The results indicated that these adzuki beans, containing numerous phenolics, showed inhibitory activities to alpha-amylase with the α-AI activities between 1.760 ± 0.044 and 3.411 ± 0.186 U/g. The resistant starch (RS) contributed predominantly to the total starch with proportions between (69.78 ± 2.45%) and (81.03 ± 0.06%); Te Xiao Li No. 1 was the highest compared with the other varieties. The adzuki beans were categorized into low- or medium-GI foods, and the IVGI ranged from (39.00 ± 0.36) to (56.76 ± 4.21). These results suggested that adzuki beans can be used as a component of low-GI foods.Regular consumption of low-glycemic index (GI) foods is a common strategy for type 2 diabetes patients. To evaluate the potential application of adzuki beans in low-GI foods, the phenolic profile and alpha-amylase inhibitor (α-AI) activity of four varieties of adzuki beans  were determined. The starch digestibility properties and   Vigna angularis) are common legumes appearing in red, white, black, and green colors . In normal human pancreatic β-cells, pancreatic insulin is stimulated by incretins, such as glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP), which are responsible for glucose homeostasis . Previouin vitro digestion model, and the contribution of endogenous components to low-GI was also discussed. All of these results provide the fundamentals for the mechanism revealing that adzuki beans regulate the blood glucose content.Adzuki bean is a staple crop in Bijie city of Guizhou province, where the typical geography conditions are hilly topographic and changeable climate which induce the production of endogenous phytochemicals , and the°C, the precipitation was approximately 780.6 mm, and the sunshine duration was 667 h. The adzuki beans were ground into flour using a universal high-speed smashing machine , and then screened using a 40-mesh sieve; the flours were stored at –20°C for further analysis.The four varieties of adzuki beans, namely G24, Te Xiao Li No. 1, Gui Nong No. 1, and Qian Xiao Hei , plantedp-hydroxybenzoic acid, and ferulic acid were purchased from Macklin Bioch. Co., Ltd . Alpha-amylase enzyme  was purchased from Solarbio Science & Tech. Co., Ltd. . Hexane, ethyl acetate, acetic acid, petroleum ether, and methanol were purchased from Tianjin Fuyu Fine Chemical Co., Ltd. .Folin-Ciocalteu’s phenol reagent, pepsin (source: gastric mucosa of a pig), and gallic acid were purchased from Sigma-Aldrich Ltd. . 3, 5-Dinitrosalicylic acid (DNS) was purchased from Sinopharm Chemical Reagent Co., Ltd. Acetone and hydrochloric acid (36–38%) were purchased from Chongqing Chuandong Chemical Group Co., Ltd. . Sodium hydroxide was purchased from Chengdu Jinshan Chemical Reagent Co., Ltd. . Deionized water was purchased from Watsons Water Co., Ltd. . HPLC grade acetonitrile and acetate were purchased from Aladdin company . Sodium carbonate was purchased from Yongda Chemical Reagent Co., Ltd. . Aluminum chloride hexahydrate was purchased from Kemio Chemical Co., Ltd. . Rutin, 2, 3, 4-trihydroxybenzoic acid, catechin, chlorogenic acid, g for 5 min to collect the supernatants. The residues were again subjected to phenolic extraction twice following the procedures mentioned above, and the collected supernatants were evaporated at 45°C using a rotary evaporator  and eventually reconstituted to 10 ml with deionized water. The phenolic extracts were stored at –20°C for further analysis, and the phenolic extraction was performed in triplicate.Phenolic extracts were obtained using the method described by Adom and Liu  and De L2CO3 solution and 0.8 ml of deionized water. After 90 min of reaction in the dark, 200 μl of the reaction mixtures were pipetted into a 96-well microplate reader  and determined at 760 nm, and the standard curves were plotted from the absorbances and concentrations of the gallic acid standards. The TP was calculated from the standard curve and expressed as mg gallic acid equivalents (GAE)/100 g DW.Total phenolics (TP) were determined using the method described by De La Parra et al. . A volum2 for 6 min; the mixtures were continuously reacted with 0.3 ml of 10% AlCl3.6H2O for 5 min. Afterward, 1 ml of 1 mol/L NaOH was vigorously mixed with the mixtures to terminate the reactions. After 15 min of reaction, 200 μl of the mixtures was pipetted into a 96-well microplate reader and determined at 510 nm. The standard curve was plotted from the absorbance and concentration of the rutin standards, and the TF was calculated from the standard curve and expressed as mg rutin equivalents (RE)/100 g DW.The total flavonoids (TF) were estimated using the methods described by Zhishen et al.  and BakaThe profile of the phenolic compounds of the adzuki beans was determined using HPLC , 35. Theg, 4°C) for 30 min to collect the water extracts, and then the volume was recomposed to 20 ml. The above process was performed in triplicate  for 10 min, and then a 5-min reaction was allowed after the addition of 0.5 ml of 1% (w/v) aqueous starch solution to the mixtures. Eventually, the mixtures were mixed with 1 ml DNS solution and subsequently heated in a boiling water bath for 10 min. After cooling to room temperature, the volume of the mixtures was reconstituted to 20 ml with deionized water and measured at 540 nm using a spectrophotometer instrument .The α-AI specific activity was determined using the method described by Yang et al. , with miThe trial groups were estimated as (1) blank tube (no addition of water extracts), (2) blank control tube (addition of neither α-amylase solution nor water extracts), and (3) inhibition control tube (no addition of α-amylase solution). PBS was used to make up for the deficiency.The inhibition rate of the α-AI in adzuki beans to α-amylase was calculated using formula (1):1, A2, A3A, and A4 are the absorbances of the blank tube, blank control tube, inhibition tube, and inhibitory control tube at 540 nm wavelength, respectively. The amount of α-amylase that catalyzes the formation of 1 mol glucose/min at 37°C and pH 6.9 was defined as an activity unit (U). The amount of α-AI that catalyzes the inhibition of 1 mol glucose formation per min in an α-amylase catalytic starch hydrolysis reaction at 37°C and pH 6.9 was defined as an activity unit (U), while the α-AI specific activity (U/g) is defined as the α-AI activity unit in 1 g of adzuki bean, and was calculated using formula (2):Where 1” represents the activity of α-amylase solution (U/ml), “n” is the water extract dilution times, “V” is the total volume of water extract (ml), and “m” is the mass of the adzuki beans powder (g).Where “The TS of adzuki beans was estimated by referring to the National Food Safety Standards of China GB 5009.9 . Adzuki g, 10 min) before glucose content determination. The DNS colorimetry method was adopted to determine glucose content. The aliquots of the digestion mixture were equivalently mixed with DNS (200 μl). In addition, the mixtures were sequentially processed by heating in boiled water for 6 min and cooling in running water to room temperature before recomposing the volume to 2 ml using deionized water. The absorbances of the reacted mixtures were measured using a spectrophotometer at 540 nm.In brief, an aliquot of bean powder comprising 50 mg of starch was mixed with 2 ml of deionized water, and then the mixture was heated for 10 min in a boiling water bath. In addition, 10 ml of HCl-KCl buffer  and 0.2 ml pepsin solution  were sequentially added to the gelatinized mixture, which was then agitated for 60 min in a water bath at 40°C. Furthermore, 15 ml of PBS (pH 6.9) and 5 ml of α-amylase solution (2.6 U/ml) (pH 6.9) were sequentially added to the mixture, which was then agitated for 180 min in a water bath at 37°C, and aliquots of the digested mixture (1 ml) were collected at intervals of 30 min  for GI determination. The white bread was subjected to the above processes except for the gelatinization process . The adzThe slowly digestible starch (SDS), rapidly digestible starch (RDS), and RS contents were calculated using formula (3), formula (4), and formula (5) , 39.C0 represents the initial glucose concentration (mg/ml) before the enzymatic hydrolysis reaction, “C20” and “C120” represent the glucose concentration (mg/ml) released within 20 and 120 min, respectively, “V” refers to the initial volume of the enzymatic hydrolysis, “0.9” is the conversion factor of glucose to starch, and “50” represents the starch content (mg) in the measured sample.Where The IVGI of adzuki beans was computed as described in previous studies , 37. TheThe starch hydrolysis rate (SR%) was calculated using formula (6):Ct” represents the glucose concentration at a time “t” (mg/ml), “C0” represents the glucose concentration at a time “0” (mg/ml), “32.2” represents the initial volume of the enzymatic hydrolysis reaction system (ml), “0.9” is the conversion factor of glucose to starch, and “50” represents the amount of starch in adzuki bean powder.“∞) (mg/ml).Formula (7) was used to calculate the enzymatic hydrolysis kinetic constant (k) and the infinite glucose content (CThe area under the hydrolysis curve (AUC) was delineated using formula (8).tf” and “t0” represent the last time point (180 min) and the initial time point (0 min) of the enzymatic hydrolysis reaction, respectively.Where “The IVGI was computed using formula (9).The calcHI-starch hydrolysis index indicates the AUC sample/AUC white bread standard.n = 3) and tested using the IBM SPSS (one-way ANOVA plus post-hoc Tukey’s b test) software . P-values less than 0.05 indicated a significant difference.The values were expressed as means ± standard deviation  to (7.46 ± 0.75) mg/100 g DW, (3.94 ± 0.27) to (7.15 ± 1.08) mg/100 g DW, and (5.96 ± 0.59) to (7.54 ± 0.16) mg/100 g DW, respectively. The catechin and rutin contents range from (12.63 ± 1.38) to (23.05 ± 1.60) mg/100 g DW and (2.41 ± 0.19) to (16.31 ± 2.09) mg/100 g DW, respectively. In Te Xiao Li No. 1, the rutin and catechin contents are dominant with 16.31 ± 2.09 and 23.05 ± 1.60 mg/100 g DW, respectively.The TP and TF of adzuki beans are shown in The α-AI specific activity indicates the inhibitory efficiency of α-AI to α-amylase in the starch hydrolysis reaction. The α-AI specific activities of adzuki beans range from (1.76 ± 0.04) to (3.41 ± 0.18) U/g. G24 shows the highest activity and Te Xiao Li No. 1 and Gui Nong No. 1 show minor fluctuations; both are ranked intermediate, while Qian Xiao Hei shows the lowest α-AI specific activity .The starch digestibility properties can be categorized using the digestive process in which the RDS can be digested in 0–20 min, the SDS can be digested in 20–120 min, and the RS cannot be digested after 120 min . In adzuin vitro digestion model coupled with DNS colorimetry, which was used to plot the starch hydrolysis curves (∞) were obtained, and both were eventually used to calculate the GI; the parameters and IVGI are presented in The SR was determined using the s curves . Using tAbundant phenolics were identified in the seed coats of beans, and intrinsic anthocyanins exist in colored beans , 41. Phep-hydroxybenzoic acid, and caffeic acid in beans have positive influences on type 2 diabetes  and 479.26 ± 10.40) μg/g , whereasdiabetes , 57. In diabetes . Further.40 μg/g diabetes , 60.Alpha-amylase is the key enzyme that hydrolyzes carbohydrates to disaccharides and oligosaccharides in the gastrointestinal tract, both of which are further degraded into monosaccharides or simple sugars by α-glucosidase, resulting in an increased level of postprandial glucose . α-AI α–66. α-AIAlpha-D-glucopyranosyl linked with α-D- (1–4) or α-D- (1–6) linkages are the basic units of starches, which are composed of amylose and amylopectin , 73. PreGI indicates the sugar level in the blood after the consumption of certain foods, and can be classified into three categories, namely low GI (<55), medium GI (56–69), and high GI (70–100) . In the p-hydroxybenzoic acid; of which catechin is the dominant phenolic compound in Te Xiao Li No. 1 and G24 varieties. The TP of Te Xiao Li No. 1 is higher than that of the other varieties, and the TF of G24 is higher than that of the other varieties. In addition, α-amylase inhibitors and RS are also present in adzuki beans. These adzuki beans are classified as low- or medium-GI foods, which may be caused by the whole effects of endogenous functional components. Phenolics, α-amylase inhibitors, and RS are the dominant contributors to the delays in starch digestion. These results indicate that these adzuki beans can be used as components of low-GI foods.In the present study, adzuki beans contain numerous phenolics, including gallic acid, 2, 3, 4-trihydroxybenzoic acid, chlorogenic acid, catechin, rutin, and The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.MM: data curation, validation, writing—original draft, software, formal analysis, and investigation. LQ: investigation, resources, methodology, supervision, conceptualization, project administration, and funding acquisition. YZ: resources, writing—review and editing, and funding acquisition. AW and NL: formal analysis. All authors contributed to the article and approved the submitted version.


