Non-enzymatic methods for isolation of stromal vascular fraction and adipose-derived stem cells:A systematic review

BACKGROUND Adipose-derived stem cells(ADSCs)and the stromal vascular fraction(SVF)have garnered substantial interest in regenerative medicine due to their potential to treat a wide range of conditions.Traditional enzymatic methods for isolating these cells face challenges such as high costs,lengthy processing time,and regulatory complexities.AIM This systematic review aimed to assess the efficacy and practicality of nonenzymatic,mechanical methods for isolating SVF and ADSCs,comparing these to conventional enzymatic approaches.METHODS Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines,a comprehensive literature search was conducted across multiple databases.Studies were selected based on inclusion criteria focused on non-enzymatic isolation methods for SVF and ADSCs from adipose tissue.The risk of bias was assessed,and a qualitative synthesis of findings was performed due to the methodological heterogeneity of the included studies.RESULTS Nineteen studies met the inclusion criteria,highlighting various mechanical techniques such as centrifugation,vortexing,and ultrasonic cavitation.The review identified significant variability in cell yield and viability,and the integrity of isolated cells across different non-enzymatic methods compared to enzymatic procedures.Despite some advantages of mechanical methods,including reduced processing time and avoidance of enzymatic reagents,the evidence suggests a need for optimization to match the cell quality and therapeutic efficacy achievable with enzymatic isolation.CONCLUSION Non-enzymatic,mechanical methods offer a promising alternative to enzymatic isolation of SVF and ADSCs,potentially simplifying the isolation process and reducing regulatory hurdles.However,further research is necessary to standardize these techniques and ensure consistent,high-quality cell yields for clinical applications.The development of efficient,safe,and reproducible non-enzymatic isolation methods could significantly advance the field of regenerative medicine.

A CuNi/C Nanosheet Array Based on a Metal–Organic Framework Derivate as a Supersensitive Non-Enzymatic Glucose Sensor

Bimetal catalysts are good alternatives for nonenzymatic glucose sensors owing to their low cost, high activity, good conductivity, and ease of fabrication. In the present study, a self-supported CuNi/C electrode prepared by electrodepositing Cu nanoparticles on a Ni-based metal–organic framework(MOF) derivate was used as a non-enzymatic glucose sensor. The porous construction and carbon scaffold inherited from the Ni-MOF guarantee good kinetics of the electrode process in electrochemical glucose detection. Furthermore, Cu nanoparticles disturb the array structure of MOF derived films and evidently enhance their electrochemical performances in glucose detection. Electrochemical measurements indicate that the CuNi/C electrode possesses a high sensitivity of17.12 mA mM^(-1) cm^(-2), a low detection limit of 66.67 nM,and a wider linearity range from 0.20 to 2.72 mM. Additionally, the electrode exhibits good reusability, reproducibility, and stability, thereby catering to the practical use of glucose sensors. Similar values of glucose concentrations in human blood serum samples are detected with our electrode and with the method involving glucose-6-phosphate dehydrogenase; the results further demonstrate the practical feasibility of our electrode.

Novel insights into conjugation of antitumor-active unsymmetrical bisacridine C-2028 with glutathione:Characteristics of non-enzymatic and glutathione S-transferase-mediated reactions

