Chemical Composition of the Essential Oils of Three Popular Sideritis Species Cultivated in Greece Using GC-MS Analysis.

(1) Background: The essential oils (EOs) of Sideritis L. have attracted great interest due to their pharmacological activities and potential applications in the cosmetic and perfume industries. The aim of this work was to study the EO chemical composition of three of the most popular, in Greece, mountain tea species: namely, these include Sideritis scardica, Sideritis raeseri, and Sideritis syriaca. (2) Methods: The EOs were obtained from the aerial parts of three Sideritis species that were cultivated in various regions of Greece by hydrodistillation, and the chemical composition was studied by gas chromatography-mass spectrometry (GC-MS) analysis. (3) Results: The EOs of the Sideritis species-S. scardica (SSC1, SSC2, SSC3), S. raeseri (SR1, SR2, SR3), and S. syriaca (SS1, SS2, SS3)-were analyzed by GC-MS, and they showed both qualitatively and quantitatively high variation in their chemical composition. (4) Conclusions: The EOs of S. scardica and S. raeseri from three different regions of Greece, and the S. syriaca from three different localities of Crete Island in Southern Greece, showed high chemical variability. Although 165 different components were found to be present in the nine samples through GC-MS analysis, only 7 (1-octen-3-ol, linalool, trans-pinocarveol, p-mentha-1,5-dien-8-ol, α-terpineol, myrtenol, and verbenone) were common components in the nine EOs, which were identified to be highly variable in different percentages among the samples. Even the EOs of SS1 and SS2, which were cultivated nearby, showed different GC profiles. The composition variation observed might be attributed to differentiations in the soil and climatic conditions.

Chemometric Study of Fatty Acid Composition of Virgin Olive Oil from Four Widespread Greek Cultivars.

Virgin olive oil (VOO) is one of the key components of the Mediterranean diet owing to the presence of monounsaturated fatty acids and various bioactive compounds. These beneficial traits, which are usually associated with the cultivar genotype, are highlighting the demand of identifying characteristics of olive oil that will ensure its authenticity. In this work, the fatty acid (FA) composition of 199 VOO samples from Koroneiki, Megaritiki, Amfissis, and Manaki cultivars was determined and studied by chemometrics. Olive cultivar greatly influenced the FA composition, namely, oleic acid (from 75.36% for Amfissis to 65.81% for Megaritiki) and linoleic acid (from 13.35% for Manaki to 6.70% for Koroneiki). Spearman's rho correlation coefficients revealed differences and similarities among the olive oil cultivars. The use of the forward stepwise algorithm identified the FAs arachidonic acid, gadoleic acid, linoleic acid, α-linolenic acid, palmitoleic acid, and palmitic acid as the most significant for the differentiation of samples. The application of linear and quadratic cross-validation discriminant analysis resulted in the correct classification of 100.00% and 99.37% of samples, respectively. The findings demonstrated the special characteristics of the VOO samples derived from the four cultivars and their successful botanical differentiation based on FA composition.

Quantitative determination of aloin, antioxidant activity, and toxicity of Aloe vera leaf gel products from Greece.

BACKGROUND: Aloe vera is a popular medicinal plant used widely by the cosmetic, pharmaceutical, and food industries. The A. vera leaf gel, which is used mostly for its positive effects on human health, contains over 75 different bioactive compounds, including aloin. Aloin is a toxic compound, and its content in A. vera leaf gel products depends on the different cultivation conditions and especially on leaf processing. RESULTS: In this study, A. vera leaf gel products, varied in terms of leaf processing, were analyzed using liquid chromatography for their aloin content, their antioxidant activity by 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) radical cation (ABTS·+ ) and the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH· ) antioxidant activity assays and their toxicity against Aliivibrio fisheri and SH-SY5Y cells. In the samples processed with industrial methods and in those filtered in the lab, the content of aloin was found below the limit (0.1 mg L-1 ) of the EU legislation however, the unprocessed and unfiltered samples were found to contain more than 10 mg L-1 . Antioxidant activity was estimated to vary from 1.64 to 9.21 µmol Trolox mL-1 for DPPH· and from 0.73 to 5.14 µmol Trolox mL-1 for ABTS·+ . Toxicity values on A. fisheri, expressed as the concentration at 50% loss of initial luminescence, ranged from 0.03 to 0.09 mg mL-1 . The cytotoxic study indicated that aloin A at low concentrations (1 and 10 µg mL-1 ) protects SH-SY5Y cells from toxicity induced by hydrogen peroxide. CONCLUSIONS: Consequently, the filtration process of A. vera leaf gels, either laboratory or industrial, resulted in aloin A content below the EU legislation detection limits. © 2020 Society of Chemical Industry.

