) in Salvia miltiorrhiza hairy root cultures. Results showed their production was greatly elevated by (1) overexpression of single gene, including cinnamic acid 4-hydroxylase (c4h), tyrosine aminotransferase (tat), and 4-hydroxyphenylpyruvate reductase (hppr), (2) overexpression of both tat and hppr, and (3) suppression of 4-hydroxyphenylpyruvate dioxygenase (hppd). Co-expression of tat/hppr produced the most abundant RA (906 mg/liter) and LAB (992 mg/liter), which were 4.3 and 3.2-fold more than in their wild-type (wt) counterparts respectively. And the value of RA concentration was also higher than that reported before, that produced by means of nutrient medium optimization or elicitor treatment. It is the first report of boosting RA and LAB biosynthesis through genetic manipulation, providing an effective approach for their large-scale commercial production by using hairy root culture systems as bioreactors.

Rosmarinic acid content in basil plants grown in vitro and in hydroponics

Abstract The accumulation of selected caffeic acid derivatives (CADs), in particular rosmarinic acid (RA), was investigated in different tissues (leaves, roots and plantlet shoots) of sweet basil (Ocimum basilicum L.) plants grown either in vitro or in hydroponic culture (floating system) under greenhouse conditions. Two cultivars with green leaves (Genovese and Superbo) and one with purple leaves (Dark Opal) were tested. The content of CADs in HCl-methanol extracts was determined by HPLC. LC-MS and LC-MS-MS were used to confirm the identification of the metabolites of interest. Apart from rosmarinic acid (RA) and a methylated form of this substance, no other CADs were detected at significant level in any of the analyzed samples. The content of RA ranged approximately from 4 to 63 mg/g DW, depending on the growing system. The highest RA content was found during the in vitro multiplication, in the acclimatized plants and in the roots of hydroponically-grown seedlings at full bloom. In vitro, 6-benzyladenine reduced the accumulation of RA in purple-leaf Dark Opal cultivar, but an opposite effect of this growth regulator was observed in the green-leaf genotypes. Our findings suggest the possibility to scale-up RA production by means of in vitro or hydroponic culture of sweet basil.

Effect of Some Biotic and Abiotic Elicitors on Phenolic Acids and Diterpenes Production from Rosemary (Rosmarinus officinalis L.) Leaf and Callus Analyzed by High Performance Liquid Chromatography (Hplc)

The yield of phenolic acids; rosmarinic acid (RA), caffeic acid (CAF) and phenolic diterpenes; carnosic acid (CA), carnosol (CAR), rosmanol (ROL) from rosemary (Rosmarinus officinalis L.) was investigated. Aqueous extraction was conducted for leaf and callus tissues, the chemical analysis for extracts was carried out using high performance liquid chromatography (HPLC). Data showed that RA and CAF production was 4.5, 2.7 µg/ml respectively. CA and CAR production reached 3.3, 2.8 µg/ml when the fungal elicitor; Fusarium oxysporum was added at the concentration 2.0 mg/l (2×10 4 CFU/ml), except ROL which was found at high levels (4.3, 4.2 and 4.6 µg/ml) only from leaf extracts, untreated callus and when callus treated with 0.4 mg/l of CaCl2 respectively.

The effects of jasmonic acid and methyl jasmonate on rosmarinic acid production in Mentha × piperita cell suspension cultures

The effects of two elicitors: jasmonic acid and methyl jasmonate on cell growth as well as on rosmarinic acid accumulation in cell suspension cultures of Mentha × piperita were investigated. The highest rosmarinic acid accumulation 117.95 mg g−1 DW (12% DW) was measured 24 h after addition of 100 μM methyl jasmonate. A similar concentration 110.12 mg g−1 DW was detected 48 h after application of 200 μM jasmonic acid. Those values were nearly 1.5 times higher compared to the control sample, without elicitation. There was no substantial influence of elicitors on rosmarinic acid secretion into the culture media. Extracellular concentrations of rosmarinic acid were similar to the values from the control variants. It was documented that suspension cultures of M. piperita treated with elicitors showed a decrease in biomass accumulation when compared to the control.

