Background: The interaction between lysosomes and mitochondria includes not only mitophagy but also mitochondrion–lysosome contact (MLC) that enables the two organelles to exchange materials and information. In our study, we synthesised a biosensor with fluorescence characteristics that can image lysosomes for structured illumination microscopy and, in turn, examined morphological changes in mitochondria and the phenomenon of MLC under pathological conditions.Methods: After designing and synthesising the biosensor, dubbed CNN, we performed an assay with a Cell Counting Kit-8 to detect CNN’s toxicity in relation to H9C2 cardiomyocytes. We next analysed the co-localisation of CNN and the commercial lysosomal probe LTG in cells, qualitatively analysed the imaging characteristics of CNN in different cells  via structured illumination microscopy and observed how CNN entered cells at different temperatures and levels of endocytosis. Last, we treated the H9C2 cells with mannitol or glucose to observe the morphological changes of mitochondria and their positions relative to lysosomes.Results: After we endocytosed CNN, a lysosome-targeted biosensor with a wide, stable pH response range, into cells in an energy-dependent manner. SIM also revealed that conditions in high glucose induced stress in lysosomes and changed the morphology of mitochondria from elongated strips to round spheres.Conclusion: CNN is a new tool for tracking lysosomes in living cells, both physiologically and pathologically, and showcases new options for the design of similar biosensors. Lysosomes, as the main sites of degradation in cells, play an important role in intracellular signal transduction, energy metabolism and homeostasis . Their mAs organelles involved in intracellular metabolism, lysosomes cannot function without interacting with other organelles, including for autophagy, for mitophagy and in mitochondrion–lysosome contact (MLC) . In partM-value  , photo-aM-value  in ordervia SIM. Because CNN can be used to track lysosomes under SIM, it stands as a powerful new tool for studying lysosome-related diseases.In our study, we designed a lysosome-targeted biosensor, CNN, to observe changes in lysosomal morphology and fluorescence signals in different cells under SIM. Afterwards, CNN was used to track the interaction of lysosomes and mitochondria in a diabetic cardiomyopathy model under conditions in high glucose at the nano-scale. Owing to its wide pH response range, low background and exceptional cell permeability, CNN can be used to detect morphological changes in mitochondria and lysosome–mitochondrion interaction under different conditions 1H NMR, 13C NMR and HRMS , the voltage-dependent anion channel 1  and the mitochondrial calcium uniporter   , a subunembrane) . When TRembrane) . HoweverWe developed a lysosome tracker, CNN, that can be used to observe the crosstalk of lysosomes and mitochondria in living cells under SIM. Using CNN, we found that MLC can not only be captured under normal conditions but also induced by glucose and mannitol. Therefore, CNN stands as a new tool for tracking lysosomes in living cells under both physiological and pathological conditions and showcases new options for the design of similar biosensors.Dulbecco’s modified Eagle’s medium , phenol-free medium (#1894117), fetal bovine serum  and Penicillin-streptomycin , Trypsin-EDTA (#25200-072) and other reagents for cell culture were obtained from Gibco BRL . Lyso-Tracker Green  and Hoechst (#C1022) were obtained from Beyotime . Lipi-Blue (LD01) were from DOJINDO Laboratories . PK Mito-Tracker Deep Red (PKMTDR) were obtained from Peking University. The Cell Counting Kit-8  Assay Kit,Chlorpromazine was obtained from MedChemExpress .2CO3  were stirred in acetone (60 ml) and then refluxed overnight. After removal of the solvent in vacuo, the crude compound was obtained and then purified by column chromatography , and then yellow liquid (QN) was obtained after dried . 1H NMR  δ (ppm) , 7.93 , 7.86 , 7.42 , 7.29 , 4.16 , 2.41 , 2.18 , 1.96–1.90 . 13C NMR  δ (ppm) (m/z (16H20NO2+ (M + H)+: 258.1489, found 258.1259.A mixture of 3-chloro-N, N-dimethylpropan-1-amine , 6-hydroxynaphthalene-2-carbaldehyde  and K δ (ppm) : 10.09 ( δ (ppm) : 192.76,pm)  was obtained after dried. 1H NMR  δ (ppm) , 4.52 , 2.86 , 1.55 , 1.46 . 13C NMR 13C NMR  δ (ppm) (m/z (29H35N2O+ (M)+: 188.1434, found 188.1494.A mixture of 4-hydrazinobenzoic acid , 3-methylbutan-2-one  and sodium acetate  were stirred in glacial acetic acid (3 ml) and then refluxed overnight. After removal of the solvent in vacuo, the crude compound was obtained and then purified by column chromatography , and then brown liquid was obtained after dried . Subsequently, a mixture of the previous product  and iodoethane  refluxed in acetonitrile (5 ml). A large amount of white powder solids precipitated after reacted 24 h. Then the crude product was obtained by filtered, following washed by cold ethanol and ethyl ester. A white compound  δ (ppm) : 8.03–7. δ (ppm) : 196.61,pm)  and CC1  were refluxed in ethanol (6 ml). A large amount of yellow powder solids precipitated after reacted overnight. Then the crude product was obtained by filtered, following washed by cold ethanol and ethyl ester. A yellow compound CNN  was obtained after dried. 1H NMR  δ (ppm) , 8.39 , 8.02 , 7.96 , 7.92 , 7.76 , 7.68–7.63 , 7.32 , 4.78 , 4.27 , 3.29–3.25 , 2.84 , 2.25–2.18 , 1.86 , 1.51 . 13C NMR  δ (ppm)  : 8.76 (s δ (ppm) : 181.80 pm) . All of them were transferred to culture flask and cultured at 37°C in 5% CO5 were inoculated into a 35 mm SIM-specific petri dish containing 2 ml complete medium. After 24 h culture, the cells were washed twice with preheated PBS. The cells were treated with CNN at 500 nM for 1 h and then incubated with or without commercial lysosomal probe LTG at 100 nM for 30 min. Then the cells were washed with phenol-free red DMEM medium for 5 times. Finally, the cells were cultured in complete medium without phenol red and observed under super resolution confocal microscope.H9C2 cells with a density of 2.5 × 10in vitro: the blank control group was treated with 5.5 mM low-glucose medium. The hypertonic group (HP group) was treated with 5.5 mM low-sugar medium and 29.5 mM mannitol for 24 h H9C2 cardiomyocytes were stimulated with 35 mM high glucose solution for 24 h in the high glucose group (HG group). After film-making, H9C2 cells were treated with CNN and PKMTDR for 1 h and 20 min, respectively. Finally, images were captured and processed by super-resolution confocal microscope.Membrane making and grouping of diabetic cardiomyopathy model 3 cells/well, and cultured for 24 h in an incubator containing 5% CO2 at 37°C. Then CNN and DMEM containing 0, 0.5, 1, 2, 5, 10 μM were used to replace the original media in 96-well plates. After 24 h, each well was added 10 μL CCK8 solution and incubated in an incubator for 2 h. Finally, the absorbance at 450 nm was determined by enzyme-linked immunosorbent assay.Cell Counting Kit-8 (CCK-8) was used to detect the cytotoxicity of probe CNN. H9C2 cells diluted with complete medium were inoculated into 96-well plates at a density of 8 × 105 H9C2 cells were seeded on a 35 mm glass-bottom microwell dish and incubated with 2 ml of DMEM medium supplemented with 10% FBS for 24 h. After treatment, the cells were washed twice with preheated PBS. The cells were treated with CNN at 500 nM for 1 h. Then the cells were washed with PBS for 3 times and phenol-free red DMEM medium for 4 times. H9C2 cells cultured in a phenol-free DMEM were observed under an OMX 3D-SIM  equipped with a 60×/1.42 numerical aperture oil-immersion objective lens and solid-state lasers. Obtained SIM images were analyzed using ImageJ software.A total of 2.5 × 10M-value is referred to the previous article (p < 0.05, ** as p < 0.01, *** as p < 0.001, *** as p < 0.0001, and n.s. as no significant difference. Sample sizes in all graphs are indicated in the corresponding figure legends.The calculation method of  article . Statist


Staphylococcus aureus remains a pathogen of high concern in public health programs worldwide due to antibiotic resistance and emergence of highly virulent strains. Many phytochemicals have demonstrated activity against S. aureus and other Gram-positive bacteria, but the minimum inhibitory concentration (MIC) values comparable to commonly used antibiotics are needed. In the present study, bio-guided fractionation of the ethanol extract of seeds of Mammea americana L.  throughout the antibacterial activity, against S. aureus strains that are sensitive and resistant to methicillin, led to the isolation of four coumarins identified as mammea B/BA, mammea B/BC, mammea A/AA cyclo D and mammea A/AA cyclo F, and a mixture of mammea B/BA cyclo F plus mammea B/BD cyclo F. The extract inhibited the growth of S. aureus with MIC values of 2–4 μg/ml and Mammea B/BA (MaBBA) presented MIC values in a range between 0.5 and 1.0 μg/ml in six methicillin-sensitive strains and eight methicillin-resistant strains evaluated. We consider MaBBA the most potent of all mammea coumarins reported to date, according to the literature review carried out at the time of writing of this article. Toxicity assessment in vivo against the nematode Caenorhabditis elegans and in vitro against human fibroblasts of the extract and the compound MaBBA indicated that both had low toxicity. Hypericum perforatum L. (St. John’s Wort) showed a minimum inhibitory concentration (MIC) of 1 μg/ml against methicillin-resistant S. aureus . In the US, mortality from MRSA remains the highest for any antibiotic-resistant pathogen , Escherichia coli (10 μg/ml), Salmonella typhimurium (15 μg/ml), Salmonella enteritidis (8.5 μg/ml), Aeromonas hydrophila (4 μg/ml), Yersinia sp. (12.5 μg/ml), Shigella sp. (6.3 μg/ml), Vibrio parahaemolyticus (12.5 