Levels Of Rosmarinic Acid Research Articles

Influence of elicitation and precursors on major secondary metabolite production in cultures of purple Orthosiphon aristatus Blume Miq

Major compound of interest in purple varieties of Orthosiphon aristatus Blume Miq (O. aristatus) are sinensetin and rosmarinic acid. Unfortunately, the content of these two compounds in this plant is only in small amount. An approach to produce more of these compounds is a plant tissue culture. Hence, leaf shoot explants of a purple variety O. aristatus were inoculated on Schenk and Hildebrandt (SH) media with variations of the growth regulator 2,4-dichlorophenoxyacetis acid (2,4-D). The calli formed were used as material for making cell suspension cultures. The precursors cinnamic acid, coumaric acid, caffeic acid, and ferulic acid with respective concentrations of 0.1; 0.5; and 1 mM and salicylic acid (14, 70, and 140 mg/L) were added. The media also contained Cu2+ ions (30, 40 and 50 μM), pectin (0.05; 0.1; 0.2% w/v) and AgNO3 (80, 100, 120 μM) respectively. Callus that grew on SH media with 2,4-D 0.4 ppm had a good profile, since it could produce callus faster than 2,4-D 1 ppm and 2 ppm with a friable texture on this medium. The cell suspension culture added with cinnamic acid 1 mM (S3) and Cu2+ 40 μM (C2) had a good growth curve compared to other elicitor and precursors. The highest levels of rosmarinic acid and sinensetin were found in cell suspension cultures adding with 1 mM cinnamic acid precursor. This study is believed to be the first report on the production of rosmarinic acid and sinensetin from suspension cultures of O. aristatus produced on SH media.

Plant tissue culture of cat whiskers (Orthosiphon aristatus Blume Miq): A review of secondary metabolite production and micropropagation

Background: The cat whiskers plant (Orthosiphon aristatus Blume Miq) is widely used as a raw material in traditional medicine for one of its many properties, i.e. antiviral activity. Three varieties of cat whiskers grow in Indonesia, classified according to the colour of their flower: white, white-purple, and purple. The purple variety of cat whiskers is endangered, so it is necessary to propagate this plant. Objectives: This research aimed to obtain appropriate protocols for plant propagation and production of active compounds of cat whiskers by in vitro culture. Methods: Data were collected from various online journal sites such as PubMed, ResearchGate, and Scopus. This review discusses several aspects, including callus induction efforts, modification of cell suspension cultures, and efforts to propagate the cat whiskers plant by in vitro culture. Results: Callus induction of white cat whiskers with purple and purple hues could take place on Murashige and Skoog (MS) media added with growth regulator 2,4-dichlorophenoxyacetic acid (2,4-D) 0.4 mg/L. Suspension culture medium (MS+ 2,4-D 1 mg/L + NAA 1 mg/L) can increase cell biomass and rosmarinic acid levels. Media MS + 6-benzyl amino purine (BAP) 3 mg/L + 1-naphthaleneacetic acid (NAA) 2 mg/L can induce the growth of shoots of white cat whiskers with purple and purple patterns. Root induction of the two varieties of cat whiskers could take place on MS + Indole-3-butyric acid (IBA) 0.75 mg/L medium. Conclusion: Efforts to produce secondary metabolites and plant propagation of cat whiskers by in vitro culture have been successful. It is necessary to enhance the production and propagation using bioreactors to yield more active compounds and raw materials of the cat whiskers plant.

Antioxidant Activity of Rosmarinic Acid Extracted and Purified from Mentha piperita.