Influence of fertilization and drought stress on the growth and production of secondary metabolites in Prunella vulgaris L.

The combined effects of fertilization and drought stress on the growth of Prunella vulgarisand the production of rosmarinic acid (RA), ursolic acid (UA) and oleanolic acid (OA) were investigated. The factors studied included two water conditions and three fertilizer regimes. The results showed that moderate drought stress dramatically decreases spica biomass production but increases the content of RA, UA and OA in spicas of P. vulgaris. Total RA, UA and OA yields were found to be significantly higher in well-watered plants than in drought-stressed plants. An appropriate amount of fertilizer could alleviate the negative effect of drought stress on the growth of P. vulgaris and its production of RA, UA, and OA.The interaction of water and fertilizer treatments significantly influences vegetative dry weight, reproductive dry weight and total RA, UA and OA yields in P. vulgaris. The results suggest that the application of the proper amount of fertilizer aids P. vulgaris production in arid and semi-arid regions and that the combined use of fertilizer and properly timed exposure to drought stress can enhance total RA, UA and OA yields in P. vulgaris. Key words: Prunella vulgaris L., N, P and K fertilizer, growth, drought, secondary metabolites.

Phenylalanine ammonia-lyase (PAL) gene activity in response to proline and tyrosine in rosemary callus culture

Phenylalanine ammonia-lyase (PAL) catalyzes the biosynthesis of rosmarinic acid (RA), tyrosine and phenylalanine are the precursors of RA, while proline drives metabolite precursors toward Shikimate and phenylpropanoid pathway ending with the production of RA. The aim of this study was to investigate the PAL gene activity in the callus of rosemary (Rosmarinus officinalis) due to exogenous application of L-proline and L-tyrosine. Four different concentrations of L-proline and L-tyrosine (0, 4, 5 and 6 mM, and 0, 0.4, 0.6 and 0.8 gm/L) respectively, were added to the basal Murashige and Skoog (MS) medium. The expression of the PAL gene was investigated and compared with the callus RA production. It was found that RA production increased significantly at low proline application (4 mM), on the other hand, tyrosine application at low concentrations had no effect on RA accumulation, while high tyrosine concentration (0.8 g/L) increased RA accumulation. When comparing PAL gene activity and RA production in callus tissues, it was found that they were correlated. Proline application alone at 4 mM or tyrosine alone at 0.8 g/L enhanced PAL gene activity, and also combining both proline at 4 mM and tyrosine at 0.8 g/L enhanced PAL gene activity and produced the highest RA accumulation in callus tissues [0.047 mg/g freash weight (fw)]. Key words: Phenylalanine ammonia-lyase (PAL), gene expression, rosmarinic acid, Rosmarinus officinalis, tyrosine, proline.

Antioxidant compounds in Salvia officinalis L. shoot and hairy root cultures in the nutrient sprinkle bioreactor

The study focused on the production of compounds with antioxidant activity in hairy root and shoot cultures of <em>Salvia officinalis</em> grown in laboratory-scale sprinkle nutrient bioreactors. HPLC analysis showed that production of rosmarinic acid in transformed roots (34.65 ±1.07 mg l<sup>-1</sup>) was higher that in shoot culture (26.24 ±0.48 mg l<sup>-1</sup>). In the latter diterpenoids: carnosic acid (1.74 ±0.02 mg l<sup>-1</sup>) and carnosol (1.34 ±0.01 mg l<sup>-1</sup>) were also found. Biomass accumulation after a growth period in the bioreactor was also studied. An 18-fold increase in hairy root biomass was recorded after 40 days of culture. In sage shoot culture, biomass increased 43 times after 21 days of bioreactor run. The current operating conditions of the bioreactor were not suitable for the propagation of <em>Salvia officinalis</em> mainly due to the hyperhydricity problem of leaves and stems.