μg/ml), and Candida albicans (15 μg/ml) , Escherichia coli (4 μg/ml), and Pseudomonas aeruginosa (8 μg/ml)  . On the 8 μg/ml) .Mammea, Mesua, and Calophyllum are good sources of coumarins , a tree native to western India and northern South America (S. aureus (Streptococcus mutans (ATCC 25175) and Porphyromonas gingivalis (ATCC 33277), bacteria from the oral cavity : Silica gel 60 , hexane (Hex), chloroform (CHCl3), ethyl acetate (AcOEt), acetone (Me2CO) and methanol (MeOH) brand analytical grade Merck. Thin layer chromatography (TLC): Silica gel 60 F254 0.2 mm plates, Merck® (Cat. 1.05729.0001); Silica gel 60 F254 0.5 mm preparative plates, Merck brand (Cat. 1.05744.0001); Vanillin/H2SO4 plate developing reagent (1% vanillin in 10% H2SO4 solution in ethanol); UV lamp 254 and 365 nm. HPLC: HPLC Waters 1,515 Isocratic-UV/VIS detector 2,489; normal phase column PhenoSphere-NEXT (Phenomenex), 250 × 4.6 mm (i.d), 5 μm; reverse phase column C18 PhenoSphere-NEXT (Phenomenex), 250 × 4.6 mm (i.d), 5 μm; HPLC grade hexane, acetone and acetonitrile (Merck); water milliQ. NMR: 1H, 13C and 2D NMR spectra were recorded on a Bruker Fourier 300 spectrometer  operating at 300 MHz for 1H and 75 MHz for 13C NMR, using CDCl3  as the solvent, and TMS as an internal standard. Chemical shifts (δ) are reported in ppm, and the coupling constants (J) are reported in Hz. HPLC-QTOF-MS/MS: Chromatographic separation was carried out using a 1,260 Infinity II HPLC (Agilent Technologies), equipped with a variable wavelength detector (VWD) using an InfinityLab Proroshell 120 EC-C18 column, 100 × 4.6 mm (i.d), 2.7 μm  with a mobile phase flow of 0.3 ml/min, composed of water and 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). The gradient profile was as follows: . Mass analysis was obtained using a 6530 Q-ToF quadrupole time-of-flight tandem mass spectrometer (Agilent Technologies), with electrospray ionization (ESI), positive ion mode operator. The mass detector conditions were as follows: Capillary voltage +3.5 kV, nitrogen gas temperature 320°C, drying gas flow rate 8.0 L/min, nebulizer gas pressure 35 PSI, shredder voltage 135 V, skimmer 65 V, and RF OCT 750 V. The mass range in the MS and MS/MS experiments was m/z 100–1,200 and 50–1,200 at 3 spectra/s, respectively. MS and MS/MS data were obtained with the Agilent MassHunter purchase software (version 10.1) and processed in the Agilent MassHunter Qualitative Analysis 10.0 with the METLIN metabolite database (metlin.scripps.edu), providing an error mass less than 5 ppm. Melting point: Electrothermal melting point apparatus . Antibacterial activity tests: cation adjusted Mueller Hinton broth (CAMHB); trypticase soy agar; trypticase soy broth (TSB); Mueller Hinton agar; dimethyl sulfoxide (DMSO); Vancomycin hydrochloride MP Biomedicals (LLC Cat No. 195540 Lot No QR14945); Gentamicin sulfate salt, Sigma-Aldrich ; Oxacillin sodium salt, Sigma-Aldrich . Optical density (OD) reading: Skanlt 4.1/Multiskan FC, Thermo Scientific spectrophotometer.M. americana were acquired from a farm  in the municipality of Turbaco, department of Bolívar in northern Colombia (10°19′05.2″N 75°24′31.3″W). The plant was identified at the herbarium of the Botanical Garden Guillermo Piñeres  as M. americana L. and the voucher No. JBC 467 was assigned. The seeds were washed and subsequently the cover was removed. The endosperm of the seed  was dried, ground and subjected to cold maceration with 96% ethanol with successive extractions and filtration until the material was exhausted, which was verified by TLC. The filtrate was subjected to rotary evaporation at 40°C and the dry extract (62 g) encoded as FD-I-34S was stored at −20°C.The fruits of ®) as stationary phase and elution gradient from hexane (100%), chloroform (100%), ethyl acetate (100%), and methanol (100%); four fractions (34S-F01 to 34S-F04) of different polarities were obtained  to hexane/ethyl acetate (98:2) and subsequently to preparative thin layer chromatography in normal phase, using as mobile phase hexane/ethyl acetate 9:1. Six bands named 34S-F16, 34S-F17, 34S-F18, 34S-F19, 34S-F20, and 34S-F21, were isolated and tested against the The yellow pasty fraction 34S-F11 was washed several times with hexane; the mother liquors were combined, and the solvent was allowed to evaporate at laboratory temperature, producing transparent needles on the walls and bottom of the container (34S-F22). The crystals were subjected to reverse phase HPLC (acetonitrile/acetic acid 0.1% (7:3), flow 1.0 ml/min, 254 and 365 nm) and two coumarins (34S-K01 and 34S-K02) were isolated from them. A third coumarin (34S-K04) was isolated by preparative TLC from the mother liquor from the hexane wash of the 34S-F11 fraction, using hexane/ethyl acetate (9:1) as mobile phase. Three other coumarins were isolated by open column chromatography from fractions 34S-F13 and 34S-F14 as shown in 1H and 13C) and 2D  NMR spectra and by comparison with data reported in the literature  were used. The bacterial strains were: ATCC 29213 (MSSA), ATCC 33591 (MRSA), ATCC 43300 (MRSA) and USA300-0114 (MRSA); ATCC 25922 (Escherichia coli), ATCC 700603 (Klebsiella pneumoniae) and ATCC 27853 (Pseudomonas aeruginosa). Clinical MSSA and MRSA strains isolated from pediatric patients from a hospital in the city of Cartagena-Colombia (ATCC (American Type Culture Collection) methicillin-sensitive and methicillin-resistant Colombia  were alsColombia . Briefly50 and MIC90) was evaluated by the broth microdilution method following the protocol M07-A9 of the Clinical and Laboratory Standards Institute  reading at 620 nm. Gentamicin, vancomycin, and oxacillin antibiotics served as positive inhibition controls, while 1% DMSO served as the vehicle control. The plates were incubated at 37°C for 18 h and read in a spectrophotometer at 620 nm. Tests were carried out three times in triplicate, at three different times. The formula of Quave et al.  and five serial dilutions from MaBBA  were prepared in CAMHB. These solutions were inoculated with a suspension of S. aureus to reach 5 × 105 CFU/ml in a final volume of 2 ml. The samples were incubated for 24 h at 37°C. 10 µl of the culture was seeded on Brain Heart Infusion (BHI) agar plates and incubated for 24–48 h; finally, the number of CFUs for each plate was recorded and the MBC determined  and vancomycin 1 μg/ml (MIC: 1 μg/ml) were used as control. 20 µl of 10−2, 10−4, 10−6, 10−8, and 10−10 dilutions, prepared from each sample at time 0 (before incubation), 4, 8, 12, and 24 h (after incubation), were spread on BHI agar plates and incubated at 37°C for 24–48 h  and two fractions, 34S-F05 and 34S-F11, was carried out on Caenorhabditis elegans N2 Bristol. Briefly, batches of 10 ± 2 young adult worms (L4 instar) were transferred to 24-well microplates. The nematodes were treated with eight two-fold concentrations of FD-I-34S, 34S-F05, and 34S-F11 from 2 μg/L to 256 μg/L. The control group was set up with K medium containing 1% of DMSO. Mortality was measured after 24 h of exposure, for this, the number of living and dead organisms in each well was recorded. Worms that did not present movement within 30 s were considered dead. Three technical replicates were established for each treatment and the tests were performed in triplicate  on different days and the mortality percentage was calculated  were seeded (150.000 cells/ml) into a 96-well plate and incubated at 37°C, 5% CO2 and 99% relative humidity for 24 h. After that, cells were exposed to different concentrations (6.25–200 μg/ml) of the extract (FD-I-34S) for 48 h. Subsequently, cells were incubated with a MTT solution (0.25 mg/ml) for 4 h, and the generated formazan crystals were dissolved using DMSO and the OD550 was measured using a microplate reader . The absorbance values were presented as percent growth of viable cells against untreated cells as control and expressed as the mean with confidence intervals (CI) of triplicate samples from three independent experiments.MRC-5 fibroblasts  and five of MaBBA  were prepared in a 96-well plate using TSB supplemented with dextrose 2.5% and, subsequently inoculated with S. aureus USA-300 0114 as described in the MIC assay. The plate was incubated for 24 h at 37°C. After incubation, the OD at 620 nm was recorded in order to calculate growth inhibition. The biofilms in the microtiter plates were processed following a published protocol . Statistical analysis was carried out with GraphPad Prism 8.0.1. The means of the different treatments and the control were compared using one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. Statistical significance was considered at M. americana (yield 11.3%). From the chromatographic fractionation of 46 g of the ethanol extract of M. americana seeds, four fractions (34S-F01 to 34S-F04) of different polarities were obtained , which, together with the fraction eluted with ethyl acetate, accounted for approximately 90% of the extract content, so it could be considered that components of the extract are mostly of medium polarity. This fraction produced four sub-fractions  confirmed the mixture of three coumarin-like compounds from mamey, according to the reviewed literature (1H and 13C spectra are shown in The reverse phase HPLC chromatogram of the clean needles (34S-F22) showed the presence of three major peaks at 23.5, 25.3, and 35 min . The 1H-terature . After e3): 14.64 , 6.87 , 6.03 , 5.23 , 3.93 , 3.50 , 2.93 , 1.88 , 1.83 , 1.61 , 1.49 , 1.25 , 1.00 . HRESIMS m/z 373,20141 [M + H]+ corresponding to the molecular formula C22H28O5 (Mammea B/BA (34S-K01): colorless crystals. Melting point: 121°C. vanillin: pink. Purity: 89%. 1H NMR : 14.67 , 6.87 , 6.02 , 5.22 , 3.49 , 3.29 , 2.92 , 1.88 , 1.83 , 1.77 , 1.64 , 1.02 . HRESIMS m/z 359,18543 [M + H]+ corresponding to the molecular formula C21H26O5 (Mammea B/BC (34S-K02): colorless crystals. Melting point: 141°C. vanillin: pink. Purity: 93%. 