Amygdalin Improves Allergic Asthma via the Thymic Stromal Lymphopoietin-dendritic Cell-OX40 Ligand Axis in a Mouse Model.

Asthma, characterized by persistent inflammation and increased sensitivity of the airway, is the most common chronic condition among children. Novel, safe, and reliable treatment strategies are the focus of current research on pediatric asthma. Amygdalin, mainly present in bitter almonds, has anti-inflammatory and immunoregulatory potential, but its effect on asthma remains uninvestigated. Here, the impact of amygdalin on the thymic stromal lymphopoietin (TSLP)-dendritic cell (DC)-OX40L axis was investigated. A BALB/c mouse model for allergic asthma was established using the ovalbumin-sensitization method. Amygdalin treatment was administered between days 21 and 27 of the protocol. Cell numbers and hematoxylin and eosin (H&E) staining in bronchoalveolar lavage fluid (BALF) were used to observe the impact of amygdalin on airway inflammation. TSLP, IL-4, IL-5, IL-13, and IFN-γ concentrations were determined via Enzyme-linked immunosorbent assay (ELISA). TSLP, GATA-3, and T-bet proteins were measured using western blotting. Cell-surface receptor expression on DCs (MHC II, CD80, and CD86) was assessed via flow cytometry. OX40L mRNA and protein levels were detected using western blotting and qRT-PCR, respectively. Amygdalin treatment attenuated airway inflammation decreased BALF TSLP levels, inhibited DC maturation, restrained TSLP-induced DC surface marker expression (MHCII, CD80, and CD86), and further decreased OX40L levels in activated DCs. This occurred together with decreased Th2 cytokine levels (IL-4, IL-5, and IL-13) and GATA3 expression, whereas Th1 cytokine (IFN-γ) levels and T-bet expression increased. Amygdalin thus regulates the Th1/Th2 balance through the TSLP-DC-OX40L axis to participate in inflammation development in the airways, providing a basis for potential allergic asthma treatments.

Effect of vitamin B17 (amygdalin) found in apricot kernel on the irradiated salivary glands of albino rats.

BACKGROUND: Radiotherapy is used as a treatment for head and neck cancers but increases the risk of salivary gland hypofunction. The management strategies include pharmacotherapies such as salivary substitutes and sialagogues which are largely temporary. In this study, we examine the regenerative potential of vitamin B17 to improve salivary gland function. OBJECTIVES: The present investigation aims to identify the effect of vitamin B17 (amygdaline) on the irradiated parotid salivary gland of albino rats. MATERIAL AND METHODS: Twenty-eight adult male albino rats were randomly divided into two groups subjected to irradiation procedure. Fourteen were in the control group, receiving a daily 5 mL saline by oral gavage (7 rats for 14 days and 7 rats for 30 days) while the other fourteen were treated with a daily dose of vitamin B17 (grounded apricot kernel; GAK) at 400 mg/kg in 5 mL of saline by oral gavage (7 rats for 14 days and 7 rats for 30 days). The parotid glands were dissected from the two groups at 14 and 30 days from the day of exposure to irradiation. The parotid gland sections were subjected to H&E stain, immunohistochemical localization of epidermal growth factor (EGF) and PCR using transforming growth factor beta 2 (TGF-ß2). RESULTS: The histological abnormalities corroborate with the immunohistochemical localization of EGF and the PCR results of TGF ß2, as their up-regulation in the control group demonstrate oxidative stresses and inflammation. The Treatment with GAK decreased oxidative stress and inflammation while promoting tissue regeneration. CONCLUSIONS: Vitamin B17 is a promising anti-inflammatory agent that boosts immunity, as the experimental group showed better histological architecture of the parotid gland than the other one.

Effects of amygdalin on ferroptosis and oxidative stress in diabetic retinopathy progression via the NRF2/ARE signaling pathway.