Rosmarinic acid was obtained from methanolic extract of Mentha piperita L. under a reflux condenser. The current study aimed to evaluate the in vitro antioxidant activities and rosmarinic acid levels of the methanol extracts of M. piperita. The analysis of the sample by high-performance liquid chromatography technique (HPLC) indicated that rosmarinic acid was present in high concentration 1.9 mg/mL in the extract. Purification was carried out by column chromatography to give 0.020 g from 1 g of crude extract, and then the antioxidant activity of purified rosmarinic acid was determined by the 2,2-diphenyl-1-picrylhydrazyl (DPPH), H2O2 scavenging, and REDOX methods. It was revealed that the anti-oxidant potential of the rosmarinic acid extract was greater than 95% (at 100 µg/mL) for DPPH assay and 87.83% (at 100 µg/ml) for H2O2 scavenging assay. This study was performed by using a reflux methanolic extraction of M. piperita. This possible instructional technique proved to be a quick and successful method for retaining the antioxidant properties of rosmarinic acid. The rosmarinic acid content was determined using HPLC.

The new rapid and accurate analytical HPLC‐ECD method for the determination of rosmarinic acid in meat products

A new rapid method has been developed for the determination of low levels of rosmarinic acid extracted from rosemary (Rosmarinus officinalis L.) and has been used as an antioxidant in meat and meat products after cold storage at 4°C. The method is a high performance liquid chromatography using a coulometric electrochemical detector. It provides a significant improvement on the limit of detection, which was 0.33 ppb, while the limit of quantification was 1 ppb of rosmarinic acid. The advantage of the method also lies in the simpler and faster sample preparation, which can quantify a very low concentration of rosmarinic acid (60 ppb), and is more than 40 ppb below the limits of previously existing methods. A coulometric method is well suited for determining low analyte concentrations and is one of the most sensitive analytical approaches available today, in addition to being time efficient and cost effective. PRACTICAL APPLICATION: A new method for determining low concentrations (60 ppb) of rosmarinic acid in meat and meat products is presented. The method is user-friendly, as it does not require complex sample preparation. It is a selective, precise, and accurate method that makes it useful for routine applications in the meat and other food industries.

Comparison of chemical and biological properties of in vivo and in vitro samples of Salvia siirtica Kahraman, Celep & Dogan extracts prepared with different solvents

The use of Salvia species among the public and their importance in the scientific world increase due to their numerous pharmacological and biological activities on a daily basis. In this study, the phytochemical contents of different parts (root, branch, leaf, flower, whole) of different specimens of Salvia siirtica (SS) (in vivo and in vitro) extracts prepared with various solvents (petroleum ether, chloroform and ethanol) were determined using different techniques (GC-MS and LC-MS/MS) and the results were compared. In addition, biological activities (antioxidant, cytotoxic, anticholinesterase antiurease, antityrosinase, antielastase and anticollagenase) of all samples were determined and compared. The antioxidant potential of the analysed samples was found to be high, and their enzyme activity potential was low. Besides, in vitro SS-TIS (Temporary Immersion System) sample showed high cytotoxic activity (viability% 2.12 ± 0.06) against MCF-7 (breast cancer) cell line. The results of GC-MS and LC-MS/MS analyses indicated that ferruginol and sugiol could be isolated from the ethanol extract of S. siirtica roots, salvigenin and β-sitosterol from the chloroform extract of the aerial parts, and phenolic compounds from the ethanol extract of the aerial parts. In addition, the amount of rosmarinic acid, caffeic acid and 12-demethylmulticauline of in vitro samples were found to be higher than those of in vivo samples. Furthermore, all samples, both in vivo and in vitro, contained high levels of rosmarinic acid and β-sitosterol. The whole chloroform extract (SSWC) could be the source extract for salvigenin (33952.13 µg/g) and β-sitosterol (16369.71 µg/g), and the root ethanol extract (SSRE) for ferruginol (17721.99 µg/g). As a result, it is understood that the nature of the plant material, the choice of an appropriate solvent and the parts of the material used are quite effective in chemical content. S. siirtica promise potential natural antioxidant agent in food and/or pharmaceutical industry due to its phytochemical content (rosmarinic and caffeic acids, ferruginol, salvigenin) and antioxidant activity.

Micropropagation and Secondary Metabolites Content of White-Purple Varieties of Orthosiphon aristatus Blume Miq.