Liquid shoot culture of Salvia officinalis L. for micropropagation and production of antioxidant compounds; effect of triacontanol

Liquid shoot culture of <em>Salvia officinalis</em> L. in MS medium containing IAA (0.1 mg l<sup>-1</sup>) and BAP (0.45 mg l<sup>-1</sup>) was developed and evaluated in relation to shoot multiplication and antioxidant compound (carnosic acid, carnosol and rosmarinic acid) accumulation. In the liquid medium, on average, 3 new shoots per explant (shoot tip) were obtained within 3 weeks. The shoots produced 8.2±0.02 mg of diterpenoids and 31.2±0.29 mg of rosmarinic acid per gram of dry weight. Shoot proliferation and diterpenoid content increased when triacontanol (5, 10 or 20 pg l<sup>-1</sup>) was added to the liquid medium. In optimum conditions (at 20 pg l<sup>-1</sup> TRIA) almost 7 shoots were formed per explant after 3 weeks. An increase in diterpenoid production (expressed as the sum of carnosol and carnosic acid) ranged from 30% to 50% and dependended on triacontanol concentration tested. The level of diterpenoids in triacontanol-treated shoots was similar to the content of compounds in commercial herbal product (dried leaves of <em>S. officinalis</em>) (10-12 mg g<sup>-1</sup> dry wt). Triacontanol did not increase rosmarinic acid production, but the content of the phenolic as compound in shoots grown in liquid culture (31 mg g<sup>-1</sup> dry wt) was even 24 times higher compared to samples of dried leaves of <em>S. officinalis</em> plants. We also demonstrated that the highest amounts of CA, Car and RA were accumulated in young, top parts of sage shoots. This observation could be useful for improving the selection of material for the extraction of natural antioxidants from <em>S. officinalis</em>.

Effects of Nitrogen and Calcium Carbonate on Growth, Rosmarinic Acid Content and Yield of Satureja hortensis L.

Because of the presence of free phenolic acids, especially rosmarinic acid, extracts of Satureja hortensis L.possess antioxidant and antimicrobial activity. The production of rosmarinic acid by plant cells can be modifiedby many factors. In the present study, the changes induced by calcium carbonate and nitrogen application onrosmarinic acid content, plant growth, and yield of S. hortensis cv. Saturn were investigated. Nitrogenfertilization significantly increased plant height, fresh and dry weights of herb as well as rosmarinic acid contentand yield. CaCO3 treatments did not affect rosmarinic acid content or yield, while the fresh and dry weights ofherb were increased by applying 5 t ha-1 CaCO3.

Environmental Factors Affecting the Accumulation of Rosmarinic Acid in Spearmint (Mentha spicata L.) and Peppermint (Mentha piperita L.)

Four spearmint, and two peppermint clonal lines, selected for enhanced rosmarinic acid content (50-120 mg g-1 rosmarinic acid DW), where up to 80% of the antioxidant activity was correlated to rosmarinic acid content, were examined to determine the effects of environmental and physiological conditions on the accumulation of rosmarinic acid in leaf tissues. Exposure to a short photoperiod of 12 hours in comparison to 16 hours reduced rosmarinic acid accumulation in two mint lines, but no significant difference was found between photoperiods of 14 and 16 hours. The physiological age of the plant strongly influenced the accumulation of rosmarinic acid with the highest levels recorded in the vegetative state, and a significant reduction in the concentration of rosmarinic acid in the leaves in both the bud initiation and flowering stages in the mint lines. Cold stress, impacted over a six week period had no effect on rosmarinic acid production. A field study of the commercial chemotype 700B indicated that soil type plays an essential role in the accumulation of rosmarinic acid in the leaf tissue, probably due to retention of moisture which favours rosmarinic acid production. For producers and extractors, taking these factors into account would significantly increase rosmarinic acid accumulation in commercially high rosmarinic acid mint and increase the quality control of plant extracts for the natural products industry.

