Saffron (Crocus sativus L.) and health outcomes: a meta-research review of meta-analyses and an evidence mapping study.

BACKGROUND: Although a number of systematic reviews and meta-analyses of saffron (Crocus sativus L.) have been published, no study has comprehensively summarized the clinical evidence from meta-analyses, or assessed the reporting or methodological quality of these reviews. PURPOSE: The present meta-research study was designed to fill the gaps in knowledge to inform future studies and allow enhanced clinical decision-making on saffron. METHODS: The PubMed, Cochrane Library, Embase, and CNKI databases were systematically searched from inception to April 3 rd, 2021, for meta-analyses of clinical trials that assessed the efficacy and safety of saffron. PRISMA 2009 and AMSTAR-2 were employed to assess the reporting and methodological quality of meta-analyses identified in the search, respectively. The present study was registered on PROSPERO with registration number CRD42020220274. RESULTS: Nineteen eligible systematic reviews with meta-analyses published in English were identified from 235 records. These meta-analyses were published in 12 peer-reviewed journals from 2013 to 2021. The heterogeneous results indicated that saffron significantly reduced fasting blood glucose, waist circumference, diastolic blood pressure, concentrations of total cholesterol and low-density lipoprotein cholesterol, and improved symptoms of depression, cognitive function and sexual dysfunction compared with controls (mainly placebos). Common side effects of saffron consumption included nausea, dry mouth, poor appetite, and headache, but no serious adverse reactions were reported. Primary analysis and sensitivity analysis confirmed that the reporting and methodological quality of reviews included in the study were highly correlated (p < 0.001). The quality of meta-analyses of saffron requires improvement by including a structured abstract, a prospective protocol and registration, explanation of the study designs within each study that is reviewed, the searches, risk of bias assessment, literature selection, and reporting of funding sources. CONCLUSION: The available evidence indicates that saffron is a safe plant for administration as a medicine and can improve diverse clinical outcomes, but the scientific quality of the published systematic reviews needs to be improved. Moreover, the clinical effects of saffron need to be confirmed through high-quality randomized trials in multiple countries with large sample sizes.

Saffron for "toning down" COVID-19-related cytokine storm: Hype or hope? A mini-review of current evidence.

AIM: To assess the potential role of saffron in downregulating inflammation and cytokine storm during COVID-19. MAIN FINDINGS: Three main compounds of saffron, i.e., crocetin esters, picrocrocin, and safranal, present strong antioxidant and anti-inflammatory action for several disease states (e.g., Alzheimer's, cancer, and depression) but have also been studied in COVID-19. In particular, based on our comprehensive review of both in vitro and in silico studies, saffron's essential oils and other constituents appear to have both immunomodulatory and anti-asthmatic actions; these actions can be particularly helpful to treat patients with respiratory symptoms due to COVID-19. Moreover, crocin appears to reduce the COVID-19-related cytokine cascade and downregulate angiotensin-converting enzyme 2 (ACE2) gene expression. Last, in silico studies suggest that saffron's astragalin and crocin could have inhibitory actions on SARS-CoV-2 protease and spike protein, respectively. CONCLUSION: Saffron represents a promising substance for toning down cytokine storm during COVID-19, as well as a potential preventive treatment for COVID-19. However, appropriate randomized clinical trials, especially those using biomarkers as surrogates to assess inflammatory status, should be designed in order to assess the clinical efficacy of saffron and allow its use as an adjunct treatment modality, particularly in resource-poor settings where access to drugs may be limited.

Encapsulation of Mentha pulegium Essential Oil in Yeast Cell Microcarriers: An Approach to Environmentally Friendly Pesticides.

A green approach for the encapsulation of Mentha pulegium essential oil in commercial baker's yeast and its evaluation as a pesticide against the insect pest Myzus persicae are presented. Upon treating aqueous yeast cell dispersion with the essential oil, the formation of essential-oil-loaded microparticles of about 9 µm is observed, with a loading capacity ranging from 29 to 36%, depending upon the encapsulation conditions. The thermal properties of the microparticles were characterized using differential scanning calorimetry and thermogravimetric analysis, confirming the protection of the essential oil from the cells. Encapsulation prolonged the insecticidal activity of the essential oil by 3 days.

Chemical composition and assessment of larvicidal and repellent capacity of 14 Lamiaceae essential oils against Aedes albopictus.

