Antihyperlipidemic effect and GC-MS analysis of phytoconstituents from Laurus nobilis essential oil in rat.

This study aimed to evaluate the effect of Laurus nobilis (L. nobilis) essential oil (EOs) (80 mg/kg) on Triton WR-1339-induced dyslipidemia in Wistar rats. The effect of L. nobilis essential oil (80 mg/kg) on lipid and lipoprotein profile was examined on Triton WR-1339-induced dyslipidemia in rats. Furthermore, the phytochemical evaluation was performed by GC-MS. In Addition, the acute toxicity of this EO was evaluated at a dose of 2 g/kg. The results revealed that the main constituents of L. nobilis EO were 1,8-cineole (39.5%), linalool (13.09%), and a-terpineol (11.55%). Furthermore, the EO did not cause any signs of toxicity or mortality, and the acute lethal dose 50 (LD50) was estimated to be higher than 2 g/kg. L. nobilis EO ameliorated lipid parameters and atherogenic indices. In conclusion, the study demonstrates that L. nobilis essential oils possess antidyslipidemic activity in acute model of hyperlipidaemia.

Antibacterial Activity of Essential Oils Combinations based on Thymus broussonnetii, and Their Synergism with some Antibiotics.

The present study aimed to evaluate the antibacterial activity of the essential oil (EO) of Moroccan endemic Thymus broussonnetii alone, and in combination with EOs obtained from selected medicinal plants, namely Myrtus communis, Artemisia herba alba, Thymus pallidus, Thymus satureioides, Teucrium polium, and Rosmarinus officinalis. The synergistic interactions between the most effective combinations based on T. broussonnetii EO with two conventional antibiotics (streptomycin and ciprofloxacin) were also investigated. T. broussonnetii EO possessed a higher inhibitory activity against tested pathogenic bacteria with inhibition zone diameter (IZ) ranging from 21.61 ± 0.03 to 40.09 ± 0.02 mm, and MIC values between 0.140 mg/mL and 0.280 mg/mL. M. communis, A. herba alba, T. pallidus, T. satureioides, T. polium, and R. officinalis EOs showed moderate to weak antibacterial activity. Among tested EO mixtures, the highest synergistic antibacterial effect was recorded with the EO combination of T. broussonnetii and T. pallidus against S. aureus, E. coli, and S. enterica (FICI = 0.258). This EO combination was also the most effective mixture to synergistically enhance the antibacterial activity of the two antibiotics with up to a 128-fold increase, particularly against the gram-negative E. coli. These findings suggest that T. broussonnetii EO may be an interesting source of natural antimicrobials, for use in combination therapies with other plant EOs, and with conventional antimicrobial drugs to tackle the emergence of multidrug-resistant bacteria.

Molecular Survey and Microscopic Examination of Haemoparasites Infecting Lizards from Morocco.

PURPOSE: Understanding the factors influencing parasite diversification, their distribution and infection patterns among hosts provides valuable information on the impact of parasitism on natural populations and the dynamics and evolution of disease outbreaks. In this study, we investigated prevalence and intensity of infection by blood parasites, the diversity, distribution, host specificity and infection patterns of apicomplexan parasites in lizards of Morocco. METHODS: Samples from lizard specimens were collected between 2019 and 2021 from different prospected localities in Morocco. In total, 1500 lizards were collected, and their blood and tail tips were screened for blood parasites. RESULTS: Using a PCR approach, we detected two distinct apicomplexan parasite groups (haemogregarines and haemococcidians) and also Stramenopiles (Proteromonas). A significant difference was identified between hosts families, with no agamids or skinks infected and with particularly high infection rates in lacertids. Twenty-four samples were sequenced and included in a phylogenetic assessment. Two samples of Atlantolacerta andreanskyi were infected with unidentified species of Proteromonas. Three samples were infected with haemococcidians, including two samples with Schellackia and another sample with Lankesterella, more closely related to others isolated from bird hosts. These are the first reports of Lankesterella and Proteromonas in these lizards from Morocco. Nineteen different isolates of the complex of Hepatozoon/Karyolysus were identical or very similar to previously sequenced parasites from North Africa and Iberian Peninsula and included new host species. CONCLUSION: Our results highlight the need for future research on the vectors involved in transmission that may influence the ecology and distribution patterns of these parasites. Given that Morocco is a hot spot of endemicity and reptile richness, our results are relevant not only in the context of their taxonomy, but can also have implications for conservation.

