Effect of Pyridoxine Derivative B6NO on Transcription Factor Nrf2 Activity and Cytotoxic Properties of Doxorubicin In Vitro.

The effect of a new pyridoxine derivative B6NO on doxorubicin cytotoxicity and Nrf2-dependent cellular processes in vitro was studied. Antioxidant B6NO enhances the cytotoxic effect of doxorubicin on tumor cells, which is associated with G2/M cell division arrest and an increase in activity of proapoptotic enzyme caspase-3. The antioxidant promotes intracellular accumulation and nuclear translocation of Nrf2 transcription factor in non-tumor and tumor cells. In non-tumor cells, B6NO increases the expression of antioxidant system proteins and reduces ROS generation in the presence of doxorubicin. In tumor cells, no activation of Nrf2-dependent processes occurs under the action of the antioxidant. Our findings demonstrate the prospect of further studies of pyridoxine derivatives as antioxidants to reduce adverse reactions during chemotherapy.

A critical review of current technologies used to reduce ginkgotoxin, ginkgotoxin-5'-glucoside, ginkgolic acid, allergic glycoprotein, and cyanide in Ginkgo biloba L. seed.

The Ginkgo biloba has astonished scholars globally with enormous bioactives, with sales exceeding $10 billion since 2017. The Ginkgo biloba seed (GBS) is an essential part of culinary culture. Nevertheless, toxins in fresh Ginkgo biloba seed (GBS) have limited GBSs' daily consumption. Ginkgotoxin and ginkgotoxin-5-glucoside cause poisoning, tonic-clonic convulsions, and neurotoxic effects. Ginkgolic acid causes cytotoxicity and allergies. Allergic glycoprotein in GBS causes nausea, seizures, dyspnea, mydriasis, vomiting, and bellyache. The amygdalin-derived hydrocyanic acid cause dizziness, vomiting, cramping, and sleeping disorders. Food products are frequently exposed to various processing techniques to increase food safety and functionality. As a result, this review focused on the technologies that have been used to minimize toxins in GBS. In addition, a comparison of these techniques was made based on their benefits, drawbacks, feasibility, pharmacological activities, and future direction or opportunities to improve current ones were provided.

Genetic Analysis Using Vitamin B6 Antagonist 4-Deoxypyridoxine Uncovers a Connection between Pyridoxal 5'-Phosphate and Coenzyme A Metabolism in Salmonella enterica.

Pyridoxal 5'-phosphate (PLP) is an essential cofactor for organisms in all three domains of life. Despite the central role of PLP, many aspects of vitamin B6 metabolism, including its integration with other biological pathways, are not fully understood. In this study, we examined the metabolic perturbations caused by the vitamin B6 antagonist 4-deoxypyridoxine (dPN) in a ptsJ mutant of Salmonella enterica serovar Typhimurium LT2. Our data suggest that PdxK (pyridoxal/pyridoxine/pyridoxamine kinase [EC 2.7.1.35]) phosphorylates dPN to 4-deoxypyridoxine 5'-phosphate (dPNP), which in turn can compromise the de novo biosynthesis of PLP. The data are consistent with the hypothesis that accumulated dPNP inhibits GlyA (serine hydroxymethyltransferase [EC 2.1.2.1]) and/or GcvP (glycine decarboxylase [EC 1.4.4.2]), two PLP-dependent enzymes involved in the generation of one-carbon units. Our data suggest that this inhibition leads to reduced flux to coenzyme A (CoA) precursors and subsequently decreased synthesis of CoA and thiamine. This study uncovers a link between vitamin B6 metabolism and the biosynthesis of CoA and thiamine, highlighting the integration of biochemical pathways in microbes. IMPORTANCE PLP is a ubiquitous cofactor required by enzymes in diverse metabolic networks. The data presented here expand our understanding of the toxic effects of dPN, a vitamin B6 antagonist that is often used to mimic vitamin B6 deficiency and to study PLP-dependent enzyme kinetics. In addition to de novo PLP biosynthesis, we define a metabolic connection between vitamin B6 metabolism and synthesis of thiamine and CoA. This work provides a foundation for the use of dPN to study vitamin B6 metabolism in other organisms.

Age-dependent alteration in metabolism of vitamin B6 , neurotransmitters, and amino acids after 4'-O-methylpyridoxine administration in rats.

