Porphyromonas gingivalis, a Gram-negative bacterium, is known to possess important pathogenic factors for periodontal disease. In this study, we investigated the anti-periodontitis effects of Magnolia kobus extract (MKE) and magnolin as a component of Magnolia kobus (MK) in murine macrophage RAW 264.7 cells stimulated with Porphyromonas gingivalis lipopolysaccharide (LPS). Effects of MKE and magnolin on the mechanism of RAW 264.7 cellular inflammation were determined by analyzing nitric oxide (NO) production and Western blot protein expression (n = 3). MKE/magnolin inhibited NO production without affecting cell survival. MKE/magnolin treatment inhibited LPS-induced pro-inflammatory cytokines, expression levels of matrix metalloproteinases , and protein levels of inflammatory mediators . MKE/magnolin also suppressed NF-κB activation by inhibiting the TLR4 signaling pathway. These findings suggest that MKE has a therapeutic effect on inflammatory periodontal disease caused by oral bacterium P. gingivalis and that magnolin is a major functional component in the anti-inflammatory effect of MKE.Periodontitis, a disease caused by inflammation of oral bacteria, contributes to the loss of alveolar bone and destruction of connective tissues.  Periodontitis is a common chronic inflammatory disease that includes gingival inflammatory response, connective tissue loss, and weakened alveolar bones that support roots of teeth, leading to tooth loss . In periA common, mild form of gingivitis causes irritation, redness, and swelling of the gum due to an inflammatory reaction. This continuative inflammatory response can cause irreversible periodontal diseases and structure changes such as the loss of teeth, which can be related to the development of chronic diseases such as cardiovascular disease, diabetes, metabolic abnormalities, and cancer ,6,7.Porphyromonas gingivalis, one of the representative periodontal disease pathogens in oral bacteria, can produce lipopolysaccharide (LPS) endotoxins that can stimulate the induction of inflammatory cytokines and contribute to the process of alveolar bone loss and connective tissue destruction [truction ,9,10. Intruction ,12. In ptruction ,14.Activation of macrophages can cause the release of extracellular matrix metalloproteinases (MMPs) responsible for periodontal tissue destruction and connective tissue damage . MMPs seMagnolia kobus (MK) is a flowering plant native to tropical America and the subtropics in Asia . Its parts such as bark, flowers, and fruits are used in traditional medicine to treat chronic rhinitis and colds [Magnoliae genus, a Streptococcus mutans-inhibiting effect of Magnolia grandiflora [Magnoliae cortex [Manoliae cortex and zea mays L complex extract have been reported [Magnolia, has various effects such as anti-cancer, anti-inflammatory, and antioxidant effects [P. gingivalis and its mechanism of action remains unclear. In order to investigate the anti-periodontitis effects of MK, macrophages were treated with MK extract (MKE) and its active compound, magnolin, and the inflammatory responses were evaluated . Primary antibodies against IL-1β was obtained from the Invitrogen Life Technologies . Anti-MMP-3, -8, -9, -13, mPGES-1, and TNF-α antibodies were purchased from Abcam Inc. . Anti-MMP-1, pTAK1, phospho-ERK, ERK, phospho-JNK, JNK, NF-κB pathway antibody sampler kit and β-actin were purchased from Cell Signaling Technology . Goat anti-Mouse lgG-HRP and Goat anti-Rabbit lgG-HRP were purchased from GenDEPOT .Magnolia kobus extract was supplied by NUON Co., Ltd., . MK was dried and extracted by aqueous water. The solution was filtered, concentrated, and dried to obtain MK extract. The magnolin content was determined at approximately 1.5% in MKE by quantitative analysis (Data not shown).The murine macrophage RAW 264.7 cells  were cultured in Dulbecco’s modified minimal essential medium  with 10% fetal bovine serum  and 1% penicillin–streptomycin  at 37 °C cell incubator humified atmosphere with 5% CO2. MKE and magnolin were dissolved in dimethyl sulfoxide . Samples dissolved in DMSO were diluted 1/1000 in the medium and treated with cells.A 3--2,5-diphenyl tetrazolium bromide  assay was performed to determine the cytotoxicity of MKE and magnolin to RAW 264.7 cells. Cells were treated with MKE  and magnolin  for 24 h. MTT solution was added to each well to a final concentration of 0.5 mg/mL, and then the plate was incubated at 37 °C for 2 h. After 2 h, the supernatant was removed, and 100 μL DMSO was added to each well to dissolve formazan crystals. The dissolved formazan was measured at a wavelength of 570 nm in a microplate plate reader .4 cells/well. After 24 h, cells were pretreated for 2 h with MKE and magnolin at concentrations that were confirmed to be nontoxic in a 24 h cell viability assay. RAW 264.7 cells were stimulated with lipopolysaccharide from Porphyromonas gingivalis  (100 ng/mL), as well as MKE  and magnolin  for 24 h, then the cell culture supernatants were harvested and briefly centrifuged. For NO concentration, 50 µL of cell culture supernatant was harvested and mixed with 100 µL of Griess reagent  according to the manufacturer’s protocol. The mixture was reacted for 10 min under dark conditions and absorbance was determined using a microplate reader at a wavelength of 425 nm.RAW 264.7 cells were seeded into a 48-well microplate at a density of 2.0 × 10After removing the culture media of macrophage RAW 264.7 cells, the cells were scraped with CelLytic buffer . The cell lysate was centrifuged at 13,000 rpm at 4 °C for 15 min. The protein concentration of the cell lysate supernatant was quantified by the Bradford assay , and all samples were diluted with CelLytic buffer to have the same concentration of protein (20 μg/20 μL). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-gel was transferred to an Immobilon-P membrane  and blocked with 5% skim milk for 30 min. After blocking, the membrane was incubated with specific primary antibodies at 4 °C overnight. Blots were washed three times with Tris-buffered saline with 0.5% Tween 20 (TBS-T) and then incubated with corresponding horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G for 1 h. ECL solution  was dispensed on the membrane, and protein bands were measured using LuminoGraph . Band images and intensities were quantified in the ImageJ program  and corrected by β-actin levels.p > 0.05) continued to be analyzed. Significance between the LPS-activated control group and vehicle control group was determined using the Student’s t-test. Significance between MKE/magnolin treatment group and LPS activated control group was determined using the Student’s t-test. Significant difference was considered at p < 0.05. All experiments were performed in triplicates.In this study, all data are expressed as mean ± standard deviation. All statistical analyses were performed using SPSS version 12.0.0 . Normality of the distribution of the results was verified using the Shapiro–Wilk test, and the homogeneity of variances using the Levene’s test. The normal data  compared to LPS-untreated cells (vehicle-treated control cells). On the other hand, MKE treatment inhibited NO production in a dose-dependent manner, with inhibition rates of 19.20 (±3.28)% and 59.43 (±1.03)% at 100 and 300 μg/mL, respectively (** p < 0.01). Magnolin at concentrations of 1.5 and 4.5 μg/mL also resulted in 21.26 (±3.90)% and 32.87 (±3.60)% reductions of NO production, respectively, compared to the control group  (** p < 0.01) (LPS-induced RAW 264.7 cells showed a significant increase ( < 0.01) . increases of inflammatory mediators such as microsomal prostaglandin E synthase-1 (mPGES-1), TNF-α, and interleukin (IL)-1β (** p < 0.01) (** p < 0.01).LPS-induced RAW 264.7 cells showed significant  (IL)-1β . MKE tre < 0.01) . In grou** p < 0.01) (** p < 0.01). These results indicate that magnolin might inhibit inflammatory mediators as a biologically active compound in MKE. MKE and magnolin did not decrease cell viability at the concentrations tested, indicating that their inhibitory effects on inflammatory mediator production were not related to their cytotoxicity.In groups treated with 0.45, 1.5, and 4.5 μg/mL magnolin, TNF-α expression levels were reduced by 9.95 (±1.38)%, 45.21 (±2.75)%, and 61.12 (±0.84)%, respectively, and IL-1β expression levels were decreased by 29.76 (±4.11)%, 34.62 (±1.68)%, and 26.73 (±4.99)%, respectively ( < 0.01) . Furtherp < 0.01) (** p < 0.01) MMP-1, -3, -8, -9, and -13 levels by about 70.18 (±4.55)%, 81.25 (±2.85)%, 66.28 (±2.37)%, 53.57 (±3.57)%, and 69.86 (±3.16)%, respectively  . Magnoliectively . This sup < 0.01). Treatment with MKE at 30, 100, and 300 μg/mL reduced TLR4 by 22.28 (±4.98)%, 46.61 (±2.89)%, and 60.80 (±1.82)%, respectively (** p < 0.01). Furthermore, in cells treated with 30, 100, and 300 μg/mL MKE, pp65/p65 ratios were inhibited by 39.95 (±0.22)%, 41.55 (±0.42)%, and 67.42 (±0.30)%, respectively, and pIκBα /IκBα ratios were inhibited by 35.74 (±0.76)%, 66.90 (±1.43)% and 69.76 (±1.11)%, respectively (** p < 0.01) (Expression level of toll-like receptor 4 (TLR4) and phosphorylation levels of p65 and IκBα were increased in LPS-induced RAW 264.7 cells (##  < 0.01) .p < 0.05, ** p < 0.01) (p < 0.01).At concentrations of 0.45, 1.5, and 4.5 µg/mL, magnolin reduced TLR4 protein expression levels by 10.63 (±5.60)%, 18.01 (±1.80)%, and 29.17(±0.70)%, respectively (*  < 0.01) . In addip < 0.01) phosphorylation levels of TAK1 and MAPK factors c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). MKE at 100 and 300 μg/mL inhibited  phosphorylation levels of pTAK1 by 13.64 (±5.05)% and 36.04 (±3.34)%, respectively. In cells treated with 30, 100, and 300 MKE μg/mL, decreases in the phosphorylation ratio of MAPK factors were by 43.23 (±0.72)%, 66.68 (±1.21)%, and 84.80 (±1.03)%, respectively, for ERK and 16.06 (±1.45)%, 27.00 (±0.71)%, and 54.85 (±0.15)%, respectively, for JNK (LPS-induced RAW 264.7 cells significantly increased (##  for JNK .** p < 0.01) reduced phosphorylation levels of pTAK1 by 70.06 (±1.77)%, 58.61 (±0.18)%, and 81.47 (±2.03)%, respectively. In groups treated with 0.45, 1.5, and 4.5 μg/mL magnolin, phosphorylation ratios of MAPK factors were decreased by 30.47 (±1.35)%, 57.85 (±0.74)%, and 50.05 (±0.90)%, respectively, for ERK and 36.50 (±0.51)%, 39.58 (±0.30)%, and 52.55 (±0.63)%, respectively, for JNK  to activate NF-κB and MAPK signaling pathways, resulting in the production of inflammatory mediators . It is kNF-κB is known as a multi-unit transcription factor that plays an important role in proinflammatory cytokine gene induction. In the present study, we confirmed that expression levels of inflammatory cytokines such as IL-1β, TNF-α, and mPGES-1 were increased as phosphorylation levels of NF- κB and MAPK factors increased in RAW 264.7 cells. TNF-α and IL-1β are known to be involved in the induction of bone resorption and inhibition of bone formation. High levels of TNF-α and IL-1β have been observed in diseased gum tissues ,43,44,45P. gingivalis-induced overexpression of TNF-α and IL-1β can additionally generate other inflammatory mediators such as MMPs [ as MMPs . Levels  as MMPs . Excessi as MMPs ,52,53. A as MMPs ,56,57,58 as MMPs ,60. ResuIn conclusion, our study confirmed the anti-inflammatory and MMP-expression-inhibiting effects of MKE on LPS-induced RAW 264.7 cells by inhibiting TLR4–TAK1-mediated activation of NF-κB and MAPK signaling pathways. Magnolin also demonstrated effects similar to MKE, demonstrating that magnolin could act as an active ingredient in MKE. The results of this study suggest that MKE can be used as a therapeutic agent for periodontitis through its anti-inflammatory and MMP-inhibiting effects, and revealed the inhibitory mechanism of MKE against oral-disease-causing microbial endotoxins. However, this study has limitations in that it used an in vitro model, and therefore, it is necessary to evaluate the anti-periodontitis effect of MKE in animal models. Additionally, the efficacy and safety of oral administration of MKE in patients with periodontitis will be evaluated.Through these additional studies, MKE can be developed as a botanical drug and health functional food for the prevention of periodontal disease, or the treatment of patients with periodontal diseases.


The advantages of using a pretargeted imaging approach are on the one hand the possibility to achieve a high signal-to-noise ratio and imaging contrast; on the other hand, the method allows the uncoupling of the biological half-life of antibodies from the physical half-life of short-lived radionuclides. A brain-penetrating antibody (mAb) specific for β-amyloid (Aβ) plaques was functionalized with TCO moieties for pretargeted labeling of Aβ plaques in vitro, ex vivo, and in vivo by a tritium-labeled Tz. The overall aim was to explore the applicability of mAbs for brain imaging, using a preclinical model system. In vitro clicked mAb–TCO–Tz was able to pass the blood–brain barrier of transgenic PS2APP mice and specifically visualize Aβ plaques ex vivo. Further experiments showed that click reactivity of the mAb–TCO construct in vivo persisted up to 3 days after injection by labeling Aβ plaques ex vivo after incubation of brain sections with the Tz in vitro. An attempted in vivo click reaction between injected mAb–TCO and Tz did not lead to significant labeling of Aβ plaques, most probably due to unfavorable in vivo properties of the used Tz and a long half-life of the mAb–TCO in the blood stream. This study clearly demonstrates that pretargeted imaging of CNS targets via antibody-based click chemistry is a viable approach. Further experiments are warranted to optimize the balance between stability and reactivity of all reactants, particularly the Tz.Bioorthogonal pretargeted imaging using the inverse-electron-demand Diels–Alder (IEDDA) reaction between a tetrazine (Tz) and a  The rapid decay of the label along with the slow pharmacokinetics of the antibody will not allow the selection of an appropriate imaging time point yielding a sufficient signal in brain tissue over a background signal in the bloodstream (limited imaging contrast). Consequently, antibodies would need to be labeled with longer-lived isotopes such as 89Zr (t1/2: 3.27 d) oxymethyl]benzoate (TCO–NHS)  was synt1H NMR  δ 8.12 , 7.51 , 5.68 , 5.53 , 4.57 , 3.69 , 2.91 , 2.38 , 2.24 , 2.07 , 1.84 , 1.54 , and 1.23 . LC-MS (ESI) m/z C20H23NO5 requires: 358.2 ([M + H]+).A colorless solid was isolated in a yield of 21% and purity of 99%. 