New host and locality records of helminths' infection of seven lizards from Morocco.

Morocco has a great diversity of reptiles; more than 26% of the herpetofauna species are considered to be endemic. Nevertheless, there is little information available on helminth parasites of Moroccan lizards. The purpose of this article is to establish a helminth list using non-invasive methods for natural populations of seven lizard species: Acanthodactylus maculatus, Chalcides mionecton, Chalcides montanus, Chalcides polylepis, Quedenfeldtia moerens, Quedenfeldtia trachyblepharus, and Tarentola mauritanica. For each species, prevalence and intensity of infection were given as well as their relationship to some determinant parameters (sex, age class, size, altitude, and seasons). Four species of nematodes, Parapharyngodon micipsae, Pharyngodon mamillatus, Spauligodon auziensis, Thelandros alatus, and unassigned Spauligodon were found. Mean helminth intensity for the seven lizard species was 7.3 ± 16.2 SD (range = 1-92 in infected lizards), with total infection prevalence of 5.6%. Mixed infections were observed in Chalcides montanus and Chalcides polylepis co-infected by Pharyngodon mamillatus and Spauligodon sp. Furthermore, the intensity of infection by nematodes were significantly different among the seven studied species. The nematode infection in Chalcides mionecton were related to sex, host size, and altitude; in Chalcides polylepis to the host age; in Quedenfeldtia trachyblepharus to elevation, age, and host size; and in Tarentola Mauritanica to age, body size, seasons, and altitude. In conclusion, our study provides data about nine new host and locality records and the effect of some determinant factors on host parasitism.

Synergistic Anticandidal Effects of Six Essential Oils in Combination with Fluconazole or Amphotericin B against Four Clinically Isolated Candida Strains.

The development of opportunistic pathogenic Candida strains insensitive to several classes of antifungals has emerged as a major health care problem during the last years. Combinational therapy of natural products (e.g., essential oils, EOs) with conventional antifungals has been suggested as a promising alternative to overcome this medical problem. The present study investigates the potential antifungal activity of EOs extracted from some selected medicinal plants, alone and in combination with two common conventional antifungals (fluconazole and amphotericin B) against four clinical Candida isolates. MIC assays indicated that EOs induced strong anticandidal activities with MIC values ranging from 0.162 to 4.950 mg/mL. The combination of amphotericin B with Thymus leptobotrys, Origanum compactum and Artemisia herba alba EOs provided a synergistic effect against C. krusei only, with MIC gain of four-fold, and additive effect against remaining strains (MIC gain = two-fold). Interesting synergistic interactions were observed by combining all studied EOs with fluconazole, with reduction rates of their MICs ranging from 16 to 512-fold. This synergistic effect was very pronounced with the combination of T. leptobotrys EO and fluconazole. These findings indicate that studied EOs can be used as anti-candidals in combination with antifungals, particularly fluconazole, to counteract the emergence of resistant Candida spp.

New insight into antimicrobial activities of Linaria ventricosa essential oil and its synergetic effect with conventional antibiotics.

The purpose of the present study was to determine for the first time the volatile constituents, the antioxidant and antimicrobial activities of the essential oil (EO) of the endemic Moroccan Linaria ventricosa, alone or in combination with four known antibiotics. The major constituents were 2-methoxy-4-vinylphenol (17.4%), α-terpinene (13.64%) and 3,5-dimethylphenyl isocyanate (12.21%). The EO had moderate antioxidant potency, as measured by DPPH free radical scavenging (1.233 ± 0.031 mg/mL), ferric reducing antioxidant power assay (0.373 ± 0.019 mg/mL) and ß-carotene/linoleic acid (0.922 ± 0.026 mg/mL). EO showed microbicidal activity against all microorganisms tested. The highest effectiveness was recorded against Candida albicans (IZ = 24 mm, MIC = 4.87 mg/mL and MMC = 9.75 mg/mL) and Candida glabrata (IZ = 22 mm, MIC = MMC = 4.87 mg/mL). Gram negative bacteria were the most resistant (MIC = MMC = 39 mg/mL). The combination of EO at sub-inhibitory concentrations with antibiotics showed a significant decrease in their individual MICs from 2 to 128 fold, being the best for ciprofloxacin and fluconazole against E. coli and C. albicans and C. glabrata, respectively.