<b>Background and Objective:</b> The cat whiskers plant (<i>Orthosiphon aristatus</i> Blume Miq) is a plant that has been widely used as raw material for traditional medicine. The population of white-purple varieties of <i>O. aristatus</i> is decreasing efforts to maintain the white-purple <i>O. aristatus</i> need to be done keeping in mind its potential as raw material for traditional medicine. This study aims to determine the composition of a suitable medium in growing plantlet <i>O. aristatus</i> white-purple varieties and the content of its secondary metabolites. <b>Materials and Methods:</b> The internode explants were induced on MS medium added by various combinations of zeatin and 2,4-Dichlorophenoxyacetic acid (2,4-D). Root induction was carried out on shoots formed on MS medium with Indole-3-Butyric Acid (IBA). The acclimatization process was carried out using soil media. Determination of secondary metabolite levels was carried out on <i>O. aristatus</i> (<i>in vitro</i> culture) and wild-type plants aged ten months using high-performance liquid chromatography (HPLC). <b>Results:</b> MS+BAP 2ppm+NAA3 ppm media was the optimal medium for growing shoots in leaf explants. Media MS+zeatin 3 ppm+2,4-D 2 ppm produced good shoot growth on internode explants. The best root induction occurred in MS+IBA media of 0.75 ppm. The acclimatization process was successful on shoots originating from the internode, while those from leaf explants had not succeeded in growing and developing. <b>Conclusion:</b> The levels of rosmarinic acid and sinensetin in the white-purple variety <i>O. aristatus</i> (<i>in vitro</i> culture) were 1.08 and 1.62% w/w and higher than those of wild varieties.

Transformed Shoots of Dracocephalum forrestii W.W. Smith from Different Bioreactor Systems as a Rich Source of Natural Phenolic Compounds.

Transformed shoots of the Tibetan medicinal plant Dracocephalum forrestii were cultured in temporary immersion bioreactors (RITA and Plantform) and in nutrient sprinkle bioreactor (NSB) for 3 weeks in MS (Murashige and Skoog) liquid medium with 0.5 mg/L BPA (N-benzyl-9-(2-tetrahydropyranyl)-adenine) and 0.2 mg/L IAA (indole-3-acetic acid). The greatest biomass growth index (GI = 52.06 fresh weight (FW) and 55.67 dry weight (DW)) was observed for shoots in the RITA bioreactor, while the highest multiplication rate was found in the NSB (838 shoots per bioreactor). The levels of three phenolic acids and five flavonoid derivatives in the shoot hydromethanolic extract were evaluated using UHPLC (ultra-high performance liquid chromatography). The predominant metabolite was rosmarinic acid (RA)—the highest RA level (18.35 mg/g DW) and total evaluated phenol content (24.15 mg/g DW) were observed in shoots grown in NSB. The NSB culture, i.e., the most productive one, was evaluated for its antioxidant activity on the basis of reduction of ferric ions (ferric reducing antioxidant power, FRAP) and two scavenging radical (O2•– and DPPH, 1,1-diphenyl-2-picrylhydrazyl radical) assays; its antibacterial, antifungal, and antiproliative potential against L929 cells was also tested (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) test). The plant material revealed moderate antioxidant and antimicrobial activities and demonstrated high safety in the MTT test—no cytotoxicity at concentrations up to 50 mg/mL was found, and less than a 20% decrease in L929 cell viability was observed at this concentration.

Extractability, stability, and accumulation of nepetoidins in Ocimum basilicum L. leaves and cell cultures

