Optimization of extraction and quantification technique for phenolics content of garlic (Allium sativum): An application for comparative phytochemical evaluation based on cultivar origin.

A range of conventional, i.e. maceration, percolation, ultrasonic assisted, Soxhlet and Soxtec extraction (STE), to advanced extraction techniques of accelerated solvent extraction (ASE) was utilized for the first time in order to optimize the extract yield and recovery of phenolics-gallic acid (GA), rutin (RT) and quercetin (QT)-quantified via ultra-high performance liquid chromatography with diode array detector (UHPLC-DAD). The effect of solvents (n-hexane, dichloromethane and methanol) and temperature (60, 80 and 100°C) upon extraction yield, phenolic content and antioxidant activity (DPPH, ABTS and DPPH) was studied, and the method was validated in commercial food samples from Saudi Arabia, China and India. A high extract yield with percentage recovery was observed for STE (1221.10 mg/5 g; 24.42%) and ASE techniques (91.50 mg/1 g; 9.15%) in methanol at 100°C. UHPLC-DAD showed retention times (min) of 0.67, 1.93 and 1.90 for GA, RT and QT, respectively in the shortest runtime of 3 min. The yield for phenolics was higher for STE/ASE (ppm): 15.27/15.29 (GA), 85.24/37.56 (RT) and 52.20/33.40 (QT), respectively. In terms of antioxidant activities, low IC50 values (µg/ml) of 1.09/1.18 (DPPH), 2.11/5.32 (ABTS) and 4.35/7.88 (phenazine methosulfate-nicotinamide adenine dinucleotide) were observed for STE and ASE, respectively. Multivariate analysis for STE showed a significant (P = 0.000) correlation for extraction type vs. extract yield and phenolics content; however, there was no significance for antioxidant activities vs. extraction type. ASE showed a positive correlation for solvent vs. extraction yield, phenolics and antioxidant activity; however, there was no correlation for extraction yield and DPPH activity. Principal component analysis for STE showed a major variability (52.02%) for extraction yield and phenolics in PC1 followed by PC2 (38.30%) for antioxidant activities. For ASE, PC1 (48.68%) showed a positive correlation for solvent vs. extraction yield and phenolics while PC2 (33.12%) showed a positive correlation for temperature and antioxidant activities. STE and ASE were the optimized extraction techniques for the garlic food sample while a significant effect of solvent and temperature was observed upon extraction yield, phenolics and antioxidant activity.

Effect of pre-storage removal of different parts of the scape top on the biochemical changes of two garlic cultivars during controlled atmosphere storage.

BACKGROUND: This study highlights the effect of removing different plant parts on the biochemical changes in the garlic scape of cultivars G064 and G025 during controlled atmosphere storage (O2 = 2%-5%, CO2 = 3%-6%) at temperature = 0 ± 0.5 °C, Relative Humidity (RH) RH = 85%-95%, for 168 day. Total polyphenols (TP), total flavonoids (TF), the concentration of allicin, ethylene production and the antioxidant potential were evaluated and analyzed by using four different assays. Statistically significant differences were observed between different treatments. RESULTS: Total polyphenols, allicin concentration, and ethylene production were the highest in G064, while the total flavonoid concentration was the highest in G025. Removing the top resulted in the highest levels of total polyphenols, total flavonoids, allicin, and ethylene in both cultivars. The antioxidant capacity was also significantly higher when the top was removed than with other treatments in both cultivars. 2,2-Diphenyl-1-picrylhydrazyl (DPPH), hydroxyl radical scavenging activity (HRSC), ferric ion reducing antioxidant power (FRAP) and metal-chelating capacity (MCC) were higher in cultivar G064 compared to G025. CONCLUSION: Overall, the results suggest that the removing the tops of the scapes of G025 and G064 is the best treatment to maintain these biochemical changes. © 2018 Society of Chemical Industry.

Microsatellite analysis and marker development in garlic: distribution in EST sequence, genetic diversity analysis, and marker transferability across Alliaceae.

