A Review of Recent Advances in Chromatographic Quantification Methods for Cyanogenic Glycosides.

Cyanogenic glycosides are naturally occurring compounds found in numerous plant species, which can release toxic hydrogen cyanide upon hydrolysis. The quantification of cyanogenic glycosides is essential for assessing their potential toxicity and health risks associated with their consumption. Liquid chromatographic techniques coupled with various detectors have been widely used for the quantification of cyanogenic glycosides. In this review, we discuss recent advances in chromatographic quantification methods for cyanogenic glycosides, including the development of new stationary phases, innovative sample preparation methods, and the use of mass spectrometry. We also highlight the combination of chromatographic separation with mass spectrometric detection for the identification and quantification of specific cyanogenic glycosides and their metabolites in complex sample matrices. Lastly, we discuss the current challenges and future perspectives in the development of reliable reference standards, optimization of sample preparation methods, and establishment of robust quality control procedures. This review aims to provide an overview of recent advances in chromatographic quantification methods for cyanogenic glycosides and their applications in various matrices, including food products, biological fluids, and environmental samples.

Dynamics of cyanogenic glycosides in apple and plum fruits, products, and byproducts: A concise review.

Apples (Malus domestica) and plums (Prunus domestica) are important fruit crops belonging to the Rosaceae family. The edible parts of fruits and seeds contain phytochemicals; however, the seeds are rich in cyanogenic glycosides (CNGs), which release toxic hydrogen cyanide (HCN) upon the loss of plant cell integrity. This review aims to explore the quantitative and qualitative CNG profiles in apples and plums, focusing on their distribution in different parts of the fruit, changes during fruit development, and environmental impacts on their biosynthesis. It also discusses the intricate dynamics of CNGs in processed fruits and waste-derived products and the effects of the processing methods on CNG content. There is considerable variation in the CNG content of fruit crops, as well as in its distribution in fruit parts other than seeds and shifts during fruit maturation. Although several studies have attempted to explain this variability by the influence of cultivars and exogenous factors, there is insufficient evidence to draw reliable conclusions. Furthermore, due to the lack of studies, the dynamics of CNGs during the storage of fresh or preserved fruit remains unaddressed. In the context of reusing plum stones from waste to produce distillates, it is recommended to monitor the HCN content in the products during storage, as it can increase significantly over time. Processing methods influence CNG levels, with strategies like seed separation, soaking, and microwave heating showing promise in reducing cyanide (CN-) presence. The insights from this review will provide direction for future detailed research.

Bacterial Degradation of Antinutrients in Foods: The Genomic Insight.

Antinutrients, also known as anti-nutritional factors (ANFs), are compounds found in many plant-based foods that can limit the bioavailability of nutrients or can act as precursors to toxic substances. ANFs have controversial effects on human health, depending mainly on their concentration. While the positive effects of these compounds are well documented, the dangers they pose and the approaches to avoid them have not been discussed to the same extent. There is no dispute that many ANFs negatively alter the absorption of vitamins, minerals, and proteins in addition to inhibiting some enzyme activities, thus negatively affecting the bioavailability of nutrients in the human body. This review discusses the chemical properties, plant bioavailability, and deleterious effects of anti-minerals (phytates and oxalates), glycosides (cyanogenic glycosides and saponins), polyphenols (tannins), and proteinaceous ANFs (enzyme inhibitors and lectins). The focus of this study is on the possibility of controlling the amount of ANF in food through fermentation. An overview of the most common biochemical pathways for their microbial reduction is provided, showing the genetic basis of these phenomena, including the active enzymes, the optimal conditions of action, and some data on the regulation of their synthesis.

A Novel UHPLC-MS/MS Based Method for Isomeric Separation and Quantitative Determination of Cyanogenic Glycosides in American Elderberry.