Unsymmetrical bisacridines(UAs) are a novel potent class of antitumor-active therapeutics.A significant route of phase II drug metabolism is conjugation with glutathione(GSH),which can be non-enzymatic and/or catalyzed by GSH-dependent enzymes.The aim of this work was to investigate the GSHmediated metabolic pathway of a representative UA,C-2028.GSH-supplemented incubations of C-2028 with rat,but not with human,liver cytosol led to the formation of a single GSH-related metabolite.Interestingly,it was also revealed with rat liver microsomes.Its formation was NADPH-independent and was not inhibited by co-incubation with the cytochrome P450(CYP450) inhibitor 1-aminobenzotriazole.Therefore,the direct conjugation pathway occurred without the prior CYP450-catalyzed bioactivation of the substrate.In turn,incubations of C-2028 and GSH with human recombinant glutathione S-transferase(GST) P1-1 or with heat-/ethacrynic acid-inactivated liver cytosolic enzymes resulted in the presence or lack of GSH conjugated form,respectively.These findings proved the necessary participation of GST in the initial activation of the GSH thiol group to enable a nucleophilic attack on the substrate molecule.Another C-2028-GSH S-conjugate was also formed during non-enzymatic reaction.Both GSH S-conjugates were characterized by combined liquid chromatography/tandem mass spectrometry.Mechanisms for their formation were proposed.The ability of C-2028 to GST-mediated and/or direct GSH conjugation is suspected to be clinically important.This may affect the patient’s drug clearance due to GST activity,loss of GSH,or the interactions with GSH-conjugated drugs.Moreover,GST-mediated depletion of cellular GSH may increase tumor cell exposure to reactive products of UA metabolic transformations.

A novel sensitive non-enzymatic glucose sensor

Cu nanoclusters were electrochemically deposited on the film of a Nafion-solubilized multi-wall carbon nanotubes (CNTs) modified glassy carbon electrode (CNTs-GCE), which fabricated a Cu-CNTs composite sensor (Cu-CNTs-GCE) to detect glucose with non-enzyme. The linear range is 7.0 × 10?7 to 3.5 × 10?3 mol/L with a high sensitivity of 17.76 μA/(mmol L), with a low detection limit 2.1 × 10?7 mol/L, fast response time (within 5 s), good reproducibility and stability.

The Non-Enzymatic Antioxidant and Level of Oxidative Stress of Tuberculosis Patients in Selected Treatment Center in Addis Ababa Ethiopia

Introduction: Non-enzymatic antioxidants are good scavengers of free radicals preventing their overproduction there by reducing the level of oxidative stress. This work was undertaken at Saint Peter TB specialized hospital and TekleHaimanot health center from March 2012 to May 2013.Aim: To determine changes in Non-Enzymatic Antioxidants and level of oxidative stress of tuberculosis Patients before and after taking anti tuberculosis treatment.Materials and Methods: In this comparative cross sectional study, a total of 210 individuals including: newly diagnosed TB patients as group-I (n = 70), TB patients who completed treatment as group-II (n = 70), and healthy volunteers as group-III (n = 70) were enrolled. Different methods were used to determine the parameters;vit-C (HPLC method), lipid peroxidation (thiobarbuituric acid method), and bilirubin (Colorimetric assay). Results: Vitamin-C (Vit-C) and of group-I showed a significant reduction (p < 0.001) as compared with both group-II and group-III whereas Malondialdehyde (MDA) level was increased. However, the total and direct bilirubin was not different among the groups. In group-III, there was a positive correlation between BMI and serum Vit-C (r = -0.305, p = 0.010). Vit-C showed a negative correlation with serum MDA in all the groups with values (r = -0.265, p = 0.027), (r = -0.389, p = 0.001) and (r = -0.375, p = 0.001) for group-I, group-II and group-III respectively. In addition to this Vit-C was negatively correlated with serum UA (r = -0.285, p = 0.017) in group-I. Conclusion: The findings of the current study suggest that the amount of Vit-C in the newly diagnosed TB patients and those who finished their treatment is much lower than the healthy volunteers. In contrast to this, the MDA value was significantly higher both in the newly diagnosed TB patients and TB patients who completed treatment than in healthy volunteers suggesting higher degree of oxidative stress.