Allium vegetables, such as garlic and onion, have understudied genomes and limited molecular resources, hindering advances in genetic research and breeding of these species. In this study, we characterized and compared the simple sequence repeats (SSR) landscape in the transcriptomes of garlic and related Allium (A. cepa, A. fistulosum, and A. tuberosum) and non-Allium monocot species. In addition, 110 SSR markers were developed from garlic ESTs, and they were characterized-along with 112 previously developed SSRs-at various levels, including transferability across Alliaceae species, and their usefulness for genetic diversity analysis. Among the Allium species analyzed, garlic ESTs had the highest overall SSR density, the lowest frequency of trinucleotides, and the highest of di- and tetranucleotides. When compared to more distantly related monocots, outside the Asparagales order, it was evident that ESTs of Allium species shared major commonalities with regards to SSR density, frequency distribution, sequence motifs, and GC content. A significant fraction of the SSR markers were successfully transferred across Allium species, including crops for which no SSR markers have been developed yet, such as leek, shallot, chives, and elephant garlic. Diversity analysis of garlic cultivars with selected SSRs revealed 36 alleles, with 2-5 alleles/locus, and PIC = 0.38. Cluster analysis grouped the accessions according to their flowering behavior, botanical variety, and ecophysiological characteristics. Results from this study contribute to the characterization of Allium transcriptomes. The new SSR markers developed, along with the data from the polymorphism and transferability analyses, will aid in assisting genetic research and breeding in garlic and other Allium.

Nutritional Value, Chemical Characterization and Bulb Morphology of Greek Garlic Landraces.

Garlic (Allium sativum L.) is an important vegetable crop throughout the world. In Greece there are many areas which have specialized in garlic cultivation through the last decades, considered the main production areas. However, despite the significance of garlic as a food product and the high annual income of this crop, there is a decreasing trend in total cultivated area in Greece, and the local landraces are gradually neglected in favor of new imported genotypes. In the present study, garlic genotypes (local landraces/varieties, imported genotypes, commercial cultivars) from the main production regions of Greece were assessed for their chemical composition and quality (total soluble solids, dry matter content, nutritional value, mineral composition, organic acids, fatty acids content and free sugars content), and bulb morphology. The results of the present study showed significant diversity in quality features and bulb morphology, not only between the genotypes from different growing regions, but also between those of the same region. This result is interesting since it could be implemented for further improvement and valorization of this important vegetable crop through extensive breeding programs within the framework of sustainability and genetic, material conservation.

Diallyl Polysulfides from Allium sativum as Immunomodulators, Hepatoprotectors, and Antimycobacterial Agents.

Mycobacterium tuberculosis remains one of the world's deadliest killers, with an annual death rate of ∼1.5 million. The medicinal effects of garlic have been well documented, and natural products have been shown to have antimycobacterial activity. The current study evaluated the efficacy of six Allium sativum L. polysulfide mixtures as antimycobacterial agents together with their cytotoxic, immunomodulatory, and hepatoprotective activities. The microtitre PrestoBlue assay was used to determine the minimum inhibitory concentrations (MIC). Cytotoxicity was evaluated by using peripheral blood mononuclear cells (PBMC). Excreted cytokine levels were determined by utilizing an enzyme-linked immunosorbent assay (ELISA), by exposing isolated PBMCs to varying concentrations of polysulfide mixtures. Human C3A liver cells were utilized in the hepatoprotective study, to assess the protective effect against the toxicity induced by acetaminophen. Samples with higher amounts of diallyl trisulfide (Sample G4) showed the highest antimycobacterial activity, exhibiting an MIC of 2.5 µg/mL against M. tuberculosis H37Rv. Five samples showed moderate toxicity in PBMC, with G1 showing no toxicity. The selective index of G4 was the highest, with a selectivity index close to one. Two samples, G3 and G6 containing higher amounts of diallyl tetrasulfide and lower amounts of diallyl trisulfide, showed >50% hepatoprotection. This is comparable to a hepatoprotective agent, Silymarin, which showed a hepatoprotective effect of 30% at the tested concentration. Diallyl tetrasulfide showed significant antimycobacterial activity. A combination of higher diallyl tetrasulfide and lower diallyl trisulfide was indicative of hepatoprotective activity.

SSR-based genetic diversity and structure of garlic accessions from Brazil.

Garlic is a spice and a medicinal plant; hence, there is an increasing interest in 'developing' new varieties with different culinary properties or with high content of nutraceutical compounds. Phenotypic traits and dominant molecular markers are predominantly used to evaluate the genetic diversity of garlic clones. However, 24 SSR markers (codominant) specific for garlic are available in the literature, fostering germplasm researches. In this study, we genotyped 130 garlic accessions from Brazil and abroad using 17 polymorphic SSR markers to assess the genetic diversity and structure. This is the first attempt to evaluate a large set of accessions maintained by Brazilian institutions. A high level of redundancy was detected in the collection (50 % of the accessions represented eight haplotypes). However, non-redundant accessions presented high genetic diversity. We detected on average five alleles per locus, Shannon index of 1.2, HO of 0.5, and HE of 0.6. A core collection was set with 17 accessions, covering 100 % of the alleles with minimum redundancy. Overall FST and D values indicate a strong genetic structure within accessions. Two major groups identified by both model-based (Bayesian approach) and hierarchical clustering (UPGMA dendrogram) techniques were coherent with the classification of accessions according to maturity time (growth cycle): early-late and midseason accessions. Assessing genetic diversity and structure of garlic collections is the first step towards an efficient management and conservation of accessions in genebanks, as well as to advance future genetic studies and improvement of garlic worldwide.