LC-MS/MS analyses have been reported as challenging for the reliable separation and quantification of cyanogenic glycosides (CNGs), especially (R)-prunasin and sambunigrin isomers found in American elderberry (Sambucus nigra L. subsp. canadensis (L.) Bolli). Hence, a novel multiple reaction monitoring (MRM)-based ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated in the present study for simultaneous separation and quantification of five CNGs, including amygdalin, dhurrin, linamarin, (R)-prunasin, and (S)-prunasin (commonly referred to as sambunigrin). Initially, the role of ammonium formate was investigated as an aqueous mobile-phase additive in developing MRM-based UHPLC-MS/MS. Later, chromatographic conditions for the resolved separation of (R)-prunasin and sambunigrin were identified. Validation studies confirmed that the developed method has good linearity and acceptable precision and accuracy. A noticeable matrix effect (mainly signal enhancement) was observed in leaf samples only. This method was used to detect and quantify CNGs, including (R)-prunasin and sambunigrin, in leaf and fruit samples of American elderberry. Among the studied CNGs, only (R)-prunasin was detected in the leaf samples. Interestingly, (S)-prunasin (sambunigrin) was not detected in the samples analyzed, even though it has been previously reported in elderberry species.

Evaluation of exposure to cyanogenic glycosides and potential hydrogen cyanide release in commercially available foods among the Korean population.

The release of hydrogen cyanide (HCN) after food ingestion can pose a serious health risk to consumers. This study aimed to simultaneously quantify four cyanogenic glycosides (lotaustralin, prunasin, taxiphyllin, and dhurrin) using liquid chromatography-tandem mass spectrometry. The analysis scope extended beyond agricultural products to various consumer foods to estimate dietary exposure to cyanogenic glycosides and assess its risk levels. The major exposure sources are cassava chips (lotaustralin), apples (seeds) (prunasin and dhurrin), and Prunus mume axis (taxiphyllin). In addition to quantifying specific cyanogenic glycosides, this study proposed the development of a preliminary risk assessment framework based on the dietary exposure assessment and the calculation of theoretical levels of HCN derived from cyanogenic glycoside concentrations. In the absence of established guidelines for the permissible intake of foods containing cyanogenic glycosides, this study provides initial guidance for assessing the risks associated with a range of commonly consumed foods.

Evidence for Reductions in Physical and Chemical Plant Defense Traits in Island Flora.

Reduced defense against large herbivores has been suggested to be part of the "island syndrome" in plants. However, empirical evidence for this pattern is mixed. In this paper, we present two studies that compare putative physical and chemical defense traits from plants on the California Channel Islands and nearby mainland based on sampling of both field and common garden plants. In the first study, we focus on five pairs of woody shrubs from three island and three mainland locations and find evidence for increased leaf area, decreased marginal leaf spines, and decreased concentrations of cyanogenic glycosides in island plants. We observed similar increases in leaf area and decreases in defense traits when comparing island and mainland genotypes grown together in botanic gardens, suggesting that trait differences are not solely driven by abiotic differences between island and mainland sites. In the second study, we conducted a common garden experiment with a perennial herb-Stachys bullata (Lamiaceae)-collected from two island and four mainland locations. Compared to their mainland relatives, island genotypes show highly reduced glandular trichomes and a nearly 100-fold reduction in mono- and sesquiterpene compounds from leaf surfaces. Island genotypes also had significantly higher specific leaf area, somewhat lower rates of gas exchange, and greater aboveground biomass than mainland genotypes across two years of study, potentially reflecting a broader shift in growth habit. Together, our results provide evidence for reduced expression of putative defense traits in island plants, though these results may reflect adaptation to both biotic (i.e., the historical absence of large herbivores) and climatic conditions on islands.

Harnessing the anti-cancer potential of linamarin: A computational study on design and hydrolysis mechanisms of its derivatives.

Cassava extracts containing cyanogenic compounds demonstrate anticancer properties. The cyanogenic glucoside linamarin found abundantly in cassava can release hydrogen cyanide (HCN) upon hydrolysis, a potent cytotoxin. However, linamarin's hydrolysis mechanism by human enzymes is poorly delineated and constitutes a bottleneck for therapeutic development. This study aimed to investigate linamarin's hydrolysis mechanism by human ß-glucosidase and identify structural derivatives with enhanced hydrolytic potential using density functional theory calculations. Results revealed α-anomeric derivatives as promising, with leaving group ability and steric bulk strongly governing hydrolysability. We identified several linamarin analogs with predicted rapid hydrolysis kinetics that may enable swift cytotoxic HCN release against cancer cells. This investigation enriches understanding of cyanogenic glycoside reactivity to facilitate their development as targeted antineoplastic agents. The identified derivatives set the groundwork for experimental evaluation of enhanced linamarin-inspired compounds as innovative cancer therapeutics.