4'-O-methylpyridoxine (MPN), a recognized antivitamin B6 compound, is a potentially poisonous substance found in Ginkgo biloba L. In this work, the effects of MPN on the metabolism of vitamin B6 , neurotransmitters, and amino acids were compared in the plasma and brain of young and adult rats under various administration times. Results showed that the contents of MPN residues in the plasma and brain of young rats were 12.72 and 14.76 µM higher than adult rats, respectively. Moreover, the levels of 5-hydroxytryptamine and dopamine in the brain of young rats have decreased by 13.78% and 7.19%, respectively, compared with the control group, at 2 h after MPN administration. Furthermore, the principal component analysis revealed that MPN was an important contributor to the amino acid composition in the brain of young rats. These results suggest that age may lead to different toxic effects of MPN. PRACTICAL APPLICATION: 4'-O-methylpyridoxine is primarily responsible for poisoning due to overconsumption of Ginkgo biloba seeds. This study will provide an exploratory understanding of the age-dependent toxicity of 4'-O-methylpyridoxine.

A Facile Method to Determine the Native Contents of 4'-O-Methylpyridoxine and 4'-O-Methylpyridoxine-5'-glucoside in Ginkgo biloba Seeds.

4'-O-Methylpyridoxine (MPN) and MPN-5'-glucoside (MPNG) are collectively known as ginkgotoxin, which are the main toxic ingredients of excessive consumption of Ginkgo biloba seeds. Water extraction is the generally adopted sample preparation method for high-performance liquid chromatography determination of ginkgotoxin. However, endogenous enzymes such as glycosidases in Ginkgo biloba seeds can hydrolyze MPNG to MPN in the process of water extraction, which will result in the measured contents of MPN and MPNG but not their natural contents in Ginkgo biloba seeds. In this work, inhibitors for the endogenous enzymes were first screened, and it was found that silver fluoride could effectively inhibit endogenous enzymes such as glucosidase and phosphatase. The optimized concentration of silver fluoride was 25 mmol/L, which could effectively inhibit the endogenous enzymes for more than 60 h. A new sample preparation method based on water extraction with 25 mmol/L silver fluoride addition was thus developed. This method was employed to determine the native contents of MPN and MPNG in the exotesta and kernel of five Ginkgo biloba seed cultivars. The result showed that the contents of MPNG in the exotesta and kernel of five cultivars were significantly higher than those of MPN. MPNG was present at high content in raw seeds, which was the main form of ginkgotoxin in seeds. The method established in this work is simple and effective and can be used to accurately quantify the native contents of MPN and MPNG.

In vivo toxicity assessment of 4'-O-methylpyridoxine from Ginkgo biloba seeds: Growth, hematology, metabolism, and oxidative parameters.

4'-O-methylpyridoxine (MPN), a recognized antivitamin B6 compound, is a potentially poisonous substance found in Ginkgo biloba seeds and leaves. In this work, the body weights, histopathological changes, plasma vitamin B6 (VB6), biochemical parameters, oxidative stress responses, and amino acids of rats were investigated after intragastric administration of MPN for 15 days. Results showed that intragastric administration of 50 mg/kg BW MPN caused pathological changes in the brain and heart tissues of rats. Administration of 10 mg/kg and 30 mg/kg BW MPN can significantly increase VB6 analogs in the plasma of rats, such as pyridoxal-5'-phosphate, pyridoxal. Results of biochemical parameters indicated that MPN can damage brains and hearts by changing the enzyme activity of these organs. These results suggest that consumption of Ginkgo biloba seeds for the long term, even in a small quantity, may lead to poisoning.

4'-O-methylpyridoxine: Preparation from Ginkgo biloba Seeds and Cytotoxicity in GES-1 Cells.