3N in heptane, 40 g column, 0% to 100% acetone in heptane in 20 min) to yield 86 mg (19%) of the desired compound Tz–biotin , 1-hydroxy-7-azabenzotriazole , and 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride  were dissolved in dry DMF (6 mL) under an argon atmosphere to give a colorless solution. Triethylamine  was added and the mixture was stirred at 22 °C for 1 h. Then, 6-(6-(pyridine-2-yl)-1,2,4,5-tetrazin-3-yl)pyridine-3-amine was added and stirring was continued for 47 h at 50 °C. All volatiles were evaporated and the crude material was purified by flash chromatography  δ 10.56 , 9.05 , 8.94 , 8.62 , 8.59 , 8.44 , 8.16 , 7.73 , 6.45 , 6.37 , 4.29–4.34 , 4.12–4.17 , 3.07–3.20 , 3.05 , 2.84 , 2.52–2.61 , 2.39–2.48 , and 1.35–1.71 . LC-MS m/z C22H23N9O2S requires: 478.1 ([M + H]+), 500.3 ([M + Na]+).2, 188 mmol hydrazine monohydrate, and 2 mL (38 mmol) acetonitrile were added to give a purple-colored solution. The tube was sealed, and the reaction solution was stirred for 16 h at 80 °C. The reaction solution was cooled to 22 °C and transferred into a 250 mL three-neck round bottom flask. An amount of 25 mL of a 3.5 M sodium nitrate solution was added slowly with ice bath cooling, followed by dropwise addition of 1 M hydrogen chloride (75 mL) until no gas evolution was observed and a pH of 3–4 was reached. The mixture was extracted 3× with ethyl acetate, the combined organic phase was separated, dried over sodium sulfate, and concentrated in vacuo. The crude residue was purified using silica-based flash chromatography to yield 38%  of the desired phenol-precursor as an orange solid in a purity of 98%.To a 25 mL sealable tube, equipped with a stir bar, 452 mg (3.8 mmol) of 4-hydroxybenzonitrile, 1.9 mmol Zn(OTf)1H NMR  δ 8.52 , 7.03 , 5.21 , and 3.07 . LC-MS (ESI) m/z C10H8N4O requires: 189.4 ([M + H]+).3H]Methyl nosylate , cesium carbonate , and 333 µg (1.77 µmol) of the phenol-precursor were dissolved in 400 μL tetrahydrofuran. The solution was stirred at 50 °C for 5 h. The reaction mixture was diluted with 1 mL dichloromethane and passed through a strong anion exchange (SAX) solid phase extraction (SPE) cartridge to remove the sulfonic acid by-product [3H]methyl nosylate, since Tz did not ionize in mass spectrometric analysis and therefore no isotope pattern could be determined.[-product . The soln = 9) with a degree of label 2.6 on average.Ten-fold molar excess of TCO–NHS (10 mM in DMSO) was added to the mAb . The solution was shaken orbitally for 2 h at 22 °C and purified using Slide-A-Lyzer Dialysis Cassettes  against PBS, pH 7.4. The buffer was changed after 30 min for 4 times in total to generate mAb–TCO in a yield of 83 ± 11%  and Tz  were mixed in a 1:1 molar ratio (2–10 μM) and incubated for 1 h at 22 °C, and purified by dialysis  against PBS (pH 7.4) to obtain mAb–TCO–Tz in a radiochemical purity of > 95%.In analogy, mAb–TCO was coupled with a biotinylated Tz derivative. For this click reaction, a 10-fold excess of Tz–biotin (5 mM in DMSO) was used to ensure complete conversion to mAb–TCO–Tz–biotin.For protein MS analysis of mAb–TCO, the protein concentration was adjusted to 0.5 mg/mL with 0.1 M Tris pH 7.8. The samples were chromatographed on a Waters nanoAcquity system  using an Agilent, Poroshell column  heated to 65 °C. Eluent A was 0.1% trifluoroacetic acid in water, and eluent B was 0.1% trifluoroacetic acid in acetonitrile. The following gradient for the separation was applied: initial: 10% B; 0.2 min: 10% B; 1 min: 20% B; 11 min: 60% B; 15 min: 90% B; 16 min: 90% B; 17 min: 10% B; and 20 min: 10% B, with a flow rate of 70 μL/min. The eluent was analyzed with a Waters LCT Premier XE mass spectrometer (Waters Corporation) using the electrospray method for ionization of the sample . The raw data were deconvoluted with the MaxEnt1 software (Waters Corporation) and displayed using standard software.Modified antibody constructs were subjected to SDS-PAGE and Western blot analysis. SDS-PAGE was performed using the XCell SureLock™ Mini-Cell Electrophoresis System  under reducing conditions. Samples were diluted in NuPAGE LDS Sample buffer  and NuPAGE Sample Reducing Agent , heated at 70 °C for 10 min, and 1–2 μg in 20 μL were separated using a Novex NuPAGE 4–12% Bis-Tris Gel (Thermo Fisher Scientific) with a 1× NuPAGe MOPS-SDS running buffer (Thermo Fisher Scientific). Separated proteins were transferred to a nitrocellulose membrane using the iBlot Gel Transfer System  in conjunction with Novex Mini iBlot Gel Transfer Stacks (Thermo Fisher Scientific). The membrane was blocked for 1 h at 22 °C with 5% TopBlock , 0.1% Tween-20 in 1× PBS pH 7.6. mAb–TCO was detected using a goat-anti-human-horseradish peroxidase (HAP) coupled antibody  and mAb–TCO–Tz–biotin was detected using a Streptavidin-HPA . Membrane was rinsed twice in blocking buffer, washed 1 × 15 min followed by 2 × 5 min in blocking buffer, 1 × 5 min in TBS-T , and 1 × 5 min in TBS pH 7.6. The modified proteins were detected using Amersham ECL Western Blotting Detection Reagents  and developed by applying standard procedures using Amersham Hyperfilm high performance chemiluminescence film.Binding of fusion constructs to fibrillary Aβ was measured by an ELISA assay. Briefly, the 40 amino acid residue peptide Aβ(1–40)  was coated at 7 µg/mL in PBS onto Maxisorp plates  for 3 days at 37 °C to produce fibrillary Aβ then dried for 3 h at 22 °C. The plate was blocked with 1% Crotein C and 0.1% RSA in PBS (blocking buffer) for 1 h at 22 °C, then washed once with wash buffer. Fusion polypeptides or controls were added at concentrations up to 100 nM in blocking buffer and incubated at 4 °C for 16 h. After 4 wash steps, constructs were detected by addition of anti-human-IgG-HRP  at 1:10,000 dilution in blocking buffer , followed by 6 washes and incubation in TMB . Absorbance was read out at 450 nm after stopping color development with 1 M HCl.4 cells were incubated with a 1.5 pM to 10 nM dilution series of the antibody fusions in 100 µL RPMI/10% FCS for 1.5 h on ice. After 2 washes with RPMI/10% FCS, cells were incubated with goat-anti-human IgG coupled to Phycoerythrin  at a dilution of 1:600 in RPMI/10% FCS for 1.5 h on ice. Cells were washed again, re-suspended in RPMI/10% FCS, and phycoerythrin fluorescence was measured on a FACS-Canto instrument .Binding of fusion constructs to murine transferrin receptor was tested by FACS analysis on mouse BA/F3 pro-B cells. Cells were harvested by centrifugation and washed once with PBS, and 5 × 10All animal experiments were performed according to the Swiss federal regulations on animal protection and to the rules of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC).Adult male or female APP751 Swedish × PS2N141I transgenic (PS2APP)  mice on Animals were injected intravenously (IV) with 20 mg/kg mAb–TCO–Tz in PBS  and sacrificed 3 days and 12 days post-injection. The brain was dissected and hemisected along the midline. One hemisphere was used for cryosections and immediately frozen on powdered dry ice. The other hemisphere was dissected to obtain brain regions including the cerebellum, hippocampus, cortex, and the rest brain, used for beta scintillation counting. The brain tissue was stored at −80 °C.Animals were injected intravenously (IV) with 20 mg/kg mAb–TCO in PBS . After 1, 3, 6, 12 d post-injection, animals were sacrificed by decapitation. The brain was hemisected and frozen in powdered dry ice. The brain tissue was stored at -80 °C.Animals were injected IV with a single dose of 20 mg/kg mAb–TCO. At 24 h and 72 h post-injection of the antibody, the animals received a single dose IV injection of 55.5 MBq/kg Tz in PBS, pH 7.4 . At 2 h after the Tz injection, the animals were sacrificed by decapitation and plasma samples were collected. The brain was hemisected. One hemisphere was frozen on powdered dry ice, the other one was dissected, and brain regions including cerebellum, cortex, hippocampus, and rest brain