Oxidative stress has been involved in the pathogenesis of diabetic retinopathy (DR). Amygdalin is an effective component of bitter almond that exhibits excellent antioxidant properties. We explored the effects of amygdalin on ferroptosis and oxidative stress in high-glucose (HG)-stimulated human retinal endothelial cells (HRECs) via the NRF2/ARE pathway. HG-stimulated HRECs were used to establish a DR model. Cell viability was evaluated using the MTT assay. The release of lactate dehydrogenase was used to evaluate cell toxicity. The protein levels of NRF2, NQO1, and HO-1 were detected using western blotting. The GSH, GSSG, GPX4, SOD, CAT, MDA, and Fe2+ levels in the HRECs were also detected. Flow cytometry was used to detect reactive oxygen species (ROS) using a fluorescent probe. Immunofluorescence staining was performed to detect NRF2 expression. The results revealed that HG stimulation decreased the levels of GSH, GPX4, SOD, and CAT but increased those of MDA, ROS, GSSG, and Fe2+ in HRECs. Ferrostatin-1 treatment reversed the effects of HG stimulation, whereas erastin aggravated these effects. Amygdalin treatment relieved HG-induced injury in HRECs. Amygdalin treatment promoted the nuclear transport of NRF2 in HG-stimulated HRECs. NQO1 and HO-1 levels were upregulated in HG-stimulated HRECs after amygdalin treatment. An inhibitor of NRF2 reversed the effects of amygdalin. Therefore, amygdalin treatment inhibited ferroptosis and oxidative stress in HG-stimulated HRECs by activating the NRF2/ARE signaling pathway.

Amygdalin epimers exert discrepant anti-pulmonary fibrosis activity via inhibiting TGF-ß1/Smad2/3 pathway.

Idiopathic pulmonary fibrosis (IPF) represents a chronic and progressive tissue repair response that leads to irreversible scarring and lung remodeling. The decoction of bitter almond usually contains amygdalin epimers in traditional clinical application for lung disease. To reveal the differences of cytotoxicity and antifibrotic effect between amygdalin epimers, and potential mechanism is also explored. The cytotoxicity of amygdalin epimers were evaluated with MRC-5 cells in vitro. Their antifibrotic activities were evaluated in bleomycin-induced C57BL/6 mice and TGF-ß1-induced MRC-5 cells. Here we demonstrated that L-amygdalin is more toxic of the amygdalin epimers in MRC-5 cells, and D-amygdalin is more effective in anti-pulmonary fibrosis among the amygdalin epimers in bleomycin-induced C57BL/6 mice. Herein, it was observed that D-amygdalin had a stronger inhibitory effect on inflammation than L-amygdalin, and had similar results in inhibiting the mRNA and protein expression levels of fibrosis-related biomarkers. The mechanism of anti-pulmonary fibrosis showed that amygdalin epimers suppressing expression of phosphorylation of Smads2/3, which implying deactivation of the TGF-ß1induced Smads2/3 signal pathway. This study evaluates the amygdalin epimers cytotoxicity and antifibrotic effect, and its mechanisms were related to the TGF-ß1/Smads2/3 signal pathway. It provides a reference for clinical safety and effectiveness of amygdalin epimers.

Amygdalin alleviated TGF-ß-induced epithelial-mesenchymal transition in bronchial epithelial cells.

OBJECTIVE: Transforming growth factor-beta TGF-ß-induced epithelial-mesenchymal transition (EMT) in bronchial epithelial cells contributes to airway wall remodeling in asthma. This study aims to explore the role of amygdalin, an active ingredient in bitter almonds, in TGF-ß-induced EMT in bronchial epithelial cells and to elucidate the possible mechanisms underlying its biological effects. METHODS: An asthmatic mouse model was established through ovalbumin induction. Primary mouse bronchial epithelial cells and a human bronchial epithelial cell line were incubated with transforming growth factor-beta (TGF-ß) to induce EMT, whose phenotype of cells was evaluated by the expressions of EMT markers [alpha-smooth muscle actin (α-SMA), vimentin, and fibronectin] and cell migration capacity. A co-immunoprecipitation assay was performed to assess the ubiquitination of heparanase (HPSE). RESULTS: In asthmatic model mice, amygdalin treatment relieved airway wall remodeling and decreased expressions of EMT markers (α-SMA and vimentin). In TGF-ß-treated bronchial epithelial cells, amygdalin treatment decreased the mRNA and protein levels of EMT markers (α-SMA, vimentin, and fibronectin) without impairing cell viability. Through the Swiss Target Prediction database, HPSE was screened as a candidate downstream target for amygdalin. HPSE overexpression further promoted TGF-ß-induced EMT while the HPSE inhibitor suppressed TGF-ß-induced EMT in bronchial epithelial cells. In addition, HPSE overexpression reversed the inhibitory effect of amygdalin on TGF-ß-induced EMT in bronchial epithelial cells. The following mechanism exploration revealed that amygdalin downregulated HPSE expression by enhancing ubiquitination. CONCLUSION: Our study showed that amygdalin inhibited TGF-ß-induced EMT in bronchial epithelial cells and found that the anti-EMT activity of amygdalin might be related to its regulatory effect on HPSE expression.