Traceability of Italian garlic (Allium sativum L.) by means of HRMAS-NMR spectroscopy and multivariate data analysis.

(1)H High Resolution Magic Angle Spinning-Nuclear Magnetic Resonance (HRMAS-NMR) spectroscopy was used to analyse garlic (Allium sativum L.) belonging to red and white varieties and collected in different Italian regions, in order to address the traceability issue. 1D and 2D NMR spectra, performed directly on untreated small pieces of garlic, so without any sample manipulation, allowed the assignment of several compounds: organic acids, sugars, fatty acids, amino acids and the nutritionally important fructo-oligosaccharides and allyl-organosulphur compounds. Application of Partial Least Squares projections to latent structures-Discrimination Analysis provided an excellent model for the discrimination of both the variety and, most important, the place origin, allowing the identification of the metabolites contributing to such classifications. The presence of organosulphurs, allicin and some allyl-organosulphurs found by HRMAS-NMR, was confirmed also by SPME-GC-MS; 11 molecules were identified, containing from one up to three sulphur atoms and with and without allyl moieties.

New microsatellite markers for garlic, Allium sativum (Alliaceae).

PREMISE OF THE STUDY: A new set of microsatellite or simple sequence repeat (SSR) markers for garlic, an important medicinal spice, was developed to aid studies of genetic diversity and to define efficient strategies for germplasm conservation. METHODS AND RESULTS: Using a (CT)(8)- and (GT)(8)-enriched library, a total of 16 SSR loci were developed and optimized in garlic. Ten loci were found to be polymorphic after screening 75 accessions. The parameters used to characterize the loci were observed and expected heterozygosity, number of alleles, Shannon Index, and polymorphism information content (PIC). A total of 44 alleles were identified, with an average of 4.4 alleles per loci. The vast majority of loci were moderate to highly informative according to PIC and the Shannon Index. CONCLUSION: The new SSR markers have the potential to be informative tools for genetic diversity, allele mining, mapping and associative studies, and in the management and conservation of garlic collections.

Changes in phenolic compounds in garlic (Allium sativum L.) owing to the cultivar and location of growth.

The contents of total phenolic compounds, flavonoids, and phenolic acids were determined in selected garlic cultivars grown at four locations. The total phenolic content varied from 3.4 mg gallic acid equivalents (GAE)/g of dry matter (dm) to 10.8 mg GAE/g of dm with a mean value of 6.5 mg GAE/g of dm. The myricetin, quercetin, kaempferol, and apigenin flavonoids were not detected in any of the samples. Caffeic acid and ferulic acid were the major phenolic acids found with mean values of 2.9 mg/kg of dm and 2.6 mg/kg of dm, respectively. The mean contents of vanillic, p-hydroxybenzoic, and p-coumaric acids were comparable (0.4-0.8 mg/kg of dm), and the level of sinapic acid was negligible (< 0.1 mg/kg of dm). There was a significant effect of location but an insignificant effect of genotype on contents of caffeic, vanillic, p-hydroxybenzoic, and p-coumaric acids. However, genotype but not location affected the contents of total phenolics and ferulic acid. On average, the white garlic cultivars and Chinese garlic cultivars contained higher contents of total phenolics and ferulic acid than the purple garlic cultivars. However, the differences in the total phenolic content between the purple and white garlic cultivars were not significant.

Molecular cloning and characterization of phenylalanine ammonia-lyase and cinnamate 4-hydroxylase in the phenylpropanoid biosynthesis pathway in garlic (Allium sativum).

The cDNAs encoding phenylalanine ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H) were cloned from garlic (Allium sativum) using reverse transcription-polymerase chain reaction (RT-PCR) with degenerate primers and 5' and 3' rapid amplification of cDNA ends (RACE) PCR. Amino acid sequence alignments showed that AsPAL and AsC4H have more than 70% amino acid identity with their homologues in other plants. The expression of AsPAL and AsC4H transcripts was highest in the roots but surprisingly low in the bulbils, where phenylpropanoid compounds are most concentrated. These results suggest that some phenylpropanoids are synthesized in the roots and subsequently transported to the bulbils of A. sativum .