1H NMR : 14.80 , 7.40 , 7.31 , 6.89 , 5.99 , 5.63 , 2.95 , 2.22 , 1.67 , 0.96 .Mammea A/AA ciclo D (34S-K04): yellow crystals. Melting point: 142°C. vanillin: green. Purity: 87%. 1H NMR : 14.52 , 7.40 , 7.31 , 7.29 , 5.95 , 4.91 , 3.32 , 2.98 , 2.83 , 3.00 , 1.44 , 1.32 , 0.98 , 0.96 .Mammea A/AA ciclo F (34S-K06): white powder. Melting point: 107°C. vanillin: colorless. Purity: 85%. 1H NMR : 14.21 , 6.00 , 4.86 , 3.90 , 3.17 , 2.84 , 1.61 , 1.45 , 1.28 , 1.24 , 1.02 , 0.97 . Mammea B/BD ciclo F: 1H NMR : 14.21 , 6.00 , 4.86 , 3.90 , 3.17 , 2.84 , 1.61 , 1.45 , 1.28 , 1.24 , 1.02 , 0.97 .Mixture of Mammea B/BA ciclo F and Mammea B/BD ciclo F (34S-K05), yellow powder. Melting point: 119–122°C. vanillin: colorless. Some characteristic signals in the NMR spectra are: Mammea B/BA ciclo F: 1H NMR  inhibited the growth of all S. aureus strains  at low concentrations, with MIC90 values between 2 and 4 μg/ml and MIC50 between 0.5 and 1 μg/ml  and highest (34S-F03 and 34S-F04) polarity fractions were found to be inactive up to a concentration of 16 μg/ml. The 34S-F05 fraction obtained from 34S-F02 remained in the MIC range of 2–8 μg/ml, but in the fractions obtained from it, coded as 34S-F10 and 34S-F11, the MIC decreased, being between 1 and 4 μg/ml. Through TLC analysis  it was possible to characterize the fractions 34S-F10 and 34S-F11, clearly distinguishable by their pink, violet and green spots when developed with 1% vanillin  and of the active compound MaBBA (tested up to a concentration of 8 μg/ml), against S. aureus ATCC 33591, indicated that these did not cause a reduction in the CFU/ml count greater than 3log10, and consequently, they are considered to have a bacteriostatic effect; this effect is also observed in the growth curves shown in The evaluation of the minimum bactericidal concentration (MBC) of the extract of S. aureus, of the extract of M. americana, it was found that at a concentration of 0.5 μg/ml (concentration at which it showed less than 50% inhibition of growth), it only produced an inhibitory effect of 21.92%. Regarding the inhibitory activity of coumarin MaBBA, it was found that it inhibited biofilm formation by 43.01%, a concentration of 0.25 μg/ml, but with a growth reduction of 34.44%. Below concentrations of 0.5 μg/ml for the extract and 0.25 μg/ml for MaBBA, both were found to inhibit biofilm between 0 and 4%  than those that inhibit S. aureus strains growth , the extract (FD-I-34S) and the fractions active as antistaphylococcals (34S-F05 and 34S-F11) did not produce mortality in the nematode s growth . The selM. americana as an anti-infective is not widely cited in the literature, however, it is mentioned that its seeds without the embryo are used to prepare an anti-helmintic infusion and it is believed that the infusion of fresh or dried leaves helps in cases of intermittent fever; leaf extracts are effective against Mycobacterium tuberculosis and, in addition, antimalarial properties have been suggested (The use of uggested .Streptococcus mutans (ATCC 25175) and Porphyromonas gingivalis (ATCC 33277); the oily phase was the most active against S. mutans (MIC: 15.6 μg/ml), showing bactericidal effect at concentrations greater than 125 μg/ml (M. americana seed extract against S. aureus was 2 μg/ml for a methicillin-sensitive strain (209P) and 4–8 μg/ml for methicillin resistant strain , Echinops kebericho Mesfin (Asteraceae), Mikania glomerata Spreng. (Asteraceae), Curcuma longa L. (Zingiberaceae), and Combretum album Pers (Combretaceae) show the best average MIC values (3.5–16 μg/ml) (M. americana reported in this article (MIC90 of 2–4 μg/ml).The antibacterial activity of mamey seed extract has been previously demonstrated in some studies against Gram-positive bacteria; in two of these investigations, some MIC values are reported. Herrera et al. reported that the oily and ethanolic fractions obtained from the extract of mamey seeds, inhibited the growth of 25 μg/ml . Yasunakt strain . The res6 μg/ml) , which aM. americana seeds against different sensitive and resistant strains of S. aureus. From the most active fraction of this extract, four mamey coumarin-type compounds were isolated, namely, mammea B/BA, mammea B/BC, mammea A/AA cycle D, mammea A/AA cycle F and a mixture of mammea B/BA cycle F + mammea B/BD cycle F. Evaluation of the antibacterial activity against S. aureus of these coumarins showed that mammea B/BA was the most active. Mammea B/BA was first isolated from M. americana by Morris M.P. and Pagán in 1952 from a sample called mamein, which also contained two other coumarins, mammea B/BB and mammea B/BC  in all strains evaluated in this study. Regarding the MICM. americana extract and the MaBBA compound, both showed a bacteriostatic effect, since the MBC:MIC ratio is greater than 16. The growth curves of the methicillin-resistant S. aureus strain ATCC 33591 in the presence of the extract and MaBBA, confirmed a bacteriostatic effect for both (p < 0.05) were found in the number of CFU/ml at each time, for the five concentrations evaluated above the MIC90. M. americana, as a new natural antibiotic against methicillin resistant S. aureus. It is observed that the two antibiotics used as positive controls, and the coumarin, inhibited growth up to 12 h of treatment; MaBBA behaved as bacteriostatic, while gentamicin and vancomycin showed a bactericidal tendency; however, at the end of 24 h of treatment, only vancomycin behaved as bactericidal. Gentamicin did not inhibit the growth of S. aureus after 12 h, reaching an increase in CFU/ml by two logarithmic units above MaBBA and six logarithmic units above vancomycin. This study demonstrates the in vitro efficacy of MaBBA, being superior to gentamicin and similar to vancomycin in inhibiting bacterial growth. Vancomycin is one of the first antibiotics to consider in deep or life-threatening MRSA infections, such as staphylococcal endocarditis . M. americana and mammea A/BA (MIC of 1–2 μg/ml), isolated from Calophyllum brasiliense Cambess. leaves (MaBBA (MIC 0.5–1 μg/ml) turned out to be the most active of all coumarins with anti-staphylococcal activity isolated from the genera . leaves .M. americana and the compound MaBBA, against E. coli, K. pneumoniae and P. aeurginosa up to a concentration of 32 μg/ml, without present activity in none of the cases. According to Mesua ferrea, evaluated against Gram-negative bacteria E. coli, E. cloacae, S. marcescens, P. aeruginosa, and Acinetobacter spp., were inactive up to a concentration 128 μg/ml; however, against the Gram-positive bacteria Enterococcus faecalis and Enterococcus faecium, the compounds mammea A/BB, mesuol, mammea A/AB, mammea A/AA showed activity in the MIC range of 8–16 μg/ml. Mammea africana G. Don, was active against two Gram-positive bacteria, Clostridium difficile (MIC: 0.25 μg/ml) and Streptococcus pneumoniae (MIC: 0.25 μg/ml), and against a Gram-negative bacterium, Campylobacter jejuni with a MIC of 0.25 μg/ml . On the other hand, the prenylated derivatives of benzophenone garcinol, isogarcinol and xanthozimol isolated from Garcinia indica (Thouars) Choisy and Garcinia subelliptica Merr., Clusiaceae, presented good anti-staphylococcal activity with a MIC range between 3.13 and 25 μg/ml , with a butyryl group at the C-8 carbon, it was less active than MaBBA which possesses a 3-methyl-butyryl. This is in some way in agreement with what was reported by Mesua, which presented a MIC range of 2–64 μg/ml , isolated from mamey seeds in the present work and from mammea A/BA (MIC: 1–2 μg/ml) isolated from usiaceae . Accordi64 μg/ml . The prein vivo on the nematode C. elegans (eukaryotic organism) and in vitro on human fibroblasts. Since the extract was innocuous when evaluated against C. elegans up to a concentration of 256 μg/ml and that the highest MIC90 value against S. aureus was 4 μg/ml, the IS in relation to the nematode should have a value higher than 64; likewise, the IS in relation to MRC-5 cells would correspond to a value greater than 12. The IS value is very important to select the promising species that can be used as a source of bioactive metabolites to develop new pharmaceutical products and we consider that the M. americana extract meets this criterion. The 34S-F11 fraction, where the compound MaBBA is present, was also innocuous against C. elegans at a concentration of 256 μg/ml; these results indicate that both, the mamey seed extract and the coumarin MaBBA could be considered relatively safe.In order to evaluate the safety of the mamey seed extract, the toxicity was evaluated M. americana against strains of S. aureus sensitive and resistant to methicillin, led to the isolation of four coumarins identified as mammea B/BA, mammea B/BC, mammea A/AA cyclo D and mammea A/AA cyclo F and a mixture of mammea B/BA cyclo F plus mammea B/BD cyclo F. Mammea B/BA was shown to be a very active compound with a MIC between 0.5 and 1.0 μg/ml, comparable to vancomycin (1.0 μg/ml), an antibiotic used clinically for the treatment of methicillin-resistant S. aureus, which allows proposing mammea BB/A as a natural alternative of plant origin for staphylococcal infections.The biodirected fractionation of the ethanolic extract of the seeds of


Scientific Reports 10.1038/s41598-020-70332-3, published online 06 August 2020.Correction to: This original version of this Article contained errors in Figures 1 and 2.In Figure 1, in chemical structure “Cimetidine, 2”, the abbreviation N was incorrectly given as NH and its CN-group was omitted.Additionally, in chemical structure “10, UR-KAT514”, thiophene was connected at the wrong position.In Figure 2, in chemical structure “15, BODIPY 630/650 NHS ester” thiophene was connected at the wrong position.The original Figures The original Article has been corrected.