Antioxidant polyphenols nepetoidin A and B are widely spread in Lamiaceae family yet often remain undetected due to their purported instability and poor extractability with polar solvents typically used for phenolics extraction. Coextraction of nepetoidins may confer additional, or enhance existing, bioactivities of polyphenol extracts. This study includes a detailed analysis of nepetoidin extractability from sweet basil (Ocimum basilicum L.) leaves, and a survey of their occurrence in basil tissue cultures and peltate trichomes. Coextraction of nepetoidins with the major polyphenol rosmarinic acid was possible with solvent mixtures, yet substantially less efficient than that with the respective preferred solvents. Nepetoidin, but not rosmarinic acid levels in extracts from dried leaves were dramatically reduced. However, this was not due to degradation suggested by earlier reports, but to reduced extractability, which could be restored by wetting the tissue with water prior to addition of nonpolar solvent. Nepetoidins were found to accumulate at high levels in basil callus and suspension cultures. Correlation analysis revealed three novel compounds that are coextracted with nepetoidins and are proposed to represent nepetoidin dimers. Candidate signals for nepetoidin and rosmarinic acid glycosides were also detected in dedifferentiated basil cells. By contrast, nepetoidins were not enriched in basil peltate trichomes. This study establishes optimized recovery procedure for nepetoidins, reports their production by dedifferentiated basil cells, and illustrates how sample preparation methodology affects the phytochemical findings. This study reports an improved method for the recovery of polyphenolic nepetoidins from fresh and dry plant tissue and demonstrates their high abundance in dedifferentiated Ocimum basilicum cells.

Establishment of hairy root cultures of Salvia bulleyana Diels for production of polyphenolic compounds

This study was to obtain stable transformed roots of Salvia bulleyana using A. rhizogenes strain A4 and then evaluate their phytochemical profile and selected the most productive clone. Our results indicated that the type of explant and medium used for bacterium and explant incubation had an influence on the frequency of hairy root formation. The best response was obtained on leaves infected with bacteria cultivated on YMB medium supplemented with acetosyringone. Of the four selected transformed root clones, after five-week cultivation in Woody Plant (WP) medium, the highest growth indexes were demonstrated for line C1: i.e. 13 for fresh and 15 for dry weight (81.4 and 8.2 g/l fresh and dry weight, respectively). The qualitative analysis of hydromethanolic extracts of hairy roots of S. bulleyana using UPLC-PDA-ESI-MS/MS method showed the presence of 10 polyphenolic compounds including predominant rosmarinic acid (RA), its derivatives (hexoside and methyl rosmarinate), caffeic acid, its derivatives and several salvianolic acids: K, E and F. Their production varied among the four root clones studied; the highest RA (39.6 mg/g dry weight) and total polyphenol (48.9 mg/g dry weight) level were found in the roots of C4 clone. These values were significantly higher than those of the roots of plants grown for several years under field conditions. The transformation of the obtained root cultures was confirmed by polymerase chain reaction using aux1, aux2, rolB, rolC and rolD primers.

Blue and White LED Lights Enhance Biosynthesis of Rosmarinic Acid in Cell Culture of Agastache rugosa

Agastache rugosa, an herbaceous Korean perennial, contains pharmacologically important phenylpropanoids such as tilianin and rosmarinic acid. However, the knowledge of biosynthesis of phenylpropanoids from A. rugosa is limited. Therefore, a study was conducted to develop an efficient protocol for in vitro regeneration of A. rugosa and to investigate the influence of three different LED light wavelengths (white, blue and red) on the biosynthesis of phenylpropanoids, including altered gene expression and variance in rosmarinic acid accumulation in calli. Transcription analyses revealed that white LED light enhances RAS expression; transcript levels were 15.7- and 10.4-fold higher for calli treated with white LED light than for those treated with red and blue light, respectively. Similarly, HPPR and C4H were also more highly expressed under blue and white LED light. In addition, HPLC quantification assays indicated that the highest levels of rosmarinic acid accumulate in calli treated with blue, red and white light (in that order) one week after cultivation. Together, our gene expression and HPLC quantification results provide evidence that rosmarinic acid in A. rugosa increases with the application of various wavelengths of LED light.