Variability of solids, organosulfur compounds, pungency and health-enhancing traits in garlic (Allium sativum L.) cultivars belonging to different ecophysiological groups.

Garlic is a vegetable mainly agamically propagated, and it has been dispersed all around the world. Garlic cultivars have been classified in different ecophysiological groups (EG) according to their bulbing requirements. The variability in organosulfur composition (ACSOs), solids content (SC), pungency (PC) and antiplatelet activity (IAA) and the correlation among these traits in garlic clones belonging to three EG was studied. We found variability for ACSOs, SC, PC and IAA between clones belonging to different EG and also among clones belonging to the same EG. Cutivars EG III presented more variability than EG IV for ACSOs, thiosulfinates, allicin and PC, while for SC, EG IV was the most variable. The correlations found suggested that IAA observed was mainly due to organosulfur composition. Finally recommendations about the most suitable cultivars for fresh consumption, pharmaceutical and dehydration industry are made.

Garlic: empiricism or science?

Garlic (Allium sativum L. fam. Alliaceae) is one of the best-researched, best-selling herbal remedies and is also commonly used as a food and a spice. Garlic constituents include enzymes (for example, alliinase) and sulfur-containing compounds, including alliin, and compounds produced enzymatically from alliin (for example, allicin). Traditionally, it has been employed to treat infections, wounds, diarrhea, rheumatism, heart disease, diabetes, and many other disorders. Experimentally, it has been shown to exert antilipidemic, antihypertensive, antineoplastic, antibacterial, immunostimulant and hypoglycemic actions. Clinically, garlic has been evaluated for a number of conditions, including hypertension, hypercholesterolemia, intermittent claudication, diabetes, rheumatoid arthritis, common cold, as an insect repellent, and for the prevention of arteriosclerosis and cancer. Systematic reviews are available for the possible antilipidemic, antihypertensive, antithrombotic and chemopreventive effects. However, the clinical evidence is far from compelling. Garlic appears to be generally safe although allergic reactions may occur.

Genetic diversity studies of Brazilian garlic cultivars and quality control of garlic-clover production.

The garlic cultivars grown in Brazil evolved from somatic mutations and clone selection by breeding programs and by the introduction of germplasm from other countries. Morphological characters have been used to differentiate these cultivars. Two hundred and six random amplified polymorphic DNA markers were utilized for a diversity analysis of the 17 most planted garlic cultivars in Brazil. Bootstrap analysis showed that the number of markers was efficient and sufficient to obtain a coefficient of variation of 10%. Similarity varied between 16 and 98% and cluster analysis showed that, in general, genetic similarities correlate with morphological characters of the cultivars and production cycle variation. High bootstrap values at most of the nodes supported the dendrogram stability. The grouping of most varieties agreed well with previous reports based on morphological characters. As a vegetative-propagated species, viral diseases are a key problem regarding production and quality of the bulbs, causing gradual loss of yield and decrease in storage capacity. To improve the health quality of garlic seed, a virus-free stock of garlic cloves of the Amarante cultivar was obtained. The ability to distinguish garlic cultivars to detect varietal mixing after in vitro multiplication is extremely important, since correct identification is not possible until bulbs are produced. Random amplified polymorphic DNA markers were also used to differentiate cultivars while they are in vitro and not amenable to morphological discrimination. No difference was identified between the fingerprints of the virus-free or of the infected bulks of Amarante, showing that there was no clove mixing in the handling of material in the clonal multiplication phase.

Genetic diversity studies of Brazilian garlic cultivars and quality control of garlic-clover production

The garlic cultivars grown in Brazil evolved from somatic mutations and clone selection by breeding programs and by the introduction of germplasm from other countries. Morphological characters have been used to differentiate these cultivars. Two hundred and six random amplified polymorphic DNA markers were utilized for a diversity analysis of the 17 most planted garlic cultivars in Brazil. Bootstrap analysis showed that the number of markers was efficient and sufficient to obtain a coefficient of variation of 10%. Similarity varied between 16 and 98% and cluster analysis showed that, in general, genetic similarities correlate with morphological characters of the cultivars and production cycle variation. High bootstrap values at most of the nodes supported the dendrogram stability. The grouping of most varieties agreed well with previous reports based on morphological characters. As a vegetative-propagated species, viral diseases are a key problem regarding production and quality of the bulbs, causing gradual loss of yield and decrease in storage capacity. To improve the health quality of garlic seed, a virus-free stock of garlic cloves of the Amarante cultivar was obtained. The ability to distinguish garlic cultivars to detect varietal mixing after in vitro multiplication is extremely important, since correct identification is not possible until bulbs are produced. Random amplified polymorphic DNA markers were also used to differentiate cultivars while they are in vitro and not amenable to morphological discrimination. No difference was identified between the fingerprints of the virus-free or of the infected bulks of Amarante, showing that there was no clove mixing in the handling of material in the clonal multiplication phase.