The most potent of these, N6-(Δ2-isopentenyl)adenosine, had a 50% inhibitory concentration (IC50) of 2 μM. These natural antiviral compounds exhibit structural and functional similarities to synthetic drugs that have been clinically examined for use against COVID-19. Our discovery of structurally diverse metabolites with anti-SARS-CoV-2 activity from screening a small fraction of the bacteria reported to be associated with the human microbiome suggests that continued exploration of phylogenetically diverse human-associated bacteria is likely to uncover additional small molecules that inhibit SARS-CoV-2 as well as other viral infections.The COVID-19 pandemic has highlighted the need to identify additional antiviral small molecules to complement existing therapies. Although increasing evidence suggests that metabolites produced by the human microbiome have diverse biological activities, their antiviral properties remain poorly explored. Using a cell-based SARS-CoV-2 infection assay, we screened culture broth extracts from a collection of phylogenetically diverse human-associated bacteria for the production of small molecules with antiviral activity. Bioassay-guided fractionation uncovered three bacterial metabolites capable of inhibiting SARS-CoV-2 infection. This included the nucleoside analogue NIMPORTANCE The continued prevalence of COVID-19 and the emergence of new variants has once again put the spotlight on the need for the identification of SARS-CoV-2 antivirals. The human microbiome produces an array of small molecules with bioactivities , but its ability to produce antiviral small molecules is relatively underexplored. Here, using a cell-based screening platform, we describe the isolation of three microbiome-derived metabolites that are able to prevent SARS-CoV-2 infection in vitro. These molecules display structural similarities to synthetic drugs that have been explored for the treatment of COVID-19, and these results suggest that the microbiome may be a fruitful source of the discovery of small molecules with antiviral activities. The rapid global spread of SARS-CoV-2 has brought on a worldwide public health crisis. While the development of vaccines provides reason for optimism, numerous factors, including the persistent prevalence of SARS-CoV-2 across the globe, the continued emergence of new variants, and high rates of vaccine hesitancy, underscore the long-term need for alternative therapeutic modalities to address COVID-19 13. The hu6-(Δ2-isopentenyl)adenosine (IPA), the 5-hydroxytryptamine receptor (5-HTR) agonist tryptamine, and the pyrazine 2,5-bis(3-indolylmethyl)pyrazine (BIP). Interestingly, there are clear structural and functional parallels between commensal bacteria-encoded and synthetic anti-SARS-CoV-2 active small molecules, as synthetic nucleosides, 5-HTR signaling modulators, and pyrazines have all been examined in either COVID-19 clinical trials or observational studies   911. The iFirmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria). We sampled species from genera that are highly represented , as well as from those with only a small number of commensal species , which allowed us to expand the phylogenetic diversity of our collection. Each strain was grown as a monoculture under the appropriate oxygen environment. After incubating at 37°C for 7 days, extracellular metabolites from these cultures were collected on Amberlite XAD7HP polymeric adsorbent. Resin-bound metabolites were then eluted to yield a library of crude bacterial extracts associated with a diverse collection of human-associated bacteria. In order to facilitate bioactive small-molecule discovery, we performed a prefractionation procedure on each extract in which the crude sample was partitioned by reverse-phase chromatography into 4 subfractions of increasing hydrophobicity (in vitro cell-based SARS-CoV-2 (USA-WA1/2020) infection assay.Although the human microbiome is composed of thousands of unique strains, metabolic redundancy across related bacteria suggests that examining a phylogenetically diverse subset of commensal bacteria is likely to uncover many of the common biologically active molecules that are produced by the microbiota . For thihobicity . The resRuminococcus gnavus, Prevotella nigrescens, Bacteroides caccae, and Micrococcus luteus) generated discrete antiviral-active HPLC fractions, from which individual anti-SARS-CoV-2 metabolite(s) were purified and structurally characterized (At the higher testing concentration (a 25-fold concentrate of culture broth), 10 bacteria produced at least 1 fraction that reduced viral infection to less than 10% . To detecterized .(i) SARS-CoV-2 active pyrazines. Partitioning of the anti-SARS-CoV-2 active fraction from a culture of M. luteus SK58 that was isolated from a human skin sample (Human Microbiome Project [HMP] ID 569) (50) of 70 μM (50 > 250 μM) (50 > 250 μM). ID 569)  revealed ID 569)  and S9C. ID 569) . Based o ID 569)  and S4. of 70 μM , while I 250 μM) . Neither10.1128/mSphere.00711-21.1FIG S11H (600 MHz) and 13C (151 MHz) NMR of 2,5-bis(3-indolylmethyl)pyrazine isolated from Micrococcus luteus (DMSO-d6). Download FIG S1, TIF file, 0.3 MB.Copyright © 2021 Piscotta et al.2021Piscotta et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00711-21.2FIG S21H (600 MHz) and 13C (151 MHz) NMR of 2-(3-indolylmethyl)-5-methylpyrazine isolated from Micrococcus luteus (DMSO-d6). Download FIG S2, TIF file, 0.3 MB.Copyright © 2021 Piscotta et al.2021Piscotta et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00711-21.3FIG S31H (600 MHz) and 13C (151 MHz) NMR of chemically synthesized 2,5-bis(3-indolylmethyl)pyrazine (DMSO-d6). Download FIG S3, TIF file, 0.3 MB.Copyright © 2021 Piscotta et al.2021Piscotta et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00711-21.4FIG S41H (600 MHz) and 13C (151 MHz) NMR of chemically synthesized 2-(3-indolylmethyl)-5-methylpyrazine (DMSO-d6). Download FIG S4, TIF file, 0.3 MB.Copyright © 2021 Piscotta et al.2021Piscotta et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00711-21.5FIG S5FIG S5, TIF file, 0.02 MB.Dose-response curve of IMP for the reduction of SARS-CoV-2 infection in Huh-7.5 cells. Download Copyright © 2021 Piscotta et al.2021Piscotta et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00711-21.9FIG S9FIG S9, TIF file, 0.1 MB.Dose-response curves of structurally related small molecules of IPA (a), tryptamine (b), and BIP (c) against SARS-CoV-2 infection in Huh-7.5 cells. Download Copyright © 2021 Piscotta et al.2021Piscotta et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the (ii) SARS-CoV-2 active tryptamine. NMR and HR-MS data indicated that the anti-SARS-CoV-2 active metabolite produced by R. gnavus CC55_001C that was isolated from a colonic biopsy specimen (HMP ID 1201)   was trypID 1201) . Its ideman host . It is aman host .10.1128/mSphere.00711-21.6FIG S61H (600 MHz) and 13C (151 MHz) NMR of tryptamine isolated from Ruminococcus gnavus (DMSO-d6). Download FIG S6, TIF file, 0.3 MB.Copyright © 2021 Piscotta et al.2021Piscotta et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 50 of 33 μM   and 5-NT  exhibited antiviral activity adenosine.(iii) SARS-CoV-2 active N Fractions from extracts of P. nigrescens CC14M that was identified in colonic biopsy specimen (HMP ID 1173)  (m/z = 336.1672). Structural elucidation by NMR and HR-MS identified these metabolites as the N6-modified adenosine derivative N6-(Δ2-isopentenyl)adenosine (IPA) . Notably, IPA also displayed cytotoxicity , something that is often observed with antiviral nucleoside analogues (ID 1173)  and B. cID 1061)  yielded ne (IPA) . Compari of 2 μM , which inalogues .10.1128/mSphere.00711-21.7FIG S71H (600 MHz) and 13C (151 MHz) NMR of 6N-(Δ2-isopentenyl)adenosine isolated from Bacteroides caccae (DMSO-d6). Download FIG S7, TIF file, 0.3 MB.Copyright © 2021 Piscotta et al.2021Piscotta et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vivo in order to function as RdRp substrates  , 22. We,bstrates . The abibstrates , indicat6-(Δ2-isopentenyl)ATP (ppp-IPA)  . In vitrA strand   (–The structure-activity relationship (SAR) of each anti-SARS-CoV-2 active metabolite was explored using collections of naturally occurring related structures. For IPA, we tested adenine and adenosine, as well as several ivatives . None of 250 μM) . Althoug 250 μM)  31–33).–33.N-mod250 μM) –, 34, 35,250 μM) –, and thu250 μM) –. Once agWe tested all three microbiome-derived anti-SARS-CoV-2 active metabolites for activity against a panel of RNA viruses, including the seasonal coronavirus HCoV-229E, yellow fever virus 17D (YFV 17D), and human parainfluenza virus 3 (hPIV-3). These are representative of both positive-sense single-strand RNA (+ssRNA) viruses  and negative-sense single-strand RNA (−ssRNA) viruses (hPIV-3). As with SARS-CoV-2, all viral infections were carried out using Huh-7.5 cells.50, 2 to 13 μM). It was most potent against the three +ssRNA viruses  with roughly the same IC50 (2 μM to 4 μM) against each . Tryptamine showed a narrower spectrum of activity  and HCoV-229E , and less effective against the −ssRNA virus hPIV-3 (IC50 218 μM). Despite also being a +ssRNA virus, tryptamine was almost five times less active against YFV 17D  than it was against SARS-CoV-2. Like IPA, BIP displayed similar antiviral activity against the three +ssRNA viruses, with IC50s of 12, 20, and 70 μM against HCoV-229E, YFV 17D, and SARS-CoV-2, respectively , IPA and tryptamine are not specialized metabolites that arise from complex and rare biosynthetic gene clusters. Similar to many of the bioactive molecules that have been characterized from the human microbiota , 38, theite rare . Tryptamite rare . Among tbacteria . Althougrmicutes , 40, 41,10.1128/mSphere.00711-21.10TABLE S1Table S1, XLSX file, 0.02 MB.Bacterial strain taxonomy. Download Copyright © 2021 Piscotta et al.2021Piscotta et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vivo production of these metabolites, we looked for tandem mass spectra matching IPA, BIP, or IMP in metabolite extract data from fecal and tissue samples submitted to the GNPS, an open-access metabolomics repository  that is typically used to treat obsessive-compulsive disorder, was associated with improved COVID-19 outcomes . While tM. luteus among the strains we examined. Small pyrazines like 2,5-dimethylpyrazine have been reported to have antifungal activity, while the larger, indole-containing BIP has been reported to be inactive as an antimicrobial    were culSARS-CoV-2  and HCoV-229E were amplified and titrated by standard plaque assay (PA) in Huh-7.5 cells . The gen4·7H2O, 100 mg/liter MnCl2·4H2O, 5 mg/liter hemin, 1 g/liter maltose, 1 g/liter cellobiose, and 0.5 g/liter l-cysteine). Bacterial fermentations were performed aerobically or anaerobically for each individual strain. For aerobic fermentations, the bacterial glycerol stock provided by BEI was used to inoculate 10 ml of LBM. This culture was grown for 24 to 48 h at 37°C with shaking at 200 rpm, and once, turbid was used to inoculate 1 liter of fresh LBM media in a 3-liter baffled Fernbach flask. The culture was fermented for 7 days at 37°C with shaking at 200 rpm, followed by metabolite extraction as described below. All anaerobic fermentations were performed in a 37°C incubator placed inside vinyl anaerobic chamber (Coy) with a gas mix of 5% CO2, 5% H2, and 90% N2. The bacterial glycerol stock provided by BEI was used to inoculate 10 ml of LBM. This culture was grown for 24 to 48 h, and once, turbid was used to inoculate 1 liter of fresh LBM media in a 1-liter glass bottle (Chemglass). Fermentation proceeded