Benactyzine is a combined anticholinergic, anti-NMDA compound. Based on previous development of novel GABA derivatives (such as prodrugs based on perphenazine for the treatment of schizophrenia and nortriptyline against neuropathic pain), we describe the synthesis and preliminary testing of a mutual prodrug ester of benactyzine and GABA. It is assumed that once the ester crosses the blood–brain-barrier it will undergo hydrolysis, releasing benactyzine and GABA, which are expected to act synergistically. The combined release of both compounds in the brain offers several advantages over the current OP poisoning treatment protocol: improved efficacy and safety profile (where the inhibitory properties of GABA are expected to counteract the anticholinergic cognitive adverse effects of benactyzine) and enhanced chemical stability compared to benactyzine alone. We present here preliminary results of animal studies, showing promising results with early gabactyzine administration.Organophosphates (OPs) are inhibitors of acetylcholinesterase and have deleterious effects on the central nervous system. Clinical manifestations of OP poisoning include convulsions, which represent an underlying toxic neuro-pathological process, leading to permanent neuronal damage. This neurotoxicity is mediated through the cholinergic, GABAergic and glutamatergic (NMDA) systems. Pharmacological interventions in OP poisoning are designed to mitigate these specific neuro-pathological pathways, using anticholinergic drugs and GABAergic agents As a consequence of this inhibition, a progression of toxic signs is evident, including hypersecretion, tremors, convulsions, respiratory distress, and, ultimately, death4. The initial phase is the result of massive acetylcholine (ACh) accumulation leading to excessive cholinergic activity6. Thus, the primary targets of medical countermeasures (MCM) against OP poisoning are to prevent further AChE inhibition and reactivation and counteracting the effects of ACh in muscarinic receptors7. However, while present MCMs for OP poisoning offer robust protection against lethality8, CNS damage, including long-lasting behavioral deficits, are not completely prevented10. Currently-used treatments fail to control the convulsions associated with glutamate excitotoxicity following the initial step of excessive cholinergic activation11. Notably, antagonists of N-methyl-d-aspartate (NMDA)12 or AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors13, two subtypes of central glutamate receptors, afforded additional protection 14. In this respect, it is important to note that non-human primates poisoned with 3–5 LD50 of soman and treated with a mixture of an oxime, atropine, and benactyzine (TAB), showed remarkable recovery16. Benactyzine is a compound with mixed anti-cholinergic and anti-glutamatergic properties, approved for emergency use in Israel in OP poisoning18.Organophosphates (OPs) are powerful inhibitors of acetylcholinesterase (AChE), with a deleterious central nervous system (CNS) effect20. It was recently suggested that the degree of brain damage and ensuing cognitive deficits depend on the severity of convulsions21.Numerous studies have shown that exposure to different OPs at different doses produces a neuronal loss in various CNS structures, particularly in the entorhinal and piriform cortex, amygdala, and the CA1 and CA3 subfields of the hippocampus23.Spatial learning and memory impairments are two major detrimental consequences of OP poisoning, with ensuing neuronal degeneration in CNS structures . Indeed, cognitive weakness, such as impaired learning and memory resulting from exposure to OPs, was reported in humans and animals25, demonstrated that caramiphen, a drug with a mixed anticholinergic and antiglutamatergic profile, affords better protection when compared to that provided by scopolamine, a pure anticholinergic agent when given in a pretreatment paradigm, and that TAB and caramiphen given to soman-exposed animals offered cognitive protection as reflected with the Morris water maze test26. These results show that drugs with a pharmacological profile consisting of anticholinergic and antiglutamatergic properties such as caramiphen and benactyzine have an advantage as post-exposure therapies against poisoning by OP agents27.Raveh et al.The lack of a side-effect-free anticonvulsant prompts current research efforts aimed at developing better MCMs for OP-induced seizures. Any such drug should be devoid of behavioral side effects as possible, to diminish incapacitation of the casualty and since the drug may be taken inadvertently. Therefore, it is critical to identify drugs that not only counteract the effects of nerve agents but also produce minimal behavioral effects when administered alone.28. It is known that benzodiazepines that bind allosterically to GABA-chloride channels, display anticonvulsant activity and are commonly administered to patients affected by OP agents. Moreover, the benzodiazepine drug midazolam was shown to be superior over other compounds in terminating OP-induced seizures, with some neuroprotective qualities. However, GABA itself does not readily cross the blood–brain-barrier (BBB) and is therefore ineffective when systemically administered.Gama-Amino-Butyric-Acid (GABA) is the major inhibitory neurotransmitter in the brain and was found to be involved in the reduction of convulsionsTherefore, a concept of a prodrug of an anticholinergic compound acting in the CNS together with GABA activity may offer a novel therapeutic approach, influencing the main receptorial systems involved in seizure initiation and propagation induced by OP poisoning. Moreover, it is anticipated that the inhibitory action of GABA receptors would attenuate some of the central anticholinergic adverse effects , thus increasing current drugs' efficacy and safety.30 and the nortriptyline-GABA amide for the treatment of neuropathic pain31, we sought to synthesize a benactyzine-GABA mutual prodrug (labeled "gabactyzine"). It is expected to be able to cross the BBB due to the lipophilic character of the benactyzine, acting as the “carrier” and transporting GABA into the brain. Once the molecule crosses the BBB it should undergo hydrolysis releasing concomitantly benactyzine and GABA, which are expected to act synergistically.Based on our previously described prodrugs development such as the perphenazine-GABA ester for the treatment of schizophrenia1H and 13C NMR spectra were obtained on Bruker Avance-200, Avance-DPX-300, Avance-400, Avance-DMX-600, and Avance-III-700 spectrometers. Chemical shifts are expressed in ppm downfield from Me4Si (TMS) used as internal standard. The values are given in δ scale. It should be noted that the compounds that have carbamates, frequently are seen in the 1H NMR and 13C NMR spectra as mixtures of pairs of rotamers. Whereby the signals for the protons of the CH2’s, and the corresponding carbons appear in close proximity as pairs of signals. In these cases. the chemical shifts of the rotameric H’s and C’s are indicated while being linked by + signs. For some compounds, the NMR data assignment was aided by several 2-dimensional spectra including COSY, HMQC, and HMBC analyses. High-resolution mass spectra (HRMS) were obtained on a 6545 QTOF instrument (Agilent). The progress of the reactions was monitored by TLC on silica gel . All the flash chromatographic procedures were carried out on silica gel . The nomenclature of the compounds was assigned according to ChemDraw Ultra version 12.0.3.1216 (Cambridge Soft).N-Boc-GABA or 2-(4-(tert-butoxycarbonylamino)butanoyloxy)-2,2-diphenylacetic acid, 7) and DBU (1 mmol) in ~ 2 mL of CH3CN (dried over molecular sieves) was stirred at room temperature overnight. The mixture was then quenched with distilled water. A white solid that precipitated was washed with ether, and the organic layer was washed with saturated KHSO4. The organic layer was dried over Na2SO4, filtered, and evaporated. The residue was purified by silica gel chromatography, or by preparative HPLC.A mixture of an alkyl halide (1 mmol), an acid (1 mmol) (To a solution of a Boc-protected amino compound in dry ether was added HCl(g). The solution was stirred for 1–2 h and the solvent was evaporated to give the hydrochloride salt of the free amino compound.33 in HBr/AcOH was kept at 0 °C overnight. The solvent was evaporated, and the residue was dried under a high vacuum. 1H NMR : δ 7.34–7.40 , 7.48–7.52 . 13C NMR : δ 71.3 (C-4°), 128.7, 129.0, 130.0 (CH ar), 142.1 (C-4°), 171.3 (C-4°).A suspension of benzilic acid1 was obtained from 6 as an oil in quantitative yield using procedure 2. 1H NMR : δ 1.16 , 1.95 , 2.68 , 2.90 , 3.03 , 3.43 , 4.54 , 7.35 , 7.52 . 13C NMR : δ 9.3 (Me), 23.7, 32.1, 39.8, 49.3, 51.2, 61.5 (six CH2), 85.6 (C-4°), 128.6, 129.59, (CH ar), 140.55 (C-4°), 170.1 (C-4°), 172.6 (C-4°). HRMS calcd for C24H33N2O4+ [MH+] 413.2435, found 413.2437.Compound 22 was obtained from 20 in quantitative yield using procedure 2. 1H NMR : δ 1.09 , 1.95 , 2.70 , 2.79 , 2.91 , 3.33 , 4.45 , 7.24 , 7.29 , 7.34 , 7.49 . 13C NMR : δ 9.14 (Me), 23.8, 32.3, 39.7, 39.7, 49.8, 51.0 (six CH2), 86.7 (C-4°), 87.0 (C-4°),128.5, 129.12, 129.22, 129.25, 129.77 (CH ar), 139.50 (C-4°), 140.3 (C-4°), 168.1 (C-4°), 169.6 (C-4°), 172.23 (C-4°).Compound 20 was obtained from 18 as an oil using procedure 1. 1H NMR : δ 1.12 , 1.41 , 1.84 , 2.68 , 3.02 , 3.11 , 3.41 , 4.55 , 6.15 , 7.23–7.29 , 7.35–7.36 , 7.55–7.57 . 13C NMR : δ 8.8 (Me), 26.0 (CH2), 28.6 (Boc Me’s), 32.4, 40.17, 47.7, 50.0, 60.8 (five CH2), 78.5 (C-4°), 85.07 (C-4°), 85.93 (C-4°), 127.87, 128.44, 128.84, 128.89, 129.09 (CH ar), 139.36 (C-4°), 139.36 (C-4°), 156.69 (CO carbamate), 167.55 (C-4°), 168.9 (C-4°), 172.15 (C-4°).Compound 6 was obtained from 7 as an oil in ~ 45% using procedure 1. 1H NMR : δ 0.94 , 1.41 , 1.85 , 2.36–2.64 , 3.14 , 4.17 , 6.05 , 7.28–7.38 , 7.57–7.61 . 13C NMR  δ 12.5 (Me), 26.1 (CH2), 28.6 (Boc Me’s), 32.4, 40.4, 48.0, 51.8, 64.8 (five CH2), 78.5 (C-4°), 84.7 (C-4°), 128.25, 128.58, (CH ar), 141.4 (C-4°), 156.7 (CO carbamate), 169.8 (C-4°), 171.9 (C-4°).Compound 21 was obtained in trace amounts from 19 as a white solid, using procedure 1. 1H NMR : δ 1.06 , 1.41 , 1.86 , 2.63 , 2.85 , 3.12 , 3.20 , 4.44 , 6.12 , 7.21–7.24 , 7.26–7.30 , 7.43–7.44 . 13C NMR : δ 8.9 (Me), 26.2 (CH2), 28.6 (Boc Me’s), 32.6 (CH2), 40.36 + 40.24 , 47.6, (CH2), 49.7 (CH2), 60.7 (CH2), 78.5 (C-4°), 85.5 (C-4°), 86.4 (C-4°), 87.1 (C-4°), 128.54, 128.59, 128.66, 128.73, 128.86, 129.02, 129.09, 129.37 (CH ar), 139.16 (C-4°), 139.38 (C-4°), 140.38 (C-4°), 156.69 + 159.73 (CO carbamate two rotamers), 166.47 (C-4°), 167.40 (C-4°), 168.98 (C-4°), 171.32 (C-4°).Compound 7 was obtained from 16 as an oil using procedure 1 and was used further without additional purification. 1H NMR : δ 1.40 , 1.84 , 2.55 , 3.12 , 6.05 , 7.29–7.35 , 7.62–7.66 . 13C NMR : δ 26.0 (CH2), 28.6 (Boc Me’s), 32.3 (CH2), 40.3 (CH2), 78.6 (C-4°), 84.4 (C-4°), 128.23, 128.60, (CH ar), 141.6 (C-4°), 156.8 (CO carbamate), 170.7 (C-4°), 171.8 (C-4°).Compound 17 was obtained from 16 as an oil in 38% using procedure 1 in refluxing benzene. 