Ginkgo biloba seeds are wildly used in the food and medicine industry. It has been found that 4'-O-methylpyridoxine (MPN) is responsible for the poisoning caused by G. biloba seeds. The objective of this study was to explore and optimize the extraction method of MPN from G. biloba seeds, and investigate its toxic effect on human gastric epithelial cells (GES-1) and the potential related mechanisms. The results showed that the extraction amount of MPN was 1.933 µg/mg, when extracted at 40 °C for 100 min, with the solid-liquid ratio at 1:10. MPN inhibited the proliferation of GES-1 cells, for which the inhibition rate was 38.27% when the concentration of MPN was 100 µM, and the IC50 value was 127.80 µM; meanwhile, the cell cycle was arrested in G2 phase. High concentration of MPN (100 µM) had significant effects on the nucleus of GES-1 cells, and the proportion of apoptotic cells reached 43.80%. Furthermore, the Western blotting analysis showed that MPN could reduce mitochondrial membrane potential by increasing the expression levels of apoptotic proteins Caspase 8 and Bax in GES-1 cells. In conclusion, MPN may induce apoptosis in GES-1 cells, which leads to toxicity in the human body.

Efficient removal of ginkgotoxin from Ginkgo biloba seed powder by combining endogenous enzymatic hydrolysis with resin adsorption.

BACKGROUND: Ginkgotoxin including 4'-O-methylpyridoxine (MPN) and MPN-5'-glucoside (MPNG) is responsible for Ginkgo seed food poisoning. The purpose of the work reported was to prepare detoxified Ginkgo seed powder and at the same time to retain the nutritional and functional components of Ginkgo seed powder to the maximum extent. RESULTS: Resin adsorption technology was firstly employed to remove ginkgotoxin from water extract of Ginkgo seed powder. Under optimal conditions, the adsorption efficiency of the optimal resin for MPN could reach 100%, and that for MPNG could only reach 85.4 ± 0.93%. Resin adsorption alone could not effectively remove MPN and MPNG simultaneously. Endogenous enzymatic hydrolysis was next attempted to transform MPNG to MPN. MPNG could be completely hydrolyzed to MPN by endogenous enzyme(s) at 40 °C and pH 5.0 in 180 min. Ginkgotoxin only in the form of MPN in the enzymatic hydrolysate was then adsorbed with resin and the conditions were statistically optimized. The adsorption efficiency of MPN reached 98.89 ± 0.99% under the optimized conditions. CONCLUSIONS: Removal of ginkgotoxin by combining endogenous enzymatic hydrolysis with resin adsorption could preserve the main nutritional and functional components of Ginkgo seed powder to the most extent, and did not change its main characteristics. The ginkgotoxin removal method developed in this work is a relatively simple and efficient approach. © 2020 Society of Chemical Industry.

Submerged fermentation of Ginkgo biloba seed powder using Eurotium cristatum for the development of ginkgo seeds fermented products.

BACKGROUND: Ginkgo biloba seeds are well known for the significant curative effects on relieving cough and asthma. However, the development of products from ginkgo seeds still falls behind at present, resulting in a great waste of ginkgo seeds' resource. In this work, submerged fermentation of ginkgo seed powder using Eurotium cristatum was studied to investigate its feasibility as a new processing method. RESULTS: To promote the growth of E. cristatum, the optimum fermentation medium was 80.0 g L-1 of ginkgo seed powder with addition of 5.0 g L-1 calcium chloride (CaCl2 ), 4.0 g L-1 magnesium sulfate (MgSO4 ), 1.25 g L-1 zinc sulfate (ZnSO4 ) and 0.65 g L-1 iron(II) sulfate (FeSO4 ). The optimum fermentation conditions were pH 5.8 ± 0.1, inoculum size 5.1 × 106 CFU mL-1 , liquid medium volume 100 mL in 250-mL Erlenmeyer flask and fermentation 4 days. Through fermentation, the production of lovastatin in fermentation broth could reach up to 32.97 ± 0.17 µg mL-1 and the total antioxidant capacity was improved by more than two-fold. In addition, 40.15% of the ginkgotoxin in ginkgo seed powder was degraded while the entire degradation of ginkgolic acids was obtained. Moreover, fermented ginkgo seed powder suspension presented pleasant fragrances, and the activities of amylase and protease were enhanced to 11.30 ± 0.10 U mL-1 and 23.01 ± 0.20 U mL-1 , respectively. CONCLUSIONS: Submerged fermentation using E. cristatum could significantly enhance the functional value and safety of ginkgo seed powder, and had great potential to become a novel processing method for the development of ginkgo seeds fermented products. © 2020 Society of Chemical Industry.

