Z-ajoene from Crushed Garlic Alleviates Cancer-Induced Skeletal Muscle Atrophy.

Skeletal muscle atrophy is one of the major symptoms of cancer cachexia. Garlic (Allium sativum), one of the world's most commonly used and versatile herbs, has been employed for the prevention and treatment of diverse diseases for centuries. In the present study, we found that ajoene, a sulfur compound found in crushed garlic, exhibits protective effects against muscle atrophy. Using CT26 tumor-bearing BALB/c mice, we demonstrate in vivo that ajoene extract alleviated muscle degradation by decreasing not only myokines secretion but also janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) and SMADs/forkhead box (FoxO) signaling pathways, thereby suppressing muscle-specific E3 ligases. In mouse skeletal myoblasts, Z-ajoene enhanced myogenesis as evidenced by increased expression of myogenic markers via p38 mitogen-activated protein kinase (MAPK) activation. In mature myotubes, Z-ajoene protected against muscle protein degradation induced by conditioned media from CT26 colon carcinoma cells, by suppressing expression of muscle specific E3 ligases and nuclear transcription factor kappa B (NF-κB) phosphorylation which contribute to muscle atrophy. Moreover, Z-ajoene treatment improved myofiber formation via stimulation of muscle protein synthesis. These findings suggest that ajoene extract and Z-ajoene can attenuate skeletal muscle atrophy induced by cancer cachexia through suppressing inflammatory responses and the muscle wasting as well as by promoting muscle protein synthesis.

Identification and Allelopathy of Green Garlic (Allium sativum L.) Volatiles on Scavenging of Cucumber (Cucumis sativus L.) Reactive Oxygen Species.

Garlic and formulations containing allicin are used widely as fungicides in modern agriculture. However, limited reports are available on the allelopathic mechanism of green garlic volatile organic compounds (VOCs) and its component allelochemicals. The aim of this study was to investigate VOCs of green garlic and their effect on scavenging of reactive oxygen species (ROS) in cucumber. In this study, green garlic VOCs were collected by HS-SPME, then analyzed by GS-MS. Their biological activity were verified by bioassays. The results showed that diallyl disulfide (DADS) is the main allelochemical of green garlic VOCs and the DADS content released from green garlic is approximately 0.08 mg/g. On this basis, the allelopathic effects of green garlic VOCs in vivo and 1 mmol/L DADS on scavenging of ROS in cucumber seedlings were further studied. Green garlic VOCs and DADS both reduce superoxide anion and increase the accumulation of hydrogen peroxide of cucumber seedlings. They can also regulate active antioxidant enzymes (SOD, CAT, POD), antioxidant substances (MDA, GSH and ASA) and genes (CscAPX, CsGPX, CsMDAR, CsSOD, CsCAT, CsPOD) responding to oxidative stress in cucumber seedlings.

Diallyl Disulfide Induces Apoptosis and Autophagy in Human Osteosarcoma MG-63 Cells through the PI3K/Akt/mTOR Pathway.

Diallyl disulfide (DADs), a natural organic compound, is extracted from garlic and scallion and has anti-tumor effects against various tumors. This study investigated the anti-tumor activity of DADs in human osteosarcoma cells and the mechanisms. MG-63 cells were exposed to DADs (0, 20, 40, 60, 80, and 100 µM) for different lengths of time (24, 48, and 72 h). The CCK8 assay results showed that DADs inhibited osteosarcoma cell viability in a dose-and time-dependent manner. FITC-Annexin V/propidium iodide staining and flow cytometry demonstrated that the apoptotic ratio increased and the cell cycle was arrested at the G2/M phase as the DADs concentration was increased. A Western blot analysis was employed to detect the levels of caspase-3, Bax, Bcl-2, LC3-II/LC3-I, and p62 as well as suppression of the mTOR pathway. High expression of LC3-II protein revealed that DADs induced formation of autophagosome. Furthermore, DADs-induced apoptosis was weakened after adding 3-methyladenine, demonstrating that the DADs treatment resulted in autophagy-mediated death of MG-63 cells. In addition, DADs depressed p-mTOR kinase activity, and the inhibited PI3K/Akt/mTOR pathway increased DADs-induced apoptosis and autophagy. In conclusion, our results reveal that DADs induced G2/M arrest, apoptosis, and autophagic death of human osteosarcoma cells by inhibiting the PI3K/Akt/mTOR signaling pathway.

