Extraction of phenolics and essential oil from dried sage (Salvia officinalis) using ethanol–water mixtures

Effects of particle size, temperature, contact time, solvent-to-sage ratio and the ethanol–water ratio on the extraction of the active compounds rosmarinic acid, carnosic compounds and essential oil from dried sage (Salvia officinalis) were studied. Optimal extraction conditions giving highest yield of all three active compounds were particle diameter 1 mm, extraction temperature 40 °C, solvent-to-sage ratio of 6:1 and 55–75 wt% ethanol for up to 3 h. This gave an extract equivalent to 14.9% of dry sage, containing 6.9% rosmarinic acid (55% recovery), 10.6% carnosic compounds (75% recovery) and 7.3% essential oil (42% recovery). Scale up of the process by a factor of 100 demonstrated that the optimised laboratory scale process can be carried out without any loss of efficiency at an industrial scale.     

Inhibitory effect of Turkish Rosmarinus officinalis L. on acetylcholinesterase and butyrylcholinesterase enzymes

In the current study, we have tested acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity of the petroleum ether, ethyl acetate, chloroform, and methanol extracts, rosmarinic acid as well as the essential oil obtained from Rosmarinus officinalis L. growing in Turkey by a spectrophotometric method of Ellman using ELISA microplate-reader at 0.2, 0.5, and 1.0 mg/mL concentrations. In addition, quantification of rosmarinic acid, a common phenolic acid found in rosemary, was carried out by reversed-phase HPLC in the methanolic extract of the plant, which was found to have 12.21 ± 0.95% (122.1 ± 9.5 mg/g extract) of rosmarinic acid. Rosmarinic acid was also tested for its AChE and BChE inhibitory effect and found to cause 85.8% of inhibition against AChE at only 1.0 mg/mL. Besides, the essential oil was analyzed by GC–MS technique, which was shown to be dominated by 1,8-cineol (44.42%) and followed by α-pinene (12.57%).     

A new rapid spectrophotometric method to determine the rosmarinic acid level in plant extracts

A novel spectrophotometric method to determine the amount of rosmarinic acid in unpurified methanol extracts of the plants was developed. Rosmarinic acid is a naturally occurring bioactive compound in plants as an ester of caffeic acid with 3,4-dihydroxyphenyl lactic acid. The developed method was based on the complexation of rosmarinic acid with Zr⁴⁺ ions, giving a maximum absorbance at 362 nm. The absorptivity coefficient at this wavelength was found to be ε₃₆₂ = 2.66 × 10⁴ L mol⁻¹ cm⁻¹. In fact, this method is also specified giving two more absorption bands in UV region at 299.5 and 263.5 nm besides 362 nm. In addition, the accuracy and sensitivity of the new developed method are compared with the direct UV and rosmarinic acid–Fe²⁺ complex spectrophotometric methods by using methanol extracts of 11 Salvia species. As a conclusion, the present method is faster, cheaper, and more selective than the conventional methods for rosmarinic acid.     

Antioxidant potentials and rosmarinic acid levels of the methanolic extracts of Salvia verticillata (L.) subsp. verticillata and S. verticillata (L.) subsp. amasiaca (Freyn & Bornm.) Bornm

This study was designed to examine the in vitro antioxidant activities and rosmarinic acid levels of the methanol extracts of Salvia verticillata subsp. verticillata and S. verticillata subsp. amasiaca. The extracts were screened for their possible antioxidant activity by two complementary test systems, namely DPPH free radical-scavenging and β-carotene/linoleic acid systems. In the first case, S. verticillata subsp. verticillata was superior to the subsp. amasiaca with an IC₅₀ value of 14.5 ± 1.21 μg mg⁻¹. In the β-carotene/linoleic acid test system, inhibition capacity of S. verticillata subsp. verticillata was 74.4 ± 1.29%. Antioxidant activities of BHT, ascorbic acid, curcumin and α-tocopherol were determined in parallel experiments. Activity of rosmarinic acid was also screened for better establishing the relationship between rosmarinic acid level and antioxidant activity for the plant extracts. S. verticillata subsp. verticillata had the highest rosmarinic acid level with a value of 28.7 ± 0.89 μg mg⁻¹. There is a strong correlation between the rosmarinic acid level and antioxidant activity potential. Our results showed that rosmarinic acid and its derivatives are more likely to be responsible for most of the observed antioxidant activities of Salvia species.     