Non-Enzymatic Glucose Sensor Based on the Novel Flower Like Morphology of Nickel Oxide

In this study, novel nickel oxide (NiO) flowers like nanostructures were fabricated onto gold coated glass substrate by hydrothermal method using high alkaline pH medium. The structural study of nickel oxide nanostructures was performed by scanning electron microscopy (SEM) and X-ray differaction (XRD) techniques. Nickel oxide nanostructures are highly dense, uniform and possess good crystalline quality. The so prepared structures were investigated for their electrochemical properties by cyclic voltammetry and amperometric techniques. The nickel oxide flower like morphology has shown good electrochemical performances for the oxidation of glucose. The presented sensing material was able to detected glucose in a wide range of concentration of 0.001 mM to 8 mM with a high sensitivity (123 μmA/mM) and regression coefficient of 0.99. Moreover, the NiO nanostructures based sensor is highly reproducible, stable, exhibiting a fast response time and selective in the response. All the obtained results indicate the potential use of this material in the development of enzyme free sensors for the detection of glucose.

Ce(Ⅲ)-modulation over non-enzymatic Pt/CeO_(2)/GO biosensor with outstanding sensitivity and stability for lactic acid detection

A series of non-enzymatic graphene functionalized biosensors was developed via deposition precipitation method for lactic acid(LA) detection,which we re characterized by transmission electron micro scopy(TEM),Raman spectroscopy,X-ray photoelectron spectroscopy(XPS),gas chromatography-mass spectrometry,liquid chromatography-mass spectro metry,and proton nuclear magnetic re sonance(~1H NMR).The electrochemical performances of the non-enzymatic biosensors were measured by means of the electrochemical impedance spectroscopy(EIS) and cyclic voltammetry(CV) method.The comprehensive analysis of structures shows that Pt,CeO_(2),and GO components interact with each other.During the storing and releasing oxygen,the valence ratio of Ce^(3+)/Ce^(4+) and the number of oxygen vacancies in CeO_(2) change accordingly,which can be conducive to increasing electronic transmission capacity and finally leads to the improvement of electrocatalytic performance.Among them,the Pt/CeO_(2)/GO biosensor containing 0.47 at% platinum exhibits an excellent electrochemical detection performance with high sensitivity of 12.3 μA·L/(mmol·cm^(2)) and a low limit of detection(LOD) of 5.12 μmol/L in a wide linear range from 10 to 900 μmol/L.In addition,the proposed biosensor possesses a promising anti-interference capability,as well as high stability and good reproducibility,which was assessed by testing the cyclic voltammogram in 0.1 mol/L lactic acid one year later.The underlying mechanism was proposed for electrochemical oxidation of LA to carbon dioxide and acetic acid with the synergistic effect among Pt,CeO_(2),and GO.Furthermore,the results of the standard addition method in real samples(human serum and urine samples) reveal that the lactic acid detection of the non-enzymatic Pt/CeO_(2)/GO biosensor is accompanied by high reliability.Thus,it will be a valuable biosensor for in vitro detection of lactic acid level in clinical samples.

Electrochemical non-enzymatic glucose sensors based on nano-composite of Co3O4 and multiwalled carbon nanotube

Nanocomposite of Co3O4 and multiwalled carbon nanotube (MCNT) was synthesised using one step solvothermal method, and an electrochemical non-enzymatic glucose sensor (Co3O4-MCNT/GCE) was successfully constructed by a dropping method. The obtained Co3O4 and Co3O4- MCNT were characterized and investigated by transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy (EDS). Quantitative analysis of glucose was performed using the amperometric (i–t) method, and plot of current difference versus concentration of glucose was linear in the range of 1.0–122μmol/L, with a linear correlation coefficient (R^2) of 0.9983 and limit of detection (LOD) of 0.28μmol/L. Sensitivity of this sensor was evaluated as 2550μA L mmol^-1 cm^-2. This new sensor produced satisfactory reproducibility and stability and was applied to monitor trace amounts of glucose in human serum samples.

Non-enzymatic covalent modifications:a new link between metabolism and epigenetics

Epigenetic modifications,including those on DNA and histones,have been shown to regulate cellular metabolism by controlling expression of enzymes involved in the corresponding metabolic pathways.In turn,metabolic flux influences epigenetic regulation by affecting the biosynthetic balance of enzyme cofactors or donors for certain chromatin modifications.Recently,non-enzymatic covalent modifications(NECMs)by chemically reactive metabolites have been reported to manipulate chromatin architecture and gene transcription through multiple mechanisms.Here,we summarize these recent advances in the identification and characterization of NECMs on nucleic acids,histones,and transcription factors,providing an additional mechanistic link between metabolism and epigenetics.