Effect of different drying methods on product quality, bioactive and toxic components of Ginkgo biloba L. seed.

BACKGROUND: Ginkgo biloba seeds are used as a functional food across Asia. However, the presence of toxic compounds has limited their application. In this study, freeze drying, infrared drying, hot-air drying and pulsed-vacuum drying were used to dry G. biloba seeds. A comprehensive analysis was performed on their product quality, antioxidant activities, bioactive and toxic components. RESULTS: Results showed that the drying methods had a significant influence on product quality with freeze drying being superior due to the minimal microstructural damage, followed by infrared drying and pulsed-vacuum drying. Infrared-dried product possessed the strongest antioxidant activities and higher bioactive compound content than hot-air-dried and pulsed-vacuum-dried product. Toxic compounds in fresh G. biloba seeds (ginkgotoxin, ginkgolic acid and cyanide) were reduced markedly by drying. Ginkgotoxin was reduced fourfold, and the contents of acrylamide, ginkgolic acid and cyanide in dried G. biloba seeds were reduced to the scope of safety. Amongst the four drying methods, infrared drying had the shortest drying time, and its product showed higher quality and bioactive compound content, and stronger antioxidant activities. CONCLUSIONS: These findings will offer salient information for selecting a drying method during the processing of ginkgo seeds. Infrared drying could be considered as a multiple-effect drying method in the processing of ginkgo seeds. © 2020 Society of Chemical Industry.

Pyridoxine-resveratrol hybrids as novel inhibitors of MAO-B with antioxidant and neuroprotective activities for the treatment of Parkinson's disease.

A series of pyridoxine-resveratrol hybrids were designed and synthesized as monoamine oxidase B inhibitors for the treatment of Parkinson's disease. Most of them exhibited potent inhibitory activities on MAO-B with high selectivity. Specifically, compounds 12a, 12g and 12l showed the most excellent inhibition to hMAO-B with the IC50 values of 0.01 µM, 0.01 µM and 0.02 µM, respectively. Further reversibility study demonstrated that 12a and 12l were reversible and 12g was irreversible MAO-B inhibitors. Molecular docking studies of MAO revealed the binding mode and high selectivity of these compounds with MAO-B. In addition, these three representative compounds also exhibited low cytotoxicity and excellent neuroprotective effect in the test on H2O2-induced PC-12 cell injury. Moreover, 12a, 12g and 12l showed good antioxidant activities and high blood-brain barrier permeability. Overall, all of these results highlighted 12a, 12g and 12l were potential and excellent MAO-B inhibitors for PD treatment.

An Adult Case of Generalized Convulsions Caused by the Ingestion of Ginkgo biloba Seeds with Alcohol.

A 64-year-old woman developed symptoms of vomiting and tonic-clonic convulsions 9.5 h after eating 50 roasted Ginkgo biloba seeds with 100 g of alcohol. The intravenous administration of pyridoxal phosphate effectively improved the symptoms. Blood samples were collected and stored over 35 h. The assessment of 4'-O-methylpyridoxine and vitamin B6 vitamers indicated high levels of both, but the pyridoxal phosphate levels were low during the acute stage. These results suggest that 4'-O-methylpyridoxine inhibits the transformation of vitamin B6 analogues to the active form, pyridoxal phosphate. In our case, alcohol may have extended the period until ginkgo intoxication appeared.

Structural attributes and substrate specificity of pyridoxal kinase from Leishmania donovani.

The enzyme pyridoxal kinase (PdxK) catalyzes the conversion of pyridoxal to pyridoxal-5'-phosphate (PLP) using ATP as the co-factor. The product pyridoxal-5'-phosphate plays a key role in several biological processes such as transamination, decarboxylation and deamination. In the present study, full-length ORF of PdxK from Leishmania donovani (LdPdxK) was cloned and then purified using affinity chromatography. LdPdxK exists as a homo-dimer in solution and shows more activity at near to physiological pH. Biochemical analysis of LdPdxK with pyridoxal, pyridoxamine, pyridoxine and ginkgotoxin revealed its affinity preference towards different substrates. The secondary structure analysis using circular dichroism spectroscopy showed LdPdxK to be predominantly α-helical in organization which tends to decline at lower and higher pH. Simultaneously, LdPdxK was crystallized and its three-dimensional structure in complex with ADP and different substrates were determined. Crystal structure of LdPdxK delineated that it has a central core of ß-sheets surrounded by α-helices with a conserved GTGD ribokinase motif. The structures of LdPdxK disclosed no major structural changes between ADP and ADP- substrate bound structures. In addition, comparative structural analysis highlighted the key differences between the active site pockets of leishmanial and human PdxK, rendering LdPdxK an attractive candidate for the designing of novel and specific inhibitors.