Potentides: New Cysteine-Rich Peptides with Unusual Disulfide Connectivity from Potentilla anserina.

Cysteine-rich peptides (CRPs), which are disulfide-constrained peptides with 3 to 5 disulfide bonds and molecular weights of 2 to 6 kDa, are generally hyperstable and resistant to thermal, chemical, and enzymatic degradation. Herein, the discovery and characterization of a novel suite of CRPs, collectively named potentides pA1-pA16 from the root of the medicinal herb Potentilla anserina L, are described. Through a combination of proteomic and transcriptomic methods, it is shown that 35-residue potentide pA3, which is the most abundant member of potentides, exhibits high stability against heat, acidic, and proteolytic degradation. Transcriptomic analysis revealed that potentide precursor sequences contained four tandem repeats in the mature domain, which is the first report on tandem repeats being found in the Rosaceae family. Disulfide mapping showed that potentide pA3 displayed a novel disulfide connectivity of C1-C3, C2-C6, and C4-C5; a cystine motif that has not been reported in plant CRPs. Transcriptomic data mining and a neighbor-joining clustering analysis revealed 56 potentide homologues and their distribution in the families of Rosaceae and Ranunculaceae in angiosperm. Altogether, these results reveal a new plant CRP structure with an unusual cystine connectivity. Additionally, this study expands the families and structure diversity of CRPs as potentially active peptide pharmaceuticals.

Cyclic sulfur-containing compounds from Allium fistulosum 'Kujou'.

Sulfur-containing compounds, allicin and ajoene, etc., were isolated from Allium species. In a recent study, some sulfur-containing cyclic compounds were isolated from A. sativum, A. cepa, and A. fistulosum. Four new compounds with multiple rings with methyl disulfide or propyl disulfide at the side chain of the 7-position, kujounins A3 (1), B1 (2), B2 (3) and B3 (4), and two new thiolane type compounds with methoxy and methyl sulfoxide moiety at the 2- and 5-positions, and allium sulfoxides A2 (5) and A3 (6), were isolated from the acetone extract of the fresh white parts of Allium fistulosum 'Kujou' with three known compounds, kujounin A1 (7) and A2 (8), and allium sulfoxide A1 (9). The chemical structures of the new compounds were elucidated on the basis of physicochemical evidence. The kujounins had a rare molecular skeleton, which was tetrahydro-2H-difuro[3,2-b:2',3'-c]furan-5(5aH)-one.

Analogues of Disulfides from Allium stipitatum Demonstrate Potent Anti-tubercular Activities through Drug Efflux Pump and Biofilm Inhibition.

Disulfides from Allium stipitatum, commonly known as Persian shallot, were previously reported to possess antibacterial properties. Analogues of these compounds, produced by S-methylthiolation of appropriate thiols using S-methyl methanethiosulfonate, exhibited antimicrobial activity, with one compound inhibiting the growth of Mycobacterium tuberculosis at 17 µM (4 mg L-1) and other compounds inhibiting Escherichia coli and multi-drug-resistant (MDR) Staphylococcus aureus at concentrations ranging between 32-138 µM (8-32 mg L-1). These compounds also displayed moderate inhibitory effects on Klebsiella and Proteus species. Whole-cell phenotypic bioassays such as the spot-culture growth inhibition assay (SPOTi), drug efflux inhibition, biofilm inhibition and cytotoxicity assays were used to evaluate these compounds. Of particular note was their ability to inhibit mycobacterial drug efflux and biofilm formation, while maintaining a high selectivity towards M. tuberculosis H37Rv. These results suggest that methyl disulfides are novel scaffolds which could lead to the development of new drugs against tuberculosis (TB).