Determinants of prevalence and co-infestation by ecto- and endoparasites in the Atlas day gecko, Quedenfeldtia trachyblepharus, an endemic species of Morocco.

The present work was undertaken to investigate the occurrence of ectoparasites (mites and ticks) and endoparasites (haemogregarines and helminths) in the Atlas day gecko, Quedenfeldtia trachyblepharus (Boettger, 1874), a high-altitude Moroccan endemic lizard. The study examinated also the effect of some host parameters (age, sex, size, body condition), in addition to the season and altitude on the prevalence and intensity of parasite infestations. The study was conducted in three localities from May to September 2019. The results indicated that 35% of juvenile geckos were found to be parasited by one type of parasite. Contrarily, up to three types of parasites were detected in the adults. The prevalence of mite infestations was 48.02%, with a mean intensity of 11.80 ± 15.69. The patterns of mite infestations was found to be mainly related to the altitude, while the prevalence and intensity of infestations were linked to the host size, and to the sex and season, respectively. Larvae and nymphs of Ixodes ricinus (Linnaeus, 1758) were the only life stages infesting geckos, with a prevalence and mean intensity of infestations of 4.41% and 2.2 ± 1.48, respectively. The tick infestations observed were mainly related to the season. The prevalence and intensity of haemogregarine infections were, respectively, 7.92% and 0.24 ± 0.15. The altitude was found to be the only factor associated with this infection. This study also revealed the presence of one helminth genus, Spauligodon sp., with a prevalence and mean intensity of 12.33% and 1.46 ± 0.88, respectively. Spauligodon infestations was significantly associated with age, host size and altitude. This finding represents the first citation of this parasite in Quedenfeldtia genus. Our study indicated that there was no significant relationship between parasite load and geckos body condition, which suggested a stable interaction between the gecko and its parasites. However, it showed a difference of infection between the localities, which could be in relation with habitat conditions.

Novel DiOC3 96-well real-time efflux assay for discovery of NorA efflux pump inhibitors in Staphylococcus aureus.

The NorA efflux pump is one of the most studied efflux systems in Staphylococcus aureus and confers multidrug resistance to a variety of dyes and antimicrobial compounds. Hence, inhibition of the NorA efflux pump might be a viable option for restoring susceptibility to antibiotics like fluoroquinolones. Fluorescent real-time efflux assays are important tools to identify putative efflux pump inhibitors. Nevertheless, the number of available compounds for usage in Staphylococcus aureus is limited. Previously, a 3-dipropyloxacarbocyanine iodide (DiOC3) efflux assay was published that circumvented problems associated with the usage of ethidium bromide, namely slow efflux and suggested mutagenicity. However, the DiOC3 assay protocol was cuvette - based and therefore needs to be adapted to the 96-well plate format. Hence, we optimized this assay for usage with 96-well plates. The new assay allows for rapid high-throughput efflux pump inhibitor screening.

Absence of Hemolivia mauritanica (Apicomplexa: Haemogregarinidae) in natural populations of Testudo graeca in Morocco.

During spring 2018, we captured 101 spur-thighed tortoises, Testudo graeca, from seven localities in central Morocco. All tortoises were examined for the presence of blood parasites Hemolivia mauritanica and Hyalomma aegyptium ticks, the known vectors. We looked for H. mauritanica infection by examination of blood smears and by genetic analysis with PCR using extractions from both tortoises and ticks. On all tortoises collected, 71.29% were infested with at least one tick, with a mean infestation intensity of 7.12 ticks/tortoise and maximum of 15.55 ticks/tortoises in Had Draa locality (Essaouira region). Although the definitive host is present and abundant in all tortoise populations, all blood samples were Hemolivia-negative. Our results support and confirm the finding of studies previously conducted in other populations of Morocco and indicate that H. mauritanica has a narrower distribution range than its tick vector.

Seaweed Essential Oils as a New Source of Bioactive Compounds for Cyanobacteria Growth Control: Innovative Ecological Biocontrol Approach.