were frozen on dry ice. The brain tissue was stored at -80 °C.Animals were deeply anaesthetized using 3–5% (vol %) isoflurane in an anesthesia box with the support gas, oxygen/air (2:1). Animals were sacrificed by decapitation. The brains were removed, rinsed in 0.9% NaCl solution, blotted dry, hemisected along the midsagittal plane, and immediately frozen on powdered dry ice for histology and/or autoradiography.Brains were cut sagittally on a cryostat . Sections (10 µm thickness) were thaw-mounted on HistoBond plus glass slides , air dried for at least 2 h, and stored at −80 °C until further use.Slides were defrosted, rehydrated with PBS , fixed with acetone  and blocked for 20 min at 22 °C .The mAb constructs, either applied in vitro or injected in vivo, were detected using a fluorescently labeled secondary antibody (goat-anti-human Alexa Fluor (AF) 488, AF 555, or AF 647, 15 µg/mL in 1% normal goat serum in PBS, 1 h, 22 °C, Thermo Fisher Scientific). The sections were counterstained with 1 μg/mL 4′,6-diamidino-2-phenylindole  in PBS for 3 min. Sections were thoroughly washed with PBS between each incubation step. Standard SudanBlack B  staining was used to quench lipofuscin autofluoresence. Microscopic, whole-slice images were acquired using the Metafer 4 slide scanner .Fixed and blocked brain sections from transgenic and wild-type animals were incubated with 1 μg/mL mAb–TCO–Tz antibody solution in 1% normal goat serum in PBS for 1 h at 22 °C or 4 °C for 16 h. After intensive washing with PBS, the sections were air- dried at 4 °C and used for autoradiography.2, 1.2 mM CaCl2, 1.2 mM Na2HPO4, 0.27 mM NaH2PO4, pH 7.4; Merck KGaA, Fluka, Darmstadt, Germany).For the in vitro and ex vivo on-slide click reaction, the brain sections of transgenic animals were defrosted, re-hydrated and blocked (no blocking step for ex vivo on-slide click reaction) as described above. In vitro staining with 1 μg/mL mAb–TCO or mAb as control in 1% normal goat serum in PBS, 4 °C for 16 h, was carried out. Slides were washed for 3 × 5 min with PBS followed by 3 × 5 min with a Ringer solution  in Ringer solution. Sections were washed 3 × 15 min with a Ringer solution and dipped briefly in distilled water at 4 °C. The slides were air-dried at 4 °C and used for autoradiography.Air-dried brain sections were exposed for 5–10 days on tritium-sensitive phosphor imaging plates . Plates were scanned using the Fujifilm BAS-5000 phosphorimager and analyzed using MCID Analysis software . Regions of interest were selected, and bound radioactivity was analyzed based on a standard  co-exposed with the brain sections. Bound radioactivity was expressed as ratios between the regions of interest in the transgenic animal divided by the bound radioactivity of the same regions in the wild-type control animal.Dissected brain regions were weighted and homogenized in ice-cold homogenization buffer  in 50 mM Tris/HCl, pH 7.4) using a Polytron PT1200  and/or ultrasound . A 100 µL aliquot was mixed with 3 mL UltimaGold scintillation cocktail  and incubated over night before beta counting in a TriCarb 2500 Beta Counter (Perkin Elmer Packard). Disintegrations per minute (DPM) were used to calculate concentrations of the radioactive tracer (fmol/mg) and %ID/g (percentage of injected dose per gram tissue weight).trans-cyclooctene modified mAb (mAb–TCO). The mAb–TCO–Tz antibody retained its functionality and specificities as demonstrated by in vitro experiments using PS2APP mouse brain sections, ELISA, FACS assays, radio-immunohistochemistry, and in vivo approaches. The radiolabeled Aβ-specific antibody crossed the blood-brain barrier and bound its target. One to three days after injection of mAb–TCO proved to be a suitable time window for in vivo pretargeting. The TCO groups remained reactive towards the tetrazine reactant since a specific radioactive labeling of Aβ plaques was evident in brain sections of PS2APP mice, using an ex vivo click reaction. However, the in vivo click reaction with intravenously injected Tz one and three days after injection of the TCO-modified antibody did not reveal a specific difference in binding intensity between the transgenic and WT animals. A radioactive signal was detected in brain tissue, but no click reaction was revealed by autoradiography. Further experiments are needed to optimize the multi-component pretargeting system, such as the TCO degree of labeling of the mAb, mode of conjugation of the TCO, concentration of Tz, and type of Tz. The right balance between stability and reactivity of both components can thus be observed for a successful in vivo pretargeting reaction. The presented study suggests that the application of PET imaging in drug development and for clinical diagnostics may be extended to targets currently considered undruggable and not approachable by a traditional small-molecule PET tracer.This study explored the suitability of an antibody-based pretargeting approach for in vivo imaging of targets in the central nervous system. As a first step, the Aβ-specific, brain shuttle version of mAb31  was successfully radiolabeled in vitro using the IEDDA reaction of a tritiated 1,2,4,5-tetrazine (Tz) group and a


As a result, the TVOC levels gradually declined in consecutive years due to the pandemic in India. The mean TVOC levels at all monitoring stations declined from 9 to 61% during the pandemic period as compared with the pre-pandemic period. In the current study, the T/B ratio values ranged from 2.16 (PG) to 26.38 (NL), which indicated that the major pollutant contributors were traffic and non-traffic sources during the pre-pandemic period. The present findings indicated that TVOC levels had positive but low correlations with SR, BP, RF, and WD, with correlation coefficients (r) of 0.034, 0.118, 0.012, and 0.007, respectively, whereas negative correlations were observed with AT and WS, with correlation coefficients (r) of −0.168 and −0.150, respectively. The lifetime cancer risk (LCR) value for benzene was reported to be higher in children, followed by females and males, for the pre-pandemic, pandemic, and post-pandemic periods. A nationwide scale-up of this study’s findings might be useful in formulating future air pollution reduction policies associated with a reduction in health risk factors. Furthermore, the present study provides baseline data for future studies on the impacts of anthropogenic activities on the air quality of a region.During the COVID-19 pandemic, governments in many countries worldwide, including India, imposed several restriction measures, including lockdowns, to prevent the spread of the infection. COVID-19 lockdowns led to a reduction in gaseous and particulate pollutants in ambient air. In the present study, we investigated the substantial changes in selected volatile organic compounds (VOCs) after the outbreak of the coronavirus pandemic and associations with health risk assessments in industrial areas. VOC data from 1 January 2019 to 31 December 2021 were collected from the Central Pollution Control Board (CPCB) website, to identify percentage changes in VOC levels before, during, and after COVID-19. The mean TVOC levels at all monitoring stations were 47.22 ± 30.15, 37.19 ± 37.19, and 32.81 ± 32.81 µg/m Coronavirus disease COVID-19) is a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and was first reported in Wuhan, China, in late December 2019 9 is a di. The Uni3 formation, with complex chemical mechanisms found in them [3 formation [Few studies have