1H NMR : δ 1.39 , 1.81 , 2.51 , 3.15 , 6.85 , 6.85 , 7.28 , 7.35 , 7.41 . 13C NMR : δ 26.1 (CH2), 28.6 (Boc Me’s), 32.0 (CH2), 40.19 + 40.32 (CH2 two rotamers), 77.4 (CH), 78.4 (C-4°), 127.6, 128.5, 129.3 (CH ar), 141.8 (C-4°), 156.7 (CO carbamate), 172.5 (C-4°).Compound 13 was obtained from 12 as an oil in 79% using procedure 1. 1H NMR : δ 1.02 , 1.51 , 1.95 , 2.55 , 2.72 , 3.25 , 4.26 , 6.20 , 7.54–7.53 , 7.63–7.65 . 13C NMR : δ 12.5 (Me), 26.1 (CH2), 28.6 (Boc Me’s), 31.7, 40.7, 48.0, 51.9, 64.6 (five CH2), 75.3 (CH), 78.5 (C-4°), 128.6, 129.4, 129.8 (CH ar), 135.2 (C-4°), 156.6 (CO carbamate), 169.4 (C-4°), 173.0 (C-4°).Compound Method 1 To a solution of 13 (1 eq) in dry EtOAc, CuBr2 (1 eq) was added. The mixture was refluxed for 1 h, and when no starting material was detected by TLC, the mixture was filtered through a short silica column. The silica was washed EtOAc/MeOH, and the combined eluent was evaporated to give compound 15 as an oil in quantitative yield. Method 2 To a solution of 13 (1 eq) in CCl4 was added N-bromosuccinimide (NBS) (2 eq) and azobisisobutyronitrile (AIBN) (0.025 eq). The solvent was evaporated after 1.3 h when starting material was not detected by TLC. 1H NMR : δ 1.38 , 1.82 , 2.51 , 3.14 , 3.46 , 3.78 , 4.66 , 6.05  6.20 , 7.54–7.53 , 7.63–7.65 . 13C NMR : δ 8.4 (Me), 25.0 (CH2), 27.6 (Boc Me’s), 30.0, 39.1, 48.6, 50.7, 59.7 (five CH2), 74.0 (CH), 78.0 (C-4°), 127.6, 128.7, 129.3 (CH ar), 137 (C-4°), 156.6 (CO carbamate), 169.8 (C-4°), 172.3 (C-4°). 11 (1 eq) and diethylaminoethanol in CH2Cl2 was stirred at room temperature overnight and was then washed with 5% aq. NaHCO3, dried over Na2SO4, filtered and evaporated to give compound 12 as a yellow oil in 72%. 1H NMR : δ 0.93 , 2.45 , 2.64 , 4.13 , 5.67 , 7.39–7.41 , 7.56–7.59 . 13C NMR : δ 12.5 (Me), 47.9 (CH2), 51.7(CH2), 59.7 (CH), 65.2 (CH2), 75.3 (CH), 128.8, 129.4, 129.8 (CH ar), 137.2 (C-4°), 168.6 (C-4°).A solution ofThe study was approved by and performed in accordance with the guidelines of The Animal Care and Use Committee of Sheba Medical Center, which is approved by the Israeli authorities for animal experimentation. The reporting in the manuscript follows the recommendations in the ARRIVE guidelines.Adult male albino Sprague–Dawley rats, weighing 300–320 g , were used in all studies, following two–three days of acclimatization in the animal facility. The animals were housed under standard laboratory conditions in plastic cages, four per cage at a temperature of 21 ± 2 °C and 50 ± 10% humidity, in controlled animal quarters, and maintained on a 12 h light–dark cycles (light on at 6.00 a.m.). Food and water were available ad labium.2O. All other drugs were diluted in normal saline. The novel compound gabactyzine was synthesized and supplied by the Medicinal Chemistry laboratory of Bar-Ilan University. Anesthesia applied before each imaging session, was administered by an intramuscular (IM) injection consisting of 450 µL of Ketamine (22.5 mg/mL) and 150 µL xylazine (0.3%).Paraoxon, atropine sulfate, obidoxime, and midazolam were purchased from Sigma (Israel). Paraoxon was diluted in a vehicle containing 40% propylene glycol, 10% EtOH, 1.5% benzyl alcohol and 48.5% distilled H34.A preliminary observational study was performed in naïve rats to evaluate the gross toxic effects of the compound during a relatively short period, serving as a go-no go approach. We used escalating doses of gabactyzine ranging from 1 to 5.2 mg/kg, injected IM, and continuously observed the treated animals for a period of 6 h, regarded as sufficient for this stage of development of an antidotal therapy, allowing to assess any potential acute adverse effects of the drug50 of paraoxon . One min after exposure, all rats were treated with atropine  and obidoxime . One min or 30 min after the clinical appearance of general tonic–clonic convulsions , gabactyzine (5.2 mg/kg) was administered IM. The use of 1- and 30-min time points were previously established in the rat model of paraoxon poisoning as allowing to test the efficacy of anticonvulsant drugs36, representing either immediate use of an autoinjector, or if delayed, representing treatment given by a medic in the field. The paraoxon dose was chosen based on previous studies, showing it to be toxic enough to induce seizures, and with the chosen atropine and oxime doses will allow survival but will not prevent the brain damage, thus allowing to test the efficacy of various centrally-acting drugs in alleviating the brain injury36. The 5.2 mg/kg gabactyzine dose was chosen as this was the highest dose tested showing no adverse effects. The animals were closely observed for post-exposure clinical signs every 15 min for a period of 4 h, and then once a day for a week. Observation included signs of cholinergic toxicity and of general distress . The clinical signs were processed into a scoring system of 0–4: 0 = no reaction, 1 = mild reaction , 2 = moderate reaction , 3 = severe reaction , 4 = mortality. The rats were weighed before exposure and daily thereafter.This study focused on evaluating the efficacy of gabactyzine in a rat model of paraoxon poisoning using two treatment schedules. Adult (8-week-old) male albino Sprague–Dawley rats weighing 300–320 g  were housed under standard laboratory conditions in plastic cages, four per cage in a controlled environment with constant temperature of 21_2C_ and a 12 h light/dark cycle. Food  and water were available ad libitum. Care and maintenance were in accordance with the principles described in the Guide for Care and Use of Laboratory Animals . The experimental protocol was examined and approved by the Institutional Committee for Animal Experimentation as required by local law. Paraoxon, atropine sulfate, and obidoxime were purchased from Sigma–Aldrich, Jerusalem, Israel. Paraoxon was diluted in a vehicle containing 40% propylene glycol. All other drugs were diluted in normal saline. 24 animals were exposed to 1.4 LD50 of Paraoxon . One min after poisoning, all rats were treated with Atropine  and Obidoxime . Either 1 or 30 min after clinical generalized seizures were evident, one of three anticonvulsant drugs—midazolam (1 mg/kg), benactyzine (1 mg/kg) or gabactyzine (1 mg/kg)—was administered IM. The clinical state was assessed and scored every 15 min until 1 h after poisoning, and then once per day. The rats were divided into 5 treatment groups:Group C served as control (n = 12): No additional treatmentGroup G1 was treated with gabactyzine 1 min post poisoning (n = 8)Group B1 was treated with benactyzine 1 min post poisoning (n = 9)Group G30 was treated with gabactyzine 30 min post poisoning (n = 18)Group M30 was treated with midazolam 30 min post poisoning (n = 13)Group B30 was treated with benactyzine 30 min post poisoning (n = 9)Sixty adult (8-week-old) male albino Sprague–Dawley rats weighing 300–320 g  were handled as in the second study depicted above. All animals were administered a dose of 1.4 LDThe animals were scanned by MRI 1-, 8-, and 15-days post-exposure. Anesthesia, applied prior to each imaging session, was administered by an intra-muscular (i.m.) injection consisting of ketamine  and Xylasine . The rats were weighed before treatment and following each MRI scan. At the end of the follow-up, the rats were sacrificed.35 and in Gudbjartsson et al.37. In short, scanning was performed using a General Electric 3.0 T MRI machine  with the HD12 operating system, gradients intensity of up to 4.3 Gauss/cm, the line scan diffusion-weighted imaging acquisition software package (LSDI)37, and a clinical phased array knee coil.MR imaging and analysis methods of T2-weighted MRI, diffusion weight MRI (DWMRI), and apparent diffusion coefficient maps followed the methods described in Rosman et al.2 field of view (FOV), repetition time (TR) of 3000 ms, echo time (TE) of 90 ms and 2 mm slices covering the full rat brain, thus containing the 3D information. LSDI DWMR images were acquired with: 256 × 128 matrix, 12 × 9 cm2 FOV, TR = 5440 ms, TE = 142 ms and 2 mm slices. Data was acquired at b = 0 and 1000 s/mm2. Volumes of the temporal tissue toxicity were calculated by plotting regions of interest (ROIs) over the entire enhancing regions in each DWMR slice, adding the number of pixels over the slices and multiplying by the volume of each pixel. Ventricular volume representing hydrocephalus was calculated by plotting ROIs over the frontal ventricles in each T2-weighted MR slice, adding the number of pixels over the slices and multiplying by the volume of each pixel.T2-weighted fast spin echo (FSE) MR images were acquired with: 256 × 128 matrix, 12 × 9 cmSame scores used in the previous study were implemented here, ranging from 0 (no reaction) to 4 . The numbers of animals are shown in parenthesis. The percentage was calculated from surviving animals in each treatment group for that day.® computer program . In the second study, the clinical score was subjected to a two-way analysis of variance (ANOVA) with a repeated measure on one variable (time following paraoxon exposure), with p < 0.05 considered statistically significant (marked with *); a 1-Way ANOVA Dunnett Multiple Comparison Test was used to analyze the change in weight, with p < 0.05 considered statistically significant. Survival in the third study was analyzed using Kaplan–Meier survival curves. Comparison between groups was performed using the unpaired two-tailed t-test, except where the changes in the Apparent Diffusion Coefficient (ADC) as a function of time were studied. In this case, the ADC of each rat was compared at different time points, thus the one-tailed paired t-test was used.Analysis was performed using the SPSSThe synthetic goal of this study was the development of a suitable process for the preparation of gabactyzine Fig. .Figure 1N-protected GABA derivatives or with GABA itself under acidic conditions, failed. A search of the literature revealed that the only esters of benactyzine reported are the acetate and propionate for which no synthetic procedures are provided42. The highly hindered character of the 3ary OH group of benactyzine is reflected in a variety of unsuccessfully attempted acylation conditions using i.e., benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (Py-Bop), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), 2,4,6-trichlorobenzoyl chloride (TCBC—Yamaguchi reagent), 1-hydroxy-7-azabenzotriazole (HOAt)/(1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolopyridinium 3-oxide hexafluorophosphate (HATU), or GABA + benactyzine in the presence of a catalytic amount of concentrated H2SO443 with N-Boc-GABA, where no evidence of esterification was detected.Although gabactyzine is the GABA ester of benactyzine, all attempts to directly esterify benactyzine with carboxy-activated-Because of our inability to carry out direct esterification of benactyzine, various approaches were undertaken to build intermediates suitable for modification, which would lead to the desired gabactyzine. The