The application of natural compounds extracted from seaweeds is a promising eco-friendly alternative solution for harmful algae control in aquatic ecosystems. In the present study, the anti-cyanobacterial activity of three Moroccan marine macroalgae essential oils (EOs) was tested and evaluated on unicellular Microcystis aeruginosa cyanobacterium. Additionally, the possible anti-cyanobacterial response mechanisms were investigated by analyzing the antioxidant enzyme activities of M. aeruginosa cells. The results of EOs GC-MS analyses revealed a complex chemical composition, allowing the identification of 91 constituents. Palmitic acid, palmitoleic acid, and eicosapentaenoic acid were the most predominant compounds in Cystoseira tamariscifolia, Sargassum muticum, and Ulva lactuca EOs, respectively. The highest anti-cyanobacterial activity was recorded for Cystoseira tamariscifolia EO (ZI = 46.33 mm, MIC = 7.81 µg mL-1, and MBC = 15.62 µg mL-1). The growth, chlorophyll-a and protein content of the tested cyanobacteria were significantly reduced by C. tamariscifolia EO at both used concentrations (inhibition rate >67% during the 6 days test period in liquid media). Furthermore, oxidative stress caused by C. tamariscifolia EO on cyanobacterium cells showed an increase of the activities of superoxide dismutase (SOD) and catalase (CAT), and malondialdehyde (MDA) concentration was significantly elevated after 2 days of exposure. Overall, these experimental findings can open a promising new natural pathway based on the use of seaweed essential oils to the fight against potent toxic harmful cyanobacterial blooms (HCBs).

Host-parasite association of Placobdella costata (Glossiphoniidae: Hirudinea) and Mauremys leprosa (Geoemydidae: Testudinoidea) in aquatic ecosystems of Morocco.

Emys orbicularis (Linnaeus, 1758) was considered as a specific host of Placobdella costata (Fr. Mûller, 1846). However, since the parasite was recorded from outside the distribution area of its host, some authors suggested a possible relationship with other hosts. Although two accidental associations were found with another turtle, Mauremys leprosa (Schweigger, 1812), the obtained data remain insufficient to better understand this discovered host-parasite ecological system. In this context, the present study was carried out to evaluate the relationship between the Mediterranean pond turtle, M. leprosa, and the freshwater rhynchobdellid leech, P. costata (Hirudinida: Glossiphoniidae), in aquatic ecosystems of Morocco. During the period from April to June 2018, we found leeches attached to turtles in five out of 30 populations sampled with a prevalence of infection significantly higher in adult than that in juvenile turtles. Moreover, the males are the most infested with 51% of the total, followed by females (33.3%) and juveniles (15.7%). The obtained results indicated that 51 turtles were infested by 139 leeches with a mean intensity of infestation of 4.17 ± 0.47 leeches/turtle (up to 10 leeches/turtle). It was higher in males than that in females in almost all sites. The posterior limbs are the most preferred attachment site, and the body condition of turtles was not affected by the intensity of infestation but it is rather a function of altitude. Our findings proved that M. leprosa-P. costata association is more than accidental and that M. leprosa is rather the main host of P. costata in aquatic ecosystems of Morocco.

Chemical Characterization and Biological Activities of Essential Oil Obtained from Mint Timija Cultivated under Mineral and Biological Fertilizers.

Cultivation of mint timija (Mentha suaveolens subsp. timija (Briq.) Harley) constitutes a promising solution to the conservation and sustainable utilization of this Moroccan endemic and threatened species. Optimized agronomic practices require mineral and/or biological fertilizer applications. The aim of this study was to determine the effects of application of a complete (N, P, and K) mineral fertilizer and vesicular arbuscular mycorrhizae (VAM) inoculation on the composition, antioxidant, and insecticidal properties of mint timija essential oils (EOs). The GC-MS analyses identified 27 components representing more than 99.9% of the total oils. Menthone (40.7-49.3%), pulegone (31.3-36.5%), and isomenthone (2.5-4.4%) were found to be the main constituents. Cultivation of mint timija with mineral fertilizer and VAM inoculation induced an increase in menthone content and a parallel decrease of pulegone. Both treatments enhanced the antioxidant activity of the investigated EOs in all assays (IC50 ranged from 2.34 ± 0.03 mg/mL to 6.82 ± 0.25 mg/mL), while no significant difference in the toxicities of these oils against Tribolium confusum du Val. has been observed. Overall, we conclude that cultivation using complete mineral fertilizer and VAM inoculation could be useful in modulating the chemical composition and enhancing the antioxidant activity of the EO of this endemic Moroccan species.