Determination of Amygdalin in Apricot Kernels and Almonds Using LC-MS/MS.

BACKGROUND: Cyanogenic glycosides are secondary metabolites in plants. In almonds and apricot kernels, amygdalin is an abundant cyanogenic glycoside. Upon consumption, amygdalin is enzymatically metabolized into hydrogen cyanide. Depending on the number of kernels consumed and the amygdalin concentration, ingestion of amygdalin-containing kernels may result in adverse effects. To better understand the US marketplace, the development and validation of analytical methods to reliably measure amygdalin in apricot kernels and almonds is needed to support the collection of occurrence and consumption data in retail products. OBJECTIVE: The aim of this study was to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitation of amygdalin in apricot kernels and almonds following the U.S. Food and Drug Administration (FDA). Foods Program Guidelines for the Validation of Chemical Methods, 3rd Edition. METHODS: Apricot kernels and almonds were cryogenically homogenized and extracted using methanol containing an internal standard (IS), geniposide, followed by filtration, dilution, and LC-MS/MS analysis. Matrix effects were minimized using dilution. Quantitation was achieved using an external, solvent-based calibration. RESULTS: The amygdalin response was linear (r2 > 0.99) over a range of 0.05-50 µg/mL. The recovery of amygdalin spiked at 10-10 000 µg/g in sweet apricot kernels, raw almond, and dry-roasted almond ranged from 90 to 107% with RSDs ≤6%. The method limit of detection and limit of quantitation was 0.8 and 2.5 ng/g, respectively. Amygdalin concentrations in 18 market samples ranged from 2 to 24 000 µg/g. Corresponding estimates of cyanide concentration ranged from 0.2 to 1420 µg/g. CONCLUSIONS: Method performance meets the acceptance criteria defined by FDA guidelines and is fit for purpose for the analysis of amygdalin in apricot kernels and almonds. HIGHLIGHTS: An LC-MS/MS method is developed for the quantification of amygdalin in apricot kernels and almonds.

Host-microbiome metabolism of a plant toxin in bees.

While foraging for nectar and pollen, bees are exposed to a myriad of xenobiotics, including plant metabolites, which may exert a wide range of effects on their health. Although the bee genome encodes enzymes that help in the metabolism of xenobiotics, it has lower detoxification gene diversity than the genomes of other insects. Therefore, bees may rely on other components that shape their physiology, such as the microbiota, to degrade potentially toxic molecules. In this study, we show that amygdalin, a cyanogenic glycoside found in honey bee-pollinated almond trees, can be metabolized by both bees and members of the gut microbiota. In microbiota-deprived bees, amygdalin is degraded into prunasin, leading to prunasin accumulation in the midgut and hindgut. In microbiota-colonized bees, on the other hand, amygdalin is degraded even further, and prunasin does not accumulate in the gut, suggesting that the microbiota contribute to the full degradation of amygdalin into hydrogen cyanide. In vitro experiments demonstrated that amygdalin degradation by bee gut bacteria is strain-specific and not characteristic of a particular genus or species. We found strains of Bifidobacterium, Bombilactobacillus, and Gilliamella that can degrade amygdalin. The degradation mechanism appears to vary since only some strains produce prunasin as an intermediate. Finally, we investigated the basis of degradation in Bifidobacterium wkB204, a strain that fully degrades amygdalin. We found overexpression and secretion of several carbohydrate-degrading enzymes, including one in glycoside hydrolase family 3 (GH3). We expressed this GH3 in Escherichia coli and detected prunasin as a byproduct when cell lysates were cultured with amygdalin, supporting its contribution to amygdalin degradation. These findings demonstrate that both host and microbiota can act together to metabolize dietary plant metabolites.