synthesis originally expected to be a straightforward one turned out to be quite challenging.4, which was esterified with diethylaminoethanol to give the corresponding α-chloro ester 544. Several unsuccessful attempts were then made to replace the chloride with N-Boc-GABA  in refluxing benzene32. However, since the reaction was carried out at reflux temperature, most of the isolated material was the product of thermal decarboxylation 17. Whereas in previous attempts, we encountered great difficulty in direct esterification of the 3ary OH group of benactyzine, herein the condensation between the N-Boc-GABA/DBU and 16 proceeded readily involving replacement of Br by the N-Boc-GABA carboxylate, to give 7, albeit in low yield. The yield of 7 was improved considerably when the reaction was carried out in DMF or CH3CN at room temperature to give 7 together with small amounts of the side products 18 and 19. It is assumed that 18 and 19 were obtained by an SN1 reaction involving the formation of the planar carbocation, which reacted with the N-Boc-GABA/DBU. Treatment of the mixture 7, 18 and 19 with 2-diethylaminoethyl chloride hydrochloride and two additional equivalents of DBU, or with 2-diethylaminoethanol/carbonyldiimidazole (CDI), led to the formation of the N-Boc-protected gabactyzine 6, together with 20 and 21. Removal of 20 and 21 by silica gel chromatography was unsuccessful and therefore these side products were removed by preparative HPLC. Final deprotection of the Boc group from 6 in the presence of dry HCl(g) gave the desired gabactyzine 1.Benzilic acid was converted into the α-bromo derivative No changes from baseline activities were noted and no clinical adverse effects were observed. We concluded that the compound does not have observable adverse effects on animals' behavior in the doses used.After paraoxon injection, all animals convulsed. In both the control group and the gabactyzine—30 min treatment group, one animal was euthanized 48 h after exposure due to extreme weight loss from baseline. The change in the rats' weights as a function of time is shown in Fig. Kaplan–Meier survival curves of the different treatment groups are shown in Fig. Significant tissue toxicity was depicted by diffusion-weighted and T2-weighted MRI 24 h post-treatment, manifested as bright temporal enhancement with low apparent diffusion coefficients. An example is shown in Fig. 3) than in all treatment groups: G1 , B1 , G30  and M30 .Figure Later toxicity, reflected by ventricular enlargement, was noted from the second follow-up and on. An example is shown in Fig. Figure There was no significant difference between the weights of the different groups before poisoning (average weight: 301 ± 2 g), and there was no significant difference between treatment groups during the different follow-ups.50. This is more pronounced with nerve agent, in which the preliminary cholinergic hyperstimulation is followed by the activation of other neurotransmitter systems, with the glutamatergic system being the most pronounced52. When exposed to moderate or high doses of OPs, casualties may develop long-lasting seizures50, and if treatment is delayed or absent, seizures could progress to status epilepticus and cause irreversible seizure-related brain damage53. The typical neuropathological damage was described as bilaterally symmetrical, with severe damage to the temporal lobe structures  and the thalamus59. The prevention of seizures following OP poisoning are regarded as a primary objective of medical treatment53. However, this objective becomes increasingly difficult to achieve as time elapses before therapy is started, and seizures become less responsive to medical treatment60.Shortly after exposure to OPs, and even in transient exposure and despite antidotal treatment, brain damage with long-lasting neuro-behavioral damage may occurAccording to the Israeli treatment doctrine of nerve agent poisoning, midazolam and Benactyzine should be co-administered for improved therapeutic effect in severe nerve agent poisoning. However, as this combination has the potential to induce side effects, we aim to test novel combinations that will provide as good survival results as this combination, with fewer side effects.Though benzodiazepine treatment shows that increasing synaptic GABAergic activity is useful, that is not the same as increasing GABA concentrations, especially as GABA itself is subject to dynamic regulation, being formed and degraded continuously.61. Thus, it was postulated that increasing the concentration of GABA in the brain might have positive effects in OP poisoning. Tiagabine is an anticonvulsant drug that directly inhibits the re-uptake of GABA into presynaptic nerve terminals and increases its concentrations, thus enhancing GABAergic transmission. Despite these features, tiagabine was found ineffective in blocking soman-induced convulsions63. Vigabatrin is an antiepileptic agent that prevents the metabolism of GABA by irreversibly inhibiting GABA transaminase. This results in increased circulating GABA concentrations by reversing the direction of the transport. As vigabatrin is an irreversible inhibitor of GABA transaminase, its duration of effect is thought to be dependent on the rate of GABA transaminase re-synthesis rather than on the rate of drug elimination. Using vigabatrin alone and in combination with atropine in a mouse model of OP poisoning has shown to protect mice against dimethoate lethality, helped to maintain the righting reflex, and delayed the onset of muscle fasciculations64.However, specifically with OPs, it has been reported that sarin directly reacts with muscarinic acetyl-choline receptors present in presynaptic GABAergic neurons in rat hippocampal slices, thereby reducing GABA releaseGabactyzine is expected to be able to cross the BBB due to the lipophilic character of its benactyzine component, helping to transport GABA into the brain. Next, it is expected to undergo hydrolysis, releasing concomitantly benactyzine and GABA, which are expected to act synergistically in reducing brain damage following exposure to nerve agents. Moreover, and as centrally-acting drugs have various side effects, we anticipate that by combining the two drugs it could potentially lead to reduction of the total dose of benactyzine being used, thus reducing its side effects while preserving its therapeutic value.66. Such an accurate, noninvasive and early assessment of brain tissue response is essential when assessing novel therapeutics against acute poisoning, enabling real time adjustments of treatment. The disadvantage is that the DWMRI is sensitive mainly to early changes in tissue characteristics and less to subtle changes in blood vessels' function which may cause later effects.Diffusion-weighted magnetic resonance imaging (DWMRI) allows noninvasive description of biological tissues based on their water diffusion characteristics, and at an early stage. It was already shown that nonspecific brain damage can be depicted early using DWMRI, days and even weeks before it can be detected in T1 and T2 weighted MR images35. The images in this study were acquired on a 3.0 T scanner in small animals using echo-planar based DWMRI sequences. We have included a LSDI sequence, enabling robust yet low-resolution DWMR images. For this reason, the images are of low resolution, appearing blur. Still, the damage induced by paraoxon is clearly depicted and quantification , showing significant effects. As we shown previously35 and in this study, the early paraoxon-induced toxicity detected in DWMRI precedes later brain atrophy, reflected by the enlarged ventricles.In a previous study we have already shown DWMRI as a valid method for monitoring paraoxon-induced brain damage in a rat model, as well as using it for the assessment of early vs late treatment, using two known compounds with beneficial CNS effects1. The preliminary animal studies showed promising results regarding the safety of gabactyzine 1. In the second study we showed preliminary clinical efficacy in OP-poisoned animals, in two administration schedules: early (1 min after seizure initiation) vs. late (30 min). This led us to further investigate the efficacy of gabactyzine 1 in comparison to the standard antidotal treatment in Israel using more elaborated tools.A successful synthesis was developed for the preparation of gabactyzine In means of survival, as shown in the Kaplan–Meier curves of the different treatment groups, early administration of gabactyzine and benactyzine had a similar effect to late administration of midazolam. When applied 30 min post-seizure onset, gabactyzine was less efficient than benactyzine in terms of survival. Early administration of gabactyzine was as good as early administration of benactyzine in preventing brain damage, as assessed by DWMRI and T2-weighted MRI. The later administration of gabactyzine (30 min) seemed to be less effective than the late administration of midazolam, although still better than no treatment at all. Brain atrophy, calculated from ventricular changes on T2-weighted MRI and from DWMRI, was consistent and showed no difference between the treatments. However, since gabactyzine is a new compound, it may be more effective at different doses. Therefore, to determine if the new drug can obtain higher treatment efficacy, dose-escalating studies should be performed. The fact that early administration of gabactyzine enables as good treatment efficacy as benactyzine is encouraging since gabactyzine is expected to have fewer side effects.A limitation of this study is the lack of correlation of brain damage with histological analysis. Though a potential advantage of using MRI is to replace the need to sacrifice animals for histology in various time point during the study, MRI—at least in such early go-no go studies—would be ideally correlated with histological analysis, at least following euthanasia.These studies were all defined as part of a preliminary proof-of-concept effort, to test whether there is merit in gabactyzine as a prodrug. Within the generic process of studying a new therapeutic agent, after showing preliminary safety and efficacy, the next steps include testing the compound in a larger set of animals, including another species, as defined in the FDA's "two animal rule", and testing the new compound within various drug combinations, comparing new combinations with known drug combinations, as it is our experience that sometimes a single drug performs quite differently than when it is co-administered with other drugs.In this set of preliminary proof-of-concept studies, we focused on the safety and preliminary efficacy of a novel pro-drug, gabactyzine, intended to protect the CNS from organophosphate poisoning. As the preliminary results were generally regarded as similar to results obtained with higher doses of other drug combinations, this will now set the way to future studies, further looking into the effects of this novel pro-drug, including at higher concentrations and doses and longer follow-up periods.