Determination of the country of origin of garlic (Allium sativum) using trace metal profiling.

A method for determining the country of origin of garlic by comparing the trace metal profile of the sample to an authentic garlic database is presented. Protocols for sample preparation, high-resolution inductively coupled plasma mass spectrometry, and multivariate statistics are provided. The criteria used for making a country of origin prediction are also presented. Indications are that the method presented here may be used to determine the geographic origin of other agricultural products.

Chemical and genomic combined approach applied to the characterization and identification of Italian Allium sativum L.

Chemotype analyses and random amplified polymorphic DNA (RAPD) genomic analyses have been applied to the characterization of Allium sativum variety from Voghiera (Ferrara, Italy), a typical Italian product actually demanding the Protected Designation of Origin (PDO). The garlic from Voghiera is characterized by peculiar morphological and composition characteristics. The proximate composition and atomic absorbance spectrometry elemental pattern of this garlic suggested as the chemical composition did not depend on the intrinsic pedologic soil features only, but it was probably connected to some peculiar genetic characters. Amplification of genomic DNA using random primers highlighted a good clustering differentiating of Voghiera Allium sativum from five commercial reference samples used in this study (Piacentino, Serena, France, China, and Adriano varieties), confirming the existence of intervarietal genetic difference. The intravarietal polymorphisms of Voghiera samples were low.

How to distinguish garlic from the other Allium vegetables.

The establishment of international monographs for herbs is in progress. Here, we propose both a marker compound and a method for its analysis for the identification of garlic bulbs and their products. The constituents in 26 kinds of fresh edible parts of Allium vegetables and three types of garlic preparations were analyzed. Sulfur compounds are the most characteristic constituents in garlic, but manufacturing processes of garlic products dramatically affect these constituents. Thus, no sulfur compound could be specified as a universal marker of identification applicable for any type of garlic. On the other hand, garlic contains other characteristic compounds, namely, saponins. After analyzing Allium vegetables and garlic preparations, we concluded that sapogenins, especially beta-chlorogenin, may be a viable candidate for identifying and distinguishing garlic from other Allium vegetables.

Wild garlic has a greater effect than regular garlic on blood pressure and blood chemistries of rats.

When groups of 10 Spontaneously Hypertensive Rats (SHR) were fed diets containing either 1% w/w regular garlic (Allium sativum) (AS) or 1% w/w wild garlic (Allium ursinum) (AU) for 45 days, the final mean systolic blood pressure (SBP) was reduced significantly compared to control (C) (C 189; AS 175; Au 173 mm Hg). Compared to C, body weight and circulating glucose and triglyceride levels were not significantly different; but circulating insulin was significantly higher (C 23.6; AS 33.9; AU 29.5 uIU/dl), and total cholesterol was significantly lower (C 133; AS 115; AU 117 mg/dl) in the two groups consuming AS or AU. HDL rose in the two garlic groups, but the differences from C were statistically significant only for the AU group. In a second study, the effects of a lower dose of dietary AS and AU (0.1% w/w) on SBP and various blood chemistries were compared head-to-head in 80 SHR-40 control and 40 test rats. Both AS and AU decreased SBP significantly compared to a control group of 10 SHR followed simultaneously. However, AU at this lower concentration produced a significantly greater SBP-lowering effect compared to the AS group. In addition, AU decreased total cholesterol significantly and tended to increase HDL compared to AS. Accordingly, the results suggest that AU has a greater therapeutic benefit compared to AS at a given concentration.

Complete nucleotide sequences of garlic viruses A and C, members of the newly ratified genus Allexivirus.

Complete genomic sequences of garlic viruses A (Gar V-A) and C (Gar V-C), members of an unassigned virus group recently identified in garlic plants, were determined. Their respective genomes consist of 8 660 and 8 405 nucleotides. The genomic structure and organization of these viruses are similar to shallot virus X (ShVX) which is the type species of the newly ratified genus Allexivirus. Phylogenetic analysis based on the amino acid sequences of the putative proteins including RNA-dependent RNA polymerase (RdRP), DNA helicase, or viral coat protein showed that the GarV-type viruses should be included in the genus Allexivirus. Furthermore, the amino acid sequence in the RdRP hypervariable region is highly divergent among the viruses in the genus Allexivirus, suggesting that they evolved independently from a hypothetical ancestor virus(es).