Accumulation of phenolic compounds in different in vitro cultures of Salvia viridis L. and their antioxidant and antimicrobial potential

Different in vitro cultures of the Turkish origin medicinal plant species, Salvia viridis, were estimated in order to investigate their potential for the accumulation of phenolic compounds. Among the seventeen detected compounds, thirteen phenolic acid derivatives and four phenylethanoids were identified in extracts from shoot and/or callus cultures using the UPLC-DAD/ESI-MS method. The accumulation of the compound depended on the type of culture (callus, shoot), the medium composition and age of culture (6, 12, 18 and 24 months in in vitro conditions). Six of the seventeen detected compounds were estimated in all the analyzed samples, however, sometimes only in trace amounts. The predominant metabolite was rosmarinic acid (RA). Its content ranged between 2.4 and 11.5 mg/g dry weight (DW). The highest levels of rosmarinic acid and total phenolic compounds were found in a half-year-old shoot culture. The content of the RA was nine times higher than that in the shoots of plant grown in field conditions. The antioxidant activity of S. viridis cultures was examined by the O2− and 1-1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging test and FRAP (ferric reducing antioxidant power) assay. In addition, the antifungal and the antibacterial activities were examined against a wide spectrum of micro-organisms. All the samples showed promising antioxidant potential; however, the shoot extract had the highest antioxidant activity in all the assays. Preliminary results demonstrated that all the analyzed extracts inhibited the growth of Gram-positive and Gram-negative bacteria. The extracts also showed strong activity against Candida and Aspergillus species.

Chemical Eustress Elicits Tailored Responses and Enhances the Functional Quality of Novel Food Perilla frutescens.

Consumer demand for fresh and functional horticultural products is on the rise. Perilla frutescens, L. Britt (Lamiaceae) is a potential specialty/niche crop for consumption and therapeutic uses with high contents of phenolic and volatile compounds. Plant growth, mineral composition, polyphenol profile and aroma volatile components of two perilla genotypes in response to salinity (non-salt control, 10, 20 or 30 mM NaCl) applied as chemical eustressor were assessed. Salinity suppressed growth and yield of both genotypes, although the red-pigmented genotype was less sensitive than the green-pigmented one. Mild (10 mM NaCl) and moderate (20 and 30 mM NaCl) salinity suppressed foliar potassium, magnesium, nitrate and chlorophyll a concentrations of both genotypes and increased the levels of rosmarinic acid, total polyphenols and target aroma volatile components. Green perilla showed higher yield and biomass production and higher content of protein, dry matter, calcium, magnesium, perilla ketone and cis-jasmone, whereas red perilla exhibited higher content of potassium, chlorophyll a, rosmarinic acid, total polyphenols, perilla aldehyde and benzaldehyde. Our findings support that chemical eustressors such as mild to moderate salinity offer valuable means to manipulate phytochemical and aroma profiles.

Review on rosmarinic acid extraction, fractionation and its anti-diabetic potential

Rosmarinic acid is a bioactive phytochemical that can be found in many herbs as ethnomedicines. It possesses remarkable pharmacological activities, and thus leading to its exploration as a therapeutic drug in diabetes treatment recently. This article reviews the extraction and fractionation techniques for plant-based natural rosmarinic acid and its anti-diabetic potential based on literature data published in journals, books, and patents from 1958 to 2017. Factors affecting the performance of rosmarinic acid extraction and fractionation such as operating temperature, time, solvent to sample ratio and eluent system are compiled and discussed in detail. The inhibitory action of rosmarinic acid against sugar digestive enzymes, and protective action towards pancreatic β-cell dysfunction and glucolipotoxicity mediated oxidative stress are also critically reviewed. The optimal parameters are largely dependent on the applied extraction and fractionation techniques, as well as the nature of plant samples. Previous studies have proven the potent role of rosmarinic acid to control plasma glucose level and increase insulin sensitivity in hyperglycemia. Although rosmarinic acid is readily absorbed by human body, its mechanism after consumption is remained unclear. Intensive studies should be well planned to determine the dosage and toxicity level of rosmarinic acid for efficacy and safe consumption.

Pharmacokinetic and Pharmacodynamic Properties of Rosmarinic Acid in Rat Cholestatic Liver Injury.