Discrimination of botanical origin of olive oil from selected Greek cultivars by SPME-GC-MS and ATR-FTIR spectroscopy combined with chemometrics.

BACKGROUND: Consumers today wish to know the botanical origin of the olive oil they purchase. The objective of the present study was the development of robust chemometric models based on gas chromatography-mass spectrometry (GC-MS) and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) for the purpose of botanical differentiation of three commercial Greek olive oil cultivars. RESULTS: Using the solid-phase microextraction technique (SPME), volatile compounds (VC) were obtained and analyzed by GC-MS. Five hydrocarbons and one ester were selected by the forward stepwise algorithm, which best discriminated the olive oil samples. From ATR-FTIR analysis, the spectral regions chosen from the forward stepwise algorithm were associated with CO stretching vibration of the esters of triglycerides and the CH bending vibrations of the CH2 aliphatic group and double bonds. Application of the supervised methods of linear and quadratic discriminant cross-validation analysis, based on VC data, provided a correct classification score of 97.4% and 100.0%, respectively. Corresponding statistical analyses were used in the mid-infrared spectra, by which 96.1% of samples were discriminated correctly. CONCLUSION: ATR-FTIR and SPME-GC-MS techniques in conjunction with the appropriate feature selection algorithm and classification methods proved to be powerful tools for the authentication of Greek olive oil. The proposed methodology could be used in an industrial setting for determination of the botanical origin of Greek olive oil. © 2020 Society of Chemical Industry.

Classification of Greek Mentha pulegium L. (Pennyroyal) samples, according to geographical location by Fourier transform infrared spectroscopy.

INTRODUCTION: Mentha pulegium L. (pennyroyal) is one of the four most commercially important Mentha species, even it is not a cultivated plant. It can be abundantly located in the Iberian Peninsula and North African countries. In Greece it grows in the wild and it is scattered all over the country. Pennyroyal is best known for its essential oil, with Spain and Morocco being the largest producers in the world. Mid-infrared spectroscopy has been applied to determine the origin of various samples. OBJECTIVES: In this work Fourier transform infrared spectroscopy (FT-IR) combined with canonical discriminant analysis has been applied to distinguish 70 Greek pennyroyal samples according to their collection areas. MATERIAL AND METHODS: Pennyroyal nonpolar organic extracts were prepared using ultrasound-assisted solvent extraction. The spectra of the extracts were recorded in the range of 4000-400 cm(-1) and the best discrimination was achieved in the spectral region 1720-1650 cm(-1) . RESULTS: Spectral features for the discrimination of pennyroyal samples among the different collection areas occur primarily in the carbonyl region and are correlated with the main volatile constituents of the extracts (menthone, isomenthone, pulegone, piperitone). All areas were easily differentiated by canonical discriminant analysis. The percentages of correct classification and validation were 94.3 and 90.0%, respectively. CONCLUSION: The combination of FT-IR spectroscopy and multivariate analysis provides a rapid and ambient method to discriminate pennyroyal samples in terms of geographical origin.

Quantification of crocetin esters in saffron (Crocus sativus L.) using Raman spectroscopy and chemometrics.