anaerobically for 7 days at 37°C followed by metabolite extraction as described below.All bacteria were cultivated in LBM media  to afford a crude metabolite extract.Amberlite XAD7HP resin (Sigma) was activated by soaking in methanol for 15 min, followed by washing five times with deionized water. Resin was stored in water until needed. Twenty grams of activated Amberlite resin was added to 1 liter of a cell suspension of bacterial culture and incubated with shaking (200 rpm) at room temperature for 4 h. Resin was then collected by filtration. We added 300 ml of acetone (Sigma), and this mixture was shaken for 2 h at room temperature. The acetone elution was collected, and an equal volume of fresh acetone was added and allowed to incubate with the resin overnight at room temperature. This second acetone elution was combined with the first and dried 18 solid-phase extraction (SPE) cartridge . The SPE cartridge was activated with two column volumes of methanol followed by washing with two column volumes of water. The crude extract was then passed over the column, and the flowthrough (fraction F1) was collected. Metabolites were then eluted (two column volumes) using a stepwise gradient of increasing methanol concentrations as follows: F2, 30%; F3, 70%; and F4, 100%. These elutions, along with the column flowthrough, were dried in vacuo, affording four prefractionated extracts from each crude metabolite pool.Each extract was resuspended in methanol (Sigma) to a concentration of 100 mg/ml. One milliliter (100 mg) of this solution was aliquoted and diluted in water to 5% methanol and spun down to prepare for fractionation on a Strata C3 cells/well into 96-well plates. The next day, serially diluted compounds were added to the wells, followed 1 h later by infections with either SARS-CoV-2 , HCoV-229E , YFV 17D Venus , or hPIV-3-GFP . Cells were incubated for 48 h at 33°C for infections performed with SARS-CoV-2 and at 37°C for infections performed with other viruses. Cells were then fixed by adding an equal volume of 7% formaldehyde to the wells and subsequently permeabilized with 0.1% Triton X-100 for 10 min. After extensive washing, SARS-CoV-2- and HCoV-229E-infected cells were incubated for 1 h at room temperature with blocking solution of 5% goat serum in phosphate-buffered saline (PBS) . A rabbit polyclonal anti-SARS-CoV-2 nucleocapsid antibody  was added to the cells at 1:1,000 dilution in blocking solution and incubated at 4°C overnight. J2, a mouse monoclonal anti-double-stranded RNA (dsRNA) antibody  was added to the cells under similar conditions to detect HCoV-229E-infected cells. Goat anti-rabbit Alexa Fluor 594  and goat anti-mouse Alexa Fluor 488  were used as secondary antibodies at a dilution of 1:2,000. Nuclei for all infections, including those with the fluorescently labeled YFV 17D and hPIV-3, were stained with Hoechst 33342  at 1 μg/ml. Next, images were acquired with an ImageXpress XLS Widefield High-Content microscope . Images were acquired using the DAPI  filter  for Hoechst staining and the fluorescein isothiocyanate (FITC) filter  for viral nucleocapsid staining. Images were analyzed using an automated custom module in the MetaXpress software (Molecular Devices). Default software analysis scripts were used for DAPI- and FITC-positive cell counting. Infection was calculated as the percentage of viable cells that were positive for viral nucleocapsid. For determining the efficacy of isolated metabolites, 10-point dose-response curves in a 1:3 dilution series were tested in triplicate using a maximum concentration of 50 μM (IPA) or 250 μM . IC50 values were calculated using a variable-slope, four-parameter nonlinear regression curve fit (GraphPad Prism 8). All SARS-CoV-2 experiments were performed in a biosafety level 3 laboratory.The day prior to infection, Huh-7.5 cells were seeded at 7.5 × 10in vacuo.Bacterial metabolite fractions were partitioned on an Agilent Series 1200 HPLC using a solvent system of water (A) and acetonitrile  connected to a Waters XSelect CSH phenyl-hexyl OBD prep column  run at a constant flow rate of 3 ml/min. The specific chromatographic method was chosen according to the identity of the prefraction as follows: for F1, 0 to 5 min, 5% B; 5 to 10 min, increase to 10% B; 10 to 20 min, 10% B; 20 to 40 min, increase to 30% B; 40 to 45 min, increase to 95% B; 45 to 55 min, 95% B; for F3, 0 to 5 min, 10% B; 5 to 30 min, increase to 30% B; 30 to 40 min, increase to 9% B; 40 to 50 min, 95% B; and for F4, 0 to 5 min, 30% B; 5 to 30 min, increase to 95% B; and 30 to 40 min, 95% B. Fractions were collected in 2-min intervals and dried 18 column  at 0.50 ml/min with the following method: 0 to 0.3 min, 10% B; 0.3 to 3.3 min, increase to 95% B; and 3.3 to 3.8 min, 95% B. All analyses were performed on SCIEX OS software. NMR data were collected on a Bruker Avance DMX 600 MHz spectrometer.All high-resolution mass spectrometry (HR-MS) data were collected on a Sciex X500B quadrupole time of flight (QTOF) mass spectrometer running in positive mode, using a 5,500-V spray voltage and 5 V collision energy. The mass spectrometer was connected to an Exion liquid chromatography system using water (A) and acetonitrile (B) solvents, each with 0.1% formic acid. Samples were run on a Kinetex PS CBacteroides caccae F3 as a single metabolite using the HPLC method described above. [M+H]+; calculated, 336.1672; observed, 336.1670. 1H NMR : δH 8.34 , 8.21 , 7.92 , 5.88 , 5.43 − 5.40 , 5.30 , 5.17 , 4.61 , 4.14 , 4.06 , 3.96 , 3.67 , 3.55 , 1.70 , 1.67 . 13C NMR : δC 154.8, 152.8, 148.7, 140.2, 133.8, 122.5, 120.3, 88.5, 86.4, 74.0, 71.1, 62.2, 25.8, 18.3.IPA was isolated from Ruminococcus gnavus F1 as a single metabolite using the HPLC method described above. [M+H]+; calculated, 161.1079; observed, 161.1075. 1H NMR : δH= 10.99 , 8.09 , 7.56 , 7.36 , 7.23 , 7.09 , 7.00 , 3.03 . 13C NMR : δC 136.8, 127.3, 123.8, 121.6, 118.9, 118.6, 112.0, 109.9, 23.5.Tryptamine was isolated from Micrococcus luteus F3 as a single metabolite using the HPLC method described above. [M+H]+; calculated, 339.1610; observed, 339.1600. 1H NMR : δH 10.87 , 8.45 , 7.44 , 7.32 , 7.18 , 7.04 , 6.91 , 4.16 . 13C NMR : δC 154.2, 143.4, 136.7, 127.3, 123.9, 121.5, 118.9, 112.1, 111.9, 31.3.BIP was isolated from Micrococcus luteus F3 as a single metabolite using the HPLC method described above. [M+H]+; calculated, 224.1188; observed, 224.1176. 1H NMR : δH 10.90 , 8.35 , 8.32 , 7.48 , 7.34 , 7.21 , 7.05 , 6.94 , 4.18 , 2.46 . 13C NMR : δC 156.0, 152.9, 141.8, 136.7, 127.3, 124.0, 121.5, 118.9, 118.9, 111.9, 31.8, 21.6.IMP was isolated from (i) Formation of Weinreb amide. Benzyloxy carbonyl (Cbz)-protected amino acid  dissolved in 10 ml dimethylformamide (DMF) was mixed with N,O-dimethylhydroxylamine (2 equiv.) and N,N-diisopropylethylamine (3 equiv.) and stirred for 1 h at room temperature. The reaction mixture was diluted with dichloromethane  and washed several times with acidified water (1% formic acid) to remove DMF. DCM was concentrated in vacuo and purified by flash chromatography using ethyl acetate-hexane as eluent to give the Weinreb amide as a yellow oil.(ii) Formation of aldehyde derivative. Lithium aluminum hydride (5 equiv.) was added and stirred for 2 h into a stirred solution of Weinreb amide  in diethyl ether (50 ml) at 0°C. The reaction mixture was quenched with ice cubes, filtered through a celite pad, and washed with water (50 ml) and diethyl ether (30 ml). The filtrate was extracted 3 times with diethyl ether and washed with hydrochloric acid (1 M), saturated sodium hydrogen carbonate solution, and brine. The extract was dried over magnesium sulfate, concentrated in vacuo, and purified by flash chromatography using ethyl acetate-hexane as eluent to give the aldehyde derivative as a yellow oil.(iii) Formation of pyrazine. Pd(OH)2  was added and stirred for 2 h at room temperature under a hydrogen atmosphere into the aldehyde derivative (100 mg) dissolved in a mixture of acetic acid, methanol, and DCM . The hydrogen source was removed, and the reaction mixture was stirred overnight while open to air. The mixture was then filtered through a celite pad, washed with methanol, dried in vacuo, and purified by flash chromatography using ethyl acetate-hexane as an eluent. Identities of the desired pyrazines were verified and characterized by HR-MS and NMR.6 cells/ml. Each well of a 6-well plate was seeded (3.0 × 106 cells), and cells were allowed to attach overnight. The medium was aspirated and replaced with 1 ml of fresh DMEM containing either IPA or remdesivir at 10 μM. After 3 h, cells were harvested. The medium was removed, and plates were immediately placed on ice. Ice-cold Hanks’ balanced salt solution (HBSS) was used to wash each well twice, and then, 500 μl extraction solution  was added to each well. Cells were allowed to lyse for 15 min on ice. Each well was then scraped with a sterile cell scraper, and the lysate was transferred to an Eppendorf tube. Lysates were centrifuged at 20,000 × g for 10 min at 4°C. Supernatants were transferred into preweighed 4-ml glass vials and dried in vacuo without heat . Dried material was resuspended in 200 μl 50:25:25 H2O/CH3CN/CH3OH and centrifuged at 16,000 × g for 10 min at 4°C. Supernatants were moved into 96-well polypropylene plates, covered with slit silicone mats, and stored at −20°C until analysis by HR-MS.Confluent HEK293 cells were detached with trypsin-EDTA (0.25%) and adjusted to a concentration of 3.0 × 10Trimethyl phosphate  (0.5 ml) was added to IPA (0.05 mmol) over 4-Å molecular sieves. The mixture was stirred for 24 h under an argon atmosphere. Phosphorus oxychloride  was added via syringe, and the mixture was stirred for 3 h at 0°C. A mixture of tributylamine  and tributylammonium pyrophosphate  dissolved in acetonitrile (0.25 ml) was added, and the mixture was stirred for an additional 30 min at 0°C. The reaction was quenched by addition of triethylammonium bicarbonate (TEAB) (1 M) (0.5 ml) and water (5 ml). The reaction mixture was purified by HPLC using the conditions described above with the following solvent gradient: 0 to 4 min, 5% B; 4 to 30 min, increase to 60% B; 35 to 40 min, increase to 95% B; and 40 to 50 min, 9% B. The identity of the IPA 5′-triphosphate was confirmed by HR-MS.CGCGUAGCAUGCUACGUCAUUCUCC UAAGAAGCUA-3′) was labeled at the 5′ end with T4 polynucleotide kinase (New England Biolabs) and [γ32-P]ATP (PerkinElmer). The labeling reaction was heat inactivated at 70°C for 20 min, and unincorporated [γ32-P] ATP was removed with a 7 molecular weight cutoff (MWCO) Zeba spin column (Thermo Fisher). The labeled RNA primer was then annealed to the template strand  by heating to 95°C for 2 min and cooling to 10°C for 30 min. Proteins of the SARS-CoV-2 replication-transcription complex were purified as previously reported  through a 7 MWCO Zeba Spin desalting column (Thermo Fisher). The holo-complex (1.25 μM) was incubated with the RNA scaffold (1 μM) at 37°C for 15 min before the addition of NTPs and inhibitors (50 μM) 3′-deoxyadenosine-5′-triphosphate (TriLink), remdesivir triphosphate (Gilead Sciences), and isopentenyl ATP in 10-μl reactions. In vitro transcription reaction mixtures were incubated at 30°C for 10 min before being quenched with an equal volume of 2× stop buffer . Elongation products were separated on 15% acrylamide-6 M urea denaturing gel alongside Decade RNA ladder (Thermo Fisher) in 1× Tris-borate-EDTA (TBE) and analyzed by phosphorimaging.RNA primer (5′-reported , 57. NspAll data that support the findings of this study are available from the corresponding author upon request.