Most plants produce chemicals that are toxic to at least some animals. Whether or not the toxins are harmful to a particular animal depends on how much they consume and the specific biochemistry that occurs during digestion. The enzymes produced in the gut both by the animal and by the microbes that reside there often help break down toxic substances into less harmful molecules. However, some products of this breakdown can be toxic themselves. While these products can harm the animal, they may also be detrimental to parasites living in the gut, resulting in an overall positive effect. Almonds and their pollen are consumed by humans and bees without apparent harmful effects. However, almonds contain amygdalin, a molecule that can produce the highly toxic compound hydrogen cyanide upon digestion. Although amygdalin can be toxic to bees in high doses, the amount usually found in almond nectar is not harmful, and indeed, it may protect bees from parasites. Motta et al. wanted to know how amygdalin is digested in the gut of bees, and whether gut microbes have a role in this digestion. To answer these questions, Motta et al. compared the effects of consuming amygdalin on normal bees and bees lacking gut microbes. Bees without gut microbes broke down amygdalin into a harmless substance called prunasin. However, only bees with gut microbes could further break down prunasin into hydrogen cyanide. Interestingly, the full metabolism of amygdalin had no detectable effect on whether the bees survived for longer times or on which microbes were found in the gut. Motta et al. also found some gut bacteria in bees that can break down amygdalin and release hydrogen cyanide, and identified the enzyme responsible for the process. When the gene encoding this enzyme was inserted into a different species of bacteria, the second species gained the ability to break down amygdalin. The findings of Motta et al. explain a role of gut microbes in processing amygdalin in bees. In the future, this may be the key to understanding how humans and other creatures process plant toxins. Future work on the relationship between animals and microbes living in their guts could help scientists understand how to manipulate the digestion and processing of toxins, nutrients, or drugs to benefit human health.

Co-opted genes of algal origin protect C. elegans against cyanogenic toxins.

Amygdalin is a cyanogenic glycoside enriched in the tissues of many edible plants, including seeds of stone fruits such as cherry (Prunus avium), peach (Prunus persica), and apple (Malus domestica). These plants biosynthesize amygdalin in defense against herbivore animals, as amygdalin generates poisonous cyanide upon plant tissue destruction.1,2,3,4 Poisonous to many animals, amygdalin-derived cyanide is detoxified by potent enzymes commonly found in bacteria and plants but not most animals.5 Here we show that the nematode C. elegans can detoxify amygdalin by a genetic pathway comprising cysl-1, egl-9, hif-1, and cysl-2. A screen of a natural product library for hypoxia-independent regulators of HIF-1 identifies amygdalin as a potent activator of cysl-2, a HIF-1 transcriptional target that encodes a cyanide detoxification enzyme in C. elegans. As a cysl-2 paralog similarly essential for amygdalin resistance, cysl-1 encodes a protein homologous to cysteine biosynthetic enzymes in bacteria and plants but functionally co-opted in C. elegans. We identify exclusively HIF-activating egl-9 mutations in a cysl-1 suppressor screen and show that cysl-1 confers amygdalin resistance by regulating HIF-1-dependent cysl-2 transcription to protect against amygdalin toxicity. Phylogenetic analysis indicates that cysl-1 and cysl-2 were likely acquired from green algae through horizontal gene transfer (HGT) and functionally co-opted in protection against amygdalin. Since acquisition, these two genes evolved division of labor in a cellular circuit to detect and detoxify cyanide. Thus, algae-to-nematode HGT and subsequent gene function co-option events may facilitate host survival and adaptation to adverse environmental stresses and biogenic toxins.

Whole-genome analysis guided molecular mechanism of cyanogenic glucoside degradation by yeast isolated from Prunus mume fruit syrup.

Consumption of fermented Prunus mume fruit (maesil) sugar syrup raise a health concern due to the presence of the cyanogenic glucoside amygdalin. The goal of the present study was to investigate the amygdalin degradation potential and genome profile of the native microbes found in maesil syrup. The microbial profile analysis revealed that yeast is the predominant microorganism native to maesil syrup and that the isolated yeast cells showed a remarkable potential for amygdalin reduction (99.7%). Moreover, the reduction in amygdalin was inversely proportional to the growth of the isolated yeast. The whole-genome analysis revealed that the isolated yeast is Zygosaccharomyces rouxii (genome size 10 Mb, 39.25% of GC content). Of the 5250 genes (64.88%) predicted in the Z. rouxii genome, 5245 (99.90%) were annotated using NCBI Non-Redundant, UniProt, and InterProScan databases. The genome of the isolated Z. ruoxii harbored 2.03% of repeats and 0.68% of non-coding RNAs. Protein prediction indicated that ß-glycosidases and hydroxynitrile lyase may play a key role in amygdalin degradation. The predicted degradation initiated by ß-glycosidases that hydrolyze α-glucosidic bonds of amygdalin results in α-hydroxy nitriles (cyanohydrins) that are subsequently converted into carbonyl compounds (benzaldehyde) and hydrogen cyanide catalyzed by hydroxynitrile lyases. Present findings provide valuable data for constructing engineered microorganisms that can degrade amygdalin. Further analysis of Z. rouxii may elucidate the exact mechanism of amygdalin reduction in the production of maesil syrup.