New cyclic sulfides extracted from Allium sativum: garlicnins P, J2, and Q.

Two atypical cyclic-type sulfides, garlicnin P (1) and garlicnin J2 (2), and one thiabicyclic-type sulfide, garlicnin Q (3), were isolated from the acetone extracts of garlic, Allium sativum, bulbs cultivated in the Kumamoto city area, and their structures characterized. Their production pathways are also discussed.

New cyclic sulfides, garlicnins I2, M, N, and O, from Allium sativum.

One atypical thiolane-type sulfide, garlicnin I2 (1), two 3,4-dimethylthiolane-type sulfides, garlicnins M (2) and N (3), and one thiabicyclic-type sulfide, garlicnin O (4), were isolated from the acetone extracts of Chinese garlic bulbs, Allium sativum and their structures were characterized. Hypothetical pathways for the production of the respective sulfides were discussed.

Garlic Organosulfur Compounds Reduce Inflammation and Oxidative Stress during Dengue Virus Infection.

Dengue virus (DENV) is a mosquito-borne flavivirus that causes significant global human disease and mortality. One approach to develop treatments for DENV infection and the prevention of severe disease is through investigation of natural medicines. Inflammation plays both beneficial and harmful roles during DENV infection. Studies have proposed that the oxidative stress response may be one mechanism responsible for triggering inflammation during DENV infection. Thus, blocking the oxidative stress response could reduce inflammation and the development of severe disease. Garlic has been shown to both reduce inflammation and affect the oxidative stress response. Here, we show that the garlic active compounds diallyl disulfide (DADS), diallyl sulfide (DAS) and alliin reduced inflammation during DENV infection and show that this reduction is due to the effects on the oxidative stress response. These results suggest that garlic could be used as an alternative treatment for DENV infection and for the prevention of severe disease development.

The redox-active drug metronidazole and thiol-depleting garlic compounds act synergistically in the protist parasite Spironucleus vortens.

Spironucleus vortens is a protozoan parasite associated with significant mortalities in the freshwater angelfish, Pterophyllum scalare. Control of this parasite is especially problematic due to restrictions on the use of the drug of choice, metronidazole (MTZ), on fish farms. Use of garlic (Allium sativum) is undergoing a renaissance following experimental validations of its antimicrobial efficiency. Ajoene ((E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide), is a stable transformation product of allicin, the primary biologically active component of garlic. In the current study, an ajoene oil crude extract had a minimum inhibitory concentration (MIC) of 40µg/ml against S. vortens. GC-MS and NMR spectroscopy revealed this ajoene extract contained a mixture of the (E) and (Z)-ajoene isomers along with diallyl disulphide (DADS) and diallyl trisulphide (DATS). The only component of the ajoene crude oil found to substantially inhibit S. vortens growth by optical density monitoring (Bioscreen C Reader) was (Z)-ajoene (MIC 16µg/ml). Ajoene oil acted in synergy with MTZ in vitro, reducing the individual MIC of this drug (4µg/ml) by 16-fold, and that of ajoene oil by 200-fold with a fractional inhibitory concentration (FIC) index of 0.263. This synergistic interaction was confirmed in vivo. S. vortens-infected Pterophyllum scalare angelfish dosed orally with 0.5% (v/w) MTZ combined with 0.05% (v/w) ajoene displayed a significant reduction in faecal trophozoite count, whilst those fed on 0.5% MTZ flakes (half the recommended oral dose) alone did not. This study demonstrates for the first time the synergistic interaction between the synthetic drug MTZ and natural ajoene oil both in vitro and in vivo. Future work should evaluate the potential synergy of ajoene and MTZ against MTZ-resistant bacteria and protists.

