Phytochemical Profile, GC-MS Profiling and In Vitro Evaluation of Some Biological Applications of the Extracts of Origanum syriacum L. and Cousinia libanotica D.C.

Indigenous to Lebanon, Origanum syriacum L. and Cousinia libanotica D.C. are notable plants in the Middle East, with O. syriacum known for its aromatic qualities and C. libanotica being less explored. Both plants have a significant role in traditional medicine for treating various ailments. This study aimed to evaluate the phytochemical composition and biological properties of the extracts from these plants. The extracts were obtained through cold maceration with solvents of increasing polarity. The ethyl acetate extract of O. syriacum exhibited the highest total polyphenol content. High-performance liquid chromatography (HPLC) identified fifteen compounds in both C. libanotica and O. syriacum extracts, whereas gas chromatography-mass spectrometry (GC-MS) analysis unveiled 179 volatile compounds. Notably, the O. syriacum-MeOH extract showed moderate antioxidant activity. Both plants' methanolic extracts demonstrated significant anti-Alzheimer's potential. The O. syriacum-dichloromethane and C. libanotica-cyclohexane extracts displayed the highest cytotoxicities against the HCT-116 cell line. For anti-proliferative activity against the Caco-2 cell line, the O. syriacum-methanol and C. libanotica-cyclohexane extracts were the most effective. This study provides valuable insights into the phytochemistry and potential therapeutic applications of extracts from these two oriental plant species.

Phenolic content, antioxidant and antimicrobial activities evaluation and relationship of commercial spices in the lebanese market.

Lebanese cuisine is renowned for its distinctive flavours and vibrant aromas. In Lebanese cuisine, spices are not just used for their flavour; they are also valued for their medicinal properties. This study consists of evaluating and comparing the total phenolic content and the antioxidant capacity of 21 samples of spices used in the Lebanese daily diet, such as cinnamon, allspice, coriander, cloves, etc. and the mixtures prepared in well-defined proportions. Several solvents were tested for the extraction of the phenolic compounds from spices, and the water and ethanol (v/v) mixture were retained for this study. Results showed that clove presented the highest polyphenol content (173.7 ± 2.98 mg Gallic Acid Equivalent (GAE)/g Dry Matter (DM)) and the highest antioxidant capacity by ABTS test (4875.68 ± 480.40 µmol trolox / g DM). and DPPH test (85.84 ± 0.5%). The examination of the results showed a positive significant correlation between the polyphenol contents and the antioxidant activity of the spices. The antimicrobial activity tested by the broth microdilution method was determined against Escherichia coli, Listeria monocytogenes, Staphylococcus aureus and Salmonella Enteritidis. The results showed high antimicrobial activity manifested by low value of minimum inhibitory concentration (MIC) (MIC < 2.4 µg/mL) for cinnamon, turmeric, white pepper, red pepper, allspice, clove and nutmeg. In conclusion, spices used in Lebanese cuisine, such as clove, cinnamon, allspice and spices, were rich in phenolic compounds and presented important potential health benefits.

Comparative Study of Phytochemistry, Antioxidant and Biological Activities of Berberis libanotica Fruit and Leaf Extracts.

Berberis libanotica Ehrenb. ex C.K. Schneider of the Berberidaceae family is an endemic Lebanese plant and is widely used in folk medicine. This study highlights the phytochemical composition and biological activities (in vitro) of fruit and leaf extracts. The two organs were extracted by cold maceration with four solvents of increasing polarity: cyclohexane, dichloromethane, ethyl acetate and methanol. The extracts were screened for their chemical composition by HPLC-DAD to identify and quantify the phenolic compounds. Volatile compounds were detected by GC-MS. The antioxidant capacity through DPPH inhibition was tested. The anti-acetylcholinesterase, antibacterial and anti-proliferative activities were evaluated. Thirteen compounds, including 12 phenolics, were detected in the fruits, whereas 8 phenolic compounds were identified in the leaves. A total of 137 volatile compounds were identified in both organs. At 50 µg/mL, the methanolic leaf extract presented the highest antioxidant capacity, with an inhibition percentage of 54.9%. The dichloromethane fruit extract reduced the acetylcholinesterase activity by 65.3%. The cyclohexane leaf extract reduced the proliferation of the HCT-116 cells by 54.8%, while the dichloromethane fruit extract exhibited the best inhibition against the Caco-2 cells (54%). Interestingly, the minimum inhibitory concentration (MIC) value of the cyclohexane fruit extract against Salmonella enterica serovar Kentucky was 2.4 µg/mL, and the MIC value of the cyclohexane leaf extract against E. coli was 9.7 µg/mL.

Chemical Characterization and Antioxidant, Antibacterial, Antiacetylcholinesterase and Antiproliferation Properties of Salvia fruticosa Miller Extracts.