Facile synthesis of CuO-Co_(3)O_(4)prickly-sphere-like composite for non-enzymatic glucose sensors

In the field of glucose sensors,the development of inexpensive and high-efficiency electrochemical glucose sensors is the current research hotspot.In this paper,CuO-Co_(3)O_(4)composite with a prickly-sphere-like morphology is prepared by the facile hydrothermal method for the non-enzymatic electrochemical glucose detection.X-ray diffraction,scanning electron microscopy,transmission electron microscopy,energy-dispersive X-ray spec-troscopy,and X-ray photoelectron spectroscopy are used to analyze the structure,composition,and morphology of the material.In addition,the electrochemical catalytic perfor-mance of CuO-Co_(3)O_(4)to glucose is obtained by cyclic voltammetry and chronoamperometry.The excellent elec-trochemical sensing performance may be attributed to the large number of catalytic sites in the prickly-sphere-like composite and the synergistic effect of Cu and Co.Under an applied voltage of 0.55 V,CuO-Co_(3)O_(4)composite shows sensitivity to glucose(1503.45μA·(mmol·L^(-1))^(-1)-cm^(-2)),a low detection limit(21.95μmol·L^(-1)),excellent selectivity,a high level of reproducibility,and good sta-bility.This indicates that the CuO-Co_(3)O_(4)composite has a broad prospect of non-enzymatic glucose sensing application.

Enzymatic and non-enzymatic isolationsystems for adipose tissue-derived cells:current state of the art

In the past decade, adipose tissue became a highly interesting source of adult stem cells for plastic surgery andregenerative medicine. The isolated stromal vascular fraction (SVF) is a heterogeneous cell population including theadipose-derived stromal/stem cells (ASC), which showed regenerative potential in several clinical studies and trials.SVF should be provided in a safe and reproducible manner in accordance with current good manufacturing practices(cGMP). To ensure highest possible safety for patients, a precisely defined procedure with a high-quality control isrequired. Hence, an increasing number of adipose tissue-derived cell isolation systems have been developed.These systems aim for a closed, sterile, and safe isolation process limiting donor variations, risk for contaminations,and unpredictability of the cell material. To isolate SVF from adipose tissue, enzymes such as collagenase are used.Alternatively, in order to avoid enzymes, isolation systems using physical forces are available. Here, we provide anoverview of known existing enzymatic and non-enzymatic adipose tissue-derived cell isolation systems, which arepatented, published, or already on the market.

Reusable electrochemical non-enzymatic glucose sensors based on Au-inlaid nanocages

Mass detection of glucose,which is required in many applications,remains challenging.The commercial enzyme-based glucose test strips cannot be reused,and current non-enzymatic glucose sensors exhibit a narrow range of detection and slow glucose oxidation kinetics.Herein,controlled etching of Prussian blue analogue(PBA)nanocubes at the vertices is conducted and Au nanoparticles(Au NPs)are subsequently inlaid in the etched cavities by in-situ reduction of HAuCl4.The unique AuNP-PBA nanocomplexes exhibit low electrochemical potential for glucose oxidation,high electrocatalytic activity,and rapid redox electron transfer rate.Covalent immobilization of the Au-inlaid nanomaterials on a fine Au wire leads to a non-enzymatic glucose sensor with a particularly wide linear detection range(10μM to 16 mM),excellent anti-interference,and fast response.More importantly,the sensor is reusable,and its sensitivity is well maintained even after 150 times of detection.This new-concept material promises to enable high-throughput glucose detection at a low cost,which is essential in diabetic management and other healthcare applications.