Composition of the Essential Oil of Allium neapolitanum Cirillo Growing Wild in Sicily and its Activity on Microorganisms Affecting Historical Art Crafts.

Essential oil of the aerial parts of Allium neapolitanum Cirillo collected in Sicily were analyzed by gas-chromatography-flame-ionization detection and gas-chromatography-mass spectrometry. Nineteen compounds were identified in the oil and the main components were found to be (E)-chrysanthenyl acetate (28.1%), (Z)-chrysanthenyl acetate (23.8%), (E)-ß-farnesene (9.6%), dimethyl trisulfide (9.6%), camphor (7.4%), methyl allyl disulfide (6.8%) and 1-methyl-3-allyl trisulfide (5.8%). The essential oil showed good antimicrobial activity against 11 strains of test microorganisms, including several species infesting historical material.

Chemical Composition and Aroma Evaluation of Essential Oils from Skunk Cabbage (Symplocarpus foetidus).

Two sample preparation methods, namely hydrodistillation (HD) and solvent-assisted flavor evaporation (SAFE), have been used to investigate the essential oils of the aerial parts (leaves and stems) of Symplocarpus foetidus, a plant with a characteristic odor, by gas chromatography mass spectrometry (GC-MS). Characteristic aroma-active compounds in the oils were detected by GC-Olfactometry (GC-O) and aroma extract dilution analysis (AEDA). From the HD method, the main compounds in the oil were found to be p-vinyl-guaiacol (15.5%), 2-pentyl-furan (13.4%), and (Z)-ligustilide (9.5%). From the SAFE method, the main compounds were 2-butoxy-ethanol (49.6%), ethyl-pentanoate (4.5%), and mesitylene (4.0%). In HD oil, the most intense aroma-active compounds were 2-pentyl-furan (flavor dilution factor (FD) = 32, odor activity value (OAV) = 57), p-vinyl-guaiacol (FD = 16, OAV = 41), and dimethyl disulfide (FD = 16, OAV = 41). In SAFE oil, the main aroma-active compounds were 2-butoxy ethanol (FD = 32, OAV = 16), and 2-methoxy thiazole (FD = 32, OAV = 25).

Applications of thin-layer chromatography in extraction and characterisation of ajoene from garlic bulbs.

Novel and inexpensive methods of thin-layer chromatography (TLC) were employed for the extraction, characterisation and mechanism of quorum sensing inhibition by ajoene, a component from toluene garlic bulb (Allium sativum L.) extract (TGE). TLC profiling of TGE was carried out using ethyl acetate as solvent. Out of total spots extracted from TLC, four spots exhibited quorum sensing inhibitory (QSI) potential. Among those, spot 5 was identified as Z-ajoene by TLC and confirmed by NMR and MS. HPLC analysis indicated 97.7% purity for purified ajoene. TLC densitometric analysis quantified 221.08 µmol/g of ajoene in TGE and indicated that ajoene is stable at 4°C and at acidic pH. HPTLC profiling showed that ajoene exhibits QSI effect by inhibiting the production of both long-chain acyl homoserine lactones and Pseudomonas quinolone signal (PQS) by P. aeruginosa and also by inactivating PQS molecules.

Investigating the responses of Cronobacter sakazakii to garlic-drived organosulfur compounds: a systematic study of pathogenic-bacterium injury by use of high-throughput whole-transcriptome sequencing and confocal micro-raman spectroscopy.