reported a relationship between air pollution and infectious disease transmission ,6. Sever in them ,27. The ormation .3 concentrations [Up to 60% of non-methane VOCs (NMVOCs) released into the atmosphere are BTEX ), and chtrations . During trations ,22,23,312), particulate matter (PM), and carbon monoxide (CO) levels in many different cities across the world [Changes in atmospheric pollutants during the COVID-19 lockdown periods have been widely investigated; researchers have reported significant reductions in nitrogen dioxide , the most toxic BTEX compound is benzene, which has been categorized as a Group 1 and class A human carcinogenic by the International Agency for Research on Cancer (IARC) and the United States Environmental Protection Agency (U.S. EPA), respectively . SeveralChanges in VOC levels due to particle and gaseous contaminants, in particular, have received more attention and several studies of various cities in India have revealed that the air quality improved significantly during the pandemic period ,24,25. NThe National Capital Territory of Delhi, India, has coordinates of 28.70° N and 77.10° E. It is situated on the Indo-Gangetic Plain of the northern region of India ,25. One Delhi is among the most polluted cities in India according to the index of global pollution . Delhi ihttps://app.cpcbccr.com/ccr/#/caaqm-dashboard-all/caaqm-landing, accessed on 5 January 2022). The measurements and technical specifications of the instruments can be found elsewhere [In the present study, hourly and daily data of volatile organic compounds, especially benzene, toluene, ethylene, and xylene (BTEX), were collected from the Central Pollution Control Board (CPCB) website  programs and detection limits of each BTEX compound through rigorous sampling, analysis, and calibration procedures .https://app.cpcbccr.com/ccr/#/caaqm-dashboard-all/caaqm-landing, accessed on 5 January 2023). The data were procured in three time periods before, during, and after COVID-19. To examine the relative and temporal changes in VOC levels in the ambient atmosphere, the time period between 1 January 2019 and 31 December 2019 represented the pre-pandemic period, the time period between 1 January 2020 and 31 December 2020 represented, the pandemic period, and the time period between 1 January 2021, and 31 December 2021, represented the post-pandemic period. All 10 industrial air quality monitoring stations in Delhi that were selected for this study, with their latitudes, longitudes, and population census data, are presented in 2), pressure (BP in kg/ms2), atmospheric temperature (AT in Celsius), rainfall (RF in mm), wind speed (WS in km/h), and wind direction  were observed on an hourly basis at all 10 monitoring sites.In the present study, data were procured from 1 January 2019 to 31 December 2021, to identify percentage changes in the VOC levels  can be performed to assess the nature and probability of different pollutants in a population based on acute and chronic exposure.The pollutants that cause major impacts on human health are considered hazardous. In this current study, VOCs such as BTEX are hazardous to human health and can cause cancer.3) represents the exposure concentration, defined as the number of TVOCs present per cubic meter; CA (µg/m3) = VOC, the average concentrations of benzene, toluene, ethylene, and xylene; ET (h/d) is the exposure time, the total time duration per day in which exposure to TVOCs takes place; EF (d/y) represents exposure frequency, defined as the number of exposures taking place in a day;An exposure assessment (EA) was performed to examine the duration and magnitude of the pollutants based on different parameters. In the present study, inhalation was the major route of exposure for the identified pollutants. We estimated the daily and annual readings of normal and acute exposure periods for different age groups, namely males (70 years), females (60 years), and children (36 years) . The val3) is the hazard quotient, the ratio of exposure to chemicals and the measure at which no defined results can occur; RfC is the reference concentration of inhalation toxicity, which refers to continuous exposure to the human population without any cancerous health risks; SF (kg d mg −1) represents the carcinogenic slope factor, defined as an upper bound, approximating a 95% confidence limit in the escalated cancer crisis from the lifetime exposure to a chemical [ED (y) represents the exposed length of working, the difference of the average age of exposure and the average age at the beginning; AT (h) is the average exposure time, during the carcinogenic assessment, the average lifetime (per capita life expectancy × 365 d/y × 24 h/d) was adopted, and during the non-carcinogenic assessment, the average period of exposure cycle (ED × 365 d/y × 24 h/d) was adopted; HQ  levels, from 2019 to 2021, in Delhi, India. At all monitoring stations, the mean TVOC levels were 47.22 ± 30.15, 37.19 ± 37.19, and 32.81 ± 32.81 µg/mectively . The res3 (BW), 408.91 µg/m3 (NL), and 467.30 µg/m3 (NL) for 2019, 2020, and 2021, respectively. The maximum TVOC levels at all stations varied from 23.40 µg/m3 (PG) to 347.27 µg/m3 (BW), from 75.06 µg/m3 (SON) to 408.91 µg/m3 (NL), and from 37.18 µg/m3 (PG) to 467.30 µg/m3 (NL) for 2019, 2020, and 2021, respectively. The maximum TVOC levels were 347.27 µg/m3 for benzene, toluene, eth-benzene, and mp-xylene, respectively, for 2019. The average concentration values for benzene at all monitoring stations varied from 1.26 (AL) to 5.43 (SP) µg/m3, whereas, for toluene, the average concentration values varied from 4.58 (PG) to 74.22 (MD) µg/m3. The trend of the mean benzene value was observed to be SP > OKH > MD > SON > WA > NL > PG > NG > AL; the trend of the mean toluene value was NL > MD > SON > WA > OKH > SP > AL > NG > PG.The BTEX concentrations for 2019–2021 at all selected monitoring regions are presented in 3, respectively, for 2020–2021. The standard deviation value was high, indicating large variations in emission sources at various monitoring stations. The average benzene values at the monitoring stations ranged from 1.47 (AL) to 98.42 (NL) µg/m3, whereas the average toluene values ranged from 2.39 (NG) to 145.22 (BW) µg/m3, respectively. The maximum mean values among all monitoring stations were reported at Narela (benzene) and Bawana (toluene) monitoring stations, which are hubs of industrial zones in Delhi. Most plastic industries in BW operated during the pandemic period due to their association with the production of sanitizing bottles. These industries might have contributed more benzene and toluene compound emissions. Considering all of the selected monitoring stations, the trend for the average benzene values was NL > NG > SP > WA > SON > OKH > MD > AL > PG > BW, and the trend for the average toluene values was BW > OKH > MD > WA > SP > SON > AL > PG > NL > NG.The mean values of benzene, toluene, eth-benzene, and mp-xylene at all monitoring stations were calculated to be 12.85 ± 9.42, 30.42 ± 19.06, 4.06 ± 7.06, and 8.60 ± 13.71 µg/m3 for benzene, toluene, eth-benzene, and mp-xylene, respectively, for 2021–2022. The average values for benzene at the monitoring stations ranged from 5.26 (SP) to 0.55 (PG), whereas the average values for toluene ranged between 164.91 (BW) and 0.91 (NG) µg/m3. Considering all of the selected monitoring stations, the trend of average benzene values was SP > OKH > MD > SON > NL > WA > AL NG > PG > PG, and the trend for average toluene