Amygdalin Induced Mitochondria-Mediated Apoptosis of Lung Cancer Cells via Regulating NF[Formula: see text]B-1/NF[Formula: see text]B Signaling Cascade in Vitro and in Vivo.

Lung cancer is the most commonly diagnosed cancer and the leading cause of cancer death worldwide. Amygdalin, a natural compound commonly distributed in plants of the Rosaceae species, owns anticancer activity, less side effects, wide source, and relatively low price. Although the apoptosis is a central process activated by amygdalin in cancer cells, the underlying molecular mechanisms through which amygdalin induces the apoptosis of lung cancer cells remain poorly understood. In this research work, amygdalin could suppress the proliferation of lung cancer A549 and PC9 cells by CCK8 assay. Amygdalin significantly promoted the apoptosis of lung cancer A549 and PC9 cells stained with Annexin V-FITC/PI by flow cytometry assay. Furthermore, amygdalin dose-dependently decreased the mitochondrial membrane potential (MMP) with JC-1 dye by flow cytometry. To investigate the underlying molecular mechanisms through which amygdalin induced mitochondria-mediated apoptosis of cancer cells, the differentially-expressed genes with a fold change >2.0 and p < 0.05 were acquired from the cDNA microarray analysis. The results of qRT-PCR further confirmed that the differentially-expressed level of the NF[Formula: see text]B-1 gene was most obviously enhanced in lung cancer cells treated with amygdalin. The results of immunofluorescence staining, Western blotting and siRNA knockdown indicated that amygdalin induced mitochondria-mediated apoptosis of lung cancer cells via enhancing the expression of NF[Formula: see text]B-1 and inactivating NF[Formula: see text]B signaling cascade and further changing the expressions of proteins (Bax, Bcl-2, cytochrome C, caspase 9, caspase 3 and PARP) related to apoptosis, which were further checked by in vivo study of the lung cancer cell xenograft mice model accompanying with immunohistochemical staining and TUNEL staining. Our results indicated that amygdalin might be a potential activator of NF[Formula: see text]B-1, which sheds more light on the molecular mechanism of anticancer effects of amygdalin. These results highlighted amygdalin as a potential therapeutic anticancer agent, which warrants its development as a therapy for lung cancer.

Amygdalin protects against acetaminophen-induced acute liver failure by reducing inflammatory response and inhibiting hepatocyte death.

Amygdalin is a natural compound from Bitter Apricot Seed which is reported to have anti-inflammatory activity. Acetaminophen (APAP) resulted in drug-induced liver injury is the main cause of acute liver failure (ALI) worldwide and only N-acetylcysteine is the accepted detoxification drug. However, there is no effective medicine to perfect the hepatocyte death and secondary inflammation injury. In this study, we aim to investigate the protective effect of Amygdalin in the APAP-induced acute liver failure mice model. We establish the ALI model via intraperitoneal APAP injection and mice were treated with Amygdalin with intraperitoneal injection. We detected liver enzyme and histological change to evaluate the liver injury. We measured oxidative damage markers and inflammatory cell infiltration of liver tissues. At last, we investigated the mechanism of Amygdalin on protecting hepatocytes. Results showed that Amygdalin reduced ALT/AST level and decreased necrotic area of liver tissue. In addition, Amygdalin reduced the count of MPO+(neutrophils) and F4/80+(macrophages) of the liver and inhibited IL-6, TNF-a, and IL-1b expression. Amygdalin reduced liver SOD and MDA levels and increased Nrf2/NQO1/HO1 protein expression. Moreover, Amygdalin reduced TUNEL+ and P-MLKL + staining cells in liver tissue. Mechanically, Amygdalin promoted phosphorylation of AKT and suppressed JNK/RIP3/MLKL signaling.