We present the results of a study using high-throughput whole-transcriptome sequencing (RNA-seq) and vibrational spectroscopy to characterize and fingerprint pathogenic-bacterium injury under conditions of unfavorable stress. Two garlic-derived organosulfur compounds were found to be highly effective antimicrobial compounds against Cronobacter sakazakii, a leading pathogen associated with invasive infection of infants and causing meningitis, necrotizing entercolitis, and bacteremia. RNA-seq shows changes in gene expression patterns and transcriptomic response, while confocal micro-Raman spectroscopy characterizes macromolecular changes in the bacterial cell resulting from this chemical stress. RNA-seq analyses showed that the bacterial response to ajoene differed from the response to diallyl sulfide. Specifically, ajoene caused downregulation of motility-related genes, while diallyl sulfide treatment caused an increased expression of cell wall synthesis genes. Confocal micro-Raman spectroscopy revealed that the two compounds appear to have the same phase I antimicrobial mechanism of binding to thiol-containing proteins/enzymes in bacterial cells generating a disulfide stretching band but different phase II antimicrobial mechanisms, showing alterations in the secondary structures of proteins in two different ways. Diallyl sulfide primarily altered the α-helix and ß-sheet, as reflected in changes in amide I, while ajoene altered the structures containing phenylalanine and tyrosine. Bayesian probability analysis validated the ability of principal component analysis to differentiate treated and control C. sakazakii cells. Scanning electron microscopy confirmed cell injury, showing significant morphological variations in cells following treatments by these two compounds. Findings from this study aid in the development of effective intervention strategies to reduce the risk of C. sakazakii contamination in the food production environment and on food contact surfaces, reducing the risks to susceptible consumers.

Enhancement of intestinal IgA production by Ajoene in mice.

We investigated the effects of ajoene on intestinal IgA production. Ajoene (1.35, 4.5, and 13.5 µg/kg/d) was administered to mice for 4 weeks. The fecal IgA level in the 13.5 µg/kg/d group increased after 3 weeks. The intestinal IgA level also increased in a dose-dependent manner upon ajoene administration. An oil-macerated garlic extract, with 1500 µg/g of ajoene, enhanced the intestinal IgA production.

Cyclic sulfoxides garlicnins B2, B3, B4, C2, and C3 from Allium sativum.

Several novel sulfides, called garlicnins B2 (1), B3 (2), B4 (3), C2 (4), and C3 (5), were isolated from acetone extracts of garlic, Allium sativum L. and characterized. These garlicnins are capable of suppressing M2 macrophage activation and they have a novel skeleton of cyclic sulfoxide. The structures of the former 3 and latter of 2 were deduced to be 2-(sulfenic acid)-5-(allyl)-3,4-dimethyltetrahydrothiophene-S-oxides and 2-(allyldithiine)-5-(propenylsulfoxide)-3,4-dimethyltetrahydrothiophene-S-oxides, respectively. The mechanism of the proposed production of these compounds is discussed. The identification of these novel sulfoxides from garlic accumulates a great deal of new chemistry in the Allium sulfide field, and future pharmacological investigations of these compounds will aid the development of natural, healthy foods and anti-cancer agents that may prevent or combat disease.

Effect of allyl sulfides from garlic essential oil on intracellular ca2+ levels in renal tubular cells.

Diallyl sulfide (1), diallyl disulfide (2), and diallyl trisulfide (3), which are major organosulfur compounds of garlic (Allium sativum), are recognized as a group of potential chemopreventive compounds. In this study, the early signaling effects of 3 were examined on Madin-Darby canine kidney (MDCK) cells loaded with the Ca(2+)-sensitive dye fura-2. It was found that 3 caused an immediate and sustained increase of [Ca(2+)](i) in a concentration-dependent manner (EC(50) = 40 µM). Compound 3 also induced a [Ca(2+)](i) elevation when extracellular Ca(2+) was removed, but the magnitude was reduced by 45%. In Ca(2+)-free medium, the 3-induced [Ca(2+)](i) level was abolished by depleting stored Ca(2+) with 1 µM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor). Elevation of [Ca(2+)](i) caused by 3 in the Ca(2+)-containing medium was not affected by modulation of protein kinase C activity. The 3-induced Ca(2+) influx was inhibited by nifedipine and nicardipine (1 µM). U73122, an inhibitor of phospholipase C, abolished ATP (but not the 3-induced [Ca(2+)](i) level). These findings suggest that 3 induced a significant [Ca(2+)](i) elevation in MDCK renal tubular cells by stimulating both extracellular Ca(2+) influx and thapsigargin-sensitive intracellular Ca(2+) release via as yet unidentified mechanisms. Furthermore, the order of the allyl sulfide-induced [Ca(2+)](i) elevation and cell viability was 1 < 2 < 3. The differential effect of allyl sulfides on Ca(2+) signaling and cell death appears to correlate with the number of sulfur atoms in the structure of these allyl sulfides.