In the current laboratory study, 14 essential oils (EOs) derived from 12 Lamiaceae plant species and their major components were screened for their larvicidal and repellent properties against Aedes albopictus, an invasive mosquito species of great medical importance. The results of toxicity bioassays revealed that the EOs from Thymus vulgaris, Ocimum basilicum, Origanum dictamnus, Origanum majorana, and Origanum vulgare, as well as their major components (terpenes), namely thymol, carvacrol, p-cymene, and γ-terpinene exerted the highest larvicidal effect. Essential oils from Mellisa officinalis, Origanum dictamus, Mentha spicata (chem. piperitenone epoxide), Origanum majorana, and Satureja thymbra were the most potent repellents, with the last two assigned as the best ones. Among the terpenes tested, piperitenone epoxide, carvacrol, thymol, and piperitenone provided the highest level of protection against Ae. albopictus adults. Chemical analysis revealed the presence of a high number of terpenes in the EOs, while in most cases, the biological action of the tested EOs and their major components was in consistency. The most effective EOs and terpenes that were identified through the current laboratory bioassays could be used as alternative agents to control larvae and repel adults of Ae. albopictus.

Integrated analytical methodology to investigate bioactive compounds in Crocus sativus L. flowers.

INTRODUCTION: The increasing interest on Crocus sativus L. over the last decades is caused by its potential employment as a source of biologically active molecules, endowed with antioxidant and nutraceutical properties. These molecules are present mainly in stigmas and tepals, these last generally considered as byproducts. OBJECTIVE: To characterise bioactive compounds in stigmas, stamens, and tepals of Crocus sativus L. for quality, cross-contamination of tissues or fraudulent addition, joining spectroscopic and chromatographic techniques. METHODOLOGY: Fourier transform infrared (FT-IR) and Raman spectroscopies were initially employed, being very rapid in response; volatiles were more appropriately investigated by gas chromatography with mass spectrometry (GC-MS), while finally nuclear magnetic resonance (NMR) and high-performance liquid chromatography with a diode array detector (HPLC-DAD) were adopted for a more thorough characterisation of secondary metabolites. NMR was also used to investigate the anthocyanins content in tepals upon acid extraction. RESULTS: The results obtained highlighted the drying method as the dominant factor affecting the content of volatile constituents and contributing to the quality of saffron, while only slight differences were observed in the most abundant metabolites of stigmas, as well as in the anthocyanin content of tepals. In particular, for the first time, delphinidin and petunidin were detected by NMR in this latter tissue. CONCLUSION: The integrated analytical methodology here proposed, allowed to achieve a deeper level of compositional and structural details of secondary metabolites in Crocus sativus L. flowers.

Comparative evaluation of an ISO 3632 method and an HPLC-DAD method for safranal quantity determination in saffron.

The aim of this work was a comparison of the ISO 3632 (2011) method and an HPLC-DAD method for safranal quantity determination in saffron. Samples from different origins were analysed by UV-vis according to ISO 3632 (2011) and by HPLC-DAD. Both methods were compared, and there was no correlation between the safranal content obtained by UV-vis and HPLC-DAD. An over-estimation in the UV-vis experiment was observed, which was related to the cis-crocetin esters content, as well as other compounds. The results demonstrated that there was no relationship between ISO quality categories and safranal content using HPLC-DAD. Therefore, HPLC-DAD might be preferable to UV-vis for determining the safranal content and the classification of saffron for commercial purposes. In addition, HPLC-DAD was adequate for determining the three foremost parameters that define the quality of saffron (crocetin esters, picrocrocin and safranal); therefore, this approach could be included in the ISO 3632 method (2011).

Sudan dyes in adulterated saffron (Crocus sativus L.): Identification and quantification by (1)H NMR.

Saffron, the dried red stigmas of Crocus sativus L., is considered as one of the most expensive spices worldwide, and as such, it is prone to adulteration. This study introduces an NMR-based approach to identify and determine the adulteration of saffron with Sudan I-IV dyes. A complete (1)H and (13)C resonance assignment for Sudan I-IV, achieved by two-dimensional homonuclear and heteronuclear NMR experiments, is reported for the first time. Specific different proton signals for the identification of each Sudan dye in adulterated saffron can be utilised for quantitative (1)H NMR (qHNMR), a well-established method for quantitative analysis. The quantification of Sudan III, as a paradigm, was performed in varying levels (0.14-7.1g/kg) by considering the NMR signal occurring at 8.064ppm. The high linearity, accuracy and rapidity of investigation enable high resolution (1)H NMR spectroscopy to be used for evaluation of saffron adulteration with Sudan dyes.