Antioxidant activity of cichoric acid and alkamides from Echinacea purpurea, alone and in combination

The antioxidant activity of extracts of the stems, leaves, and roots of Echinacea purpurea was compared with the antioxidant activity of purified cichoric acid and alkamides, both constituents of Echinacea purpurea. The antioxidant activity was determined using different methods: effect on oxygen consumption rate of a peroxidating lipid emulsion, and scavenging of radicals, i.e. 2,2-diphenyl-1-picrylhydrazyl (DPPH), measured by two different techniques. The efficacy of the extracts in the reaction with DPPH correlated well with the amount of cichoric acid present in the various extracts. The alkamides alone showed no antioxidant activity in any of the tests. Alkamides present in the extract increased, however, the antioxidative effect of cichoric acid in the peroxidating lipid emulsion. The activity was further compared with that of rosmarinic acid, a well-characterised antioxidant, and the extracts as well as cichoric acid were found to be efficient scavengers of radicals with an activity comparable to that of rosmarinic acid. Cichoric acid was found to have a stoichiometric factor of 4.0 in scavenging DPPH and to react in a second-order reaction with DPPH with a rate constant of 40 l/mol/s at 25 °C in methanol.     

Assessment of in vitro antioxidant capacity of stem and leaf extracts of Rabdosia serra (MAXIM.) HARA and identification of the major compound

The leaf and stem of Rabdosia serra were extracted by different solvents in this work. The results indicated that 60% ethanol was the most suitable solvent for efficiently extracting antioxidants with strong free radical-scavenging capacity and ferrous ion chelating capacity. The butanol and 60% ethanol extracts of stem, methanol and 60% ethanol extracts of leaf were analysed by HPLC–PDA. A similar chemical composition was observed for these stem and leaf extracts. The major compound was obtained by column chromatography and identified by mass spectrometry and nuclear magnetic resonance spectroscopy, which was confirmed to be rosmarinic acid. The contents of rosmarinic acid were in a descending order: butanol extract of stem > 60% ethanol extract of leaf > 60% ethanol extract of stem > methanol extract of leaf.     

Relationship between the solubility,dosage and antioxidant capacity of carnosic acid in raw and cooked ground buffalo meat patties and chicken patties

Antioxidant capacity of oil soluble and water dispersible carnosic acid (CA) extracted from dried rosemary leaves using HPLC was evaluated at two different dosages (22.5 ppm vs 130 ppm) in raw and cooked ground buffalo meat patties and chicken patties. Irrespective of total phenolic content, CA extracts reduced (p < 0.05) the thiobarbituric acid reactive substances (TBARS) by 39%–47% and 37%–40% in cooked buffalo meat and chicken patties at lower dosage (22.5 ppm) relative to control samples. However, at higher dosage (130 ppm) the TBARS values were reduced (p < 0.05) by 86%–96% and 78%–87% in cooked buffalo meat and chicken patties compared to controls. The CA extracts were also effective in inhibiting (p < 0.05) peroxide value and free fatty acids in cooked buffalo meat and chicken patties. The CA extracts when used at higher dosage, were also effective in stabilizing raw buffalo meat color.     

Antioxidant activities of Sideritis congesta Davis et Huber-Morath and Sideritis arguta Boiss et Heldr: Identification of free flavonoids and cinnamic acid derivatives

Different solvent extracts of endemic Sideritis (Labiatae) species, Sideritis congesta Davis et Huber-Morath and Sideritis arguta Boiss et Heldr, were analyzed for free flavonoids (quercetin, apigenin, myricetin and kaempferol) and cinnamic acid derivatives (rosmarinic acid, ferulic acid, caffeic acid, p-coumaric acid and chlorogenic acid) using HPLC-DAD. All the phenolics were quantified in acid-hydrolyzed extracts, except rosmarinic acid, chlorogenic acid and myricetin which were quantified in raw samples. Antioxidant activities of extracts of these two plants and many of their components in pure form were evaluated based on DPPH^. and ABTS^.+ assays. In general, S. arguta extracts displayed higher antioxidant activity than S. congesta extracts possibly due to their richness in antioxidant components of strong activity. Acetone extract of S. arguta, with its strikingly high TEAC value of 3.2 mM trolox and low IC₅₀ value of 38.3 ??g/mL showed the highest antioxidant potency among all extracts. ??-tocopherol, the positive control, displayed IC₅₀ and TEAC values of 33.8 ??g/mL and 2.9 mM trolox, respectively. No direct correlation was found between antioxidant activities and total phenolic contents of the plant extracts studied.     