Elderberry, an Ancient Remedy: A Comprehensive Study of the Bioactive Compounds in Three Sambucus nigra L. Subspecies.

Elderberry, the fruit of Sambucus nigra, has become a popular inclusion in foods, beverages, supplements, and more in recent years. Although the European subspecies, S. nigra ssp. nigra, has been widely studied for its composition, particularly for phenolic and volatile profiles, other subspecies, such as the American elderberry S. nigra ssp. canadensis and the blue elderberry S. nigra ssp. cerulea, have also become contenders in the elderberry supply chain. For the first time, the composition (including micronutrients, macronutrients, organic acids, titratable acid, soluble solids, phenolic compounds, and cyanogenic glycosides) of these three subspecies of elderberry is compared, highlighting the unique qualities of each subspecies and identifying gaps in the available data on the three subspecies.

Content and distribution of cyanogenic compounds in cassava roots and leaves in association with physiological age.

BACKGROUND: Cassava roots are widely consumed in tropical regions of Asia, Africa, and Latin America. Although the protein, vitamin, carotenoid, and mineral content in the leaves makes them a nutritionally attractive option, their consumption is limited due to their high levels of cyanogenic compounds (CCs). In this study, the CC content in different parts of the plant (leaves, storage root cortex, and parenchyma) was assessed at harvest for 50 landrace genotypes representative of cassava diversity in Latin America. The changes in CC in leaves at different physiological ages (3, 6, 9, and 11 months after planting) were also investigated. RESULTS: The average CC was higher in the cortex (804 ppm) and leaves (655 ppm) than in root parenchyma (305 ppm). Genotypes from different regions of Latin America, as identified by seven genetic diversity groups, differed significantly in CC levels. The Andean and Amazon groups had, respectively, the lowest (P = 0.0008) and highest (P < 0.0001) CC levels in all three parts of the plants. Cyanogenic compound concentrations were higher in leaves from young plants (P < 0.0001) and decreased with increasing physiological age. CONCLUSION: The results help to guide the selection of parental lines with low CC levels for breeding and to contribute to the expanded use of cassava and its by-products for food and feed. Cassava for fresh consumption, especially, requires varieties with low total CC content, especially in the root cortex and parenchyma. COL1108 (204, 213, and 174 ppm, respectively, in the parenchyma, cortex, and leaves) and PER297 (83, 238, and 299 ppm, respectively, in the parenchyma, cortex, and leaves) can fulfill this requirement. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Cassava (Manihot esculenta Crantz): A Systematic Review for the Pharmacological Activities, Traditional Uses, Nutritional Values, and Phytochemistry.

Cassava (Manihot esculenta Crantz) is considered one of the essential tuber crops, serving as a dietary staple food for various populations. This systematic review provides a comprehensive summary of the nutritional and therapeutic properties of cassava, which is an important dietary staple and traditional medicine. The review aims to evaluate and summarize the phytochemical components of cassava and their association with pharmacological activities, traditional uses, and nutritional importance in global food crises. To collect all relevant information, electronic databases; Cochrane Library, PubMed, Scopus, Web of Science, Google Scholar, and Preprint Platforms were searched for studies on cassava from inception until October 2022. A total of 1582 studies were screened, while only 34 were included in this review. The results of the review indicate that cassava has diverse pharmacological activities, including anti-bacterial, anti-cancer, anti-diabetic, anti-diarrheal, anti-inflammatory, hypocholesterolemic effects, and wound healing properties. However, more studies that aim to isolate the phytochemicals in cassava extracts and evaluate their pharmacological property are necessary to further validate their medical and nutritional values.