Assessing saffron (Crocus sativus L.) adulteration with plant-derived adulterants by diffuse reflectance infrared Fourier transform spectroscopy coupled with chemometrics.

Saffron, the dried red stigmas of the plant Crocus sativus L., is well-known as one of the most important and expensive spices worldwide. It is thus highly susceptible to fraudulent practices that employ, among others, plant-derived adulterants. This study presents an application of diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) and chemometric techniques for evaluating adulteration of saffron with six characteristic adulterants of plant origin, i.e. C. sativus stamens, calendula, safflower, turmeric, buddleja, and gardenia. The proposed method involved a three-step process for the detection of adulteration as well as for the identification and quantification of adulterants. Partial least squares discriminant analysis (PLS-DA) was applied to perform authentication of saffron based on mid-infrared fingerprints (4000-600cm-1), resulting in 99% correct classification of pure saffron and saffron adulterated at 5-20% (w/w) levels. Adulterant identification in positive samples was performed with high sensitivity and specificity by a six-class PLS-DA model, with spectroscopic data from the region 2000-600cm-1. Subsequently, partial least squares (PLS) regression models were built for the quantification of each adulterant. By using synergy interval PLS (siPLS) for variable selection, models with improved performance were developed, with detection limits ranging from 1.0% to 3.1% (w/w). The results obtained illustrate that this strategy based on DRIFTS has the potential to complement existing methodologies for the rapid and cost-effective assessment of typical saffron frauds.

Biocidal Compounds from Mentha sp. Essential Oils and Their Structure-Activity Relationships.

Essential oils from Greek Mentha species showed different chemical compositions for two populations of M. pulegium, characterized by piperitone and pulegone. Mentha spicata essential oil was characterized by endocyclic piperitenone epoxide, piperitone epoxide, and carvone. The bioactivities of these essential oils and their components have been tested against insect pests (Leptinotarsa decemlineata, Spodoptera littoralis and Myzus persicae), root-knot nematodes (Meloydogine javanica) and plants (Lactuca sativa, Lolium perenne, Solanum lycopersicum). The structure-activity relationships of these compounds have been studied including semi-synthetic endocyclic trans-carvone epoxide, exocyclic carvone epoxide, a new exocyclic piperitenone epoxide and trans-pulegone epoxide. Leptinotarsa decemlineata feeding was affected by piperitenone and piperitone epoxide. Spodoptera littoralis was affected by piperitone epoxide and pulegone. The strongest nematicidal agent was piperitenone epoxide, followed by piperitone epoxide, piperitenone and carvone. Germination of S. lycopersicum and L. perenne was significantly affected by piperitenone epoxide. This compound and carvone epoxide inhibited L. perenne root and leaf growth. Piperitenone epoxide also inhibited the root growth of S. lycopersicum. The presence of a C(1) epoxide resulted in strong antifeedant, nematicidal and phytotoxic compounds regardless of the C(4) substituent. New natural crop protectants could be developed through appropriate structural modifications in the p-menthane skeleton.

Genetic and Epigenetic Approaches for the Possible Detection of Adulteration and Auto-Adulteration in Saffron (Crocus sativus L.) Spice.

Saffron (Crocus sativus L.) is very expensive and, because of this, often subject to adulteration. Modern genetic fingerprinting techniques are an alternative low cost technology to the existing chemical techniques, which are used to control the purity of food products. Buddleja officinalis Maxim, Gardenia jasminoides Ellis, Curcuma longa L., Carthamus tinctorius L. and Calendula officinalis L. are among the most frequently-used adulterants in saffron spice. Three commercial kits were compared concerning the ability to recover PCR-grade DNA from saffron, truly adulterated samples and possible adulterants, with a clear difference among them, mainly with the processed samples. Only one of the three kits was able to obtain amplifiable DNA from almost all of the samples, with the exception of extracts. On the recovered DNA, new markers were developed based on the sequence of the plastid genes matK and rbcL. These primers, mainly those developed on matK, were able to recognize saffron and the adulterant species and also in mixtures with very low percentages of adulterant. Finally, considering that the addition of different parts of saffron flowers is one of the most widespread adulterations, by analyzing the DNA of the different parts of the flower (styles, stamens and tepals) at the genetic and epigenetic level, we succeeded in finding differences between the three tissues that can be further evaluated for a possible detection of the kind of fraud.

Evaluation of saffron (Crocus sativus L.) adulteration with plant adulterants by (1)H NMR metabolite fingerprinting.