Magnoliae officinalis cortex, which has been widely used in traditional Chinese and Japanese herbal medicine and possesses a wide range of pharmacological activities. In recent years, attention has been drawn to this component due to its potential as an anti-inflammatory and antitumor drug. To summarize the new biological and pharmacological data on MG, we screened the literature from January 2011 to October 2020. In this review, we provide an actualization of already known anti-inflammatory, cardiovascular protection, antiangiogenesis, antidiabetes, hypoglycemic, antioxidation, neuroprotection, gastrointestinal protection, and antibacterial activities of MG. Besides, results from studies on antitumor activity are presented. We also summarized the molecular mechanisms, toxicity, bioavailability, and formulations of MG. Therefore, we provide a valid cognition of MG.Magnolol (MG) is one of the primary active components of  Magnoliae officinalis cortex, which was first recorded in “Shennong Herbal Classic” , is the dry bark, root bark, and branch bark of Magnolia officinalis Rehd. et Wils. or Magnolia officinalis Rehd. et Wils. var. biloba Rehd. et Wils. In traditional medicine, Magnoliae officinalis cortex mainly acts to dry dampness and disperse phlegm, lower Qi, and eliminate fullness. Clinically, it is commonly used to treat asthma, constipation, edema, abdominal distension, malaria, and other diseases by combining different traditional Chinese medicines. For example, the Da Houpo Pill is used to treat abdominal distension (Song Ji Zonglu). The Xiaochengqi decoction is used for the treatment of tidal fever, constipation, and abdominal pain (Treatise on Febrile Diseases). The Banxia Houpo decoction has therapeutic effects on chronic pharyngitis, chronic bronchitis, and esophageal fistula (Synopsis of the Golden Chamber). Recent studies have shown that Magnoliae officinalis cortex has multiple pharmacological activities on the nervous system  and extraction method , signal transduction and activator of transcription (STAF), and notch signaling pathways  and honokiol  are biphenyl-type neolignans. They have been recognized as the principal active components of magnolia bark extract, usually accounted for 1–10% of dry bark, depending on the n method . Talarekhonokiol . Woodburisorders . Ong et pathways . The met . The source information of etc. Furthermore, NF-κB consists of isotype or heteromorphic p50 and p65 protein, which affects the expression of inflammatory and growth factors, chemokines, COX-2, and iNOS involved in the processes of inflammation, apoptosis, tumorigenesis, etc.  and cyclooxygenase-2 (COX-2) and excessive synthesis of nitric oxide (NO) and prostaglandins (PGEs) . Mitogenis, etc. . MG exhiis, etc. .MG (5–15 μM) could exhibit anti-inflammatory activity in lipopolysaccharide (LPS)-induced RAW 264.7 cells. It decreased the translocation of p50 and p65 subunits and downstream NF-κB transcription through downregulating inhibitor kappa B (IκB) degradation and phosphorylation. Additionally, MG blocked the phosphorylation of ERK1/2, JNK1/2, and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signal, interfered with the activation of PI3K/Akt, MAPK, and NF-κB pathway, and thus inhibited iNOS and COX-2 protein and gene expression . MG 5–2.−1, intraperitoneal (i.p.) injection) played roles in significantly ameliorating pathological characteristics and inhibiting the inflammatory reaction of acute lung injury in male Sprague Dawley (SD) rats. It could attenuate pneumonic edema, neutrophil infiltration, reactive oxygen species (ROS) production, iNOS and COX-2 expression, and NF-κB activation and upregulate PPAR-γ expression  exhibited therapeutic effect for pathological retinal angiogenesis and glial dysfunctions by decreasing the expression of inflammatory cytokines and inactivating the HIF-1α/VEGF pathway , and Wnt/β-catenin signaling pathways  showed the activity of suppressing the proliferation of PC3 cells  inhibited the growth of GBC-SD tumor in BALB/c nude xenograft model. It significantly increased caspase-3 activation and inhibited cell division cycle gene (CDC) 2 expression  in the nude immune-deficient mice, it could be observed that the growth of nude immune-deficient MDA-MB-231 and MCF-7 tumors was inhibited, and the level of MMP-9 was decreased -dependent pathway and inhibiting ROS production .MG (10 μM) reduced the accumulation of HIF-1α protein by enhancing the activity of prolyl hydroxylase and reducing the synthesis of HIF-1α protein . MG 20 . MG 10 .−1, i.p. injection) inhibited angiogenesis, tumor proliferation, and the expression of HIF-1α, VEGF, endothelial cell marker CD31, and endogenous hypoxia biomarker carbonic anhydrase IX by suppressing HIF-1α/VEGF-dependent pathway (In the T24 xenograft mouse (C57BL/6 mice), MG , such as antiplatelet, antithrombotic, and antihypertensive via inhibiting MAPK family activation, Akt/ERK1/2/GSK3 β-catenin pathway, and angiotensin-converting enzyme (ACE)/angiotensin II (Ang II)/Ang II type 1 receptor (AT-1R) cascade and upregulating PPAR-β/γ and NO/guanosine 3′,5′-cyclic phosphate/PKG pathways .50 value of 5.78 μM  caused attenuation of neointima formation, intimal area, and intimal/medial ratio and increase of luminal area via significantly decreasing the expression of cyclin D1/E and CDK4/2 mRNA and protein  exerted a therapeutic effect of PHA by altering the Akt/ERK1/2/glycogen synthase kinase 3β (GSK3β)-catenin pathway. It upregulated ACE2 and significantly downregulated the expression of iNOS, endothelin-1 (ET-1), and ETA receptors and O2- production  and protein expression  inhibited the death of RIN-m5F cells and the decrease of insulin secretion induced by methylglyoxal, thereby exerting hypoglycemic activity . It coulpression . It upreectively . Moreoveectively . The inhC57BL/6J mice were fed a high-fat diet  with or without honokiol  or MG  for 16 weeks. The results showed that honokiol and MG significantly lowered the weight of white adipose tissue, adipocyte size, and proinflammatory gene expression, protected against insulin resistance, and elevated plasma IL-10 level. In particular, honokiol could significantly decrease the plasma resistin level and increase the plasma adiponectin level compared to the control group .It can be seen that MG and honokiol have similar mechanisms to play a hypoglycemic role, such as inhibition of α-glucosidase and stimulation of glucose uptake. The difference is that MG has a better inhibitory effect on α-glucosidase, while honokiol can significantly decrease the plasma resistin level and increase the plasma adiponectin level.In vitro, MG (3–100 μM) inhibited the spontaneous contraction, acetylcholine (ACh)- and Bay k8664-induced contraction, L-type Ca2+ current, and the contraction of colonic smooth muscle through decreasing L-type Ca2+ channel activity  significantly inhibited diarrhea, reduced small intestinal transport, and increased catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px)  and honokiol  regulated the release of IP3-Ca2+ storage, suppressed SK channel, and facilitated the opening of BKα1 as well as BKβ3 channels and the closing of BKβ4 channel by blocking the IP3-Ca2+ channel, inhibiting the activation of IP3 receptor 1 and CaM, and regulating protein kinase C (PKC)  . Zeng ethonokiol . The rea C (PKC) . In thisIn conclusion, both MG and honokiol can exhibit gastrointestinal protective activity with similar mechanism, while MG’s antidiarrheal activity is better than that of honokiol.50 = 18.3 ± 8.6 µM) and CB2 (EC50 = 3.28 ± 2.10 µM), while honokiol was a full agonist of CB1 (EC50 > 10 µM) and an inverse agonist of CB2. 4′-O-Methylhonokiol was a CB2 receptor agonist and a potent COX-2 SSI (50 = 0.93 ± 0.91 µM) and RXRα (EC50 = 3.91 ± 1.08 µM)  . A greatOX-2 SSI . In addiOX-2 SSI . MG showOX-2 SSI . MG playOX-2 SSI . MG was 1.08 µM) . In addi1.08 µM) . Honokio1.08 µM) . In α1β2honokiol . MG exhihonokiol .50 = 3.49 μM) and honokiol (EC50 = 2.65 μM) promoted the transcriptional activities of PPAR-γ in a dose-dependent manner. They also dose-dependently increased the luciferase activity of PPAR-γ-LBD. MG and honokiol could fit into the protein pocket of PPAR-γ-LBD with IC50 values of 3.745 and 16.13 μM, respectively. What is more, MG had two hydrogen bonds at Glu343, which maintained the binding stability, while honokiol had one hydrogen bond at Glu343 and SER342, respectively, indicating that MG was more effective in enhancing PPAR-γ luciferase levels than honokiol  attenuated Aβ-induced AD by inhibiting the luciferase activity of NF-κB and the target gene of inflammatory cytokines, activating luciferase and liver X receptor activity, reducing ROS production induced by Aβ, upregulating apolipoprotein E (ApoE), and promoting microglial phagocytosis and Aβ degradation . MG  level and increasing NE, 5-hydroxytryptamine (5-HT), and BDNF protein levels  downregulated the levels of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) in the prefrontal cortex, suppressed the activation of microglia and the proliferation of HPA axis and oxidative stress, and reversed malondialdehyde increase and SOD as well as GSHPx decrease to produce antidepressant-like effect  downregulated the expression of bax and Ac-FOXO1 and production of NOS, 4-HNE, iNOS, phosphorylated p38MAPK, and C/EBP homologs, while upregulated the expressions of Bcl-2 and SIRT1. The regulation effect of MG on ischemic damage factors may be through inhibiting the production of ROS and upregulating p-Akt and NF-κB (−1) exhibited antiepileptic activity by prolonging the latency of seizure onset and decreasing the number of seizure spikes, through acting on GABAA/benzodiazepine receptor , CYP2C19 (Ki: 0.449 μM), CYP2C (Ki: 10.0–15.2 μM), 3A (Ki: 93.7–183 μM), and CYP1A2 (Ki: 10.0 μM) were competitive inhibition. The IC50 values of honokiol on human CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A, and CYP3A4 were 3.5, 18.8, 40.8, 9.6, 32.9, >50, >50, and 43.9 μM, respectively. Moreover, the IC50 values of honokiol on rat CYP2C, CYP2A6, CYP2D6, CYP2E1, CYP3A4, CYP1A2, and CYP2B6 were 41.86 ± 4.24, >100, 43.43 ± 2.34, 58.10 ± 3.02, >100, 95.24 ± 7.81, and 53.22 ± 0.66 μM, respectively. The inhibition type of honokiol on CYP1A2 (Ki: 6.2 μM) was competitive inhibition, and the inhibition types of honokiol on CYP2E1 (Ki: 11.1 μM) and CYP2C19 (Ki: 0.702 μM) were noncompetitive inhibition  value range of 32–64 μg ml−1 (Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA), with the MIC/minimal bactericidal concentration (MBC) value range of 16–64 mg L−1  had better bactericidal activity against S. mutans biofilm than honokiol (50 μg ml−1) and chlorhexidine (500 μg ml−1) at 5 min after exposure  Keissl, Penicillium expansum (Link) Thom, and Alternaria dauci F.Sp. solani, MG inhibited their growth by more than 57%  against 32 Fusarium spp. strains. The terbinafine (0.1–10 μg ml−1) and fluconazole (1–50 μg ml−1) were used as positive controls. The results revealed that MG had similar bactericidal activity compared with fluconazole, whereas honokiol had a better effect of inhibiting the mycelium growth compared to this fungicide. Compared to terbinafine, honokiol exhibited similar antifungal activity, whereas MG was less effective at all selected concentrations  and four peroxyl radicals in acetonitrile (kinh = 9.5 × 103 M−1 s−1). Their different behavior was due to the combination of intramolecular hydrogen bonding among the reactive OH groups (in MG) and of the OH groups with the aromatic and allyl π-systems  protected against acrolein-induced