Rosmarinic acid production from transformed root cultures of Nepeta cataria L.

Rosmarinic acid, a natural polyphenol antioxidant carboxylic acid, is an important secondary metabolite in Nepeta cataria, a member of the mint family (Labiatae) and has antioxidant, anti-inflammatory and antimicrobial activities. To investigate,in vitro production of rosmarinic acid, hairy root cultures of N. cataria were established by infecting leaf and stem explants with Agrobacterium rhizogenesR1000, and tested the growth and rosmarinic acid production of these cultures. Hairy roots were cultured in Murashige and Skoog liquid medium and maximum growth (11.2 g dry wt/l) was attained after 15 days of culture, at which time the content of rosmarinic acid was 13.6 mg/g dry wt. These results demonstrate that hairy root culture of N. cataria is a valuable alternative approach for the production of rosmarinic acid. Key words: Agrobacterium rhizogenes, catnip, hairy root, Nepeta cataria L., rosmarinic acid.

PAL Gene Activity and Rosmarinic Acid Production in Rosemary Genotypes

Phenylalanine ammonia-lyase (PAL) catalyzes the biosynthesis of rosmarinic acid (RA) in Rosmarinus officinalis. Increases in RA content in plant tissue in vitro coincided with increases in PAL activity. The activity of the gene responsible for the production of the PAL enzyme in five rosemary genotypes was investigated to better understand the accumulation of RA. The expression of the PAL gene was relatively high in genotypes “Majorca,” R. officinalis, and “Madeline Hill” and low in “Pine Scented” and “Arp.” The highest genotype in PAL gene expression, “Madeline Hill,” had the lowest RA production in the leaves. This could be due to tissue specific regulation inside plant tissues; however, inside callus tissues, tissue specific regulation no longer existed, and RA in callus was produced in repetitively large amounts in genotypes with the high PAL gene expression.

Influence of growth regulators and sucrose concentrations on growth and rosmarinic acid production in calli and suspension cultures of Coleus blumei

Rosmarinic acid, which is reported to have adstringent, antibacterial, antiviral and antioxidant activities, is one of the most prominent secondary compounds in Coleus blumei (Lamiaceae). Rosmarinic acid (RA) production in different hybrids of C. blumei was estimated by HPLC. Conditions for HPLC were as follows: column, 150 × 4.6 mm; solvent system, methanol −0.1% phosphate (45 : 55); flow rate, 0.9 mL/min; detection: 325 nm. Two out of four hybrids of C. blumei (hy1; hy2) contain better rosmarinic acid production (0.9 and 1.0% dry weight, respectively) and the leaves have the highest rosmarinic acid production, followed by stems and roots. The hydroxyphenylpyruvate reductase (HPPR) gene expression levels were analysed by semi-quantitative RT-PCR. Hy3 shows highest level of HPPR gene expression out of four hybrids on genotype-specific patterns, and stems represent the highest level of HPPR gene expression among leaves, roots and stems. This was probably a result of the fact that the RA biosynthetic pathway was regulated by interactions of several enzymes necessary for biosynthesis. The explants from the hy1 leaves were used in subsequent studies on the effect of different growth regulators (2.0 mg L−1 6-benzyl-aminopurine (6-BA), different 2,4-dichlorophenxyaretic acid (2,4-D) and α-naphthaleneacetic (NAA) concentrations) and sucrose contents (1, 2, 3, 4, 5 or 6%) on culture growth and rosmarinic acid accumulation. On the effect of different growth regulators, the best result is obtained when the B5-medium supplemented is with 2.0 mg L−1 6-BA, 0.5 mg L−1 NAA, 0.8 mg L−1 2,4-D and 2% sugar, and solidified with 0.8% agar. In this case, both growth index and rosmarinic acid accumulation reach a maximum, which is 49.7 and 25.3% (dry weight), respectively. The optimal medium for suspension culture growth contains 2.0 mg L−1 6-BA, 0.5 mg L−1 NAA, 0.8 mg L−1 2,4-D, 600 mg L−1 inositel and 2% sugar, and the rosmarinic acid production is 1.7% (dry weight). The rosmarinic acid accumulation is enhanced by the sucrose concentrations. When the sucrose content is 6%, the calli and cell culture accumulate 33.7% (dry weight) and 10.1% (dry weight) rosmarinic acid, respectively. Higher sucrose content (≥5%) has a negative influence on culture growth. B5-medium supplemented with 2.0 mg L−1 6-BA, 0.5 mg L−1 NAA, 0.8 mg L−1 2,4-D, 4% sugar and 0.8% agar is best for both culture growth and rosmarinic acid accumulation. On the basis of the above results, we can establish a two-phase culture system by calli of C. blumei for the biosynthesis of RA. In the growth phase, the calli grow well, but with lower RA production in a modified B5-1A medium with 2% sucrose. The calli are then subcultured to B5-1A medium with 6% sucrose where the calli start to accumulate RA, but grow slowly.