The objective of this study was to evaluate the hepatoprotective and metabolic effects of rosmarinic acid (RA) in rats. RA [100 mg/kg body weight (BW)] was intragastrically (i.g.) administered to Sprague-Dawley (SD) rats once a day for seven consecutive days. The rats were then i.g. administered α-naphthylisothiocyanate (ANIT) (80 mg/kg once on the 5th day) to induce acute intrahepatic cholestasis after the last administration of RA. Blood samples were collected at different time points (0.083 h, 0.17 h, 0.33 h, 0.5 h, 0.75 h, 1 h, 1.5 h, 3 h, 4 h, 6 h, 8 h, 12 h, 20 h) after administration, and the levels of RA were estimated by HPLC. Plasma and bile biochemical analysis, bile flow rate, and liver histopathology were measured to evaluate the hepatoprotective effect of RA. The PK-PD curves showed obviously clockwise (AST and ALT) or anticlockwise (TBA, TBIL). Pretreatment with RA at different doses significantly restrained ANIT-induced pathological changes in bile rate, TBA, TBIL, ALT, AST (p < 0.05 or p < 0.01). The relationship between RA concentration and its hepatoprotective effects on acute cholestasis responses was assessed by PK-PD modeling.

Liposomal Incorporation to Improve Dissolution and Stability of Rosmarinic Acid and Carvacrol Extracted from Oregano (O. onites L.).

The potential antimicrobial benefit of high levels of rosmarinic acid (RA) and carvacrol (CA) in oregano (O. onites L.) extract has been limited until now by poor bioavailability arising from the low aqueous-phase solubility and slow dissolution behaviour of the lyophilized extract (E). To address this issue, various ratios of phospholipon 90H (P90H) and 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol), sodium salt (DMPG) were sonicated, yielding four empty liposomes (L1, L2, L3, and L90). After an initial selection process, Turkish oregano extract was internalized into the more promising candidates. Each empty liposome, extract-loaded liposome (LE1, LE2, and LE3), and freeze-dried control (E) was assessed in terms of structure, composition, RA and CA dissolution profile, storage stability, and, when relevant, zeta potential. Empty liposome L1, which was prepared using P90H and DMPG in a 1:1 ratio, displayed the most convenient encapsulation traits among the four unloaded types. Loaded liposome LE1, obtained by combining oregano extract and L1 in a 1:1 ratio, proved superior as a vehicle to deliver RA & CA when compared against control freeze-dried E and test liposomes LE2 and LE3. Dissolution profiles of the active compounds RA and CA in loaded liposomes were determined using a semi-automated dissolution tester. The basket method was applied using artificial gastric juice without pepsin (AGJ, 50rpm, 500mL). The pH value was maintained at 1.5 (37 ± 0.5°C). Aliquots (5ml) were manually extracted from parallel dissolution vessels at 1, 3, 5, 7, 10, 15, 20, 25, 30, 45, and 60-minute time points. Dissolution tests, run to completion on LE1, showed that approximately 99% of loaded CA and 88% of RA had been released. Shorter dissolution times were also noted in using LE1. In particular, the release profile of CA and RA had levelled off after only 25 minutes, respectively, depicting an impressive 3.0–3.3 and 2.3-2.6 rate increase compared to the freeze-dried control extract. The improved dispersibility of RA and CA in the form of LE1 was supported by particle size and zeta potential measurements of the liposome, yielding 234.3nm and −30.9mV, respectively. The polydispersity index value was 0.35, indicating a reasonable particle size distribution. To study storage stability, liposomes were stored (4°C, 6 months) in amber coloured glass containers (4 oz.). Each container held 30 capsules, which were stored according to the ICH guidelines prescribed for long-term storage (25°C ± 2°C; 60% ± 5% RH). Triplicate samples were withdrawn after 0, 3, 6, 9, and 12 months for analysis. Lastly, LE1 displayed good storage stability. The results imply that RA and CA can be conveniently and routinely delivered via oral and mucosal routes by first internalizing oregano extracts into appropriately engineered liposomes.

Rosmarinic acid ameliorates the negative effects of salinity in in vitro-regenerated potato explants (Solanum tuberosum L.)