Sandwich structure confined gold as highly sensitive and stable electrochemical non-enzymatic glucose sensor with low oxidation potential

The preparation of highly sensitive and stable non-enzymatic glucose sensors is critical to the prevention and treatment of diabetes.Fe_(3)O_(4)@Au@Co Fe-LDH is prepared through a spontaneous galvanic displacement reaction.A series of structural characterizations testify the successful formation of Fe_(3)O_(4)@Au@Co FeLDH electrocatalyst,with the Au intercalating between Fe_(3)O_(4)and LDH to form the sandwich structure.Cyclic voltammetry tests indicate that Au is responsible for the electrocatalytic oxidation of glucose.The characterizations of the electrochemical sensor for glucose detection indicate that Fe_(3)O_(4)@Au@Co FeLDH possesses high sensitivity of 6342μA m M^(-1)cm^(-2),with an extremely low oxidation potential of 0.82 V vs.RHE.Even with the high glucose concentration of 15 m M,the sensitivity remains at 4359μA m M^(-1)cm^(-2).Due to the broad linear detection range(0.0375 to 15.64 m M)and the low limit of detection(12.7μM),Fe_(3)O_(4)@Au@Co Fe-LDH is applicable towards practical application.Thanks to the sandwich structure,which confines the Au in between Fe_(3)O_(4)and Co Fe-LDH,the Fe_(3)O_(4)@Au@Co Fe-LDH glucose sensor shows high long-term stability and satisfactory selectivity.The successful synthesis of the sandwichstructured Fe_(3)O_(4)@Au@Co Fe-LDH provides a new conception for the design of highly sensitive and stable non-enzymatic glucose electrodes.

Evaluation of In Vitro Enzymatic and Non-Enzymatic Antioxidant Properites of Leaf Extract from Alpinia Purpurata(Vieill.) K. Schum.

Objective： To evaluate the enzymatic and non-enzymatic antioxidants of leaf extract from Alpinia purpurata. Methods： One gram of fresh leaf of Alpinia purpurata was grinded in 2 mL of 50% ethanol and centrifuged at 10,000×g at 4 ℃ for 10 min. The supernatant obtained was used within 4 h for various enzymatic antioxidants assays like superoxide dismutase（SOD）, catalase（CAT）, glutathione peroxidase（GPx）, glutathione S-transferase（GST）, ascorbate oxidase, peroxidase, polyphenol oxidase（PPO） and non-enzymatic antioxidants such as vitamin C, total reduced glutathione（TRG） and lipid peroxidation（LPO）. Results： The leaf extract of Alpinia purpurata possess antioxidants like vitamin C 472.92±6.80 μg/mg protein, GST 372.11±5.70 μmol of 1-chloro 2,4 dinitrobenzene（CDNB）-reduced glutathione（GSH） conjugate formed/min/mg protein, GPx 281.69±6.43 μg of glutathione oxidized/min/mg protein, peroxidases 173.12±9.40 μmol/g tissue, TRG 75.27±3.55 μg/mg protein, SOD 58.03±2.11 U/mg protein, CAT 46.70±2.35 μmol of H_2O_2 consumed/min/mg protein in high amount whereas ascorbate oxidase 17.41±2.46 U/g tissue, LPO 2.71±0.14 nmol/L of malondialdehyde formed/min/mg protein and PPO 1.14±0.11 μmol/g tissue in moderate amount. Conclusion： Alpinia purpurata has the potential to scavenge the free radicals and protect against oxidative stress causing diseases. In future, Alpinia purpurata may serve as a good pharmacotherapeutic agent.

Comprehensive overview of human serum albumin glycation in diabetes mellitus

The presence of excess glucose in blood is regarded as a sweet hurt for patients with diabetes.Human serum albumin(HSA)is the most abundant protein in human plasma,which undergoes severe non-enzymatic glycation with glucose in patients with diabetes;this modifies the structure and function of HSA.Furthermore,the advanced glycation end products produced by glycated HSA can cause pathological damage to the human body through various signaling pathways,eventually leading to complications of diabetes.Many potential glycation sites on HSA have different degrees of sensitivity to glucose concentration.This review provides a comprehensive assessment of the in vivo glycation sites of HSA;it also discusses the effects of glycation on the structure and function of HSA.Moreover,it addresses the relationship between HSA glycation and diabetes complications.Finally,it focuses on the value of non-enzymatic glycation of HSA in diabetes-related clinical applications.