oxidative stress in human SH-SY5Ycells via acting on JNK/mitochondrial/caspase and PI3K/MEK/ERK/Akt/FoxO1 signaling pathways and inhibiting intracellular glutathione consumption as well as ROS accumulation  could significantly reduce MPO activity and the expression of iNOS, TNF-α, and IL-6 to inhibit oxidative stress and reduce mesenteric reperfusion caused lung injury in male C3H/HeOuJ mice  significantly suppressed alveolar bone resorption, the number of osteoclasts on the bony surface, and the expression of RANKL. Moreover, it could reduce the expression of MMP-1, MMP-9, iNOS, and COX-2 and TNF-α activation  inhibited the activities of osteogenic factors runt-related transcription factor 2(RUNX2), OCN, and bone morphogenetic protein 2 (BMP2) in male SD rats. Moreover, it inhibited ossification of tendon ossification by reducing heterotopic ossification of Achilles tendon (MG (0.1 μM) significantly downregulated the production of osteoclast differentiation-inducing factors such as RANKL, TNF-α, and IL-6 and inhibited mitochondrial electron transport . In RANKtivation . MG , HBV e antigen (HBeAg), and replication of HBV DNA were 2.03, 3.76, and 8.67 μM, respectively, and without cytotoxicity to HBsAg and HBeAg (−1) and honokiol (3.18 ± 0.61 μg ml−1) stimulated the expression of immune-related genes to resist grass carp reovirus infection in Ctenopharyngodon idella kidney (CIK) cells. MG significantly increased the expression of interferon (IFN) regulatory factor (IRF) 7 and IL-1β to activate type I IFN (IFN-I) but failed to induce the molecules in NF-κB pathways. The difference was that honokiol promoted the expression of IL-1β, TNFα, NF-κB, IFN-β, promoter stimulator 1, IRF3, and IRF7 but failed to increase IFN-I expression, showing that it could enhance the host innate antiviral response to grass carp reovirus infection by regulating NF-κB pathway  reduced the multidrug resistance of cancer cells to antitumor drugs through downregulating P-glycoprotein expression in a concentration- and time-dependent manner and increased the intracellular accumulation of calcein in NCI/ADR-RES cells .−1, MG could cause toxicity and inhibit MMEC survival  for 24 and 48 h. MG induced cell death in a dose- and time-dependent manner. Treatment with 50 μM MG for 24 h resulted in significant decreases in cell viability with 75.3% of the cells surviving after 24 h and 81.7% of the cells surviving after 48 h. Moreover, MG (50 µM) induced apoptosis in 76.1% of the cells after 24 h, indicating that MG inhibited cell proliferation and induced apoptosis in HCT-116 cells  and honokiol (5 μg/per plate) could inhibit against UV-induced mutations by scavenging ·OH generated by UV irradiation. The relative mutagenic activities of MG and honokiol were 62 ± 1% and 62 ± 4%, respectively, while that of control was 100%  was studied by Li et al., which was composed of 94% MG and 1.5% honokiol. The results revealed that MBE was not genotoxic under the conditions of the in vitro bacterial reverse mutation test and in vivo micronucleus test and supported the safety of MBE for dietary consumption , the MG sulfates and glucuronides were predominant in the bloodstream. And MG was mainly distributed in the liver, kidney, brain, lung, and heart; among these organs, the concentration of MG and MG glucuronides in the liver was the highest , maximum concentration-time (Tmax), and maximum concentration (Cmax) of MG were 0.63 h, 2.33 h, 1.12 h, and 0.16 μg ml−1, respectively. The water solubility and gastrointestinal absorption of MG were poor, with the oral bioavailability of only 4.9% 0-t and AUC0-∞ were significantly improved. Moreover, compared with raw MG, their relative bioavailability increased to 1.38, 2.12, and 3.45 times, respectively  and studied its bioavailability by oral administration (50 mg kg−1). The results indicated that compared with raw MG, the solid dispersion of MG with PVP significantly increased the systemic exposures of MG and MG sulfates/glucuronides by 80.1 and 142.8%, respectively   . Stefana release .−1) in male SD rats, the 1.20 h average plasma concentration of MG was 426.4727 ng ml−1, and the absolute bioavailability was 17.579%, indicating that preparing an emulsion could improve the bioavailability of MG  to prepare MG-HP-β-CD inclusion complex (HP-β-CD: MG 10:1). The stoichiometric ratio of the inclusion compound was 1:1, and the stability constant (Kc) was calculated as 2206 M−1. In addition, the aqueous solubility of the MG-HP-β-CD inclusion complex was more than 500-fold that of free MG, and it had better stability and stronger antitumor activity in vitro  as the MG carrier. After oral or intraperitoneal administration of 100 mg kg−1 of MG and MG@Uio-66(Zr), the AUC0-720 of MG@Uio-66(Zr)  was significantly higher than the AUC0-720 of free MG . The relative bioavailability of MG increased almost twofold by using Uio-66(Zr)  and 1,2-dipalmitoyl- Sn-glycero-3-phosphocholine (DPPC) liposomes to encapsulate MG with entrapment efficiencies of 74.13 ± 1.97% and 64.26 ± 2.92%, respectively. The results showed that EPC and DPPC liposomes enhanced the inhibitory effect of MG on VSMCs, and the inhibitory effect of EPC liposome-encapsulated MG on VSMCs was better than that of DPPC liposome . Qiu et in vitro . Santos o-66(Zr) . The druigration .et al. also prepared MG-M by pluronic F127 and L61, and its drug loading efficiency and entrapment efficiency were 81.57 ± 1.49% and 27.58 ± 0.53%, respectively. In vitro release test showed that MG had sustained release behavior after being encapsulated in micelles. The permeability of MG through the Caco-2 cell monolayer was enhanced, and the relative bioavailability of oral MG-M was 2.83 times higher than that of the raw MG (Mixed Soluplus (SOL) and Solutol HS15 (HS15), SOL, and D-alpha-tocopheryl polyethylene glycol 1,000 succinate (TPGS) were used to prepare MG-loaded mixed micelles (MG-M) MG-H and MG-T, respectively. The relative oral bioavailability of MG-T and MG-H were increased by 2.39- and 2.98-fold, respectively, compared to that of raw MG, indicating that MG-H and MG-T could promote the absorption of MG in the gastrointestinal tract . Shen ete raw MG . It can In general, the existing formulations can not only improve the water solubility and bioavailability of MG but also improve its stability, enhance its pharmacological effects, and enable MG to have a sustained release behavior, which will provide strategies for future clinical applications of MG.et al. summarized the pharmacological activities and molecular mechanisms of MG. According to the review, MG could exhibit anti-inflammatory activity by inhibiting the production of inflammatory enzymes/cytokines and activation of NF-κB and leukocyte. It also exerted antitumor effects by inhibiting cell proliferation and metastasis and inducing apoptosis. The molecular mechanisms mainly include the increase of p21, p27, caspase-3, caspase-8, and caspase-9 expression, inhibition of PI3K/PTEN/AKT pathway, ERK1/2, NF-κB, P38, iNOS, and COX2 activation, CYP1A1, CYP1A2, MMP-9 as well as MMP-2 activity and Bcl-2 expression, induction of cytochrome C, and AIF release and activation of the mitochondrial death receptor pathway. MG could attenuate VCAM-1, ICAM-1, MCP-1, and MMP9, inhibit the proliferation of smooth muscle cells and fibroblasts, and obtain arrhythmia from I/R injury to show cardiovascular protection. It could also exert neuroprotective activities by inhibiting the production of PGE2, regulating (GABA)A receptor subtypes and central serotonergic activity, retaining cholinergic neurons in the forebrain, and inhibiting cortical 5-HT release. MG had a therapeutic effect on gastrointestinal diseases by regulating serotonergic and gastrointestinal system functions and relaxing gastrointestinal smooth muscles. Moreover, it exhibited hypoglycemic activity by activating PPAR and increasing basal and insulin-stimulated glucose uptake (In 2011, Chen e uptake .in vivo and in vitro studies demonstrated that MG has a wide range of pharmacological activities including anti-inflammatory, antitumor, antioxidant, hypoglycemic, cardiovascular protection, antiangiogenesis, and antibacterial. MG inhibited TLR2/TLR4/NF-κB/MAPK/PPAR-γ pathways and decreased the expression of inflammatory cytokines to exhibit anti-inflammatory activity. It suppressed the growth, migration, and invasion of tumor cells and promoted apoptosis as well as autophagy, through acting on caspase-8, caspase-3, and other proteins participated in the p53, MAPK, NF-κB, TLR, PI3K/Akt/mTOR, and Wnt/β-catenin signaling pathways. It also protected the nervous system through multiple systems and multiple targets. Moreover, it has a wide range of antibacterial activity. MG is a candidate drug for anti-inflammatory, anticancer, and neuroprotective activities. However, MG’s in vivo effect with CYP enzymes is not clear yet, and there is no clinical research on MG, which cannot fully provide the pharmacological activities of it.In this review, Aggregatibacter actinomycetemcomitans, S. mutans, S. aureus, MRSA, Escherichia coli, and Fusarium spp.MG and honokiol have similar pharmacological activities. Both of them can exhibit antitumor activities by regulating MAPK, NF-κB, HIF-α, PI3K/Akt/ERK/mTOR, and Wnt/β-catenin signaling pathways. MG shows antitumor activity by regulating TLR signaling pathways, and honokiol can regulate STAF, EGFR, and notch signaling pathways to exhibit antitumor activities. They have inhibitory activity on α-glucosidase and stimulation of glucose uptake to play a hypoglycemic role, while MG has a better inhibitory effect of α-glucosidase. Moreover, both MG and honokiol exhibit gastrointestinal protective activity with similar mechanism, while MG’s antidiarrheal activity is better than that of honokiol. MG is a partial agonist of CB1 and CB2; however, honokiol is a full agonist of CB1 and an inverse agonist of CB2. MG has no activity on GPR-55, while honokiol is an antagonist of GPR-55. Honokiol has a stronger positive regulatory effect on GABAA receptors than MG; however, MG is more effective in enhancing PPAR-γ luciferase levels than honokiol. What is more, the inhibition types of MG on CYP1A, CYP2C19, CYP2C, CYP3A, and CYP1A2 were competitive inhibition. The inhibition type of honokiol on CYP1A2 was competitive inhibition, and the inhibition types of honokiol on CYP2E1 and CYP2C19 were noncompetitive inhibition. Both honokiol and MG have antimicrobial activity. The difference is that honokiol exhibits better antimicrobial activity than MG on etc., the bioavailability and stability of MG significantly improved, which will greatly promote its clinical application. Aside from its formulations, structural modification is becoming an increasingly promising method for obtaining MG derivatives with better therapeutic effects and higher bioavailability. The synthesis and research of MG derivatives are beyond the scope of this study, so we will not go into details. Consequently, the design and research of MG derivatives are of great significance in the future.MG is nontoxic and is used in dietary supplements and cosmetic products, such as added to toothpaste to play antibacterial and antiperiodontitis effects. However, the low water solubility, poor bioavailability, and skin irritation hamper its application. To overcome this problem, numerous studies have been conducted. By preparing solid dispersions, nanoparticles, phospholipid complexes, liposomes, emulsions, In summary, this article comprehensively reviews the pharmacology, toxicity, bioavailability, and formulations of MG.