The Salvia fruticosa (Mill.) is the most medicinal plant used in Lebanon. The aim of this study is to investigate the phytochemical composition and the biological activities (in vitro) of its extracts. The plant was extracted by cold maceration with four solvents presenting an increasing polarity: cyclohexane (CHX), dichloromethane (DCM), ethyl acetate (EtOAc) and methanol (MeOH). The extracts were screened for their chemical composition by a HPLC-DAD detector for phenolic compounds identification and quantification and by GC-MS for volatile compounds detection. The antioxidant capacity (DPPH inhibition) was tested. Biological activities, mainly anti-Alzheimer activity (acetylcholinesterase inhibition), the antiproliferation of two human colon cancer cell lines (HCT-116 and Caco-2 cells) and antibacterial activity, were evaluated. Ten aromatic compounds were quantified by HPLC-DAD analysis. A total of 123 compounds were detected by GC-MS analysis. The MeOH extract showed a very interesting antioxidant activity with an inhibition percentage (IP) of 76.1% and an IC50 of 19.4 µg/mL. The EtOAc extract exhibited the strongest inhibition against the acetylcholinesterase activity (IP = 60.6%) at 50 µg/mL. It also strongly inhibited the proliferation of the HCT-116 cells (IP = 87.5%), whereas the DCM extract gave the best result with the Caco-2 cells (IP = 72.3%). The best antibacterial activity was obtained with the MeOH extract against Staphylococcus aureus (MIC = 1.2 µg/mL) and with the EtOAc extract against Escherichia coli (MIC = 2.4 µg/mL). This study highlights the chemical composition and therapeutic potential of S. fruticosa. It is important to mention that the following chemical compounds were identified for the first time in plant extracts: 2,6,11,15-tetramethyl-hexadeca-2,6,8,10,14-pentaene; 4,5,6,7-tetrahydroxy-1,8,8,9-tetramethyl-8,9-dihydrophenaleno [1,2-b]furan-3-one; podocarpa-1,8,11,13-tetraen-3-one,14-isopropyl-1,13-dimethoxy; podocarpa-8,11,13-trien-3-one,12-hydroxy-13-isopropyl-,acetate; 3',8,8'-trimethoxy-3-piperidin-1-yl-2,2'-binaphthyl-1,1',4,4'-tetrone; and 2,3-dehydroferruginol, thus underlining the originality of this study.

Effect of geographical origin on yield and composition of cone essential oils of Cedrus libani A. Rich. growing in Lebanese protected areas and variability assessment in comparison with literature survey.

Gas chromatography-mass spectrometry analysis together with principal component analysis revealed that geographical origin influenced the yield and composition of the essential oils (EOs) extracted by hydrodistillation performed for 3 h using a Clevenger-type apparatus, from the cones of Cedrus libani A. Rich., growing wild at four Lebanese natural reserves and protected areas: Bsharri, Chouf, Ehden, and Tannourine, and from a cultivated cedar growing in Qartaba. Essential oil chemical variability established between the different studied provenances suggested the involvement of abiotic factors such as geographical conditions, cultivation conditions, soil composition, and environmental factors in the chemical polymorphism of C. libani cones EOs. α-Pinene/ß-pinene characterized Ehden (ß-pinene 35.6%/α-pinene 27.7%), Chouf (α-pinene 37.3%/ß-pinene 26.1%), Bsharri (α-pinene 27.7%/ß-pinene 21.4%), and Tannourine (α-pinene 25.1%/ß-pinene 16.0%) samples, whereas Qartaba EO was distinguished by the dominance of myrcene (30.6%), α-pinene(26%), and limonene (14.1%). Comparison with the existing literature reinforced the chemical variability of C. libani EOs. This current study helped the estimation of a best harvest location for a good EO quality production, resource optimization, and pharmacological properties evaluation, according to the market demand.

Evolution of Polyphenols during Syrah Grapes Maceration: Time versus Temperature Effect.

The effect of maceration time and temperature on the phenolic compounds of Syrah grape musts was studied. Pre-fermentation cold (10 °C) and heat maceration (60, 70 and 80 °C) were applied and compared to traditional maceration (control, 25 °C). The macerations were monitored and the kinetic profile of the maceration was studied by taking samples at 0, 2, 4, 8, 24 and 48 h. The results showed that heat treatment had the most significant effect on the extraction of total polyphenol. A significant loss of anthocyanin content was observed when the maceration was extended beyond eight hours at high temperatures, while longer maceration times seemed to favor the extraction of tannins. A principal component analysis showed that independently of the vinification technique, and for the same grape varieties, different winegrowing regions and harvest years affected the phenolic composition of the grape skin.

Identification of multiple-derived peptides produced by Saccharomyces cerevisiae involved in malolactic fermentation inhibition.