The inhibitory effect of Plectranthus barbatus and Plectranthus ecklonii leaves on the viability,glucosyltransferase activity and biofilm formation of Streptococcus sobrinus and Streptococcus mutans

Aqueous extracts of Plectranthus barbatus and Plectranthus ecklonii are traditionally used as anti-inflammatory and anti-fungal agents. The effect of these extracts and of its main component, rosmarinic acid, on the viability of the cariogenic bacteria, Streptococcus sobrinus and Streptococcus mutans, was determined by MIC and MBC. The influence of these extracts on the biofilm formation as well as on the inhibition of glucosyltransferase enzyme, produced by these species, was also analysed. Aqueous extracts of P. barbatus and P. ecklonii were stronger inhibitors than rosmarinic acid. MIC values of 3.8 and 4.7 mg/ml for S. sobrinus and 2.9 and 5.0 for S. mutans were obtained, while rosmarinic acid presented MIC values of 8.4 and 7.3 mg/ml. P. barbartus, P. ecklonii and rosmarinic acid presented MBC values of 9.5, 9.0 and 12.0 mg/ml for S. sobrinus, and 9.5, 10.0 and 12.5 mg/ml for S. mutans. The inhibition of biofilm formation by P. barbatus, P. ecklonii and rosmarinic acid presented IC₅₀ values of, respectively, 0.6, 1.0 and 3.1 mg/ml for S. sobrinus and 1.4 and 2.7 and 1.3 mg/ml for S. mutans. The glucosyltransferase inhibition activity by theses extracts and rosmarinic acid was calculated and IC₅₀ values presented were, respectively, 1.1, ca 1.2 and 2.1 mg/ml for S. sobrinus and 3.1, 1.6 and 3.9 mg/ml for S. mutans were obtained. These extracts may be useful in the prevention of dental carie.     

Profiling of in vitro neurobiological effects and phenolic acids of selected endemic Salvia species

The ethyl acetate and methanol extracts from 16 Salvia L. species were screened for their inhibitory activity against acetylcholinesterase, butyrylcholinesterase, lipoxygenase, and tyrosinase; the enzymes linked to neurodegeneration. Their antioxidant activity was also tested using DPPH radical scavenging, metal-chelation, and ferric-reducing antioxidant power (FRAP) assays. Total flavonoid content of the extracts was determined by AlCl₃ reagent, while HPLC technique was applied for analysis of various phenolic acids in the extracts. The extracts exerted weak cholinesterase and tyrosinase inhibition, and remarkable inhibition against lipoxygenase (13.07 ± 2.73-74.21 ± 5.61%) at 100 μg ml⁻¹. The methanol extracts showed higher antioxidant activity in DPPH radical scavenging and FRAP assays. The extracts were analyzed for their gallic, protocateuchic, p-hydroxy-benzoic, vanillic, caffeic, chlorogenic, syringic, o- and p-coumaric, ferulic, rosmarinic, and tr-cinnamic acid contents and the methanol extract of Salvia ekimiana (153.50 mg 100 g⁻¹) was revealed to be the richest in terms of rosmarinic acid.     

Antioxidant activity and rosmarinic acid changes in salicylic acid-treated Thymus membranaceus shoots

In order to characterise the antioxidant activity of Thymus membranaceus Boiss. subsp. membranaceus, an endemic species in Southeast Spain, five different analytical methods were used. Water, methanol and hexane extracts obtained from 60-day-old in vitro-grown shoots were assayed for their antioxidative properties using DPPH (1,1-diphenyl-2-picrylhydrazyl radical), ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonate), FRAP (ferric reducing antioxidant power), reducing power and conjugated diene assays. Total soluble phenol content, as well as rosmarinic acid, in the different extracts were also determined. Methanolic extracts exhibited the best antioxidant activity and the highest amounts of total soluble phenolics. A single application of salicylic acid (10 μM) on culture media resulted in an increase in rosmarinic acid and phenolic levels, which in turn improved the extracts’ antioxidant properties. These current findings open new opportunities for obtaining valuable natural antioxidants for commercial exploitation by using tissue culture systems.     