Acute monocytic leukemia belongs to type M5 of acute myeloid leukemia (AML) classified by FAB, which appears a high incidence of extramedullary infiltration (EMI) and poor prognosis. In this study, we observed the inhibitory effect of ginsenoside Rk3 on the EMI of monocytic leukemia cells and initially explored its related mechanism of targeting the miR-3677-5p/CXCL12 axis.  The MTT assay and colony formation assay were used to detect the inhibitory effect of Rk3 on proliferation. Both cellular migration and invasion were observed by the Transwell assay. The expression levels of miR-3677-5p, CXCL12, and CXCR4 were detected by RT-qPCR and Western blot, as well as overexpression of miR-3677-5p by transfected with lentivirus and detection of a dual luciferase reporter gene. The expression of MMP2 and TIMP2 was detected by immunofluorescence.  Rk3 effectively inhibits the proliferation, migration, and invasion associated with EMI of leukemia. The leukemia cells of M5 patients with EMI showed low expression of miR-3677-5p but high expression of the mRNA of CXCL12 and CXCR4. Overexpression of miR-3677-5p or intervention of CXCL12 effectively inhibited the proliferation, migration, and invasion of SHI-1 cells. The luciferase assay showed that CXCL12 was the downstream target gene of miR-3677-5p. After overexpression of miR-3677-5p or intervention of CXCL12 in combination with Rk3, the inhibitory effect on the proliferation, migration, and invasion of SHI-1 cells was more obvious. Importantly, Rk3 significantly regulated the expression levels of miR-3677-5p, CXCL12, CXCR4, and EMI-related functional proteins including MMP2 and TIMP2. Overexpression of miR-3677-5p or intervention of CXCL12 also regulated the expression of MMP2 and TIMP2.  The leukemia cells of M5 patients with EMI appeared to have low expression of miR-3677-5p and high expression of the mRNA of CXCL12 and CXCR4, which may be used as indicators of EMI and poor prognosis. Rk3 is effective in inhibiting the EMI of SHI-1 cells by targeting the miR-3677-5p/CXCL12 axis. Acute myeloid leukemia (AML) is a clonal, malignant disease of the hematopoietic tissues that is characterized by the accumulation of abnormal leukemic blast cells, principally in the marrow, and impaired production of normal blood cells. Thus, the leukemic cell infiltration in the marrow is accompanied, nearly invariably, by anemia and thrombocytopenia. The absolute neutrophil count may be low or normal, depending on the total white cell count . Acute mGinseng is a Chinese medicine herb, and its main ingredient is ginsenosides, which are characteristics of anti-inflammatory properties, antitumor and other pharmacological effects –9. Our pMicroRNA (miRNA) is a single-stranded RNA molecule of about 22 nucleotides, located at the core of the ncRNA regulatory network. Its function is to participate in the maintenance of cellular proliferation, differentiation, and apoptosis. Abnormal expression or regulation of miRNAs is closely related to the occurrence and progression of leukemia/tumor –16. StudTherefore, we studied the effects of miR-3677-5p and CXCL12 expression on the proliferation, migration, and invasion of SHI-1 cells and explored the active mechanism of Rk3 for anti-EMI in monocytic leukemia cells.2 atmosphere at 37°C. The structure of ginsenoside Rk3  was identified by nuclear magnetic resonance, mass, infrared spectrum, and ultraviolet absorption spectra. The purity was >98%, and the batch No. 140606. Polybrene transfection reagent was used for the transfection of miR-NC  and miR-3677-5p mimic  according to the manufacturer's instructions. The AMD3100 (the inhibitor of the CXCL12/CXCR4 axis) was purchased from Selleck Chemicals , and the batch No. S803002.Human monocytic leukemia SHI-1 cells were donated by Professor Chen Zixing, Institute of Hematology, and the First Affiliated Hospital of SooChow University. The cells were maintained in Iscove's Modified Dulbecco's Medium  supplemented with 15% new bovine serum  in a humidified 5% COThe bone marrow samples from 20 patients diagnosed with AML-M5 were collected in our hospital from April 2021 to March 2022; the diagnostic criteria were based on the 2016 World Health Organization Classification of AML . The bon4 SHI-1 cells were plated into each well of a 96-well plate, and treated with Rk3 at the final concentration of 60, 120, and 180 mol/L for 48 hours at 37°C in a 5% CO2 incubator, and each group of Rk3 concentration was repeated planting for 5 wells. Then, 20 μl MTT solutions (5 mg/mL) was added and incubated for 4 hours. After the supernatant was removed by centrifugation, 150 μl DMSO was added, the absorbance of the solution was read by a microplate reader ELx808 , using a test wavelength of 490 nm. The inhibition rate of Rk3 on the proliferation of SHI-1 cells was calculated, the inhibition rate (%) = [/control group A value] × 100%. The same experiment was repeated for 5 times.About 2 × 10μM of AMD3100, were treated for 12 hours. They were planted to the semisolid culture system by the method above. The experiments were performed in triplicate with 103 cells per well in a 24-well plate, and incubated at 37°C in a humidified atmosphere supplemented with 5% CO2 for 7 days. The colony number (≥40 cells) was counted under the microscope. The inhibition rate of Rk3/miR-3677-5p mimic/AND3100 on proliferation of SHI-1 cells (%) = [/colony number in control group] × 100%. The same experiment was repeated for 5 times.SHI-1 cells were cultured in IMDM consisting of 30% new bovine serum, 1% L-glutamine, penicillin-streptomycin, and 0.3% agar as viscous support, with the final concentrations of Rk3 were 60, 120, and 180 mol/L. In addition, the other three groups, including miR-3677-5p mimic, miR-NC, and 2.5 μM of AMD3100, which were collected for the Transwell assay. In the migration experiment, 1 × 105 cells (200 μl) were seeded into the upper chamber of a Transwell insert , and a medium with 15% NBS (600 μl) was added in the lower chamber. In the invasion experiment, Matrigel  was coated on the upper chamber seeded with 1 × 105 cells, and the lower chamber contained 15% NBS medium. After incubation at 37°C, 5% CO2 for 24 hours, the Transwell chamber was taken out and washed with PBS. The cells on the membrane were fixed with 4% paraformaldehyde for 20 mins, and stained with crystal violet for 30 mins. The upper unmigrated cells were gently wiped off with a cotton swab, and five fields were randomly selected under the microscope  to take photos and count cell numbers.SHI-1 cells were treated with Rk3 at a final concentration of 60, 120, and 180 mol/L for 48 hours. In addition, the other three groups included miR-3677-5p mimic, miR-NC, and 2.5 https://gepia.cancer-pku.cn/detail.php###) was used to analyze the difference in CXCL12 and CXCR4 expression levels between the AML patients group (num = 173) and the normal control group (num = 70).The Gene Expression Profiling Interactive Analysis (GEPIA) website . PowerUp™ SYBR Green Master Mix  was applied to the thermocycling reaction in the QuantStudio 7 Flex real-time PCR system. The forward and reverse primers of β-actin were 5′-CATCAAGGAGAAACTGTGT-3′ and 5′-AGGCAACTCGTAACTCTT-3′; the forward and reverse primers of U6 were 5′-AGAGAAGATTAGCATGGCCCCTG-3′ and 5′-CAGTGCAGGGTCCGAGGT-3′, the forward and reverse primers of miR-3677-5p were 5′-GCAGTGGCCAGAGCCCT-3′ and 5′-AGTGCAGGGTCCGAGGTATT-3′, the forward and reverse primers of CXCL12 were 5′-ATTCATAACACTCCAAACTGTGC-3′ and 5′-ACTTTAGCTTCGGGTCAATGC-3′, the forward and reverse primers of CXCR4 were 5′-ACTACACCGAGGAAATGGGCT-3′ and 5′-CCCACAATGCCAGTTAAGAAGA-3′. The thermal cycler conditions were performed as follows: Cycle 1 (95°C for 3 mins), Cycle 2 , and Cycle 3 (72°C for 4 mins). The relative gene expression quantification was calculated with the 2−ΔΔCt method. All experiments were done in triplicate. The RNAs of leukemia cells from patients with M5 were handled by the same method. The same experiment was repeated for 3 times. β-actin was used as an internal control.SHI-1 cells were incubated with 60, 120, and 180 mol/L of Rk3 for 48 hours, in addition, the other three groups, including miR-3677-5p mimic, miR-NC, and 2.5 β-actin  overnight at 4°C. After being washed with 1 × TBST buffer for three times, the membranes were incubated with secondary antibody  for 1 hour at room temperature. The special bands from the conjugation reaction of protein antigen and antibodies were visualized by using Luminol from the enhanced chemiluminescence (ECL) kit . Specific protein bands were measured with the densitometric intensity . The experiment above was repeated 3 times.Cells were washed twice with ice PBS, and total cell lysate samples were extracted and subjected to SDS-PAGE gel, then, transferred to a nitrocellulose membrane , and blocked with Protein-Free Rapid Blocking Buffer . The membranes were then reacted with primary antibodies including CXCL12 , CXCR4 , and https://www.targetscan.org). Wild-type GP-miRglo-CXCL12 (CXCL12-3′ UTR-Wt) and mutant-type GP-miRglo-CXCL12 (CXCL12-3′ UTR-Mut) dual luciferase reporter plasmids were designed and synthesized based on binding sites. The miR-3677-5p mimic/NC mimic and CXCL12-3′ UTR-Wt/CXCL12-3′ UTR-Mut were transfected into 293T cells using GP-transfected transfection reagent. The luciferase activity was detected after a 48 h reaction. The calculation formula of the relative fluorescence value was = firefly luciferase fluorescence value/ranilla luciferase fluorescence value. The same experiment was repeated for three times.The binding sites of miR-3677-5p and CXCL12 were predicted using a biological information database  and TIMP2  were incubated over night at 4°C, and secondary antibodies were incubated for 1 hour in the dark. The nuclei were counterstained with DAPI. The cellular fluorescence intensity on slides was observed under the