In the present work, a preliminary study for the detection of adulterated saffron and the identification of the adulterant used by means of (1)H NMR and chemometrics is reported. Authentic Greek saffron and four typical plant-derived materials utilised as bulking agents in saffron, i.e., Crocus sativus stamens, safflower, turmeric, and gardenia were investigated. A two-step approach, relied on the application of both OPLS-DA and O2PLS-DA models to the (1)H NMR data, was adopted to perform authentication and prediction of authentic and adulterated saffron. Taking into account the deficiency of established methodologies to detect saffron adulteration with plant adulterants, the method developed resulted reliable in assessing the type of adulteration and could be viable for dealing with extensive saffron frauds at a minimum level of 20% (w/w).

FTIR spectroscopic evaluation of changes in the cellular biochemical composition of the phytopathogenic fungus Alternaria alternata induced by extracts of some Greek medicinal and aromatic plants.

In this study, the biological activity of aquatic extracts of selected Greek medicinal and aromatic plants to the phytopathogenic fungus Alternaria alternata was investigated. Lamiaceae species (Hyssopus officinalis L., Melissa officinalis L., Origanum dictamnus L., Origanum vulgare L. and Salvia officinalis L.) were found to enhance significantly the mycelium growth whereas Crocus sativus appears to inhibit it slightly. M. officinalis and S. officinalis caused the highest stimulation in mycelium growth (+97%) and conidia production (+65%) respectively. In order to further investigate the bioactivity of plant extracts to A. alternata, we employed Fourier Transform Infrared Spectroscopy (FTIR). Differences of original spectra were assigned mainly to amides of proteins. The second derivative transformation of spectra revealed changes in spectral regions corresponding to absorptions of the major cellular constituents such as cell membrane and proteins. Principal component analysis of the second derivative transformed spectra confirmed that fatty acids of the cell membranes, amides of proteins and polysaccharides of the cell wall had the major contribution to data variation. FTIR band area ratios were found to correlate with fungal mycelium growth.

Bioefficacy of acyclic monoterpenes and their saturated derivatives against the West Nile vector Culex pipiens.

Twenty acyclic monoterpenes with different functional groups (acetoxy, hydroxyl, carbonyl and carboxyl) bearing a variable number of carbon double bonds were assayed as repellent and larvicidal agents against the West Nile vector Culex pipiens. Seven of them were derivatives that were synthesized through either hydrogenation or oxidation procedures. All repellent compounds were tested at the dose of 1mgcm(-2) and only neral and geranial were also tested at a 4-fold lower dose (0.25mgcm(-2)). Repellency results revealed that geranial, neral, nerol, citronellol, geranyl acetate and three more derivatives dihydrolinalool (3), dihydrocitronellol (5) and dihydrocitronellyl acetate (6) resulted in no landings. Based on the LC50 values the derivative dihydrocitronellyl acetate (6) was the most active of all, resulting in an LC50 value of 17.9mgL(-1). Linalyl acetate, citronellyl acetate, neryl acetate, geranyl acetate, dihydrocitronellol (5), dihydrocitronellal (7), citronellol, dihydrolinalyl acetate (2), citronellic acid and tetrahydrolinalyl acetate (1) were also toxic with LC50 values ranging from 23 to 45mgL(-1). Factors modulating toxicity have been identified, thus providing information on structural requirements for the selected acyclic monoterpenes. The acetoxy group enhanced toxicity, without being significantly affected by the unsaturation degree. Within esters, reduction of the vinyl group appears to decrease potency. Presence of a hydroxyl or carbonyl group resulted in increased activity but only in correlation to saturation degree. Branched alcohols proved ineffective compared to the corresponding linear isomers. Finally, as it concerns acids, data do not allow generalizations or correlations to be made.

Increased seizure latency and decreased severity of pentylenetetrazol-induced seizures in mice after essential oil administration.

The effect of pretreatment with essential oils (EOs) from eight aromatic plants on the seizure latency and severity of pentylenetetrazol- (PTZ-) induced seizures in mice was evaluated. Weight-dependent doses of Rosmarinus officinalis, Ocimum basilicum, Mentha spicata, Mentha pulegium, Lavandula angustifolia, Mentha piperita, Origanum dictamnus, and Origanum vulgare, isolated from the respective aromatic plants from NE Greece, were administered 60 minutes prior to intraperitoneal (i.p.) injection of a lethal dose of PTZ to eight respective groups of Balb-c mice. Control group received only one i.p. PTZ injection. Motor and behavioral activity of the animals after EOs administration, development of tonic-clonic seizures, seizure latency and severity, and percentage of survival after PTZ administration were determined for each group. All groups of mice treated with the EOs showed reduced activity and stability after the administration of the oil, except for those treated with O. vulgare (100% mortality after the administration of the oil). After PTZ administration, mice from the different groups showed increased latency and reduced severity of seizures (ranging from simple twitches to complete seizures). Mice who had received M. piperita demonstrated no seizures and 100% survival. The different drastic component and its concentration could account for the diversity of anticonvulsant effects.