Determination of polyphenolic compounds in commercial herbal drugs and spices from Lamiaceae: thyme,wild thyme and sweet marjoram by chromatographic techniques

HPLC and HPTLC methods were used for a qualitative and quantitative determination of luteolin-7-O-β-glucuronide, lithospermic acid, rosmarinic acid and mthyl rosmarinate, together with other known compounds, in commercial herbal drugs and spices from lamiaceous species: Thymi herba, Serpylli herba and Majoranae herba. The contents of analyzed compounds in the studied hydrophilic extracts, prepared form herbal sources, were established using a C18 column with acetonitrile–water–formic acid as a mobile phase. The HPLC method was validated for linearity, precision and accuracy. Luteolin-7-O-β-glucuronide and lithospermic acid were identified as new wild thyme constituents, luteolin-7-O-β-glucuronide and methyl rosmarinate as new compounds in sweet marjoram. Methyl rosmarinate was isolated for the first time from thyme. The investigated herbal drugs and spices provide polyphenols in high amounts, even up to 84.3 mg per 1 g of a dried herb.     

Isolation of ursolic acid from apple peels by high speed counter-current chromatography

Cuticular waxes of four varieties of Malus domestica were investigated regarding their content of ursolic acid. Peels from Fuji, Gala, Smith and Granny Smith apples were extracted with chloroform, ethyl acetate and/or ethanol. The crude extracts were purified by high speed counter-current chromatography (HSCCC), by using mobile and stationary phases derived from the two-phase solvent system composed by n-hexane:ethyl acetate:methanol:water in the proportion of 10:5:2.5:1. The phase proportions and the relative distribution of ursolic acid between the two-phases were optimized by TLC and optical densitometry, by comparison with an authentic sample of ursolic acid. The amount of ursolic acid present in the extracts as well as the characterization of the isolated compound were made by high resolution gas chromatography coupled to mass spectrometry (GC–MS), ¹³C nuclear magnetic resonance (¹³C NMR), Infrared; and by comparing thin layer chromatography and flame ionization detection gas chromatography (GC–FID) patterns with the commercial sample. The average content of ursolic acid of 0.8 mg/cm² in the peel (around 50 mg per medium sized fruit with a surface area of 50–70 cm²) was found in the Fuji and Smith varieties, whereas 0.5 mg/cm² and 0.2 mg/cm² were the amounts calculated for Granny Smith and Gala, respectively. The HSCCC technique was shown to be a good method to purify free ursolic acid from apple peels and could represent a new technological tool to be developed to exploit industrially this source of product.     

Distribution of caffeic acid derivatives in Gundelia tournefortii L.

The caffeic acid derivatives including neochlorogenic acid (3-COA), cryptochlorogenic acid (4-CQA), chlorogenic acid (5-CQA) and caffeic acid (CA) have been characterised in Gundelia tournefortii using reference compounds, chemical, spectral evidences and chromatographic data. In addition, the total phenolic content and chlorogenic acid were measured in the leaf, hull-less seed, and skin extracts of this herb by the Folin–Ciocalteu reagent and high performance liquid chromatography (HPLC), respectively. The sample analysis was carried out on a C₁₈ column with 5% (v/v) aqueous acetic acid and methanol as the mobile phase, under gradient elution at ambient temperature, at 325 nm. The amount of chlorogenic acid in the leafs (at the flowering stage and after it) and hull-less seed were 984, 466 and 199 mg per 100 g dry plant sample and the total phenolic content in their dry extract were 128.4, 103.8 and 76.3 μg/mg as CGA equivalent, respectively.     

Phytochemical composition and in vitro antimicrobial and antioxidant activities of some medicinal plants

Different parts of three plants (Primula auriculata, Fumaria vaillantii and Falcaria vulgaris) were extracted with three different solvents to yield 72 crude extracts. The phytochemical analysis (chemical screening, GC–MS) of three plants was investigated for their antioxidant and antibacterial activity using nine Gram-positive and Gram-negative bacteria. The principal antioxidant and antimicrobial components were determined using HPLC with UV detection. All extracts possessed antibacterial activity especially methanolic extracts from flowers of P. auriculata. The DPPH-radical scavenging assay exhibited high antioxidant activities in three plants (more than 80% at 50 μg). The F. vulgaris showed high content of carvacrol (29.8%) as main component. The contents of carvacrol and fumaric acid in the methanolic–water extracts were 1119 and 1966 mg/l respectively. Our results indicate that these plants would be able to promise sources of natural products with potential antibacterial and antioxidant activity.     