Biotechnological applications for rosmarinic acid production in plant

Rosmarinic acid, an important phenolic compound, is commonly found in species of the Boraginaceae and the subfamily Nepetoideae of the Lamiaceae. However, it is also found in species of other higher plant families and in some fern and hornwort species. Rosmarinic acid has a number of interesting biological activities, e.g. antiviral, antibacterial, anti-inflammatory, and antioxidant. The presence of rosmarinic acid in medicinal plants, herbs and spices has beneficial and health promoting effects. In plants, rosmarinic acid is supposed to act as a preformed constitutively accumulated defence compound. The biosynthesis of rosmarinic acid starts with the amino acids phenylalanine and tyrosine. Plant cell cultures, e.g. from Coleus blumei or Salvia officinalis, accumulate rosmarinic acid in amounts much higher than in the plant itself (up to 36% of the cell dry weight). Similarly some other biotechnological researches for production of rosmarinic acid were done in the past i.e. from shoot culture, producing hairy root, using bioreactor, and the treatment of elicitors. As a review paper the aim of this study is to gather all the possible biotechnological ways to produce rosmarinic acid, thus will help the scientists to take action for future study in this discipline.

Somatic embryogenesis, embyogenic cell suspension from Ocimum sanctum leaf callus cultures and its rosmarinic acid accumulation

The accumulation of rosmarinic acid (RA) in Ocimum sanctum (L.) somatic embryogenic callus and its cell suspension cultures was investigated. For callus induction, leaves, stems and inflorescence explants excised from field-grown plants were cultured on Murashige and Skoog (MS) medium containing 2,4-D (1 mg l-1) combined with kinetin (0.1-0.5 mg l-1), 2,4-D (0.5-2.5 mg l-1) individually, IAA (0.5-2.5 mg l -l) individually, and NAA (0.5-2.5 mg l -l) individually. Due to better proliferation ability and high RA content of leaf callus, it was chosen to induce somatic embryogenesis by supplementing with different concentration and combination of BAP + NAA + kinetin. Among the different combination tested, BAP (1 mg l -l) + NAA (1 mg l -l) + kinetin (0.5 mg l -l) exhibited a maximum embryogenic potential (73%) and number of globular embryo (34.3) per callus. In order to study the RA production, the proliferation of embryogenic suspension culture was tested by transferring embyogenic callus at stationary growth phase from MS solid to liquid medium containing the same composition and hormonal concentration. After five subcultures, the suspensions were maintained continuously for six weeks without subculturing. Suspension cultures showed progressive callus growth and RA accumulation throughout the experimental period. The highest RA (82.1 mg l -1) content and biomass growth (11.6 g l -1) was observed in the cultures supplemented with BAP (1 mg l -l) + NAA (1 mg l -l) + kinetin (0.5 mg l -l). It was 40 fold higher than the leaf callus.