Salinity causes massive loss of economic crops due to the build-up of toxic salts in shoot. Rosmarinic acid (RA) has been known to prevent the inevitable damage to vital biological molecules under stress. There is an increasing interest, therefore, in the use of polyphenols in crop stress physiology studies. An in vitro regeneration protocol was established using explants of the potato cultivar White Desiree to assess tolerance to salinity at a morphological, cellular, and biochemical level. Additionally, we examined the potential ameliorative effects of RA on growth in salt-stressed potato explants. Based on our observations, we propose a model for the ameliorative effects of a caffeic acid conjugate of α-hydroxyhydrocaffeic acid. Explants of White Desiree showed differential responses to salt and RA supplementation. Salt-stressed and control explants grown in RA-supplemented media showed a pronounced expansion of leaf area, suggesting direct effects of RA on cell elongation in potato explants. Root elongation was less affected by salinity in comparison to shoot elongation. Moderate levels of RA resulted in stimulatory effects on growth. The microscopic examination of leaves and stems indicated the presence of unique trichomes at the early stages of explant growth under RA supplementation, which varied considerably in size, shape, and distribution in response to stress. Our data indicated that RA significantly decreased osmolyte content under stress. Salinity resulted in the biosynthesis of high levels of free proline, carbohydrates, and malondialdehyde (MDA). However, the lower contents of MDA in salt-stressed explants treated with RA indicate that RA possesses anti-lipid peroxidation properties. Our observations provide evidence for the stimulatory effects of RA on cellular growth and the protection of membranes from ion toxicity.

A new validated high-performance liquid chromatography method for standardization of rosmarinic acid in Salvia extracts

Introduction: Tea bags or infuses of Salvia species from Lamiaceae family are traditionally used for the treatment of cough, and throat inflammations. They are known for antioxidant properties mainly related to the presence of rosmarinic acid (RA). Therefore it is necessary to develop a reliable analytical method for RA assay for standardization of Salvia species and also other plants containing RA like Melissa, Origanum, Lavandula, Rosmarinus, Thymus, Zataria, Mentha, Perovskia, Zhumeria, and Satureja species. In this study using a suitable extraction method by removing unwanted components present in crude methanol extract, phenolic content containing RA was extracted from dry powders of six Salvia species. Then, a suitable high-performance liquid chromatographic (HPLC) method was optimized for quantification of RA in Salvia species. Methods: HPLC analysis was done on a Waters system, equipped with 515 HPLC pump and waters 2487 dual wavelength absorbance detector. The column was a Nova-Pak C18 (3.9 × 150 mm), and Millenium software was used for the determination of the compounds and processing the data. The method was validated according to USP 32 requirements. Results: Among the investigated 6 species, S. virgata was the richest in RA level, demonstrating 3.50 ± 0.12 mg/g, followed by S. sclarea and S. chloroleuca showing 1.65 ± 0.08 and 1.65 ± 0.21 mg/g. S. ceratophylla with 0.10 ± 0.01 mg/g of RA in dried plant powder was the poorest. Conclusion: The validated HPLC method allows determination of amounts as low as 2.5 µg/ mL of RA and linearity in the ranges of 2.5-25 µg/mL and 100–600 µg/mL, which is suitable for standardization of Salvia species in traditional and pharmaceutical formulations

Chemical composition and biological activities of extracts from three Salvia species: S. blepharochlaena, S. euphratica var. leiocalycina, and S. verticillata subsp. amasiaca