Catalytic efficiency and thermal stability promotion of the cassava linamarase with multiple mutations for better cyanogenic glycoside degradation.

In our previous study, we found that cassava cyanogenic glycosides had an acute health risk. Therefore, to solve this problem, the improvement of specific degradation of cyanogenic glycosides of cassava linamarase during processing is the key. In this study, the catalytic activity and thermal stability of enzymes were screened before investigating the degradation efficiency of cyanogenic glycosides with a cassava linamarase mutant K263P-T53F-S366R-V335C-F339C (CASmut) -controlled technique. The CASmut was obtained with the optimum temperature of 45 °C, which was improved by 10 °C. The specific activity of CASmut was 85.1 ± 4.6 U/mg, which was 2.02 times higher than that of the wild type. Molecular dynamics simulation analysis and flexible docking showed there were more hydrogen bonding interactions at the pocket, and the aliphatic glycoside of the linamarin was partially surrounded by hydrophobic residues. The optimum conditions of degradation reactions was screened with CASmut addition of 47 mg/L at 45 °C, pH 6.0. The CASmut combined with ultrasonication improved the degradation from 478.2 ± 10.4 mg/kg to 86.7 ± 7.4 mg/kg. Those results indicating the great potential of CASmut in applying in the cassava food or cyanogenic food. However, challenges in terms of the catalytic mechanism research is worthy of being noticed in further studies.

Resequencing of global Lotus corniculatus accessions reveals population distribution and genetic loci, associated with cyanogenic glycosides accumulation and growth traits.

BACKGROUND: Lotus corniculatus is a widely distributed perennial legume whose great adaptability to different environments and resistance to barrenness make it an excellent forage and ecological restoration plant. However, its molecular genetics and genomic relationships among populations are yet to be uncovered. RESULT: Here we report on a genomic variation map from worldwide 272 L. corniculatus accessions by genome resequencing. Our analysis suggests that L. corniculatus accessions have high genetic diversity and could be further divided into three subgroups, with the genetic diversity centers were located in Transcaucasia. Several candidate genes and SNP site associated with CNglcs content and growth traits were identified by genome-wide associated study (GWAS). A non-synonymous in LjMTR was responsible for the decreased expression of CNglcs synthesis genes and LjZCD was verified to positively regulate CNglcs synthesis gene CYP79D3. The LjZCB and an SNP in LjZCA promoter were confirmed to be involved in plant growth. CONCLUSION: This study provided a large number of genomic resources and described genetic relationship and population structure among different accessions. Moreover, we attempt to provide insights into the molecular studies and breeding of CNglcs and growth traits in L. corniculatus.

Antineoplastic effects of cassava-cyanide extract on human glioblastoma (LN229) cells.

Several natural compounds reduce tumour cell growth and metastasis by inducing programmed cell death. Cassava (Manihot esculenta Crantz) contains cyanogenic glycosides such as, linamarin and lotaustralin, can be enzymatically cleaved by linamarase to release hydrogen cyanide (HCN), which can have therapeutic benefits against hypertension, asthma, and cancer. We have developed a technology for isolating bio-active principles from cassava leaves.The present study is designed to analyze the cytotoxic effect of cassava cyanide extract (CCE) against human glioblastoma cells (LN229). The treatment of CCE demonstrated a dose dependent toxicity on glioblastoma cells. At higher concentration tested, the CCE (400 µg/mL) was found to be cytotoxic, reducing the cell viability to 14.07 ± 2.15% by negatively influencing the mitochondrial activity, and lysosomal and cytoskeletal integrity. Coomassie's brilliant blue staining confirmed cells' morphological aberration after 24 h of treatment with CCE. Moreover, DCFH-DA assay and Griess reagent showed an increase in ROS but a decrease in RNS production at a concentration of CCE. Flow cytometry analysis revealed that CCE interfered with G0/G1, S, and G2/M stages of the cell cycle of glioblastoma, and Annexin/PI staining indicated a dose-dependent increase in cell death, confirming the toxic nature of CCE on LN229 cells. These findings suggest that cassava cyanide extract has potential as an antineoplastic agent against glioblastoma cells, which is an aggressive and difficult-to-treat type of brain cancer. However, it is important to note that the study was conducted in vitro, and further research is necessary to assess the safety and efficacy of CCE in vivo. Additionally, it is essential to establish the optimal dose and potential side effects before considering its use as a therapeutic agent.