values was BW > NL > OKH > SP > MD > AL > SON > WA PG > NG. The mean values for individual VOCs at all monitoring stations were 2.89 ± 2.66, 43.01 ± 22.26, 505 ± 5.01, and 6.23 ± 8.14 µg/mThe average TVOC values at all of the monitoring stations declined from 9 to 61% during the pandemic period compared with those of the pre-pandemic period. The highest decline was observed at the SON monitoring station (−61%) and the lowest decline was at the NG monitoring station (−9%); the reason could be that the SON monitoring station was observed to restrict measures during the pandemic period, which caused significant changes in TVOC values compared with the pre-pandemic period . HoweverThe average TVOC values continued to decline even during the post-pandemic period due to restricted measures in a few of the monitoring stations. The government declared restricted measures for the post-pandemic period, under which schools, colleges, cinemas, and gyms were to remain closed, shops dealing in non-essential items were opened only on an odd–even basis, and metro trains and buses in the city ran at 50% seating capacity.The decreases in TVOC levels varied from −77% (BW) to −22% (PG), whereas there were significant increases in TVOC levels with changes at 64%, 62%, and 11% at the NL, OKH, and SP monitoring stations. The highest increase in TVOC levels was reported at the NL monitoring station because the NL monitoring station is located near plastic industries for making shoe soles and other plastic goods, such as Rexine, adhesives, and other highly inflammable items, which could be a significant source of emissions during the reopening of industrial activities. Similarly, the OKH monitoring station witnessed a further increase in the amount of waste generated from domestic and industrial sectors, from BTEX pollutant sources that included plastics, paints, resins, rubber, adhesives, lubricants, and detergents .A study in Maharashtra reported that TVOC levels declined by 84% during the lockdown period as compared with those of the previous year . The aveIdentification and estimation of VOC emission sources can be assessed using diagnostic ratios. The toluene/benzene ratio can be used to evaluate the impact of traffic and non-traffic sources . The ratIn the current study, the T/B ratio values ranged from 2.16 (PG) to 26.38 (NL), indicating that the major VOC contributors were the traffic and non-traffic sources during the pre-pandemic period. The calculated T/B ratios at some monitoring stations, such as the WZ, SON, AL, MD, and NL monitoring stations, were reported to be 10.44, 11.39, 12.00, 16.49, and 26.38, respectively, indicating the activities of industries and factories were the primary causes of VOC emissions . During Correlations among VOC levels can help one to understand the source of origin of the different constituents. If the correlation between different pollutants is similar, it depicts that their source of origin might be the same. The correlation value was found by using the Pearson correlation coefficient for the mean VOC levels on a daily basis. These correlation data were classified into various categories depending on the coefficient value (from −1 to 1). A coefficient value from 0.8 to 1.0 indicated a very strong correlation, a coefficient value from 0.6 to 0.8 indicated a strong correlation, a coefficient value from 0.4 to 0.6 indicated a moderate correlation, a coefficient value from 0.2 to 0.4 indicated a weak correlation, and a coefficient value from 0 to 0.2 indicated irrelevant data . In the During the pandemic period, there was a strong positive correlation between the SP and AL monitoring stations (0.736) and between the SP and OKH monitoring stations (0.619). For the post-pandemic period, strong positive correlations were shown for the BW and WA (0.813), BW and NG (0.646), PG and OKH (0.725), SP and AL (0.654), OKH and SON (0.612), SON and SP (0.617), and WZ and NG (0.648) monitoring stations. Apart from this, various monitoring stations showed moderate correlations, such as WA and AL (0.572), and WA and SP (0.587) monitoring stations. p = 0.05) with solar radiation, pressure, atmospheric temperature, rainfall, wind speed, and wind direction. In the present study, we investigated the correlations between total VOC (TVOC) levels and meteorological parameters during the period from 2019 to 2021. Our results indicated that TVOC level had positive but low correlations with SR, BP, RF, and WD with correlation coefficients (r) of 0.034, 0.118, 0.012, and 0.007, respectively, whereas negative correlations were observed with AT and WS with correlation coefficients (r) of −0.168 and −0.150, respectively. These observations indicated that VOC levels were lower during high AT and WS, possibly due to photodegradation and wind dispersion, which played crucial roles in the VOC levels. Similarly, RF showed a strong negative correlation with BP (−0.992) and a lesser correlation with AT (−0.070). A similar study in Delhi reported variations in pollutant concentrations associated with meteorological parameters ,52,53.The hazard quotient (HQ) defines the ratio of the exposure concentration for the specific VOC species to an acute reference concentration (RfC) of non-carcinogenic compounds ,55. An H−5, 5.51 x 10−5, and 1.24 × 10−5, whereas the values for toluene were calculated as 1.5 × 10−4, 1.57 × 10−4, and 1.5 × 10−4, for 2019, 2020, and 2021, respectively. The LCR values for benzene at all monitoring stations varied from 5.41 × 10−6 to 2.32 × 10−5, from 1.24 × 10−6 to 4.22 × 10−4, and from 2.36 × 10−6 to 2.26 × 10−5 for 2019, 2020, and 2021, respectively. The LCR values for benzene at all monitoring stations were estimated as the lowest for AL and the highest for SP in 2019, while in 2020 they were the lowest for BW and the highest for NL, and in 2021 they were the lowest for PG and the highest for SP. The results indicated that LCR values were higher during the pandemic period than those in the pre- and post-pandemic periods. For benzene, some monitoring stations had LCR values that exceeded the standard LCR value as prescribed by the CPCB (1.0 × 10−6), such as values of 1.23 × 10−5, 1.78 × 10−5, 1.92 × 10−5, 1.93 × 10−5, 1.93 × 10−5, and 2.33 × 10−5 for NL, WZ, SON, MD, OKH, and SP, respectively, in 2019; 1.08 × 10−5, 1.27 × 10−5, 1.35 × 10−5, 1.35 × 10−5, and 1.99 × 10−5 for MD, OKH, SON, WZ, and SP, respectively, in 2020; 1.11 × 10−5, 1.28 × 10−5, 1.55 × 10−5, 1.69 × 10−5, 1.73 × 10−5, and 2.26 × 10−5 for WZ, NL, SON, MD, OKH, and SP, respectively, in 2021.For males, the total LCR values for benzene at all monitoring stations were calculated as 1.49 × 10−5, 6.42 × 10−5, and 1.44 × 10−5, whereas the values for toluene were calculated to be 1.82 × 10−4, 1.82 × 10−4, and 1.82 × 10−4 in 2019, 2020, and 2021, respectively. The LCR values for benzene at all of the monitoring stations varied from 6.31 × 10−6 to 2.71 × 10−5, from 1.45 × 10−6 to 4.92 × 10−4, and from 2.76 × 10−6 to 2.63 × 10−5 for 2019, 2020, and 2021, respectively. The LCR values for benzene, at all of the monitoring stations, were estimated as the lowest for AL and the highest for SP in 2019, while they were the lowest for BW and the highest for Narela in 2020, and the lowest for PG and the highest for SP in 2021. For females, the totals of LCR values for benzene at all monitoring stations were calculated to be 1.73 × 10−5, 1.43 × 10−5, 2.07 × 10−5, 2.24 × 10−5, 2.25 × 10−5, 2.25 × 10−5, and 2.71 × 10−5 for PG, NL, WZ, SON, MD, OKH, and SP, respectively, in 2019; 1.26 × 10−5, 1.48 × 10−5, 1.57 × 10−5, 1.58 × 10−5, 2.32 × 10−5. 