Oxidative Stress and Antioxidant Status in Acute and Chronic Myeloid Leukemia Patients

Oxidative stress, a pervasive condition of increased number of reactive oxygen species, is now recognized to be prominent feature of various diseases and their progression. The relationship between antioxidants and levels of well-known markers of oxidative stress, measured as lipid peroxides and oxidized proteins reflect health indices. The aim of this study is to evaluate the extent of oxidative stress and antioxidant status in acute and chronic myeloid leukemia patients. The present study included 60 patients selected using standard questionnaire based on age, family history, Body Mass Index (BMI), dietary intake, with no other complications and 30 age and sex-matched healthy subjects. The median age of myeloid leukemia patients was 43 years and that of controls was 42 years. Out of 60 myeloid leukemia patients, 30 were in acute and 30 were in chronic state. Oxidative stress and antioxidant status were evaluated in the patients and in the controls by assessing standard oxidative stress markers viz. plasma and erythrocyte lipid peroxide levels in terms of malondialdehyde and oxidized proteins as protein carbonyls whereas antioxidant status was assessed in terms of serum non enzymatic antioxidant levels. There was a significant increase (p 0.01) in plasma and erythrocyte lipid peroxidation and protein oxidation in acute and chronic myeloid leukemia patients as compared to healthy subjects. Antioxidant status as indicated by the levels of non-enzymatic antioxidants viz. erythrocyte reduced glutathione (GSH), serum β carotene, vitamin A & C and ceruloplasmin was found to be significantly decreased (p 0.01) in both the leukemia patients as compared to healthy participants. However, chronic myeloid leukemia patients had significantly (p 0.05) higher oxidative stress and lower antioxidant status as compared to acute myeloid leukemia patients.

Spectrophotometric Assay for the Quantification of Plasma Ethanol Levels in Mice through Chromium-Ethanol Oxidation-Reduction Reaction

The quantification of blood/plasma ethanol concentration (BEC/PEC) is of great importance in experiments involving basic research, clinical studies, and bioethanol production. Traditional methods commonly used to measure BEC can be expensive and require high-cost equipment and qualified labor. The aim of this study was to develop a low-cost method that can be performed with simple infrastructure commonly available in research laboratories. For this, we developed a protocol to quantify PEC in mice, using the method of reduction of potassium dichromate by ethanol. However, this oxidation-reduction (redox) reaction is not specific to ethanol. Thus, the PEC was measured following a sequence of chemical reactions to eliminate the reductive interfering substances presented in the samples. Firstly, we evaluated the sensitivity of the dichromate reactive to ethanol and to different reducing substances found in the plasma, in order to determine which the main interfering substances are. Next, once the main interfering substances were determined in the dichromate reduction, plasma was assayed for PEC. First, mice received intraperitoneally (i.p.) saline (basal reading, 0% ethanol) or ethanol injections (0.5, 1, 2, 3, and 4 g/kg) and had their plasma collected. After plasma deproteinization and plasma glucose oxidation, it was mixed with the dichromate/acetic acid reactive, and then the products of the redox reaction were determined by the spectrophotometric method. Then, we determined the PEC with the same plasma samples using a commercial ethanol assay kit as a positive control. We found an excellent correlation between the administered ethanol doses and PECs in both the methods analyzed. The values of PEC found in the dichromate reaction method were similar to those obtained in the literature with the same ethanol doses, and to the commercial enzyme activity assay. Therefore, despite the need for a background reading, this method can be successfully applied to determine PEC using low-cost chemical reagents.