fluorescence microscope of ZEISS. The fluorescence intensity was calculated using Image J software.SHI-1 cells were collected after being treated with 60, 120, and 180 mol/L of Rk3 for 48 hours,and the other groups included miR-3677-5p mimic, miR-NC, and 2.5 t-test or One-way ANOVA, and P < 0.05 was considered as statistically significant.The data were analyzed using GraphPad Prism software  and shown as mean ± Standard error of the mean. The data comparison was conducted by using the Student's P < 0.01). The inhibition rates of Rk3 were 24.3%, 46.7%, and 59.7%, in a dose-dependent manner, with 136.02 ± 15.03 mol/L of the IC50 in SHI-1 cells in vitro, as shown in 3, 78.4 ± 7.1/103, and 53.2 ± 7.3/103 cells after being treated by Rk3 at 60, 120, and 180 mol/L for 7 days, which were significantly lower than the 164.6 ± 7.1/103 cells of the control group (P < 0.01). The inhibition rates were 37.4%, 52.3%, and 67.7%, respectively, in a dose-dependent manner with 119.7 ± 7.2 mol/L of the IC50, as shown in The results of the MTT assay showed that the absorbance values of SHI-1 cells treated with Rk3 60, 120, and 180 mol/L were 0.73 ± 0.07, 0.51 ± 0.05, and 0.39 ± 0.04, respectively, which were significantly lower than the 0.95 ± 0.05 of the control group (P < 0.01). The inhibition rates of Rk3 on the migration of SHI-1 cells were from 23.7% to 74.4%. The similar results were also observed in the invasion assay, the number of cell invasions in the 60, 120, and 180 mol/L groups were 98.7 ± 7.4, 69.5 ± 4.3, and 42.2 ± 3.7, respectively, which was lower than the 115.5 ± 6.2 in the control group (P < 0.01). The inhibition rates of Rk3 on the invasion of SHI-1 cells were from 14.5% to 63.4%, as shown in The inhibition effect of Rk3 on migration and invasion were observed by the Transwell assay. The number of cell migrations in Rk3 groups of 60, 120, and 180 mol/L were 83.6 ± 6.4, 55.8 ± 6.3, and 28.4 ± 5.7, respectively, which were significantly lower than the 110 ± 6.0 in the control group (P < 0.05), as shown in P < 0.01). Meanwhile, the relative expression level of miR-3677-5p in EMI group was 0.36 ± 0.10, which was lower than the 0.73 ± 0.11 in N-EMI group (P < 0.01). The mRNA relative expression levels of CXCL12 and CXCR4 in AML-M5 patients was significantly higher than those in the normal control group (P < 0.01). Meanwhile, the mRNA relative expression levels of CXCL12 and CXCR4 in the EMI group were 2.92 ± 0.37 and 2.63 ± 0.66, respectively, which were significantly higher than 1.79 ± 0.24 and 1.60 ± 0.22 in N-EMI group (P < 0.01), as shown in The results of GEPIA website showed that the expression levels of CXCL12 and CXCR4 in AML patients were significantly higher than those in healthy people (P < 0.01), indicated that lentivirus miR-3677-5p mimic was successfully transfected, as shown in P < 0.01), as shown in μM and Rk3 120 mol/L alone or combination, the expression of CXCL12 in each group were 0.73 ± 0.08, 0.57 ± 0.06, and 0.35 ± 0.02, respectively, which were significantly lower than 1.00 ± 0.06 in the control group (P < 0.01). Meanwhile, the expression of CXCR4 in each group were 0.81 ± 0.10, 0.71 ± 0.05, and 0.37 ± 0.06, which were significantly lower than 1.00 ± 0.03 in the control group (P < 0.05 or 0.01), as shown in P < 0.05 or 0.01), as shown in RT-qPCR showed that the relative expression levels of miR-3677-5p in NC and miR-3677-5p mimic groups were 1.00 ± 0.12 and 2.29 ± 0.15, respectively. The miR-3677-5p mimic group showed significantly higher expression of miR-3677-5p than that of the NC group , and the inhibition rates were 14.1%, 50.5%, and 72.0% (P < 0.01), respectively, as shown in P < 0.01), and the inhibition rates were 49.3%, 41.2%, and 72.4%, respectively. The number of cell invasions in the above three groups were 44.3 ± 6.7, 52.2 ± 6.0, and 26.6 ± 6.2, respectively, which were also significantly lower than the 85.6 ± 6.0 in the control group (P < 0.01), and the inhibition rates were 47.8%, 38.0%, and 68.9%, respectively, as shown in The colony numbers in miR-3677-5p mimic, NC + Rk3 120 mol/L, and miR-3677-5p mimic + Rk3 120 mol/L groups were 142.8 ± 5.8/10P < 0.01). CXCL12 3′ UTR-Mut and miR-3677-5p mimic were co-transfected into 293T cells, but there was no significant difference between the two groups, as shown in P < 0.01), as shown in The specific binding sites between miR-3677-5p and CXCL12 were predicted by using biological information database, as shown in 3, 76.8 ± 6.3/103, and 32.8 ± 3.5/103, respectively, which was significantly lower than 154.4 ± 7.0/103 in the control group (P < 0.01), and the inhibitory rates were 22.4%, 50.2%, and 78.8% (P < 0.01), respectively, as shown in P < 0.01), and the inhibition rates were 20.6%, 42.2%, and 72.2%, respectively. The number of cell invasions in the above three groups were 67.8 ± 5.6, 52.0 ± 5.6, and 23.8 ± 6.2, respectively, which were also significantly lower than 85.0 ± 4.0 in the control group (P < 0.01), and the inhibition rates were 21.2%, 39.1%, and 73.0%, respectively, as shown in The colony forming numbers of AMD3100, Rk3 120 mol/L, and AMD3100 + Rk3 120 mol/L groups were 119.6 ± 7.5/10P < 0.05 or 0.01), as shown in P < 0.01). The result of the Western blot was consistent with that of the RT-qPCR above. The protein levels of both CXCL12 and CXCR4 in Rk3 treated cells were significantly lower than those in the control group (P < 0.05), as shown in RT-qPCR showed that the expression level of miR-3677-5p in SHI-1 cells treated by Rk3 at 60, 120, and 180 mol/L was significantly higher than that in the control group, but the mRNA expression level of CXCL12 was significantly lower than that in the control group , characterized by abnormal proliferation of immature monocytes in peripheral blood and bone marrow, eventually resulting in ineffective hematopoiesis and bone marrow failure. Clinical complications are anemia, infection, bleeding, and so on. M5 patients often have EMI in the liver, spleen, lymph nodes, skin, and gums. Infiltration of the central nervous system occurs mostly in the spinal cord membrane, brain parenchyma, and spinal cord. That is, leukemia cells directly spread or hematogenous transfer into the central nervous system, which is one of the important factors leading to recurrence and death . The mecGinsenoside Rk3 is isolated from total saponins of panax ginseng, it has been reported that Rk3 is effective to inhibit the proliferation and induce apoptosis of tumor cells in esophageal cancer, liver cancer, nonsmall cell lung cancer, and other tumors. For instance, Rk3 could decrease the expression of inflammatory factors, arrest cell cycle, promote apoptosis of hepatocellular carcinoma cells, and therefore, inhibit the occurrence and development of hepatocellular carcinoma by targeting the LPS-TLR4 signaling pathway . Rk3 couSome studies indicated that targeted upregulation of miR-3677 expression level would inhibit the proliferation and migration of human osteosarcoma cells . Our stuThe literature reported that CXCL12/CXCR4 axis was extensively involved in the migration and invasion of colorectal cancer and other cancers , 31. AdhEMI and N-EMI have the same pathogenesis, but they are two manifestations of AML. The reason for the difference between EMI and N-EMI was the heterogeneity and complexity of leukemia cells. That is, leukemia cells in the EMI group had a higher degree of malignancy and possessed extremely strong proliferation potential, so the proportion of bone marrow blasts in patients with the EMI group was significantly higher than that in the N-EMI group. The potency of both extramedullary migration and invasion was very strong for leukemia cells in the EMI group, which is one of the important factors for poor prognosis.The MMPs are zinc-dependent proteolytic enzymes of the extracellular matrix (ECM) . As a meIn summary, our results indicated that the ginsenoside Rk3 is effective to inhibit proliferation, migration, and invasion by targeting miR-3677-5p/CXCL12 axis in SHI-1 cells, it provided experimental evidence for the role of Rk3 on antimonocytic leukemia, but to elucidate the exact mechanism remains to be further explored.


Patients with depression are likely to eventually develop Cardiovascular disease(CVD) and have a higher mortality rate than general population. In addition, anxiety disorders, especially Generalized Anxiety Disorder (GAD), may be associated with mortality and other adverse cardiac outcomes.Evaluation of depression and anxiety control in Greek patients with Major Depressive Disorder (MDD) with/without GAD and CVD, under 6 months of treatment with citalopram, and/or quetiapine, and/or pregabalin.1) and after 6 months (V3) respectively.565 patients with MDD with/without GAD, enrolled in this observational, study (NCT03317262). The subgroup of 133(24%) patients had CVD. Severity of MDD and GAD symptoms was evaluated using the HAM-D and HAM-A Scores at baseline (V1 and V3 was 23.94±7.51 and 8.14±4.65 respectively (p<0.0001). Similar results were observed in patients without CVD without GAD (HAM-D score 26.67±8.79 at V1 and 7.44±4.40 at V3). Mean HAM-A score in patients with CVD and GAD at V1 and V3 was 25.64±6.38 and 8.98±3.93, respectively (p<0.0001). Same magnitude reduction in HAM-A score was observed in patients without CVD and GAD, 26.27±8.16 at V1 and 9.28±6.48 at V3 (p<0.0001). Patients’ depression symptoms with/without CVD and GAD showed also a significant reduction between V1 and V3.Mean HAM-D score in patients with CVD without GAD, at VMDD patients with CVD without GAD, had a marginally lower baseline HAM-D score versus patients with GAD. After 6 months of treatment with citalopram, and/or quetiapine, and/or pregabalin the improvement of depressive and anxiety symptoms was almost equal between MDD patients with/without GAD regardless of the presence of coexisting CVD.Employee of ELPEN Pharmaceutical Co. Inc.