5.69 × 10−5, and 4.92 × 10−4 for MD, OKH, SON, WZ, SP, NG, and NL, respectively, in 2020; 1.06 × 10−5, 1.29 × 10−5, 1.49 × 10−5, 1.81 × 10−5, 1.98 × 10−5, 2.02 × 10−5, and 2.63 × 10−5 for AL, WZ, NL, SON, MD, OKH, and SP, respectively, in 2021. For the pre-lockdown period, a value of LCR was established to be similar to 2.15 −5 and 2.05 −5 for male and female residents, respectively, in China, which showed discernibly higher carcinogenic risks for male and female residents [For benzene, some monitoring stations had LCR values that exceeded the standard LCR value as prescribed by the CPCB, such as values of 1.06 × 10esidents .−5, 1.07 × 10−4, and 2.41 × 10−5, whereas the values for toluene were calculated to be 3.05 × 10−4, 3.05 × 10−4, and 3.05 × 10−4, for 2019, 2020, and 2021, respectively. The LCR values for benzene for children ranged from 1.05 × 10−6 to 4.52 × 10−5, from 2.42 × 10−6 to 8.21 × 10−4, and from 4.59 × 10−6 to 4.39 × 10−5 in 2019, 2020, and 2021, respectively. The LCR values for benzene were estimated for all of the monitoring stations with the lowest LCR value for AL and the highest LCR value for SP in 2019, while in 2020, the lowest LCR value was for BW and the highest LCR value was for NL, and in 2021, the lowest LCR value was for PG and the highest LCR value was for SP. For benzene, some monitoring stations had LCR values that exceeded the standard LCR value as prescribed by the CPCB, such as in 2019; all stations exceeded the LCR value ranging from 1.05 × 10−5 for AL to 4.52 × 10−5 for SP; in 2020, the LCR values were 1.22 × 10−5, 2.10 × 10−5, 2.46 × 10−5, 2.61 × 10−5, 2.63 × 10−5, 3.86 × 10−5, 9.49 × 10−5, and 8.20 × 10−4 for AL, MD, OKH, SON, WZ, SP, NG, and NL, respectively; in 2021, the LCR values were 1.77 × 10−5, 2.16 × 10−5, 2.49 × 10−5, 3.01 × 10−5, 3.29 × 10−5, 3.36 × 10−5, and 4.39 × 10−5 for AL, WZ, NL, SON, MD, OKH, and SP, respectively. For all monitoring stations, the LCR values for benzene, for the pre-lockdown, lockdown, and post-lockdown periods, were higher than the authorized value (1 × 10−6), except during the lockdown period, which is a guideline limit value in some circumstances [For children, the total LCR values for benzene at all of the monitoring stations were calculated to be 2.89 × 10mstances .3 for 2019, 2020, and 2021, respectively. As a result, the level of TVOCs gradually deteriorated over consecutive years due to the pandemic. During the lockdown, the major factors behind the crucial decrease in TVOC levels were complete and partial restrictions on industrial activities, transport, and marketplace openings. The average TVOC values at all the monitoring stations declined from 9 to 61% throughout the pandemic period in contrast to the pre-pandemic period. The change in TVOC levels was reported to be the highest in NL, because NL is renowned for plastic manufacturing industries that create shoe soles and other additional plastic goods, such as adhesive, Rexine, and other tremendously explosive items, which could be significant sources of emissions during the reopening of industrial activities. During 2020, the T/B ratio was estimated in the range of 0.03–13.47, indicating that the major contributors were traffic and non-traffic sources, whereas, during 2021, it ranged from 3.54 to 42.10, where high ratios stipulated non-traffic sources and much higher ratios indicated industries, factories, and petrol pumps as the predominant contributors. The correlation results revealed that TVOC levels had negative relationships with wind speed and atmospheric temperature, which might play a significant role in the dispersion of TVOCs. Comparatively, the lifetime cancer risk (LCR) value for males and females was estimated to be higher throughout the lockdown period than in the pre- and post-lockdown periods. The reason could be the longer exposure time to increase the production of plastic and resin manufacturing units during the pandemic period. Further, the present study aims to increase the scientific accuracy of research on VOCs. The quantifications of the selected volatile organic compounds (VOCs) were executed in various industrial areas in Delhi, India, from 2019 to 2021. The VOC data from 2019 to 2021 were acquired from the Central Pollution Control Board (CPCB) website, with reference to the pre-pandemic, pandemic, and post-pandemic periods. Using statistical analysis, the current study concluded that anthropogenic activities were considerable sources of emission for VOCs in industrial areas. At all monitoring stations, the mean VOC levels were 47.22 ± 30.15, 37.19 ± 37.19, and 32.81 ± 32.81 µg/m


Botryris cinerea. The combined bio-fungicide achieved an inhibition rate of 100% for mycelial growth using ε-PL at 500 μg/mL + wuyiencin at 50 μg/mL and for spore germination using ε-PL at 200 μg/mL + wuyiencin at 80 μg/mL in vitro. This synergistic spore and mycelia-damaging effect of the combination was confirmed using scanning electron microscopy. In vivo assays with combined bio-fungicide (1500 μg/mL ε-PL + 60 μg/mL wuyiencin) on detached leaves showed depressed growth and development of the spores of B. cinerea. The synergistic effect was further tested in combinations of ε-PL with wuyiencin by measuring the fractional inhibition concentration index (FICI) value below 0.5. Moreover, ε-PL and wuyiencin inoculation before B. cinerea infection significantly increased the superoxide dismutase, peroxidase, catalase, and phenylalanine ammonia-lyase activities, which suggested their involvement in tomato defense responses to disease to minimize damage to B. cinerea. These findings revealed that a combined bio-fungicide comprising ε-PL and wuyiencin had a good prospect for controlling plant fungal disease.This study mainly evaluated the broad-spectrum fungicidal activity of ε-poly L lysine (ε-PL) against 12 pathogenic fungi. We further demonstrated synergistic antifungal activity of ε-PL combined with wuyiencin against  Worldwide, crops are severely damaged by plant pathogens, causing reductions in the quantity and quality of crops and inducing significant economic losses, thereby posing a threat to global food security ,2. To maStreptomyces produces the natural antimicrobial, ε-Poly-L-lysine (ε-PL), a poly-cationic peptide comprising 25–30 L-lysine residues, which are connected via one amido bond between the ε-amino group and the α-carboxy group [Fermentation of xy group . ε-PL shxy group . Furtherxy group ,9. Mechaxy group ,11,12.