To evaluate GT0918, a 2nd-generation AR antagonist, for its AR down-regulation activity among breast cancer patients.The effect of GT0918 on AR protein expression was evaluated in AR expression breast cancer cells and in breast cancer xenograft model. A 3 + 3 phase I dose-escalation study was launched in Peking University Cancer Hospital. The endpoints included dose finding, safety, pharmacokinetics, and antitumor activity.GT0918 was demonstrated to effectively suppress the expression of AR protein and the growth of AR-positive breast cancer tumors in mouse xenograft tumor models. All patients treated with GT0918 were at a QD dose-escalation of five dose levels from 100 to 500 mg. The most common treatment-related AEs of any grade were asthenia, anemia, decreased appetite, increased blood cholesterol, increased blood triglycerides, decreased white blood cell count, and increased low-density lipoprotein. Grade 3 AEs were fatigue , aspartate aminotransferase increase , alanine aminotransferase increase , and neutrophil count decrease . Clinical benefit rate (CBR) in 16 weeks was 23.1% (3/13). Among 7 AR-positive patients, 6 can evaluate efficacy, and 2 completed 23.5- and 25-cycle treatment, respectively (as of 2020/1/20). PK parameters showed a fast absorption profile of GT0918 in the single-dose study. GT0918 and its major metabolite reached steady-state serum concentration levels at day 21 after multiple dosing.GT0918 can effectively inhibit AR-positive breast cancer tumor growth. GT0918 was demonstrated well tolerated with a favorable PK profile. The suitable dose of GT0918 was 500 mg QD and may provide clinical benefits for AR-positive mBC. The activation of androgen receptor (AR) signaling plays a critical role in driving the initiation and progression of prostate cancer . AR-targAR has been identified in 10%–35% of triple-negative breast cancer (TNBC) –17. The Here, we reported the preclinical and clinical data of GT0918 in mBC, including in vitro and in vivo activities of GT0918 in AR-positive metastatic breast cancer models and phase I study results in mBC . Our data suggest that GT0918 may have therapeutic benefits in patients with mBC.2 at 37℃.Human Breast cancer lines, MCF-7, BT-474, and MDA-MB-468, were obtained from culture collection of Chinese Academy of Sciences, Shanghai, China. The MCF-7 and MDA-MB-468 cells were cultured in DMEM supplemented with 10% fetal bovine serum and the BT474 was cultured in RPMI1640 supplemented with 10% fetal bovine serum. The cells were cultured in incubator containing 95% air and 5% CO7 cells), BT-474 (1.5 × 107 cells), and MDA-MB-468 (1.5 × 107 cells) breast cancer cells were suspended in 200 μL of PBS with 50% Matrigel, and then, the cells were injected orthotopically into the right axial mammary gland to initiate tumor growth. The mouse was then divided . The mouse was given daily oral dose of GT0918  or MDV3100 (20 mg/kg BID) for 21 days, and 8 mg/kg of cisplatin was injected intraperitoneally once weekly. The body weight and the tumor size were measured twice a week using caliper and tumor volume were calculated according the formula: L × S2 × 0.5, in which L represents the longest diameter and S2 represents the shorter diameter of tumor.The 6–8 weeks old female BALB/c nude mouse  was provided by Vital River Laboratory Animal Technology . Mouse was supplemented with 1.7 mg E2-pellets . MCF-7 .The primary objective endpoints were the safety, the maximum-tolerated dose (MTD), the recommended dose for expansion (RDE), and dose-limiting toxicities (DLTs) of oral GT0918 in female mBC patients with progression after systemic treatments. The secondary objectives were pharmacokinetics and pharmacodynamics of GT0918 with single and multiple dosages.The starting dose was 100 mg daily, followed by dose-escalation of 200, 300, 400, and 500 mg. The dose-escalation, in 3 + 3 design, was determined by the safety and tolerability assessments. GT0918 was administered orally once daily, followed by a 7-day off treatment period for single-dose PK analysis of drug elimination. Then, the oral administration of GT0918 was resumed once daily for 28 consecutive days and multiple-dose PK analysis was assessed at the end of first cycle (28 days). The first 28 days on treatment (cycle 1) was defined as DLT period. Patients with an objective response, stable disease, or potential clinical benefit continued the GT0918 treatment until they experienced one of following events of intolerable toxicities, disease progression, or withdrew consent.Eligible patients were ≥ 18 years old women with an Eastern Cooperative Oncology Group (ECOG) performance status of 0–1 and with histologically confirmed metastatic breast cancer . All patients had been progressing after either chemotherapy, hormonal or targeted therapy, or could not tolerate currently standard treatment. Additional eligibility criteria included measurable disease per the Response Evaluation Criteria in Solid Tumors (RECIST 1.1) and adequate hematologic, coagulation, renal, and liver function.Exclusion criteria included treatments of chemotherapy, radiotherapy, targeted therapy, endocrine therapy, and Chinese traditional medicine therapy in 4 weeks prior the enrollment. Patients were ineligible for enrollment if they had known or suspected central nervous system (CNS) metastases or had a history of seizure, significant cardiovascular disease.The safety assessments were performed in all patients who received at least one dose of GT0918. Safety was determined by assessment of adverse events (AEs), vital signs, physical examination, 12-lead electrocardiograms, echocardiography, and laboratory tests during the time periods of the first GT0918 dose until 30 days after the last dose. The severity of AEs and abnormal laboratory values were graded using NCI CTCAE version 4.03.A series of blood samples were collected from each patient for pharmacokinetic analysis at the following timings: PK after single dose: 0 (predose) and at 0.5, 1, 2, 3, 4, 5,6, 8, 12, 24, 48, 72, 96, 120, and 144 h postdose on Day 1, and PK after multiple dosing: 0 (predose) and at 0.5, 1, 2, 3, 4, 5,6, 8, 12, and 24 h postdose on Day 28. In addition, pretreatment samples were collected on days 3, 5, 7, 14, and 21 during cycle 1.Cmax), time of maximum observed plasma concentration (Tmax), area under the plasma concentration–time curve (AUC) from time 0 to time of last quantifiable concentration (AUC0-t) and AUC from time 0 to 24 h postdose (AUC0-24), terminal elimination half-life (T1⁄2), apparent total clearance of the drug (CL/F), and apparent volume of distribution (Vz/F).PK analysis for all parameters was performed using Phoenix WinNonlin  with standard noncompartmental analysis methods. Parameters analyzed included maximum observed plasma concentration . Most patients (94.4%) had an ECOG PS of 0, and 5.6% had an ECOG PS of 1 at baseline. Overall, 16.7% (3 of 18 patients) had HER2-positive disease, 16.7% (3 of 18 patients) were triple-negative, 77.8% (14 of 18 patients) had HR-positive disease, and 38.9% (7 of 18 patients) had AR-positive disease.18 patients in total were enrolled in the study: 3 patients in 100 mg, 500 mg group, respectively, and 4 patients in 200 mg, 300 mg, or 400 mg group, respectively. 14 patients were evaluable for DLT (1 experienced progressive disease and 3 withdrew from the study during DLT assessment period). No DLT was observed and MTD has not been reached. Patients complain fatigue and upset but did not reach DLT in 400 mg and 500 mg/day groups. Thus, GT0918 200 mg and 300 mg/day were selected for further testing.All patients were evaluable for safety analysis. Table One patient discontinued the treatment because of AST increase (during the first cycle) which was unlikely treatment-related with GT0918 and probably was related with the disease progression. A total of 5 (27.8%) patients experienced SAEs 0.1 (5.6%) patient died during this study; the primary cause of death for the patient was disease progression and was considered to be unrelated with the study drug. Patients complaining fatigue were more frequent in the 500 mg group.Tmax) was between 1.02 and 3.01 h for single oral dose and between 0.00 and 5.13 h for multiple doses. With a single dosing at 100 mg–500 mg  and maximum concentration (Cmax) were increased with dose proportionally after a single dose and multiple doses ranging from 100 to 500 mg. The ratio of AUC from 0 to 24 h postdose (AUC0-24) between day 28 after daily dosing and day 1 after single dosing was from 8.35 to 11.0, suggesting a drug accumulation after multiple dosing. Terminal half-life (T1/2) ranged from 25.1 to 97.4 h across the dose levels for single administration. Mean apparent clearance values (CL/F) ranged from 0.217 to 1.1 L/h for single administration and from 0.10 to 0.2 L/h for consecutive administration, suggesting that the elimination of GT0918 was slow.PKs were evaluated in 18 patients, and the PK parameters of GT0918 with single and consecutive administrations are shown in Tables  and 4. TIn the dose-escalation phase, 18 patients were exposed to study drug with doses ranging from 100 to 500 mg QD on a 28-day cycle. All patients progressed from more than two lines of therapies, and 83.3% (15/18) patients progressed three lines or more. There were 13 patients can evaluate efficacy, four patients obtained SD  in a total of 18 patients. The primary objectives were to characterize the PK properties of GT0918 and to assess the safety and tolerability of GT0918 in order to support further clinical studies in patients with breast cancer.Results from this clinical study in breast cancer patients proved GT0918 to be well tolerated at a maximum dose of 500 mg/day. And the pharmacokinetics of single- and multiple-dose GT0918 in women with breast cancer was found to be similar to the pharmacokinetics of GT0918 in men with prostate cancer . Oral abThis study demonstrated that GT0918 had an acceptable safety profile in patients with advanced breast cancer, with mild and moderate level of AEs. The most common drug-related adverse events (> 15%) were asthenia (72.2%), increased blood cholesterol (44.4%), increased blood triglycerides (33.3%), anemia (33.3%), decreased appetite (22.2%), decreased white blood cell count (22.2%), increased aspartate aminotransferase (22.2%), increased low-density lipoprotein (22.2%), increased alanine aminotransferase (16.7%), weight lost (16.7%), nausea (16.7%), and constipation (16.7%). The adverse events (≥ 3 Grade) were fatigue (11.1%), increased aspartate aminotransferase (5.6%), increased alanine aminotransferase (5.6%), and decreased neutrophil count (5.6%).This incidence of fatigue is similar to the reported fatigue AE in other studies of AR inhibitors. Importantly, no seizures were observed yet at any dose level.Preliminary signs of clinical activity of GT0918 in patients with AR + mBC were observed. CBR in 16 weeks was 23.1% (3/13); among 7 AR + patients, 6 can evaluate efficacy; among AR + patients, CBR in 16 weeks was 33.3% (2/6) Fig. . Out of Cmax at 200 mg, 300 mg, and 400 mg dosage were 7.4 μg/mL, 15.1 μg/mL, and 12.7 μg/mL, respectively (Table According to the safety, tolerability, and pharmacodynamics analysis, GT0918 shows preliminary signs of clinical activity in AR + breast cancer, thereby warranting further evaluation. Because we did not see 16 weeks response in the 400 mg and 500 mg cohort, two longer duration treatment patients both in 200 mg group obtained 23.5 and 25 cycles and one of the patient had 4.5 cycles in 300 mg group Fig. . As the Based on these results, an expanded/phase Ib and Ic trials have been initiated in patients with AR-positive mBC, being progressed from multiple treatment lines.GT0918, the 2nd-generation AR antagonist, has potential antitumor effect in AR-positive breast cancer. GT0918 is well tolerated with a favorite PK profile and reveals a prospective antitumor activity in AR-positive mBC; the suitable dose of GT0918 was 500 mg QD; the presented results indicate a promising alternative treatment for AR-positive mBC patients in the future.