Chemical characterization and insecticidal properties of essential oils from different wild populations of Mentha suaveolens subsp. timija (Briq.) Harley from Morocco.

The present study is the first investigation of the volatile-oil variability and insecticidal properties of the endemic Moroccan mint Mentha suaveolens subsp. timija (mint timija). The yield of essential oils (EOs) obtained from different wild mint timija populations ranged from 0.20±0.02 to 1.17±0.25% (v/w). GC/MS Analysis revealed the presence of 44 oil constituents, comprising 97.3-99.9% of the total oil compositions. The main constituents were found to be menthone (1.2-62.6%), pulegone (0.8-26.6%), cis-piperitone epoxide (2.9-25.5%), piperitone (0.3-35.5%), trans-piperitone epoxide (8.1-15.7%), piperitenone (0.2-9.6%), piperitenone oxide (0.5-28.6%), (E)-caryophyllene (1.5-11.0%), germacrene D (1.0-15.7%), isomenthone (0.3-7.7%), and borneol (0.2-7.3%). Hierarchical-cluster analysis allowed the classification of the EOs of the different mint timija populations into four main groups according to the contents of their major components. This variability within the species showed to be linked to the altitude variation of the mint timija growing sites. The results of the insecticidal tests showed that all samples exhibited interesting activity against adults of Tribolium castaneum, but with different degrees. The highest toxicity was observed for the EOs belonging to Group IV, which were rich in menthone and pulegone, with LC50 and LC90 values of 19.0-23.4 and 54.9-58.0 µl/l air in the fumigation assay and LC50 and LC90 values of 0.17-0.18 and 0.40-0.52 µl/cm(2) in the contact assay.

Comparative evaluation of antioxidant and insecticidal properties of essential oils from five Moroccan aromatic herbs.

This study describes the antioxidant and insecticidal activities of essential oils (EOs) of Mentha suaveolens subsp. timija, Thymus satureioides, Achillea ageratum, Cotula cinerea and Salvia officinalis widely used in Morocco as flavorings, food additives and preservatives. Sixty seven components were identified accounting for more than 95.0 % of the total oils. M. suaveolens subsp. timija oil had as main components menthone and pulegone. A. ageratum oil was particularly rich in artemisyl acetate and yomogi alcohol. The essential oil of T. satureioides was characterized by high contents of carvacrol and borneol. C. cinerea oil contained trans-thujone and cis-verbenyl acetate as major constituents, whereas S. officinalis oil was characterized by trans-thujone and camphor. Antioxidant activities were examined by means of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), reducing power, ß-carotene/linoleic acid bleaching and ABTS radical tests. In all assays, the highest antioxidant potency was observed in T. satureioides EO with IC50 values ranging from 0.15 ± 0.36 µg mL(-1) to 0.23 ± 0.67 µg mL(-1) across the four assays. The in vitro evaluation of the insecticidal activity showed that M. suaveolens subsp. timija EO present the highest insecticidal efficiency against adults of Tribolium castaneum with LD50 and LD90 values of 0.17 µL cm(-2) and 0.26 µL cm(-2), respectively and LT50, LT90 values ranged from 44.19 h to 2.98 h and 98.14 h to 6.02 h, respectively. Our data support the possible use of T. satureioides oil as potential antioxidant agent, while M. suaveolens subsp. timija oil can be developed as a new natural bio-insecticide.

Insecticidal properties and chemical composition of essential oils of some aromatic herbs from Morocco.