Isolation and Quantification of Mandelonitrile from Arabidopsis thaliana Using Gas Chromatography/Mass Spectrometry.

Mandelonitrile is a nitrogen-containing compound, considered an essential secondary metabolite. Chemically, it is a cyanohydrin derivative of benzaldehyde, with relevant functions in different physiological processes including defense against phytophagous arthropods. So far, procedures for detecting mandelonitrile have been effectively applied in cyanogenic plant species such as Prunus spp. Nevertheless, its presence in Arabidopsis thaliana , considered a non-cyanogenic species, has never been determined. Here, we report the development of an accurate protocol for mandelonitrile quantification in A. thaliana within the context of A. thaliana -spider mite interaction. First, mandelonitrile was isolated from Arabidopsis rosettes using methanol; then, it was derivatized by silylation to enhance detection and, finally, it was quantified using gas chromatography-mass spectrometry. The selectivity and sensitivity of this method make it possible to detect low levels of mandelonitrile (LOD 3 ppm) in a plant species considered non-cyanogenic that, therefore, will have little to no cyanogenic compounds, using a small quantity of starting material (≥100 mg).

Solid-state fermentation of cassava (Manihot esculenta Crantz): a review.

Solid-state fermentation (SSF) is a promising technology for producing value-added products from cassava (Manihot esculenta Crantz). In this process, microorganisms are grown on cassava biomass without the presence of free-flowing liquid. Compared to other processing methods, SSF has several advantages, such as lower costs, reduced water usage, and higher product yields. By enhancing the content of bioactive compounds like antioxidants and phenolic compounds, SSF can also improve the nutritional value of cassava-based products. Various products, including enzymes, organic acids, and biofuels, have been produced using SSF of cassava. Additionally, SSF can help minimize waste generated during cassava processing by utilizing cassava waste as a substrate, which can reduce environmental pollution. The process has also been explored for the production of feed and food products such as tempeh and cassava flour. However, optimizing the process conditions, selecting suitable microbial strains, and developing cost-effective production processes are essential for the successful commercialization of SSF of cassava.

Effects of Aspergillus niger on cyanogenic glycosides removal and fermentation qualities of ratooning sorghum.

Introduction: Cyanogenic glycosides (CNglcs) are bioactive plant products involving in plant defense against herbivores by virtue of their abilities to release toxic hydrogen cyanide (HCN). Aspergillus niger has been shown to be effective in producing ß-glucosidase, which could degrade CNglcs. However, whether A. niger could remove CNglcs under ensiling conditions is still unknown. Methods: In this study, we first investigated the HCN contents in ratooning sorghums for two years, then the sorghums were ensiled with or without the addition of A. niger. Results: Two years' investigation indicated that the contents of HCN in fresh ratooning sorghum were larger than 801 mg/kg FW (fresh weight), which could not be reduced by silage fermentation under safety threshold (200 mg/kg FW). A. niger could produce ß-glucosidase over a range of pH and temperature, which degraded the CNglcs and removed the hydrogen cyanide (HCN) at early days of ratooning sorghum fermentation. The addition of A. niger (2.56 × 107 CFU/ml) altered the microbial community, increased bacterial diversity, improved the nutritive qualities, and reduced the HCN contents in ensiled ratooning sorghum lower than 100 mg/kg FW after 60 days of fermentation. Overall, the addition of 150 ml A. niger + 50 ml sterile water per 3 kg silage could efficiently remove CNglcs from ratooning sorghum silage. Conclusion: In conclusion, A. niger could produce ß-glucosidase which degraded the CNglcs during the early days of fermentation, benefiting the ensiling process and improving the utilization of ratooning sorghum.

Depletion of cyanogenic glycosides in whole flaxseed via Lactobacillaceae fermentation.