An oenological strain of Saccharomyces cerevisiae was previously shown to produce a 5-10 kDa peptidic fraction responsible for the inhibition of malolactic fermentation (MLF). In the present study, we aim to further purify the anti-MLF peptides of this fraction. The yeast fermented synthetic grape juice medium was fractionated by ammonium sulfate precipitation combined with ultrafiltration. The 5-10 kDa fraction recovered at a saturation degree of 60%-80% was the only fraction that inhibited both the bacterial growth and the malate consumption in vivo. It also inhibited the malolactic enzyme activity in vitro at a pH range between 3.5 and 6.7. Therefore, it was purified by both anion and cation exchange chromatography. The eluates that inhibited the malolactic enzyme activity in vitro were migrated on Tricine SDS-PAGE and the protein bands were excised and sequenced by LC-MS/MS. The sequencing revealed nine peptides originating from eight proteins of S. cerevisiae. Two GAPDH cationic fragments of 0.9 and 1.373 kDa having a pI of 10.5 and 11 respectively, Wtm2p and Utr2p anionic fragments of 2.42 kDa with a pI of 3.5 and 4 respectively were thought to contribute the most to the MLF inhibition.

Impact of inhibitory peptides released by Saccharomyces cerevisiae BDX on the malolactic fermentation performed by Oenococcus oeni Vitilactic F.

A previous study has shown that the malolactic fermentation (MLF) was inhibited during sequential fermentations performed with the pair Saccharomyces cerevisiae BDX/Oenococcus oeni Vitilactic F in synthetic grape juices. A yeast peptidic fraction with an apparent MW of 5-10kDa was involved in the inhibition. In the present study, the MLF was also inhibited in Cabernet Sauvignon and Syrah wines. The inhibition due to the peptidic fraction was maintained despite high phenolic contents. Kinetic studies showed that the peptidic fraction was gradually released during the alcoholic fermentation (AF). Its highest anti-MLF effect was reached when isolated from late stages of the AF stationary phase. The peptidic fraction was tested in vitro on cell-free bacterial cytosolic extracts containing the malolactic enzyme in a pH range between 3.5 and 6.7. Results showed that it was able to directly inhibit the malolactic enzyme activity with an increasing inhibitory kinetic correlated to the AF time at which it was collected.

Chemical composition, antibacterial and antioxidant activities of six essentials oils from the Alliaceae family.

Six essential oils (EOs) from the Alliaceae family, namely garlic (Allium sativum), onion (Allium cepa), leek (Allium porrum), Chinese chive (Allium tuberosum), shallot (Allium ascalonicum) and chive (Allium schoenoprasum) were characterized by GC and GC-MS and evaluated for their functional food properties. Antibacterial properties were tested on five food-borne pathogens: Two Gram-positive Staphylococcus aureus (ATCC 25923), Listeria monocytogenes (ATCC 19115) and three Gram-negative Salmonella Typhimurium (ATCC 14028), Escherichia coli (ATCC 8739) and Campylobacter jejuni (ATCC 33291) bacteria. Antioxidant and radical-scavenging properties were tested by means of Folin-Ciocalteu and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays. Garlic, Chinese chive and onion EOs had the highest antibacterial activity whereas shallot and leek EOs were the strongest antioxidants. Heating caused a decrease in the antioxidant activity of these Eos, as shown in the Total Polar Materials (TPM) test. Suggestions on relationships between chemical composition and biological activities are presented. Results show that the EOs could be of value in the food industry as alternatives to synthetic antioxidants.

Screening of congenital heart disease patients using multiplex ligation-dependent probe amplification: early diagnosis of syndromic patients.

Recurrent copy number variants (CNVs) are found in a significant proportion of patients with congenital heart disease (CHD) and some of these CNVs are associated with other developmental defects. In some syndromic patients, CHD may be the first presenting symptom, thus screening of patients with CHD for CNVs in specific genomic regions may lead to early diagnosis and awareness of extracardiac symptoms. We designed a multiplex ligation-dependent probe amplification (MLPA) assay specifically for screening of CHD patients. The MLPA assay allows for simultaneous analysis of CNVs in 25 genomic regions previously associated with CHD. We screened blood samples from 402 CHD patients and identified 14 rare CNVs in 13 (3.2%) patients. Five CNVs were de novo and six where inherited from a healthy parent. The MLPA screen led to early syndrome diagnosis in two of these patients. We conclude that the MLPA assay detects clinically relevant CNVs and suggest that it could be used within pediatric cardiology as a first tier screen to detect clinically relevant CNVs and identify syndromic patients at an early stage.

Evidence for genetic heterogeneity in Carvajal syndrome.