The essential oils (EOs) of Artemisia herba alba, Rutachalepensis and Satureja calamintha aerial parts were analysed by GC/MS and the EOs were tested for their toxicity against two flour beetles, Tribolium castaneum and Tribolium confusum. EO composition showed that the major compounds were pulegone, menthone and menthol for S. calamintha, camphor and ß-thujone for A. herba alba and 2-undecanone for R. chalepensis. Contact bioassay showed that all EOs were toxic to adults of T. castaneum and T. confusum. Higher toxicity was found with the EOs from S. calamintha and R. chalepensis (LD50 of 0.09-0.13 µL/cm(2) and LD90 of 0.17-0.29 µL/cm(2)). In fumigant toxicity test, A. herba alba and S. calamintha EOs produced insecticidal activity with S. calamintha EO being more toxic with LD50 and LD90 values of 10.5 and 19.1 µL/L air, respectively, for T. confusum against 7.8 and 17.4 µL/L air, respectively, for T. castaneum.

DNA barcoding reveals limited accuracy of identifications based on folk taxonomy.

BACKGROUND: The trade of plant roots as traditional medicine is an important source of income for many people around the world. Destructive harvesting practices threaten the existence of some plant species. Harvesters of medicinal roots identify the collected species according to their own folk taxonomies, but once the dried or powdered roots enter the chain of commercialization, accurate identification becomes more challenging. METHODOLOGY: A survey of morphological diversity among four root products traded in the medina of Marrakech was conducted. Fifty-one root samples were selected for molecular identification using DNA barcoding using three markers, trnH-psbA, rpoC1, and ITS. Sequences were searched using BLAST against a tailored reference database of Moroccan medicinal plants and their closest relatives submitted to NCBI GenBank. PRINCIPAL FINDINGS: Combining psbA-trnH, rpoC1, and ITS allowed the majority of the market samples to be identified to species level. Few of the species level barcoding identifications matched the scientific names given in the literature, including the most authoritative and widely cited pharmacopeia. CONCLUSIONS/SIGNIFICANCE: The four root complexes selected from the medicinal plant products traded in Marrakech all comprise more than one species, but not those previously asserted. The findings have major implications for the monitoring of trade in endangered plant species as morphology-based species identifications alone may not be accurate. As a result, trade in certain species may be overestimated, whereas the commercialization of other species may not be recorded at all.

Botanical identification of medicinal roots collected and traded in Morocco and comparison to the existing literature.

BACKGROUND: A literature review revealed heavy reliance on a few key publications for identification of medicinal plant species from local or vernacular names and a lack of citation of voucher specimens in many publications. There is a need for more reliable and standardized data on the identity of species used for medicine, especially because local names vary from region to region. This is especially true in the case of medicinal roots, for which identification of species is difficult. This paper contributes to existing data on the species sold as medicinal roots (and other underground plant parts such as bulbs, corms, rhizomes and tubers) in Morocco. METHODS: Data were collected in collaboration with herbalists in Marrakech and collectors in rural regions near Marrakech where species are collected from the wild. The ethno-medicinal uses of these species were also recorded. RESULTS: We identified the vernacular names for 67 medicinal roots (by free listing) used to treat a variety of human diseases. We were able to collect and identify one or more species for 39 of the recorded vernacular names. The ones we were not able to identify were either imported or no longer available in the markets. We collected more than one species for some of the vernacular names for a total of 43 species. We identified six new vernacular names and four species which had not been previously described in the literature. Our botanical identification matched at least one of the names listed in the literature 63% of the time and did not match any species listed in the literature 37% of the time. Of the three most commonly cited pieces of literature we compared to, we found the greatest overlap with the broader, more comprehensive work of Bellakhdar 1997 (as opposed to Benchâabane and Abbad 1997 which worked in a similarly focused geographical area). However there was only 63% agreement between Bellakhdar 1997 and our botanical identifications, and 29% of the time our identification didn't match even the genus of any of the species listed in any of the 3 most commonly cited pieces of literature. CONCLUSIONS: More rigorous methodology and reporting are needed for medicinal plant research in Morocco. This will ensure that studies are comparable, help to protect traditional medicine users from negative health effects, and, support efforts to conserve overharvested wild medicinal plants.

Cytotoxic withanolides from the leaves of Moroccan Withania frutescens.