Rapid identification of herbal toxins using electrospray laser desorption ionization mass spectrometry for emergency care.

The unintentional ingestion of toxic compounds in herbs is not uncommon in many parts of the world. To provide timely and life-saving care in the emergency department, it is essential to develop a point-of-care analytical method that can rapidly identify these toxins in herbs. Since electrospray laser desorption ionization mass spectrometry (ELDI/MS) has been successfully used to characterize non-volatile chemical compounds without sample preparation, it was used to identify toxic herbal compounds in this study. The herbal toxins were collected either by sweeping a metallic probe across the surface of a freshly cut herb section or by directly sampling extracts of ground herbal powder. The analytes on the probe were then desorbed, ionized and detected using ELDI/MS, wherein analysis of the herbal toxins was completed within 30 s. This approach allows for the rapid morphological recognition of herbs and early point-of-care identification of herbal toxins for emergency management and is promising in providing important toxicological information to ensure appropriate medical treatment.

[Food Poisoning by Ginkgo Seeds through Vitamin B6 Depletion].

Overconsumption of Ginkgo biloba seeds induces food poisoning characterized by tonic-clonic convulsions and vomiting. The primary toxic component, 4'-O-methylpyridoxine (MPN), was purified from the seeds in 1985. This review includes the following aspects of ginkgo seed poisoning: 1) toxicity related to the content of MPN and MPN glucoside in G. biloba seeds; 2) the effect of MPN on vitamin B6 analogs, including an increase in pyridoxal and pyridoxic acid and decrease in pyridoxal-5'-phosphate plasma concentrations; 3) case reports of ginkgo seed poisoning in Asia, North America, and Europe, and their effective treatment via vitamin B6 administration. Considering the increase in the use of G. biloba seeds, it is essential to raise global awareness of their potential toxicity.

Interactions of pharmacokinetic profiles of Ginkgotoxin and Ginkgolic acids in rat plasma after oral administration.

Ginkgolic acids (GAs) and Ginkgotoxin (4'-O-methylpyridoxine, MPN) are main toxic compounds in Ginkgo biloba seeds which are widely used in the treatment of coughing in China. To evaluate the pharmacokinetics of GAs, MPN and their metabolites in rat plasma, a highly sensitive method followed by ultra-high-pressure liquid chromatography coupled with linear ion trap-Orbitrap tandem mass spectrometry (UHPLC-LTQ-Orbitrap-MS) has been developed and validated. The proposed method is selective, precise and accurate enough of MPN and its metabolites (4-pyridoxic Acid, pyridoxal, and pyridoxine) for the pharmacokinetic study. After oral administration of MPN, the plasma concentrations of MPN and its metabolites were increased rapidly. Meanwhile, an investigation was carried out to compare the interactions of the pharmacokinetic profiles of MPN and GAs. Five GAs and main metabolites of GA (15:1) and GA (17:1) were also analyzed by using our previous method. After coadministration GAs with MPN, Tmax of MPN delayed and Cmax decreased. Meanwhile, Tmax of 4-pyridoxic Acid, pyridoxal, and pyridoxine were also showed a certain degree of delay. The concentrations of hydroxylation products of GA (15:1) and GA (17:1) increased at a slower rate and the area under the curves was significantly reduced. However, glucuronidation metabolites of GA (15:1) and GA (17:1) were increased faster than administered of GAs alone. The interactions of the pharmacokinetic profiles of GAs and MPN in rat plasma after oral administration were obviously observed.

Concentrations of various forms of vitamin B6 in ginkgo seed poisoning.