The feasibility of Raman spectroscopy for predicting the content of crocetin esters (crocins), and coloring strength was assessed. 114 samples from Greece, Iran, Italy and Spain were divided into two sets: a calibration set with 49 samples and a validation one with 65 samples. Calibration models for crocetin esters (r 0.97, RMSEC 0.92, RMSEP 0.97, RPD 3.46) and coloring strength (r 0.95, RMSEC 12.2, RMSEP 11.3, RPD 2.59) were built in the spectral region 1700-955 cm(-1) using partial least-squares (PLS) regression. The calibration models were validated using cross-validation, leaving one sample out (r 0.97, RMSECV 1.09 for crocetin esters and r 0.93, RMSECV 14.5 for coloring strength). The crocetin esters content as determined by liquid chromatography fluctuated between 18.8 and 31.7 mg/100 g saffron. The corresponding values, as calculated using the Raman method, fluctuated between 19.2 and 32.0 mg/100 g saffron. The coloring strength determined by the reference method ranged from 177.0 to 296.7 units, while with the Raman method the values were between 186.8 and 297.6 units. The results, as compared to the reference methods (liquid chromatography and UV-vis spectrophotometry), show that the proposed methodology gives data with acceptable accuracy. The proposed models can be used as a tool for rapid screening of quality in saffron samples.

Effects of mild temperature conditions during dehydration procedures on saffron quality parameters.

BACKGROUND: The dehydration procedure is responsible for saffron sensorial properties: colour, taste and aroma. Changes in the compounds responsible for these characteristics have been studied when dehydration processes at high and low temperature are employed. However, the evolution of these changes at mild temperatures is not available in the current bibliography. In this paper the effect of different mild conditions (18-20 degrees C for 24 h, 40-50 degrees C for 75 min and 55 degrees C for 75 min) applied to 45 saffron samples with the same origin was investigated. RESULTS: Crocetin esters, the compounds responsible for saffron colour, increased their content with no significant differences from other processes when high temperatures (55 degrees C) were used, thus producing a noticeable increment in saffron colouring capability. Similar behaviour was obtained for picrocrocin, the compound responsible for saffron taste, with higher average content at the highest temperature (55 degrees C) but without significant differences with the inferior conditions (40-50 degrees C). However, more volatile compounds were generated, especially safranal,at higher temperatures, e.g. 55 degrees C, during the dehydration procedure. CONCLUSIONS: The results found support the idea for employing mild to high temperatures during the dehydration process of saffron.

Picrocrocin content and quality categories in different (345) worldwide samples of saffron ( Crocus sativus L.).

In this paper, 345 saffron samples were analyzed from different countries to study their picrocrocin content using different analytical techniques. The E1cm 1% of 257 nm results from all samples are inflated in comparison by the high-performance liquid chromatography (HPLC) data, because of the interferences with the crocetin ester pool and especially with those with a lower trans/cis relation. A picrocrocin range update is proposed for International Organization for Standardization (ISO) 3632 normative because category III should be incremented up to 50 units, while category II should be incremented up to 60 units. More accurate data are achieved when the deltaEpic measurement is carried out. Consequently, improvements to the ISO method are suggested. Fourier transform (FT)-near-infrared spectrometry analysis has also been carried out, showing excellent results from the calibration with HPLC data. This spectrophotometric technique could be used by saffron enterprises to obtain quick and more accurate data for picrocrocin determination.

Effects of the active constituents of Crocus sativus L., crocins on recognition and spatial rats' memory.

Crocus sativus L. is a plant cultivated in various parts of the world. Its involvement in learning and memory processes has been proposed. Crocins are water-soluble carotenoids and are among the active components of C. sativus L. The present study was designed to investigate in the rat the effects of crocins on recognition and spatial memory. For this aim, the object recognition task which evaluates non-spatial working memory and a novel version of the radial water maze which assesses spatial reference and spatial working memory were chosen. In a first study, crocins (15 and 30mg/kg) counteracted delay-dependent recognition memory deficits in the normal rat, suggesting that these carotenoids modulate storage and/or retrieval of information. In a subsequent study, treatment with crocins (30mg/kg and to a lesser extent also 15mg/kg) attenuated scopolamine (0.2mg/kg)-induced performance deficits in the radial water maze test. The present results support and extend the enhancing effects of crocins on memory and, then, to our knowledge, for the first time, demonstrate its implication in the mechanisms underlying recognition and spatial memory.