Flaxseed is categorized as a functional food due to its abundance in oil, α-linolenic acid, dietary fibre, and lignan. However, flaxseed contains cyanogenic glycosides (CGs). Ingestion of CGs can influence nutrient absorption and induce adverse health effects. Due to the presence of CGs in flaxseed many countries prohibit the import and sale of flaxseed and flaxseed-based foods. In this study, whole flaxseed was fermented with a mixed culture of Lactobacillaceae (i.e., Lactobacillus sp., Limosilactobacillus sp., and Lactiplantibacillus sp.) and the concentration of CGs was determined. This process succeeded in completely removing CGs within 72 h in both bench-scale and scale-up studies. In addition, fatty acid composition in flaxseed remained unchanged and concentrations of flaxseed oil, and SDG in flaxseed were increased after fermentation. CG-free flaxseed products are beneficial, as they can be sold as health product ingredients, or as animal feed in markets that currently restrict the use of materials that contain CGs.

Content and distribution of prunasin in Perilla frutescens.

Perilla frutescens var. crispa (Lamiaceae) is an annual plant that is the botanical origin of the natural medicine "Soyo" listed in the Japanese Pharmacopoeia and is also used as a fragrant vegetable. Its characteristic components are essential oils and anthocyanins. Cyanogenic glycosides have also been isolated from perilla, but no reports have clarified which cyanogenic glycosides are abundant or differences in cyanogenic glycoside content according to the extent of perilla leaf growth or growth stage. Here, for the first time we determined the content and distributions of cyanogenic glycosides in perilla. The picric acid test, a common qualitative test for cyanogenic compounds, was used to quickly and semi-quantitatively detect cyanogenic compounds in perilla. Prunasin was the most abundant cyanogenic glycoside. The prunasin content per unit mass of perilla leaves varied by strain, regardless of leaf color or the main compound in the essential oils of each strain. Prunasin was higher in fresh leaves than in dried leaves and higher in young leaves than in mature leaves. When perilla was cultivated in an outdoor field, the prunasin content was initially high during the vegetative stage in summer before decreasing and then increasing until flower buds were beginning to form, and then gradually decreased again after flowering.

Separation of Dihydro-Isocoumarins and Dihydro-Stilbenoids from Hydrangea macrophylla ssp. serrata by Use of Counter-Current Chromatography.

Previously, different Hydrangea macrophylla ssp. serrata cultivars were investigated by untargeted LC-MS analysis. From this, a list of tentatively identified and unknown compounds that differ significantly between these cultivars was obtained. Due to the lack of reference compounds, especially for dihydro-isocoumarins, we aimed to isolate and structurally characterise these compounds from the cultivar 'Yae-no-amacha' using NMR and LC-MS methods. For purification and isolation, counter-current chromatography was used in combination with reversed-phase preparative HPLC as an orthogonal and enhanced purification workflow. Thirteen dihydro-isocoumarins in combination with other metabolites could be isolated and structurally identified. Particularly interesting was the clarification of dihydrostilbenoid glycosides, which were described for the first time in H. macrophylla ssp. serrata. These results will help us in further studies on the biological interpretation of our data.

Highly sensitive analysis of cyanogenic glycosides in cold-pressed flaxseed oil by employing cigarette filter fiber-based SPE coupled with ultra-performance liquid chromatography-tandem mass spectrometry.

In this study, a highly sensitive method for analysis of 4 cyanogenic glycosides (CNGs) in cold-pressed flaxseed oil was developed by using cigarette filter fiber-based SPE and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The cold-pressed flaxseed oil was diluted with 5% (v/v) isopropanol/n-hexane solution and loaded to a cigarette filters fiber-based SPE column for CNG enrichment and purification. Under optimized conditions, four CNGs could be detected with limits of detection ranging from 1.3 to 4.4 pg/mL. The linear range was 0.05-50 ng/ml with a linear correlation coefficient (r) > 0.9935. CNG recovery ranged from 113% to 133%, and the relative standard deviation was between 0.8% and 20.5%. Finally, the proposed method was applied to the determination of CNGs in nine cold-pressed flaxseed oils.