A 2-year-old girl required medical attention for a sudden onset of repetitive tonic-clonic convulsions after ingesting 20-30 ginkgo seeds. Concentrations of the major forms of circulating vitamin B6, pyridoxal-5'-phosphate (PLP), pyridoxal (PL), and 4-pyridoxic acid, as well as the known ginkgo seed toxin 4'-O-methylpyridoxine (MPN) were measured in the serum and cerebrospinal fluid (CSF). PLP is an active form of vitamin B6 and necessary for γ-aminobutyric acid (GABA) production. High MPN concentrations were observed in both the serum and CSF. As the PLP to PL ratio was markedly decreased in serum and CSF examinations, we suspected the ratio to be important in GABA production. This case report provides novel information on the metabolism of vitamin B6 in humans as a result of ginkgo seed poisoning.

Protective role of vitamin B6 (PLP) against DNA damage in Drosophila models of type 2 diabetes.

Growing evidence shows that improper intake of vitamin B6 increases cancer risk and several studies indicate that diabetic patients have a higher risk of developing tumors. We previously demonstrated that in Drosophila the deficiency of Pyridoxal 5' phosphate (PLP), the active form of vitamin B6, causes chromosome aberrations (CABs), one of cancer prerequisites, and increases hemolymph glucose content. Starting from these data we asked if it was possible to provide a link between the aforementioned studies. Thus, we tested the effect of low PLP levels on DNA integrity in diabetic cells. To this aim we generated two Drosophila models of type 2 diabetes, the first by impairing insulin signaling and the second by rearing flies in high sugar diet. We showed that glucose treatment induced CABs in diabetic individuals but not in controls. More interestingly, PLP deficiency caused high frequencies of CABs in both diabetic models demonstrating that hyperglycemia, combined to reduced PLP level, impairs DNA integrity. PLP-depleted diabetic cells accumulated Advanced Glycation End products (AGEs) that largely contribute to CABs as α-lipoic acid, an AGE inhibitor, rescued not only AGEs but also CABs. These data, extrapolated to humans, indicate that low PLP levels, impacting on DNA integrity, may be considered one of the possible links between diabetes and cancer.

Determination and Comparison of 4'- O-Methylpyridoxine Analogues in Ginkgo biloba Seeds at Different Growth Stages.

The antivitamin B6, 4'- O-methylpyridoxine (MPN); its glucoside, 4'- O-methylpyridoxine-5'-glucoside (MPNG); and vitamin B6 compounds, including pyridoxal (PL), pyridoxamine, pyridoxine, pyridoxal-5'-phosphate (PLP), and pyridoxamine-5'-phosphate, exist in Ginkgo biloba seeds, which are widely used as food and medicine. This work aimed to determine the MPN analogues in G. biloba seeds at different growth stages in terms of cultivars and ages of trees. The highest total MPN contents of 249.30, 295.62, and 267.85 µg/g were obtained in the mature stages of three selected G. biloba samples. The total contents of vitamin B6 compounds decreased significantly in the entire growth period of the three samples. Principal-component analysis revealed that MPN and MPNG were important contributors in the MPN-analogue metabolism of G. biloba seeds. The influence of the cultivar on the content and composition of MPN analogues was greater than that of the age of the G. biloba tree.

Effect of heat treatment on 4'-O-methylpyridoxine (MPN) content in Ginkgo biloba seed extract solution.

BACKGROUND: A vitamin B6 derivative, 4'-O-methylpyridoxine (MPN), is responsible for food poisoning by Ginkgo biloba seeds. In this study, we investigate the content of pyridoxine and MPN in MPN standard solution and G. biloba seed extract solution upon heat treatment in order to evaluate the reduction of toxic components in G. biloba seed by such treatment. RESULTS: Heat treatment was conducted at 90-150 °C for 0-60 min, and all samples were adjusted to the same concentration of 1 g L-1 . The MPN content decreased to 994.92-563.69 mg kg-1 for MPN standard solution and to 371.56-76.84 mg kg-1 for G. biloba seed extract solution, and in both cases decreased even further with increasing heat treatment time. However, in all samples, except for the 90 °C heat treatment group, the pyridoxine content in MPN standard solution increased with increasing heat temperature and time; in addition, the extract solution showed a similar tendency. This may be the result of thermal degradation of MPN into pyridoxine. CONCLUSION: We can expect to improve the utilization of functional food materials by applying suitable heat treatment conditions and decreasing the MPN content of the G. biloba seed. © 2018 Society of Chemical Industry.