Amygdalin promotes the activity of T cells to suppress the progression of HBV-related hepatocellular carcinoma via the JAK2/STAT3 signaling pathway.

BACKGROUND: Hepatitis B virus (HBV) infection is a high-risk factor of hepatocellular carcinoma (HCC). Cellular immune responses are essential for HCC development, and the CD4+ and CD8+ T subtypes are identified as the primary anti-tumor immune cells. In the study, we investigated the effect and mechanism of amygdalin in the cellular immune response in HBV-related HCC and HCC progression. METHODS: The cell proliferation was examined by MTT analysis. Cells metastasis ability was detected by Invasion and migration assays. Quantification of apoptotic cells was performed with Flow cytometer assay. The protein levels of p-STAT3, STAT3, p-JAK2, JAK2, caspase-3, cleaved caspase-3 were detected by performing immunoblotting assays. RESULTS: We demonstrate that amygdalin treatment could rescue the HBV-T cell viability and IFN-γ and TNF-αproduction. In HBV-T cells, the MFI levels of CD8+ are lower than that in NC-T cells. Moreover, the phosphorylation levels of STAT3 and JAK2 are higher in HBV-T cells, compared to those in NC-T cells, and then reduced by amygdalin treatment. Co-culture with HBV-T cells could reduce IFN-γ and TNF-α, production while increase IL-6 and IL-10 production in HepG2.2.15 cells; these alterations could be partially reversed by amygdalin pretreatment. Finally, co-culture with HBV-T cells significantly promoted the cell viability, inhibited the apoptosis, and promoted the migration of HepG2.2.15 cells, and these alterations could be partially reversed by amygdalin treatment. CONCLUSION: Our findings provide a rationale for further studies on the functions and mechanism of amygdalin inhibiting HBV-related HCC cell proliferation, invasion, and migration via T cell-mediated tumor immunity.

Rebound metabolic acidosis following intentional amygdalin supplement overdose.

Introduction: Amygdalin, marketed misleadingly as supplement "Vitamin B17," is a cyanogenic glycoside. When swallowed, it is hydrolyzed into cyanide in the small intestine, which causes histotoxic hypoxia via inhibition of cytochrome c oxidase. It remains available for purchase online despite a ban from the US Food and Drug Administration. We report a case of massive intentional amygdalin overdose resulting in recurrent cyanide toxicity after initial successful antidotal therapy.Case summary: A 33-year-old woman intentionally ingested 20 g of "apricot POWER B17 Amygdalin" supplements. She presented five hours post-ingestion with vital signs: P 127 bpm, BP 112/65 mmHg, RR 25/min, temperature 98.1 °F, and SpO2 98% RA. She was in agitated delirium, diaphoretic, and mydriatic. Her VBG was notable for a pH of 7.27 (rr 7.32-7.42) and lactate 14.1 mmol/L (rr 0.5-2.2), with ECG demonstrating QTc 538 ms (normal <440 ms). She was empirically treated with hydroxocobalamin and supportive care, but worsened clinically, requiring intubation and additional hydroxocobalamin and sodium thiosulfate, which resolved her toxicity. Twelve hours later, she developed recurrent hypotension, acidemia, and QTc prolongation that resolved with repeat hydroxocobalamin and sodium thiosulfate dosing.Discussion: Our case demonstrates rebound metabolic acidosis after massive amygdalin overdose. Toxicity was associated with prolonged QTc, which warrants further investigation into clinical significance. Redosing of combination antidotal therapy suggested efficacy without adverse effects.

Transport properties of paeoniflorin and amygdalin across caco-2 cell monolayer model and their modulation of cytochrome p450 metabolism.

Paeoniflorin and amygdalin are two major active saponins constituents in some Chinese herbal formulas used for cardio-cerebrovascular diseases. However, their intestinal absorption property and metabolic characteristics have not been clarified. The aim of this work was to study the absorption property of Paeoniflorin and Amygdalin across Caco-2 cell monolayer and their metabolic characteristics on the activity of cytochrome P450 (CYP450) enzyme. The results showed that the transport amount of Paeoniflorin and Amygdalin was positively correlated with the time and concentrations, and the transport amount from AP side to BL side was higher than that from BL to AP. The absorptions of Paeoniflorin and Amygdalin were reduced by P-glycoprotein, which provided the pharmacokinetic basis for their clinical application. Furthermore, we demonstrated that Paeoniflorin and Amygdalin had obvious inhibiting effects on CYP2C9 and CYP2E1. The transports of Paeoniflorin and Amygdalin across Caco-2 cell monolayer model were deduced as the passive transport, which indicated that the present bioassay system was appropriate and reliable for the evaluation of the transport characteristics and metabolic characteristics of active ingredient groups in Bu-yang-huan-wu decoction. Moreover, this research method may also be suitable for the appropriate bioactivity and metabolic characteristics analysis of other plant extracts.