The complexities of proanthocyanidin biosynthesis and its regulation in plants

Proanthocyanidins(PAs)are natural flavan-3-ol polymers that contribute protection to plants under biotic and abiotic stress,benefits to human health,and bitterness and astringency to food products.They are also potential targets for carbon sequestration for climate mitigation.In recent years,from model species to commercial crops,research has moved closer to elucidating the flux control and channeling,subunit biosynthesis and polymerization,transport mechanisms,and regulatory networks involved in plant PA metabolism.This review extends the conventional understanding with recent findings that provide new insights to address lingering questions and focus strategies for manipulating PA traits in plants.

NAMPT-targeting PROTAC promotes antitumor immunity via suppressing myeloid-derived suppressor cell expansion

Nicotinamide phosphoribosyl transferase(NAMPT) is considered as a promising target for cancer therapy given its critical engagement in cancer metabolism and inflammation.However,therapeutic benefit of NAMPT enzymatic inhibitors appears very limited,likely due to the failure to intervene nonenzymatic functions of NAMPT.Herein,we show that NAMPT dampens antitumor immunity by promoting the expansion of tumor infiltrating myeloid derived suppressive cells(MDSCs) via a mechanism independent of its enzymatic activity.Using proteolysis-targeting chimera(PROTAC) technology,PROTAC A7 is identified as a potent and selective degrader of NAMPT,which degrades intracellular NAMPT(iNAMPT) via the ubiquitin-proteasome system,and in turn decreases the secretion of extracellular NAMPT(eNAMPT),the major player of the non-enzymatic activity of NAMPT.In vivo,PROTAC A7 efficiently degrades NAMPT,inhibits tumor infiltrating MDSCs,and boosts antitumor efficacy.Of note,the anticancer activity of PROTAC A7 is superior to NAMPT enzymatic inhibitors that fail to achieve the same impact on MDSCs.Together,our findings uncover the new role of enzymatically-independent function of NAMPT in remodeling the immunosuppressive tumor microenvironment,and reports the first NAMPT PROTAC A7 that is able to block the pro-tumor function of both iNAMPT and eNAMPT,pointing out a new direction for the development of NAMPT-targeted therapies.

Sulfite as the substrate of C-sulfonate metabolism ofα,β-unsaturated carbonyl containing andrographolide:analysis of sulfite in rats'intestinal tract and the reaction kinetics of andrographolide with sulfite

One-sixth of the currently known natural products containα,β-unsaturated carbonyl groups.Our previous studies reported a rare C-sulfonate metabolic pathway.Sulfonate groups were linked to theβ-carbon ofα,β-unsaturated carbonyl-based natural compounds through this pathway.However,the mechanism of this type of metabolism is still not fully understood,especially whether it is formed through enzyme-mediated biotransformation or direct sulfite addition.In this work,the enzyme-mediated and non-enzymatic pathways were studied.First,the sulfite content in rat intestine was determined by LC-MS/MS.The results showed that the amount of sulfite in rat intestinal contents was from 41.5 to 383μg·g^(-1),whereas the amount of sulfite in rat feed was lower than the lower limit of quantitation(20μg·g^(-1)).Second,the reaction kinetics of sulfite-andrographolide reactions in phosphate buffer solutions(pH 6-8)was studied.The half-lives of andrographolide ranged from minutes to hours.This was suggested that the C-sulfonate reaction of andrographolide was very fast.Third,the C-sulfonate metabolites of andrographolide were both detected when andrographolide and L-cysteine-S-conjugate andrographolide were incubated with the rat small intestine contents or sulfite,indicating that the sulfite amount in rat intestine contents was high enough to react with andrographolide,which assisted a significant portion of andrographolide metabolism.Finally,the comparison of andrographolide metabolite profiles among liver homogenate(with NADPH),liver S9(with NADPH),small intestine contents homogenate(with no NADPH),and sulfite solution incubations showed that the C-sulfonate metabolites were predominantly generated in the intestinal tract by non-enzymatic pathway.In summary,sulfite can serve as a substrate for C-sulfonate metabolism,and these results identified non-enzymatically nucleophilic addition as the potential mechanism for C-sulfonate metabolism of compounds containingα,β-unsaturated carbonyl moiety.