Interaction of tRNA with Safranal, Crocetin, and Dimethylcrocetin.

Saffron is the red dried stigmas of Crocus sativus L. flowers and used both as a spice and as a drug in traditional therapeutic. The biological activity of saffron in modern medicine is in development. Its numerous applications as an anti-oxidant and anti-cancer agent are due to its secondary metabolites and their derivatives (safranal, crocins, crocetin, dimethylcrocetin). The aim of this study was to examine the interaction of transfer RNA with safranal, crocetin, and dimethylcrocetin in aqueous solution at physiological conditions. Constant tRNA concentration (6.25 mM) and various drug/tRNA (phosphate) molar ratios of 1/48 to 1/8 were used. FT-IR and UV-Visible difference spectroscopic methods have been applied to determine the drug binding mode, the binding constants and the effects of drug complexation on the stability and conformation of tRNA duplex. External binding mode was observed for safranal crocetin and dimethylcrocetin, with overall binding constants K(safranal) = 6.8 (+/- 0.34) x 10(3) M(-1), K(CRT) = 1.4 (+/- 0.31) x 10(4) M(-1), and K(DMCRT) = 3.4 (+/- 0.30) x 10(4) M(-1). Transfer RNA remains in the A-family structure, upon safranal, crocetin and dimethylcrocetin complexation.

Crocetin, dimethylcrocetin, and safranal bind human serum albumin: stability and antioxidative properties.

Crocetin (CRT) and dimethylcrocetin (DMCRT) are derived from crocins which are found in the stigmas of saffron (Crocus sativus L.), while safranal is the main component of saffron's essential oil. The aim of the present study was to examine their interaction with human serum albumin in aqueous solution at physiological conditions using constant protein concentration and various ligand contents. FT-IR and UV-visible spectroscopic methods were used to determine the ligands' binding mode, the binding constant, and the effects of ligand complexation on protein secondary structure. Structural analysis showed that crocetin, dimethylcrocetin, and safranal bind nonspecifically (H-bonding) via protein polar groups with binding constants of Kcrt =2.05 (+/-0.30) x 103 M-1, Kdmcrt = 9.60 (+/-0.35) x 104 M-1, and Ksaf = 2.11 (+/-0.35) x 103 M-1. The protein secondary structure showed no major alterations at low ligand concentrations (1 microM), whereas at higher content (1 mM), decrease of alpha-helix from 55% (free HSA) to 43-45% and increase of beta-sheet from 17% (free HSA) to 18-22% and random coil 7% (free HSA) to 10-14% occurred in the ligand-HSA complexes. The results point to a partial unfolding of protein secondary structure at high ligand content. The antioxidant activity of CRT, DMCRT, and safranal was also tested by the DPPH* antioxidant activity assay, and their IC50 values were compared to that of well-known antioxidants such as Trolox and butylated hydroxy toluene (BHT). The IC50 values of CRT and safranal were 17.8 +/- 1 microg/mL and 95 +/- 1 microg/mL, respectively, while the inhibition of DMCRT reached a point of 38.8%, which corresponds to a concentration of 40 microg/mL, and then started to decrease. The IC50 values of Trolox and BHT were 5.2 +/- 1 microg/mL and 5.3 +/- 1 microg/mL, respectively.

DNA interaction with saffron's secondary metabolites safranal, crocetin, and dimethylcrocetin.