Ajoene, a sulfur-rich molecule from garlic, inhibits genes controlled by quorum sensing.

In relation to emerging multiresistant bacteria, development of antimicrobials and new treatment strategies of infections should be expected to become a high-priority research area. Quorum sensing (QS), a communication system used by pathogenic bacteria like Pseudomonas aeruginosa to synchronize the expression of specific genes involved in pathogenicity, is a possible drug target. Previous in vitro and in vivo studies revealed a significant inhibition of P. aeruginosa QS by crude garlic extract. By bioassay-guided fractionation of garlic extracts, we determined the primary QS inhibitor present in garlic to be ajoene, a sulfur-containing compound with potential as an antipathogenic drug. By comprehensive in vitro and in vivo studies, the effect of synthetic ajoene toward P. aeruginosa was elucidated. DNA microarray studies of ajoene-treated P. aeruginosa cultures revealed a concentration-dependent attenuation of a few but central QS-controlled virulence factors, including rhamnolipid. Furthermore, ajoene treatment of in vitro biofilms demonstrated a clear synergistic, antimicrobial effect with tobramycin on biofilm killing and a cease in lytic necrosis of polymorphonuclear leukocytes. Furthermore, in a mouse model of pulmonary infection, a significant clearing of infecting P. aeruginosa was detected in ajoene-treated mice compared to a nontreated control group. This study adds to the list of examples demonstrating the potential of QS-interfering compounds in the treatment of bacterial infections.

Studies on purification of allicin by molecular distillation.

BACKGROUND: With recent improvements in living standards, people have been giving more consideration to the healthcare effects of foods. In this respect, allicin, which is the most important organosulfur compound in garlic and plays a key role in physiological function, has been receiving much attention. RESULTS: Allicin obtained from garlic by supercritical CO(2) extraction was purified by molecular distillation (MD). The effects of operating conditions such as absolute pressure (AP), distillation temperature (DT) and feed flow rate (FFR) on allicin purity and yield were studied. The optimal AP, DT and FFR levels in first-stage MD were found to be 200 Pa, 50 °C and 15 mL min(-1) respectively. After three stages of MD the contents of allicin, diallyl disulfide (DADS) and diallyl trisulfide (DATS) were 68.04, 9.19 and 5.91% (w/w) respectively. CONCLUSION: This study has provided a safe and effective method for the purification of allicin.

Investigation of volatiles emitted from freshly cut onions (Allium cepa L.) by real time proton-transfer reaction-mass spectrometry (PTR-MS).

Volatile organic compounds (VOCs) in cut onions (Allium cepa L.) were continuously measured by PTR-MS during the first 120 min after cutting. The headspace composition changed rapidly due to the very reactive volatile sulfurous compounds emitted from onion tissue after cell disruption. Mass spectral signals corresponding to propanethial S-oxide (the lachrymatory factor) and breakdown products of this compound dominated 0-10 min after cutting. Subsequently, propanethiol and dipropyl disulfide predominantly appeared, together with traces of thiosulfinates. The concentrations of these compounds reached a maximum at 60 min after cutting. Propanethiol was present in highest concentrations and had an odor activity value 20 times higher than dipropyl disulfide. Thus, propanethiol is suggested to be the main source of the characteristic onion odor. Monitoring the rapid changes of VOCs in the headspace of cut onion necessitates a high time resolution, and PTR-MS is demonstrated to be a very suitable method for monitoring the headspace of freshly cut onions directly after cutting without extraction or pre-concentration.