The genus Salvia has recently attracted great attention due to its notable biological activities. Within this context, in this study, the chemical characterization and biological effects of three extracts (dichloromethane (DCM), methanol (MeOH), and water) from three Salvia species (S. blepharochlaena (SB), S. euphratica var. leiocalycina (SE), and S. verticillata subsp. amasica (SV)) were assessed. For the chemical characterization, the qualitative and quantitative analysis of phenolic components in the methanol extracts was carried out by high-performance liquid chromatography with electrospray ionization mass spectrometry detection (HPLC-UV-ESI-MSn). Total phenolic, flavonoid and phenolic acid contents were also studied. Concerning the biological effects, antioxidant (DPPH and ABTS free radical scavenging; ferric (FRAP) and cupric (CUPRAC) reducing power; phosphomolybdenum, and metal chelating assays), enzyme inhibitory (cholinesterase, tyrosinase, amylase, glucosidase, lipase, and elastase) and cytotoxic effects (A-549 and MCF-7 cell lines) were evaluated. After the evaluation of the phytochemical profile by HPLC-UV-ESI-MSn, it was observed that the main compound in the analyzed extracts was rosmarinic acid, which was present at high concentrations, particularly in SV, which presented rosmarinic acid levels higher than the usual levels found in other Salvia species or related plants. Generally, the SV-water extract presented the strongest antioxidant abilities with higher levels of total bioactive compounds. However, the studied DCM extracts had higher enzyme inhibitory potentials compared with MeOH and water extracts. SE-DCM exerted the most potent cytotoxic effects, followed by SB-water and SB-MeOH extracts.

Rosmarinic Acid Accumulation and Antioxidant Potential of Dracocephalum moldavica L. Cell Suspension Culture

Dracocephalum moldavica L. (Lamiaceae) is known for its medicinal properties, however greater yields can potentially be achieved by in vitro cultivation. A cell suspension culture of D. moldavica L. (Lamiaceae) derived from root-derived callus was established in liquid MS medium supplemented with 2,4-D 0.5 mg/l and BAP 0.2 mg/l. The biomass and rosmarinic acid (RA) content were analyzed during the 15-day growth cycle of the culture. The highest fresh and dry weight (14.29 g/flask and 1.14 g/flask, respectively) and RA level (27.2 mg/g DW) were reached at day 12 of culture. Methanolic extracts of the culture were assayed for total phenolic content using the Folin-Ciocalteau method, and antioxidant activities using three in vitro tests: ABTS radical scavenging, ferric ion reduction (FRAP) and lipid peroxidation (LPO). RA content and antioxidant potential were found to be higher in cell suspension culture than in root-derived callus. The cell suspension culture also exhibited higher concentrations of RA and ABTS radical scavenging activity than those of the aerial parts of six-month-old field-grown plants of D. moldavica. The overall results show a significant correlation between antioxidant activity, total phenolic content and RA content of the examined extracts. The study presents for the first time the use of cell cultures of D. moldavica for production of therapeutically-valuable metabolites. Our results suggest that the obtained culture could be considered as a potential source of rosmarinic acid, a compound known for its strong antioxidant activity.

Response of Different &lt;i&gt;Agrobacterium Rhizogenes&lt;/i&gt; Strains for &lt;i&gt;in vitro&lt;/i&gt; Hairy Root Induction and Accumulation of Rosmarinic Acid Production in &lt;i&gt;Agastache Rugosa&lt;/i&gt;

In this study a total of seven different Agrobacterium rhizogenes strains were evaluated for their ability to transform the plant Agastache rugosa and to produce the secondary metabolite rosmarinic acid. All the strains of A. rhizogenes i.e., 13333, 15834, A4, LBA9402, R1000, R1200 and R1601 strains tested here in this study, were able to induce hairy root formation in leaf tissue explants. The strain A4had the highest rate of infection (94.1±8.3%) and the strain R1000 had the lowest rate (88.6±6.9%). The highest frequency of hairy roots per explant (13.6±1.4) was found for strain R1601 and the tallest root length (20.4±1.7 mm) was found for strain 13333. We also evaluated dry weight and level of rosmarinic acid in the hairy roots and found that the highest growth (310.1±14.6 mg/flask) was occurred after infection with strain R1200, while the highest production of rosmarinic acid (68.2±3.8 mg/g dry weight) was noted using strain 13333. Our study showed that A. rhizogenes strain 13333 was the most effective of the 7 tested strains for production of transformed root cultures as well as rosmarinic acid in the hairy roots.