Changes of amygdalin and volatile components of apricot kernels during the ultrasonically-accelerated debitterizing.

Ultrasound has been regarded as an efficient novel technique for debitterizing of the apricot kernels, but its influence is severely concerned on the possible epimerization of d-amygdalin to the l-amygdalin, a more potentially toxigenic compound. Considering this, the experiments were conducted to investigate the epimerization of d-amygdalin and the volatile components in the debitterizing water, which were separated and identified by the high performance liquid chromatography (HPLC) and gas chromatography with a mass spectrometer (GC-MS), respectively. The results indicate that the ultrasonically-debitterizing did not cause the epimerization of d-amygdalin to the l-amygdalin, while the procedure can be greatly accelerated due to the rapid mass transfer and degradation of d-amygdalin induced by ultrasound irradiation. In addition, the water from the ultrasonically-debitterizing of apricot kernels exerted more aromas compared with that of the conventional debitterizing, which might have more applications about this kind of water. In a word, ultrasound can be safely applied in the debitterizing industry of apricot kernels.

Mutation of a bHLH transcription factor allowed almond domestication.

Wild almond species accumulate the bitter and toxic cyanogenic diglucoside amygdalin. Almond domestication was enabled by the selection of genotypes harboring sweet kernels. We report the completion of the almond reference genome. Map-based cloning using an F1 population segregating for kernel taste led to the identification of a 46-kilobase gene cluster encoding five basic helix-loop-helix transcription factors, bHLH1 to bHLH5. Functional characterization demonstrated that bHLH2 controls transcription of the P450 monooxygenase-encoding genes PdCYP79D16 and PdCYP71AN24, which are involved in the amygdalin biosynthetic pathway. A nonsynonymous point mutation (Leu to Phe) in the dimerization domain of bHLH2 prevents transcription of the two cytochrome P450 genes, resulting in the sweet kernel trait.

The anti-cancer effect of amygdalin on human cancer cell lines.

Derived from rosaceous plant seed, amygdalin belongs to aromatic cyanogenic glycoside group, and its anticancer effects have been supported by mounting evidence. In this study, we objected to investigate amygdalin effect on two antiapoptotic genes (Survivin, XIAP) and two lncRNAs (GAS5, MALAT1) in human cancer cells (A549, MCF7, AGS). Employing RT-qPCR analysis, we compared the mRNA levels of the genes related to apoptosis in A549, MCF7, and AGS cancer cells between amygdalin-treated (24, 48 and 72 h) and un-treated groups. RNA was extracted from both cell groups and then cDNAs were synthesized. The changes in the gene expression levels were specified using ΔΔCt method. RT-qPCR analysis has revealed that the expression of Survivin, XIAP, GAS5 and MALAT1 in amygdala-treated cancer cells were significantly different, compared to the un-treated cells. However, these expressions were different depending on the treatment time. According to the results, amygdalin significantly inhibited the expression level of Survivin, and XIAP genes in treated via untreated group. Our findings suggest that amygdalin might have an anticancer effect due to the various gene expressions in A549, MCF7, and AGS human cancer cells, showing it's potential as a natural therapeutic anticancer drug.

Elucidation of the Amygdalin Pathway Reveals the Metabolic Basis of Bitter and Sweet Almonds (Prunus dulcis).