Herbal infusions; their phenolic profile, antioxidant and anti-inflammatory effects in HT29 and PC3 cells.

In this survey, we analyzed the phenolic profile of six herbal infusions namely Cretan marjoram, pink savory, oregano, mountain tea, pennyroyal and chamomile by LCDAD-MS and by GC-MS. Further, we investigated their anticarcinogenic effect as to their ability to (a) scavenge free radicals (b) inhibit proliferation (c) decrease IL-8 levels and (d) regulate nuclear factor-kappa B in epithelial colon cancer (HT29) and prostate (PC3) cancer cells. All herbal infusions exhibited antiradical activity correlated positevely with total phenolic content. Further, infusions exhibited the potential to inhibit cell proliferation and to reduce IL-8 levels in HT29 colon and PC3 prostate cancer cells. The molecular target for chamomile in HT29 seemed to be the NF-κB, while for the other herbal infusions needs to be identified. This study is the first to show the potential chemopreventive activity of infusions prepared from the examined herbs.

Direct determination of rosmarinic acid in Lamiaceae herbs using diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) and chemometrics.

For the determination of rosmarinic acid (RA) directly in pulverized plant material, a method is developed using diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) without any physicochemical pretreatment of samples. The RA content of 11 samples of eight different Lamiaceae herbs, as determined by high performance liquid chromatography (HPLC), varied between 86 ± 1 mg/g (in lemon balm) and 12.0 ± 0.8 mg/g (in hyssop) of dried plant material. The 11 samples and 50 other additional samples, which were prepared by mixing initial samples with KBr, were measured using DRIFTS. The second derivative of the spectral region 1344-806 cm(-1) was used and the corresponding data were analyzed using partial least squares (PLS) regression. The correlation between infrared spectral analysis and HPLC measurements shows that the DRIFTS method is sufficiently accurate, simple, and rapid. The RA content of the 11 Lamiaceae samples determined by DRIFTS ranged from 81 ± 4 mg/g (in lemon balm) to 12 ± 3 mg/g (in hyssop) of dried plant material.

Incidence of Bacteriocins Produced by Food-Related Lactic Acid Bacteria Active towards Oral Pathogens.

In the present study we investigated the incidence of bacteriocins produced by 236 lactic acid bacteria (LAB) food isolates against pathogenic or opportunistic pathogenic oral bacteria. This set of LAB contained several strains (≥17%) producing bacteriocins active against food-related bacteria. Interestingly only Streptococcus macedonicus ACA-DC 198 was able to inhibit the growth of Streptococcus oralis, Streptococcus sanguinis and Streptococcus gordonii, while Lactobacillus fermentum ACA-DC 179 and Lactobacillus plantarun ACA-DC 269 produced bacteriocins solely against Streptococcus oralis. Thus, the percentage of strains that were found to produce bacteriocins against oral bacteria was ~1.3%. The rarity of bacteriocins active against oral LAB pathogens produced by food-related LAB was unexpected given their close phylogenetic relationship. Nevertheless, when tested in inhibition assays, the potency of the bacteriocin(s) of S. macedonicus ACA-DC 198 against the three oral streptococci was high. Fourier-transform infrared spectroscopy combined with principal component analysis revealed that exposure of the target cells to the antimicrobial compounds caused major alterations of key cellular constituents. Our findings indicate that bacteriocins produced by food-related LAB against oral LAB may be rare, but deserve further investigation since, when discovered, they can be effective antimicrobials.

Effects of Americium-241 and humic substances on Photobacterium phosphoreum: bioluminescence and diffuse reflectance FTIR spectroscopic studies.