Chicoric acid from Syringodium filiforme

Detrital and living leaves of the tropical seagrass Syringodium filiforme Kütz. were screened for their phenolic contents. For the first time, the major polyphenols were identified, as chicoric acid (CA) and caftaric acid (CAF), by means of NMR and LC/MS. They were quantified in methanolic and aqueous crude extracts using HPLC. Highest amounts were obtained for extracts sequentially prepared under reflux of methanol, then aqueous-methanol. The concentrations found, respectively, ranged from 0.51 to 1.68 mg (g dw⁻¹) for CAF and 0.94–5.26 mg (g dw⁻¹) for CA (of plant dry mass g dw)⁻¹. This is the first report of CAF in a seagrass and CA in S. filiforme. Considering the demonstrated therapeutic applications of CA, its high value-added on the nutraceutical market, and its rare occurrence in the plant kingdom, the significant content found in S. filiforme leaves makes this abundant renewable raw material of interest for the pharmaceutical and food industries.     

Development and validation of an HPLC-method for determination of free and bound phenolic acids in cereals after solid-phase extraction

Whole cereal grains are a good source of phenolic acids associated with reduced risk of chronic diseases. This paper reports the development and validation of a high-performance liquid chromatography–diode array detection (HPLC–DAD) method for the determination of phenolic acids in cereals in either free or bound form. Extraction of free phenolic acids and clean-up was performed by an optimised solid-phase extraction (SPE) protocol on Oasis HLB cartridges using aqueous methanol as eluant. The mean recovery of analytes ranged between 84% and 106%. Bound phenolic acids were extracted using alkaline hydrolysis with mean recoveries of 80–95%, except for gallic acid, caffeic acid and protocatechuic acid. Both free and bound phenolic extracts were separated on a Nucleosil 100 C₁₈ column, 5 μm (250 mm × 4.6 mm) thermostated at 30 °C, using a linear gradient elution system consisting of 1% (v/v) acetic acid in methanol. Method validation was performed by means of linearity, accuracy, intra-day and inter-day precision and sensitivity. Detection limits ranged between 0.13 and 0.18 μg/g. The method was applied to the analysis of free and bound phenolic acids contents in durum wheat, bread wheat, barley, oat, rice, rye, corn and triticale.     

Rosmarinic acid from beach waste: Isolation and HPLC quantification in Zostera detritus from Arcachon lagoon

Detritus of Zostera noltii and Zostera marina collected from the beaches of Arcachon lagoon (France) over a 30-month period were screened as a new source of rosmarinic acid (RA), a phenolic acid and an economically important metabolite. The seasonal variation of the RA content was quantified in methanolic crude extracts using high-performance liquid chromatography. Concentrations of RA ranged from 2.2 to 18.0 mg g⁻¹ (dw) for Z. noltii and 1.3 to 11.2 mg g⁻¹ (dw) for Z. marina. This is the first time RA has been isolated from Z. noltii; detrital leaves of Zostera have never before been screened for their bioactive substances. The high RA content of Zostera flotsam is of interest for both the cosmetic and herbal industries. These results show that there is a real potential for harvesting beachcast Zostera.     

Bactericidal activities against pathogenic bacteria by selected constituents of plant extracts in carrot broth

HPLC-DAD analysis provided evidence for the certain identification of some constituents of hydroalcoholic extracts from Lithospermum erythrorhizon, Rheum palmatum, Thymus vulgaris, Lippia citriodora, and a mixture of Rosmarinus officinalis, Salvia lavandulifolia and Thymus mastichina. Their inhibitory and bactericidal activities in vitro against Escherichia coli and Staphylococcus aureus were evaluated either in Luria–Bertani (LB) broth or a model food system, Tyndallised carrot broth (TCB). Shikonin exhibited high inhibitory activity against both microorganisms, but to a lesser extent than the basic unit naphthazarin (5,8-dihydroxy-1,4-naphthoquinone). Rhein and carnosic acid showed noticeable inhibitory effects on S. aureus. In inoculated TCB containing 15 mg/L naphthazarin or 47 mg/L shikonin there was about 6 log reductions in E. coli counts, while 20 mg/L naphthazarin, 26.4 mg/L rhein, 45 mg/L shikonin or 68.7 mg/L carnosic acid caused 1.6, 2.4, 3.1 and 3.1 log reductions in S. aureus inocula, respectively. Log reductions obtained for S. aureus in TCB compared unfavourably with those found in LB broth.