CONTEXT: Withania species are a rich source of interesting phytochemical substances (withanolides) which have shown several biological properties. OBJECTIVE: To investigate the cytotoxic potential of Withania frutescens (L.) Pauquy (Solanaceae) leaf extracts and isolated active compounds against cultured tumor cell lines. MATERIALS AND METHODS: The crude methanol extract of W. frutescens leaves was partitioned with dichloromethane, ethyl acetate and n-butanol. MeOH extract and its fractions were tested for their cytotoxic activity against cancer cell lines (HepG2 and HT29) using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Bioassay-guided fractionation was performed for the active CH2Cl2 fraction employing column chromatography and preparative high-performance liquid chromatography. Structural elucidation of the isolated active compounds was carried out mainly by 1D and 2D NMR and mass spectrometry. The compounds were then tested for their cytotoxic activity. RESULTS: The CH2Cl2 fraction was the most active against HT29 cell line. The fractionation procedure resulted in the isolation of 4ß,17α,27-trihydroxy-1-oxo-22-R-witha-2,5,24-trienolide (1), 5ß,6ß-epoxy-4ß,17α,27-trihydroxy-1-oxowitha-2,24-dienolide (2) and 2,3-dihydroxywithaferin A-3ß-O-sulfate (3). The latter exhibited the strongest cytotoxic activity against HT29 cancer cell lines (IC50 of 1.78 ± 0.09 µM) which was comparable to that of 5-fluorouracil (5-FU) used as the positive antimitotic control. DISCUSSION AND CONCLUSION: Compounds 2 and 3 were isolated from W. frutescens for the first time. Data obtained suggest that the sulfated steroidal lactone (3) can be considered as a compound with potential application in the new anticancer drugs development field.

Intraspecific chemical variability of essential oil from leaves of Cupressus atlantica Gaussen, an endemic and endangered coniferous species in Morocco.

The composition of essential oils isolated from leaves of 11 natural populations of Cupressus atlantica, an endemic and endangered coniferous species from Morocco, was investigated by GC-MS. In total, 42 essential oil components were identified, accounting for 73.1-97.7% of the total oil. Monoterpene (25.2-84.9%) and sesquiterpene hydrocarbons (12.2-46.8%) were the principal subclasses of compounds, with α-pinene (15-65.4%), germacrene D (5.9-30.5%), δ-3-carene (2-16.6%) and γ-cadinene (1.3-9.8%) as the main constituents. The results of the oil composition were analysed by hierarchical cluster and principal component analysis that established three main groups of essential oils. These oils were differentiated by the content of the major constituents (α-pinene, germacrene D, δ-3-carene and γ-cadinene), geographical location and climatic characteristics.

Molecular identification of commercialized medicinal plants in southern Morocco.

BACKGROUND: Medicinal plant trade is important for local livelihoods. However, many medicinal plants are difficult to identify when they are sold as roots, powders or bark. DNA barcoding involves using a short, agreed-upon region of a genome as a unique identifier for species- ideally, as a global standard. RESEARCH QUESTION: What is the functionality, efficacy and accuracy of the use of barcoding for identifying root material, using medicinal plant roots sold by herbalists in Marrakech, Morocco, as a test dataset. METHODOLOGY: In total, 111 root samples were sequenced for four proposed barcode regions rpoC1, psbA-trnH, matK and ITS. Sequences were searched against a tailored reference database of Moroccan medicinal plants and their closest relatives using BLAST and Blastclust, and through inference of RAxML phylograms of the aligned market and reference samples. PRINCIPAL FINDINGS: Sequencing success was high for rpoC1, psbA-trnH, and ITS, but low for matK. Searches using rpoC1 alone resulted in a number of ambiguous identifications, indicating insufficient DNA variation for accurate species-level identification. Combining rpoC1, psbA-trnH and ITS allowed the majority of the market samples to be identified to genus level. For a minority of the market samples, the barcoding identification differed significantly from previous hypotheses based on the vernacular names. CONCLUSIONS/SIGNIFICANCE: Endemic plant species are commercialized in Marrakech. Adulteration is common and this may indicate that the products are becoming locally endangered. Nevertheless the majority of the traded roots belong to species that are common and not known to be endangered. A significant conclusion from our results is that unknown samples are more difficult to identify than earlier suggested, especially if the reference sequences were obtained from different populations. A global barcoding database should therefore contain sequences from different populations of the same species to assure the reference sequences characterize the species throughout its distributional range.