Addressing this knowledge gap could support the design of antiviral agents as well as further our understanding of viral-host protein interactions, infectivity, pathogenicity, and innate immune system clearance. Lipidomics revealed that the virus envelope comprised mainly phospholipids (PLs), with little cholesterol or sphingolipids, indicating significant differences from host membranes. Unlike cellular membranes, procoagulant amino-PLs were present on the external side of the viral envelope at levels exceeding those on activated platelets. Accordingly, virions directly promoted blood coagulation. To investigate whether these differences could enable selective targeting of the viral envelope in vivo, we tested whether oral rinses containing lipid-disrupting chemicals could reduce infectivity. Products containing PL-disrupting surfactants (such as cetylpyridinium chloride) met European virucidal standards in vitro; however, components that altered the critical micelle concentration reduced efficacy, and products containing essential oils, povidone-iodine, or chlorhexidine were ineffective. This result was recapitulated in vivo, where a 30-s oral rinse with cetylpyridinium chloride mouthwash eliminated live virus in the oral cavity of patients with coronavirus disease 19 for at least 1 h, whereas povidone-iodine and saline mouthwashes were ineffective. We conclude that the SARS-CoV-2 lipid envelope  The lipid envelope is critical to the structure and function of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), as for all enveloped viruses, such as influenza, HIV, herpes simplex virus, Middle East respiratory syndrome, and SARS-CoV , 2. Yet Coronaviruses bud from the endoplasmic reticulum (ER)/Golgi intermediate complex and exit via lysosomal secretion , 6, 7, 8https://www.who.int/publications/i/item/who-2019-nCoV-oral-health-2020.1). However, despite all these encouraging studies, the relative efficacy and the persistence of mouthwashes in vivo is currently unknown. Importantly, in order to most effectively target the virus in the oropharynx, a detailed knowledge of the lipid composition is required, so that the most appropriate formulation is selected.Following on from public health advice on handwashing, which disrupts the lipid envelope, we considered whether similar approaches using formulations that are nontoxic in vivo could represent potential antiviral strategies directed at reducing SARS-CoV2 transmission and published an evidence review on this topic in 2020 . The lipTo address these questions, we used lipidomics to provide the amount and molecular diversity of envelope lipids and the levels of external-facing aPL in virus cultured from two different cell lines. Our data provide the first complete characterization of a viral lipid envelope and show a PL-rich membrane that also contains several lyso-PL but is relatively low in cholesterol, SM, and other lipids. Sufficient aPLs were present to enhance coagulation of plasma in vitro using live virus. Following this, in vitro studies tested the interaction of varying lipid-membrane disrupting mouthwash formulations and components. Importantly, only a subset of rinses demonstrated efficacy, specifically those containing surfactant and polar components. Furthermore, a randomized controlled clinical study in patients with COVID-19 showed the virucidal effect of a surfactant-containing rinse against SARS-CoV2 in hospitalized patients. These studies demonstrate the accessibility and importance of lipids as a potential target for antiviral approaches, which is unlikely to be impacted by mutation of the virus. They also suggest that targeting virus lipids in the oropharynx may be an important component of risk management in health care during the COVID-19 pandemic and in the context of other enveloped respiratory viruses including coronaviruses and seasonal influenza viruses in the future.N-LAE hydrochloride  in deionized water (Elga Purelab Flex), adjusting the pH to 7 using an NaOH solution  and making up the solution to 50 ml. The required LAE solutions were then prepared by mixing 1 ml of the stock solution and 9 ml of deionized water (3.3 mM), 3 ml of the stock solution and 7 ml of deionized water (9.9 mM), and 1 ml of the stock solution, 6.7 ml of deionized water, and 2.3 ml of ethanol (3.3 mM LAE and 23 vol % ethanol). For dodecylbenzensulfonate, 807.4 mg 4-dodecylbenzenesulfonic acid and mixture O  were dissolved in deionized water (Elga Purelab Flex). The pH was adjusted to 6 using an NaOH solution  followed by making up the solution to 1 l. The solutions of CPC in combination with citrate and benzoate were prepared by dissolving ∼0.11 g of CPC with 0.12 g of citric acid and/or 0.07 g of benzoic acid in deionized water, adjusting the pH as before, and making up to 100 ml. The solution containing deionized water, ethanol, and thymol was prepared by dissolving 0.1675 g of thymol in 50 ml of ethanol; 2.3 ml of this solution was combined with 7.7 ml deionized water. All commercial mouthwash preparations are listed in CPC was dissolved in deionized water at 0.07% or 0.1% (w/v). For aqueous solutions containing ethyl lauroyl arginate (LAE), a 33 mM stock solution was prepared by dissolving 693.2 mg of 2.Virucidal assays utilized VeroE6 or A549 cells, a gift from the University of Glasgow/MRC Centre for Virology, UK. To enhance infectivity and produce a more sensitive cell line for detection of virus, both cell types were transduced with lentiviruses encoding angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) and then drug selected as described . The Engg. A 10-fold serial dilution was then made of the flow-through in DMEM containing 10% FCS. Virus was titrated by plaque assay; serial dilutions were used to infect VeroE6/ACE2/TMPRSS2 cells for 1 h. Following this, cells were overlaid with DMEM containing 2% FCS and 1.2% Avicel®. After 72 h, the overlay was removed, and the monolayer was washed and fixed with 100% methanol. Monolayers were stained with a solution of 25% (v/v) methanol and 0.5% (w/v) crystal violet and then washed with water, and plaques were enumerated. For measurement of toxicity, monolayers were similarly incubated, stained with crystal violet, and scored by eye for live cells.Virucidal activity of mouthwash was studied in media containing 100 μl mucin type I-S, 25 μl BSA fraction V, and 35 μl yeast extract to mimic oral secretions. About 100 μl of this mixture was added to 100 μl of virus suspension and 800 μl of the test product or medium as control. After 30 s, virucidal activity was neutralized by 10-fold serial dilution in ice-cold DMEM (containing 10% FCS). Alternatively, virus was purified by size-exclusion chromatography (SEC) to prevent direct cytotoxic effects of the products on the cell monolayer; 100 μl of the mixture was added to a microspin S-400 HR column and centrifuged for 2 min at 700 g, 5 min), and then virus pelleted through a 30% sucrose cushion . Pellets were resuspended in PBS, purified on a 20–60% sucrose gradient , before being pelleted , and resuspended as outlined later. All preparations were analyzed for purity and abundance by nanoparticle tracking analysis using Nanocyte®  and by Western blot. For PS externalization, samples were used immediately. For lipidomic profiling, they were used immediately or stored for a few days at −80° as snap-frozen pellets.Cells were infected with SARS-CoV2 at multiplicity of infection = 0.01, when cells were 70% confluent, in either serum-free media (Vero cells) or at 2% FCS (A549). At 96 h postinfection, supernatants were harvested, cellular debris pelleted , containing d18:1-18:1(d9) SM (296 ng), 15:0-18:1(d7) phosphatidylcholine (PC) (1.506 μg), 15:0-18:1(d7)PE (53 ng), 15:0-18:1(d7) phosphatidylglycerol (PG) (267 ng), 15:0-18:1(d7) phosphatidylinositol (PI) (85 ng), 18:1(d7) Lyso PC (238 ng), 18:1(d7) Lyso PE (49 ng), cholesterol-d7 (984 ng), cholesteryl ester (CE) 18:1-d7 (3.291 μg), triglyceride (TG) 15:0/18:1-d7/15:0 (528 ng), 15:0-18:1(d7) PS (39 ng), and 20 ng of 17:1 Lyso PG. Samples were also spiked with 5 μl of Cer/sphingoid internal standardmixII (Avanti Polar Lipids) containing 56.99 ng of d18:1/12:0 Cer. Samples were then extracted using a Bligh and Dyer method. Briefly, 1.9 ml of solvent mixture, 2:1 methanol:chloroform v:v, was added to 0.5 ml sample. Samples were vortexed for 30 s, and then 0.625 ml of chloroform was added. Samples were vortexed again (for 30 s), and 0.625 ml of water was then added. Samples were vortexed for 30 s and centrifuged at 1500 rpm, at 4°C, for 5 min. Lipids were recovered from the lower layer and evaporated to dryness using a Labconco RapidVap® (Labconco). Extracted lipids were reconstituted in 200 μl methanol and stored at −80°C until analysis.i) Vero: For preps 1 and 2, three technical replicates of the same extracted lipid samples were analyzed and averaged to give one set of mean values per prep (n = 1 per prep). For prep 3, three separate extractions and analyses were performed (different virus particles in each extraction) and then values averaged to give one value per lipid (n = 1). Combining preps 1–3 gave n = 3 values for lipid molecular species. Standards for Cers and dihydroceramides (DHCers) were only included in prep 3, although these lipids were detected as present in all preparations. Data for Cer and DHCer are from three separate virus isolate extractions using prep 3. ii) A549. All samples had Cer and DHCer standards included. Prep 1 was generated from an individual isolate, whereas preps 2 and 3 arose from a larger scaled-up culture preparation, separated into preps 2 and 3, prior to lipid extraction. Preps 1–3 were analyzed, giving n = 3, which was averaged to generate mean values. A full list of all lipids analyzed is shown in Targeted assays were performed on three separate culture preparations of gradient-purified SARS-CoV2 virus, from either Vero or A549 cells. Hydrophilic interaction liquid chromatography (HILIC) LC-MS/MS was used for PLs and SLs on a Nexera liquid chromatography system (Shimadzu) coupled to an API 6500 qTrap mass spectrometer (Sciex). Liquid chromatography was performed at 35°C using a Waters XBridge Amide column, 3.5 μm, 4.6 × 150 mm, at a flow rate of 0.7 ml/min over 24 min. Mobile phase A was water/acetonitrile , and mobile phase B was water/acetonitrile . The following linear gradient for B was applied: 0.1% B–6% B over 6 min, 6–25% B over 4 min, 25–98% B over 1 min, and 98–100% B over 2 min. At 13.5 min, the flow rate changed to 1.5 ml/min and remained at 100% B until 18.7 min where it returned to 0.1% B. Flow rate then returned to 0.7 ml/min at 23.5 min. Source conditions for positive mode were ionization voltage (IS) 5.5 kV, curtain gas (CUR) 35, temperature (TEM) 550°C, source gas 1 (GS1) 50, and source gas 2 (GS2) 60. Negative-mode source conditions were IS −4.5 kV, CUR 35 psi, TEM 550°C, GS1 50 psi, and GS2 60 psi. Dwell time was calculated in Analyst  automatically based on the number of multiple reaction monitorings (MRMs). This is a scheduled method with pos/neg switching throughout. PLs and Cers were quantified using an external calibration with the following standards, based on a single standard per class (Splash mix) since structurally related lipids tend to closely elute on HILIC: d18:1-18:1(d9)SM, 15:0-18:1(d7)PC, 15:0-18:1(d7)PE, 15:0-18:1(d7)PG, 15:0-18:1(d7)PI, 18:1(d7) Lyso PC, 18:1(d7) Lyso PE, 17:1 Lyso PG, and 17:1 Lyso PI. PCs, PEs, PIs, PGs Lyso PGs, Lyso PEs, and Lyso PCs were quantified from standard curves containing two primary standards each  . Cers were calculated from a standard curve generated by serially diluting the internal standard. SMs were calculated based on the following equation: (area A/area IS) ∗ (nanofram IS added). For confirming the absence of serum contamination of lipids in purified virus cultured from A549 cells, blank isolates (medium + 2% serum) were extracted and then analyzed using direct injection precursor scanning MS/MS for the presence of PE , PC , and CE , comparing with virus lipid extracts.m/z 87. These were quantified against 15:0–18:1(d7) PS, present in the splash mix. MRMs were as follows: m/z [M-H]− 758.6–671.5 (PS 34:2), 760.6–673.5 (PS 34:1), 774.6–687.5 (PS O-36:1), 786.6–699.5 (PS 36:2), 788.6–701.6 (PS 36:1), 810.7–723.6 (PS 38:4), 812.7–725.6 (PS 38:3), 814.7–727.6 (PS 38:2), 816.7–729.6 (PS 38:1), 834.7–747.6 (PS 40:6), 836.7–749.6 (PS 40:5), and 842.7–755.6 (PS 40:2).PS does not resolve well using the applied HILIC and was instead analyzed using a shotgun method to generate bulk species data. A neutral loss scan (NL 87) was acquired in negative ion mode on the Sciex 6500 platform to obtain a list of precursor PS species present in the virus lipids. Samples were injected (10 μl for preps 1 and 3 and 5 μl for prep 2) under flow , with source and MS conditions as follows: CUR 35, IS −4500, TEM 500, GS1 40, GS2 30, DP −50, CE −36, and CXP −29. Once the main PS species were identified, an MRM approach was used, monitoring precursor ions (as determined by the NL precursor scan) to the NL fragment of 4]+. MS conditions were as follows: TEM 150°C, GS1 25 psi, GS2 50 psi, CUR 35 psi, IS 5 kV, declustering potential 70 V, entrance potential 10 V, collision energy 20 V, and collision cell exit potential 25 V. Dwell time was 100 ms for each transition. Cholesterol and CEs were quantified using external calibration curves against the internal standards, with the following primary standards: cholesterol, CE 14:0, CE 16:0, CE 18:0, CE 18:1, CE 20:4, and CE 22:6. For all targeted assays, inclusion criteria for peaks were those at least 5:1 signal-to-noise ratio and with at least seven points across the peak.LC-MS/MS for free cholesterol and CEs and LC-MS analysis of TGs was performed on a Nexera liquid chromatography system (Shimadzu) coupled to an API 4000 qTrap mass spectrometer (Sciex). Liquid chromatography was performed at 40°C using a Hypersil Gold C18 (Thermo Fisher Scientific) reversed phase column  at a flow rate of 0.4 ml/min over 11 min. Mobile phase A was water/solvent B , and mobile phase B was acetonitrile/isopropanol . The following linear gradient for B was applied: 90% for 1 min, 90–100% from 1 to 5 min and held at 100% for 3 min followed by 3 min at initial condition for column re-equilibration. Samples were spiked with cholesterol-d7 (984 ng), CE 18:1-d7 (3.291 μg), and TG 15:0/18:1-d7/15:0 (528 ng) prior to extraction. TGs were analyzed in selected ion monitoring positive mode, covering a range from TG 32:0 up to TG 56:0 including also unsaturated TGs. MS conditions were as follows: TEM 450°C, GS1 35 psi, GS2 50 psi, CUR 35 psi, IS 5 kV, declustering potential 60 V, and entrance potential 10 V. Dwell time was 10 ms. Triacylglycerides (TAGs) were quantified using an external calibration with TG 15:0/18:1-d7/15:0. Free cholesterol and CEs were analyzed in MRM mode monitoring the precursor to product transitions of 12 CEs and free cholesterol, as  flow diagram). Following this, 78 individuals were randomized to receive a mouthwash. On final analysis, 51 of 78 patients had no live SARS CoV2 in baseline salivary samples and were excluded (see CONSORT flow diagram). No other patients were excluded, and no outlying data were removed, leaving 27 patients. The primary and secondary end points established prior to the study were viral load of SARS CoV2 at 30 min and viral load of SARS CoV2 at 1, 15, and 60 min (https://doi.org/10.1186/ISRCTN25647404). Sample collection was stopped after 6 months because of falling patient numbers, and the data were independently analyzed.A four-arm randomized controlled trial was conducted to study the effectiveness of antimicrobial mouthwashes in vivo. Multicentre Research Ethics Committee approval was obtained ; Videne (PVP-I); SCD Ultra (CPC), or Normasol®  using a balanced randomization scheme . Baseline saliva was collected into 30 ml Universal containers. The patient then rinsed their mouth for 30 s with 10 ml of the mouthwash. Saliva samples were then collected after 1, 15, 30, and 60 min into sterile 30 ml Universal containers. Anonymized samples were transported and stored at −802 fold change in from baseline to 60 min post-treatment is presented. The viral load from baseline to 60 min post-treatment was used to calculate a geometric mean ratio (mean change from baseline to post-treatment). Data were then log transformed. Where virus was not detected, zero values were replaced with 4, just below the lower limit of detection for the assay (5 PFU/ml). Lower and upper confidence limits from the relevant mean and SD on the transformed scale and t and P values were determined using unpaired t-test  who was not involved in the design of the clinical trial or randomization. All original data on viral load , together with the logd t-test .SARS-CoV2 England 2 strain was grown in VeroE6 or A549 monolayers and purified using density gradient centrifugation. A549 stably expressed ACE2 and TMPRSS2 to enhance infectivity . Purity insets).Viral lipid extracts were analyzed using lipidomics, including targeted (virus from both cell types) and untargeted (Vero cell virus only) to provide a comprehensive map of molecular composition and abundance. First, targeted LC/MS/MS was used to analyze ∼500 individual molecular species, in triplicate for each cell type. Across the two separate preparations (Vero vs. A549), ∼260 lipids were reproducibly detected. The full list of species analyzed and the dataset are provided in Next, mol% was calculated for PL and SLs only, since this allows comparison with older studies on composition of intracellular membranes of mammalian cells, which used thin layer chromatography coupled with phosphate analysis to measure these lipid categories . UnfortuNext, the individual molecular species within categories were compared. Within each cell type, the levels of specific lipids were very similar, indicating that the viral lipidome is relatively stable  and 3A. Next, to generate an untargeted dataset, virus lipids from Vero cultures were separated using HILIC in triplicate, subtracting extracted blanks, scanning from 50 to 2,000 amu, at 20 K resolution. Retention time windows were identified using internal standards for PC, PE, PG, PI, lysoPC, lysoPE, lysoPG, lysoPI, glycerides, SM, and Cer. Since electrospray ionization high-resolution mass spectroscopy runs contain large numbers of artifactual ions including in source fragments, isotope peaks, common contaminant ions in blanks, salt clusters, ion stacks, and other features, two informatics approaches were applied to clean up the dataset. First, XCMS was used to align and integrate peaks, before a full clean up using LipidFinder 2.0 was performed , 38. LipOverall, our lipidomics data establish SARS-CoV2 membrane as highly enriched in PL, particularly PC, PE, and PI, but with rather low levels of cholesterol, PS, and SM. They also show that the membrane is influenced to some extent by the host cell of origin in relation to specific molecular species of lipids detected.Mammalian membranes maintain asymmetry via the action of flippases and floppases, which retain aPL on the inner membrane . Here, w2, and the activated partial thromboplastin time was measured as outlined in 5 PFU/ml  exclude potential for mouthwash to interfere with plaque assay through direct toxicity toward host cells, ii) prevent persistence of effect on virus beyond the 30 s exposure time, and iii) consider the choice of soil load to best model human oropharynx conditions. An important refinement was the use of VeroE6, which stably overexpress ACE2 and TMPRSS2. This significantly improves viral infectivity, with SARS-COV2 entering >1log10 more efficiently than parental VeroE6, significantly enhancing assay sensitivity (The assay was optimized to sitivity . Rather sitivity ) to bettsitivity C. To addsitivity D. When msitivity C. SEC waNext, the ability of mouthwash to reduce virus infectivity, after a 30 s exposure in a soil load, was tested using the optimized plaque assay. Two CPC-containing mouthwashes  and a mouthwash containing 23% v/v ethanol/LAE (Listerine® Advanced DGT) eradicated the virus completely, giving >5-log10 reduction in viral titers and thus met EN14476 as a virucide. In contrast, only moderate effects (∼3-log10 fold reduction) were observed with PVP-I (Videne), CPC/sodium citric acid/benzoate (SCD Ultra), and 21% v/v alcohol/essential oils (Listerine® Cool Mint) E, which 50s  from the relevant rinses, using concentrations found in individual formulations . CPC, thListerine® Advanced DGT contains ethanol at 23% v/v and LAE (3.3 mM), whereas other formulations  contain ethanol with essential oils: thymol, menthol, and eucalyptol. Whilst 23% v/v ethanol alone had no consistent impact, the addition of thymol (5 mM) resulted in a 3-log reduction in virus titers B. This iOne mouthwash (SCD Ultra) showed only a 3-log reduction in virus titers despite containing CPC E. This fhttps://coronavirus.data.gov.uk), making further recruitment impossible. Amongst patients with live virus, saliva was collected before rinsing (baseline), and at 1, 15, 30, and 60 min postrinsing, with mouthwashes containing either containing CPC/IPM , CPC/benzoate , PVP-I , or 0.9% w/v NaCl . Data are shown as both log2 fold reduction from baseline (7 PFU/ml (2 fold reduction from baseline) and the largest Dentyl Dual Action where six of seven patients recorded no live virus (median 14.3 log2 reduction from baseline) . Impressively, in three-eighths of patients treated with Dentyl Dual Action, no live virus was recovered at any time point after the initial rinse   and 5. Tal rinse .Fig. 6InWhile vaccines and antivirals have targeted the proteins or replication cycle of SARS-CoV2, there has been very little research into the lipid envelope to date. Indeed, there is very little known about lipid membranes of any enveloped viruses hindering development of strategies targeted directly at the lipids themselves. To address this information gap, a lipidomic analysis of the viral membrane using untargeted and targeted approaches was undertaken. Old studies using thin layer chromatography and total phosphorous analysis, reporting mol% values of rat liver membranes suggest that PS and SM are enriched in mammalian plasma membranes versus ER membrane , 35 Tab. On the Here, we found that the SARS-CoV2 membrane is primarily comprised of PC, PE, and PI, while having low levels of free cholesterol, PS, and SM. This appears most similar to ER, although with even lower proportion of PS, SM, and cholesterol than previously reported for that membrane compartment, using older methods. In the mammalian plasma membrane, cholesterol is often concentrated in specialized regions that support receptor-dependent signaling, called lipid rafts . Our datFurthermore, we demonstrate that the virion membrane contains lysoPL, from PG, PE, and PC, noting that these are known bioactive lipid signaling mediators, and their presence in the envelope could impact on host inflammatory responses to infection. It was recently reported that coronaviruses exit via lysosomal secretion instead of the biosynthetic secretory pathway . Lysosom2+ ions and various proteins . A receHaving determined the composition of the lipid membrane, we next tested the impact of common mouthwash formulations, focusing on surfactants that we reasoned would effectively target a PL-rich membrane. Our data significantly extend other recent studies on enveloped viruses. For example, dequalinium/benzalkonium chloride, PVP-I, and ethanol/essential oils reduced SARS-CoV2 infectivity in vitro by up to 3-log10 , whereasImportantly, efficacy was not dependent on “classical” antibacterial components of mouthwashes but instead was critically dependent on the presence of surfactant (CPC/LAE). Whether any of the lipid-disrupting components would function in this way was not predictable from the outset; efficacy is determined by both molecular makeup and concentration, with some detergents  not inactivating SARS-CoV2 even at 0.5%, whereas others  lyse it completely at 0.1% . Thus, oWhilst surfactants are highly antiviral in isolation, other mouthwash components may reduce their effectiveness. Benzoate can bind with CPC, dramatically reducing its cmc, without changing its micellar morphology , 70. Suchttps://www.statista.com/statistics/286902/usage-mouthwash-dental-rinse-us-trend/). While CPC-containing mouthwashes can show cytotoxicity against monolayers in vitro, the lack of observed toxicity in vivo likely reflects the complex, differentiated, and multicellular nature of primary oral epithelia  and showed a strong impact of surfactant-containing oral rinses on live virus load in saliva. Only three other studies have attempted to address these effects in vivo during the pandemic, and both were inconclusive because of small numbers of patients and the use of quantitative PCR rather than live virus titration to determine virus load , 78, 79.https://www.who.int/publications/i/item/who-2019-nCoV-oral-health-2020.1). However, hydrogen peroxide only weakly inactivates SARS-CoV2 , during a pandemic. They have the potential to lessen the risk of transmission from asymptomatic carriers to health care professionals performing oropharynx investigations, as well as transmission within the wider population, in a similar manner as to how they are being tested against pathogenic oral bacteria , 84, 85.All data are included within the article.This article contains Venture Life Group plc and Johnson & Johnson provided information on mouthwash formulations employed in the in vitro study. Venture Life Group partfunded the clinical study but had no input in study design, data analysis, or drafting of the article. The authors declare that they have no conflicts of interest with the contents of this article.