Carvajal syndrome is a rare syndrome with woolly hair, palmoplantar keratosis and dilated cardiomyopathy. The inheritance of the mutation is autosomal recessive. As a causal gene, the desmoplakin gene (DSP) has so far been identified; it encodes an essential component of desmosomes, a cell-cell structure aimed at keeping cells attached to each other in tissues in which cells are often exposed to strong shear forces. Recently, familial cases of an autosomal dominant Carvajal syndrome were documented with a new feature: hypo/oligodontia. A mutation in the DSP gene was also evidenced in these latter cases. A patient was seen for cardiogenetic consultation at the University Hospital of Lyon with cardiac failure involving first degree atrioventricular block, complete left bundle branch block, non-compaction of the apex of the left ventricle and a dilated cardiomyopathy. A coronarography disclosed a complete thrombosis of the right coronary artery. At examination, he had also woolly hair, mild palmoplantar keratosis and missing teeth (essentially molars and premolars). His family history was uninformative. His DNA was screened for mutations in the DSP and plakoglobin genes but no mutation could be found. This case suggests that Carvajal syndrome with hypo/oligodontia is a heterogeneous condition in which genes other than DSP might be involved, although we cannot rule out a mutation in this gene consisting in a deletion of a single exon or a gene rearrangement.

1Novel MEFV transcripts in Familial Mediterranean fever patients and controls.

BACKGROUND: Familial Mediterranean fever is a recessive autoinflammatory disease frequently encountered in Armenians, Jews, Arabs and Turks. The MEFV gene is responsible for the disease. It encodes a protein called pyrin/marenostrin involved in the innate immune system. A large number of clinically diagnosed FMF patients carry only one MEFV mutation. This study aims at studying the MEFV gene splicing pattern in heterozygous FMF patients and healthy individuals, in an attempt to understand the mechanism underlying the disease in these patients. METHODS: RNA was extracted from peripheral blood leucocytes of 41 FMF patients and 34 healthy individuals. RT-PCR was then performed, and the amplified products were migrated on a polyacrylamide electrophoresis gel, characterized by gel extraction of the corresponding bands followed by sequencing. RESULTS: Five novel splicing events were observed in both patients and controls deleting either exons 3, 4 (del34), or exons 2, 3, 4 (del234), or exons 2, 3, 4, 5 (del2345) or exon7 (del7) or exons 7 and 8 (del78). CONCLUSIONS: The observation of such qualitative variability in the expression of the MEFV gene suggests a complex transcriptional regulation. However, the expression of these novel transcripts in both patients and controls is not in favour of a severe pathogenic effect.

Impact of the co-culture of Saccharomyces cerevisiae-Oenococcus oeni on malolactic fermentation and partial characterization of a yeast-derived inhibitory peptidic fraction.

The present study was aimed to evaluate the impact of the co-culture on the output of malolactic fermentation and to further investigate the reasons of the antagonism exerted by yeasts towards bacteria during sequential cultures. The Saccharomyces cerevisiae D strain/Oenococcus oeni X strain combination was tested by applying both sequential culture and co-culture strategies. This pair was chosen amongst others because the malolactic fermentation was particularly difficult to realize during the sequential culture. During this traditional procedure, malolactic fermentation started when alcoholic fermentation was achieved. For the co-culture, both fermentations were conducted together by inoculating yeasts and bacteria into a membrane bioreactor at the same time. Results obtained during the sequential culture and compared to a bacterial control medium, showed that the inhibition exerted by S. cerevisiae D strain in term of decrease of the malic acid consumption rate was mainly due to ethanol (75%) and to a peptidic fraction (25%) having an MW between 5 and 10 kDa. 0.4 g l(-1) of L-malic acid was consumed in this case while 3.7 g l(-1) was consumed when the co-culture was applied. In addition, there was no risk of increased volatile acidity during the co-culture. Therefore, the co-culture strategy was considered effective for malolactic fermentation with the yeast/bacteria pair studied.

Quantitative study of interactions between Saccharomyces cerevisiae and Oenococcus oeni strains.

This study examines the interactions that occur between Saccharomyces cerevisiae and Oenococcus oeni strains during the process of winemaking. Various yeast/bacteria pairs were studied by applying a sequential fermentation strategy which simulated the natural winemaking process. First, four yeast strains were tested in the presence of one bacterial strain leading to the inhibition of the bacterial component. The extent of inhibition varied widely from one pair to another and closely depended on the specific yeast strain chosen. Inhibition was correlated to weak bacterial growth rather than a reduction in the bacterial malolactic activity. Three of the four yeast strains were then grown with another bacteria strain. Contrary to the first results, this led to the bacterial stimulation, thus highlighting the importance of the bacteria strain. The biochemical profile of the four yeast fermented media exhibited slight variations in ethanol, SO(2) and fatty acids produced as well as assimilable consumed nitrogen. These parameters were not the only factors responsible for the malolactic fermentation inhibition observed with the first bacteria strain. The stimulation of the second has not been reported before in such conditions and remains unexplained.