Almond (Prunus dulcis) is the principal Prunus species in which the consumed and thus commercially important part of the fruit is the kernel. As a result of continued selection, the vast majority of almonds have a nonbitter kernel. However, in the field, there are trees carrying bitter kernels, which are toxic to humans and, consequently, need to be removed. The toxicity of bitter almonds is caused by the accumulation of the cyanogenic diglucoside amygdalin, which releases toxic hydrogen cyanide upon hydrolysis. In this study, we identified and characterized the enzymes involved in the amygdalin biosynthetic pathway: PdCYP79D16 and PdCYP71AN24 as the cytochrome P450 (CYP) enzymes catalyzing phenylalanine-to-mandelonitrile conversion, PdUGT94AF3 as an additional monoglucosyl transferase (UGT) catalyzing prunasin formation, and PdUGT94AF1 and PdUGT94AF2 as the two enzymes catalyzing amygdalin formation from prunasin. This was accomplished by constructing a sequence database containing UGTs known, or predicted, to catalyze a ß(1→6)-O-glycosylation reaction and a Basic Local Alignment Search Tool search of the draft version of the almond genome versus these sequences. Functional characterization of candidate genes was achieved by transient expression in Nicotiana benthamiana Reverse transcription quantitative polymerase chain reaction demonstrated that the expression of PdCYP79D16 and PdCYP71AN24 was not detectable or only reached minute levels in the sweet almond genotype during fruit development, while it was high and consistent in the bitter genotype. Therefore, the basis for the sweet kernel phenotype is a lack of expression of the genes encoding the two CYPs catalyzing the first steps in amygdalin biosynthesis.

Reconfigured Cyanogenic Glucoside Biosynthesis in Eucalyptus cladocalyx Involves a Cytochrome P450 CYP706C55.

Cyanogenic glucosides are a class of specialized metabolites widespread in the plant kingdom. Cyanogenic glucosides are α-hydroxynitriles, and their hydrolysis releases toxic hydrogen cyanide, providing an effective chemical defense against herbivores. Eucalyptus cladocalyx is a cyanogenic tree, allocating up to 20% of leaf nitrogen to the biosynthesis of the cyanogenic monoglucoside, prunasin. Here, mass spectrometry analyses of E. cladocalyx tissues revealed spatial and ontogenetic variations in prunasin content, as well as the presence of the cyanogenic diglucoside amygdalin in flower buds and flowers. The identification and biochemical characterization of the prunasin biosynthetic enzymes revealed a unique enzyme configuration for prunasin production in E. cladocalyx This result indicates that a multifunctional cytochrome P450 (CYP), CYP79A125, catalyzes the initial conversion of l-phenylalanine into its corresponding aldoxime, phenylacetaldoxime; a function consistent with other members of the CYP79 family. In contrast to the single multifunctional CYP known from other plant species, the conversion of phenylacetaldoxime to the α-hydroxynitrile, mandelonitrile, is catalyzed by two distinct CYPs. CYP706C55 catalyzes the dehydration of phenylacetaldoxime, an unusual CYP reaction. The resulting phenylacetonitrile is subsequently hydroxylatedby CYP71B103 to form mandelonitrile. The final glucosylation step to yield prunasin is catalyzed by a UDP-glucosyltransferase, UGT85A59. Members of the CYP706 family have not been reported previously to participate in the biosynthesis of cyanogenic glucosides, and the pathway structure in E. cladocalyx represents an example of convergent evolution in the biosynthesis of cyanogenic glucosides in plants.

Metabolomics and Biochemical Approaches Link Salicylic Acid Biosynthesis to Cyanogenesis in Peach Plants.

Despite the long-established importance of salicylic acid (SA) in plant stress responses and other biological processes, its biosynthetic pathways have not been fully characterized. The proposed synthesis of SA originates from chorismate by two distinct pathways: the isochorismate and phenylalanine (Phe) ammonia-lyase (PAL) pathways. Cyanogenesis is the process related to the release of hydrogen cyanide from endogenous cyanogenic glycosides (CNglcs), and it has been linked to plant plasticity improvement. To date, however, no relationship has been suggested between the two pathways. In this work, by metabolomics and biochemical approaches (including the use of [13C]-labeled compounds), we provide strong evidences showing that CNglcs turnover is involved, at least in part, in SA biosynthesis in peach plants under control and stress conditions. The main CNglcs in peach are prunasin and amygdalin, with mandelonitrile (MD), synthesized from phenylalanine, controlling their turnover. In peach plants MD is the intermediary molecule of the suggested new SA biosynthetic pathway and CNglcs turnover, regulating the biosynthesis of both amygdalin and SA. MD-treated peach plants displayed increased SA levels via benzoic acid (one of the SA precursors within the PAL pathway). MD also provided partial protection against Plum pox virus infection in peach seedlings. Thus, we propose a third pathway, an alternative to the PAL pathway, for SA synthesis in peach plants.