The integral bioluminescence (BL) intensity of live Photobacterium phosphoreum cells (strain 1883 IBSO), sampled at the stationary growth stage (20 h), was monitored for further 300 h in the absence (control) and presence of (241)Am (an α-emitting radionuclide of a high specific activity) in the growth medium. The activity concentration of (241)Am was 2 kBq l(-1); [(241)Am]=6.5×10(-11) M. Parallel experiments were also performed with water-soluble humic substances (HS, 2.5 mg l(-1); containing over 70% potassium humate) added to the culture medium as a possible detoxifying agent. The BL spectra of all the bacterial samples were very similar (λ(max)=481±3 nm; FWHM=83±3 nm) showing that (241)Am (also with HS) influenced the bacterial BL system at stages prior to the formation of electronically excited states. The HS added per se virtually did not influence the integral BL intensity. In the presence of (241)Am, BL was initially activated but inhibited after 180 h, while the system (241)Am+HS showed an effective activation of BL up to 300 h which slowly decreased with time. Diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy, applied to dry cell biomass sampled at the stationary growth phase, was used to control possible metabolic responses of the bacteria to the α-radioactivity stress (observed earlier for other bacteria under other stresses). The DRIFT spectra were all very similar showing a low content of intracellular poly-3-hydroxybutyrate (at the level of a few percent of dry biomass) and no or negligible spectroscopic changes in the presence of (241)Am and/or HS. This assumes the α-radioactivity effect to be transmitted by live cells mainly to the bacterial BL enzyme system, with negligible structural or compositional changes in cellular macrocomponents at the stationary growth phase.

Insecticidal activity of plant essential oils against the vine mealybug, Planococcus ficus.

The vine mealybug, Planococcus ficus (Signoret) (Hemiptera: Pseudococcidae), is a pest in grape vine growing areas worldwide. The essential oils from the following aromatic plants were tested for their insecticidal activity against P. ficus: peppermint, Mentha piperita L. (Lamiales: Lamiaceae), thyme-leaved savory, Satureja thymbra L., lavender, Lavandula angustifolia Mill, and basil, Ocimum basilicum L. Essential oils from peels of the following fruits were also tested: lemon, Citrus limon L. (Sapindales: Rutaceae), and orange, C. sinensis L. The reference product was paraffin oil. Bioassays were conducted in the laboratory by using spray applications on grape leaves bearing clusters of P. ficus of one size class, which mainly represented either 3rd instar nymphs or pre-ovipositing adult females. The LC50 values for each essential oil varied depending on the P. ficus life stage but did not significantly differ between 3(rd) instar nymphs and adult females. The LC50 values of the citrus, peppermint, and thyme-leaved savory essential oils ranged from 2.7 to 8.1 mg/mL, and the LC50 values of lavender and basil oil ranged from 19.8 to 22.5 and 44.1 to 46.8 mg/mL, respectively. The essential oils from citrus, peppermint and thymeleaved savory were more or equally toxic compared to the reference product, whereas the lavender and basil essential oils were less toxic than the paraffin oil. No phytotoxic symptoms were observed on grape leaves treated with the citrus essential oils, and low phytotoxicity was caused by the essential oils of lavender, thyme-leaved savory, and mint, whereas the highest phytotoxicity was observed when basil oil was used.

Evaluation of bioefficacy of three Citrus essential oils against the dengue vector Aedes albopictus (Diptera: Culicidae) in correlation to their components enantiomeric distribution.

Laboratory experiments were conducted to study the bioefficacy against Ae. albopictus of three Citrus essential oils, derived from peels of Citrus sinensis, Citrus limon, and Citrus paradise and of their components. Chiral gas chromatography analysis revealed the dominant occurrence of R-(+)-limonene and (-)-ß-pinene in all three essential oils while in the case of lemon oil γ-terpinene, neral, and geranial detected also among other components. The tested Citrus essential oils were toxic against mosquito larvae with LC(50) values ranging from 25.03 to 37.03 mg l(-1). Among citrus essential oils components tested, γ-terpinene was the most toxic (LC(50) = 20.21 mg l(-1)) followed by both enantiomeric forms of limonene (LC(50) = 35.99 and 34.89 mg l(-1), for R-(+)-limonene and S-(-)-limonene, respectively). The delayed toxic effects after exposure of larvae to sublethal (LC(50)) doses were also investigated for citrus essential oils and their major component R-(+)-limonene, indicating a significant reduction of pupal survival. In repellent bioassays, lemon essential oil, S-(-)-limonene, citral (mixture of neral\geranial) and (+)-ß-pinene were the most effective compared with other citrus essential oils and components against adult mosquitoes. Repellent bioassays also revealed that limonenes and ß-pinenes showed an isomer dependence repellent activity. Finally, according to enantiomeric distribution of limonene and α- and ß-pinene, the repellency of lemon essential oil is possibly attributed to the presence of citral.