Saffron comes from the dried red stigmas of the Crocus sativus L. flower. Except for its use in cooking and in traditional medicine, it has numerous applications as an antitoxic, antioxidant, and anticancer agent due to its secondary metabolites and their derivatives (safranal, crocins, crocetin, dimethylcrocetin). However, there has been no information on the interactions of these secondary metabolites with individual DNA at molecular level. This study was designed to examine the interaction of safranal, crocetin (CRT), and dimethylcrocetin (DMCRT) with calf-thymus DNA in aqueous solution at physiological conditions, using constant DNA concentration (6.25 mM) and various drug/DNA(phosphate) molar ratios from 1/48 to 1/2. FTIR and UV-visible difference spectroscopic methods are used to determine the drug binding sites, the binding constants, and the effects of carotenoids and safranal complexation on the stability and conformation of DNA duplex. Both intercalative and external binding modes were observed, with overall binding constants K(safranal) = 1.24 x 10(3) M(-1), K(CRT) = 6.2 x 10(3) M(-1) and K(DMCRT) = 1.85 x 10(5) M(-1) A partial B- to A-DNA transition occurs at high carotenoids and safranal concentrations.

Interaction of antioxidant flavonoids with tRNA: intercalation or external binding and comparison with flavonoid-DNA adducts.

Antioxidants are essential to good health. Flavonoids are powerful antioxidants, and prevent DNA damage. The antioxidative protections are related to their binding modes to a DNA duplex and complexation with free radicals in vivo. Recently we reported the interaction of flavonoids with DNA in vitro (Kanakis et al., J. Biomol. Struct. Dyn. 22, 719-724, 2005), where polyphenol different binding modes were discussed. The aim of this study was to examine the interaction of transfer RNA with quercetin (que), kaempferol (kae), and delphinidin (del) in aqueous solution at physiological conditions and to make a comparison with the corresponding pigment-DNA adducts. Constant tRNA concentration (6.25 mM) and various drug/RNA(phosphate) molar ratios of 1/48 to 1/8 were used. FTIR and UV-visible difference spectroscopic methods have been applied to determine the drug binding mode, the binding constants, and the effects of drug complexation on the stability and conformation of tRNA duplex. Both intercalative and external binding modes were observed. Structural analysis showed que, kae, and a del intercalate tRNA duplex with minor external binding to the major or minor groove and the backbone phosphate group with overall binding constants K (que) = 4.80 x 10(4) M(1), K (kae) = 4.65 x 10(4) M(1), and K (del) = 9.47 x 10(4) M(1). The stability of adduct formation is in the order of del > que > kae. A comparison with flavonoids-DNA adducts showed both intercalation and external bindings with the stability order K (que) = 7.25 x 10(4) M(1), K (kae) = 3.60 x 10(4) M(1), and K (del) = 1.66 x 10(4) M(1). Low flavonoid concentration induces helical stabilization, whereas high pigment content causes helix opening. A partial Bto A-DNA transition occurs at high drug concentration, while tRNA remains in the A-family structure.

Qualitative determination of volatile compounds and quantitative evaluation of safranal and 4-hydroxy-2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde (HTCC) in Greek saffron.

Safranal (2,6,6-trimethyl-1,3-cyclohexadiene-1-carboxaldehyde) is the main component of saffron's essential oil. It was obtained using microsimultaneous hydro distillation-extraction (MSDE) and by ultrasound-assisted extraction (USE), which is a mild method. 4-Hydroxy-2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde (HTCC) is a precursor of safranal and was obtained in considerable amounts only by USE. Five C(13)-norisoprenoids were found in saffron for the first time. Using a gas chromatography technique, safranal and HTCC were quantified from Greek saffron samples. The quantity of safranal isolated by MSDE ranged between 288.1 and 687.9 mg/100 g of saffron, whereas in the case of USE safranal and HTCC ranged between 40.7 and 647.7 mg/100 g of saffron and between 41.7 and 397.7 mg/100 g of saffron, respectively. Freeze-drying was also tested as an alternative drying method. Over years of storage at 4 degrees C the quantity of safranal remained mostly constant while the quantity of HTCC decreased over the same periods.