ε-PL is usually used as a biological antimicrobial in the post-harvest preservation of fruit and vegetables. However, some pathogenic infections infect crops and vegetables not only in post-harvest storage but also in before-harvest storage. In fact, because of its antibacterial activity and non-toxicity, ε-PL has great potential for controlling plant diseases in cultivation as an effective and environmentally safe biocontrol strategy.Tobacco Mosaic Virus (TMV) RNA accumulation and induced the host defense response, suggesting ε-PL as a natural microbial to treat plant virus diseases [Alternaria alternata disease progression by reducing germ tube elongation and spore germination and downregulating the expression of crucial fungal development-related genes [Botryris cinerea) on a variety of fruit and vegetables was effectively inhibited by ε-PL [Streptomyces albulus CK-15 and is widely used as an antifungal agent to control various fungal diseases of vegetables and field crops in agriculture [Streptomyces to inhibit plant diseases.Treatment with ε-PL in tobacco BY-2 protoplasts significantly downregulated diseases . In addied genes . In addi by ε-PL ,16. Takeiculture . HoweverB. cinerea by suppressing mycelial growth and spore germination in vitro and in vivo; it induced plant disease resistance, as demonstrated by defense-related enzyme activity assays.Herein, we aimed to test the antifungal activity of ε-PL combined with wuyiencin as bio-fungicides against 12 species of pathogenic fungi. We revealed that ε-PL combined with wuyiencin had synergistic effects against Streptomyces albulus CK-15, which was provided by the China General Microbiological Culture Collection Centre (CGMCC no. 0703), was used to prepare ε-PL. Briefly, Sephadex G-25 column chromatography was used to purify ε-PL from S. albulus CK-15 fermentation broth. Then, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) were used to characterize the purified ε-PL. The ε-PL preparation was over 99% pure and contained approximately 30 residues. The crude powder of wuyiencin was extracted from the fermentation broth. Wuyiencin was separated and purified by Sephadex G-25 and ODS column chromatography and semipreparative high-performance liquid chromatography. The wuyiencin preparation was over 95% pure.Physalospora piricola, Rhizopus stolonifer, Botrytis cinerea, Fulvia fulva, Sclerotinia sclerotiorum, Gibberella sanbinetti, Colletotrichum lagenarium, Phytophthora infestans, Valsa mali, Alteraria alternata, Helminthosporium maydis, and Phomopsis asparagi, which were stored in the Institute of Plant Protection, Chinese Academy of Agriculture Science, Beijing, China. These fungi were stored at 4 °C on potato dextrose agar (PDA) medium. Prior to the antagonist experiments, the fungi were activated at 25 °C for 5–7 days to determine their mycelial growth rates. Tomato plants were grown in a 1:1 mixture of nutritional soil and sand, by volume, in 20 cm-diameter plastic pots and incubated under greenhouse conditions at 25 °C, 90% relative humidity, and a 12 h light and 12 h dark photoperiod.A total of 12 pathogenic fungi were used: 50) values of the agent toward the pathogens were calculated according to the linear regression of the colony diameters for log10-transformed standard concentrations, repeated 3 times.The antifungal activity of ε-PL toward the 12 pathogens listed in B. cinerea mycelial growth were assessed using the radial growth test on PDA. The ε-PL solution at 5 different concentrations  and wuyiencin solution at 3 different concentrations  were tested. Different solutions were mixed with PDA medium, and medium lacking both ε-PL and wuyiencin solution was used as a control. Mycelial growth was assayed as in B. cinerea inoculation was replicated three times, and the inhibition rate was calculated as in The synergistic effects of ε-PL combined with wuyiencin on B. cinerea mycelia sampled from the edge of 5 day-old colonies were added to plates lined with cellophane containing PDA medium with ε-PL at 500 μg/mL, wuyiencin at 25 μg/mL, and ε-PL at 500 μg/mL + wuyiencin at 25 μg/mL, respectively. Plates without any treatment served as controls. The plates were incubated at 25 °C in the dark for 72 h. Thereafter, we observed the morphology of the top layer of the fungus under light microscopy . The experiment was carried out independently three times.B. cinerea was cultured at 25 °C in an incubator for 7 days until the fungus had grown to completely fill the dish. We made a spore suspension, which was subjected to filtration to achieve a concentration of 50–60 conidia per microscopic field (magnification = 10×). ε-PL was prepared at 50, 100, 200, 300, 400, and 500 μg/mL. Wuyiencin was diluted to 10, 20, 40, 60, 80, and 120 μg/mL. In addition, combination treatments were as follows: ε-PL 200 μg/mL + wuyiencin 10 μg/mL, ε-PL 200 μg/mL + wuyiencin 20 μg/mL, ε-PL 200 μg/mL + wuyiencin 40 μg/mL, ε-PL 200 μg/mL + wuyiencin 60 μg/mL, ε-PL 200 μg/mL + wuyiencin 80 μg/mL, and ε-PL 200 μg/mL + wuyiencin 120 μg/mL. A total of five milliliters of the B. cinerea spore suspension was added to the different concentrations of ε-PL, wuyiencin, and their combinations. Then, five milliliters of the B. cinerea spore suspension were added to distilled water as a control, and each treatment was repeated three times. Samples (20 μL) of the different treatments were cultured on slides with absorbent paper moistened with distilled water in a 24 °C constant temperature incubator, and the spore germination of the samples was observed every day until spore germination was over 90%. The spore germination rate was calculated for ≥200 spores each time. The experiment was performed three times.To evaluate the effect of ε-PL, wuyiencin, and their combination on spore germination, B. cinerea, and cultured in an incubator with 95% humidity and a 12 h light and 12 h dark photoperiod with daily water spraying to retain moisture. After 3 days, the diameter of the leaf lesion formed around each inoculated mycelial plug was measured, and the percent inhibition of each treatment was calculated. A total of 20 leaves were inoculated with each concentration. The assays were carried out three times independently.Tomato seeds were grown under the conditions of a 12 h light–dark cycle at 25 °C. When the tomato seedlings grew to the 5–6 leaf stage, leaves with consistent growth were selected, washed with sterile water to remove surface impurities, and the petiole was covered with cotton to retain moisture. We sprayed the leaves with various ε-PL solutions: 1500, 2000, 2500, and 3000 μg/mL; wuyiencin solution (60 μg/mL); and various combinations: wuyiencin 60 μg/Ml + ε-PL 1500 μg/mL, wuyiencin 60 μg/mL + ε-PL 2000 μg/mL, wuyiencin 60 μg/mL + ε-PL 2500 μg/mL and wuyiencin 60 μg/mL + ε-PL 3000 μg/mL. Control leaves were sprayed using distilled water. Then, 24 h later, the leaves were sprayed with a 2% glucose solution, inoculated with Percent inhibition (PI) = (dc − dt)/dc × 100%, where dc represents the diameter of the control (untreated) colony and dt represents the diameter of the treated colonies.B. cinerea-inoculated tomato leaves were sprayed with distilled water (control), ε-PL at 1500 μg/mL, wuyiencin at 60 μg/mL, and ε-PL at 1500 μg/mL + wuyiencin at 60 μg/mL), respectively. The leaves were collected at 72 h post-inoculation and fixed using glutaraldehyde following the standard protocol [B. cinerea morphology and ultrastructure.protocol . Leaf saB.cinerea. The concentration of B.cinerea spores was adjusted to 1.0 × 104 CFU/mL by hemocytometer. The concentration of wuyiencin was diluted to 0–80 μg/mL and ε-PL to 0–100 μg/mL in PDB medium. Each well added 100 μL of the diluted drug concentrations and 100 μL of conidia . The wells of 100 μL conidia + 100 μL media and 100 μL sterile water + 100 μL media were set up as controls. The plate was scored at 23 °C for 72 h. Optical density readings were performed at a wavelength of 600 nm after 72 h and were preceded with an automated 120 s shaking to ensure uniform distribution of well contents. The assays were carried out three times independently.The antimicrobial combinations of wuyiencin and ε-PL were determined using the method of Itamar Shalit et al.  with sliε-PL and MICwuyiencin indicate the minimum inhibitory concentrations of ε-PL and wuyiencin when used alone.To assess possible synergistic activity, the fractional inhibitory concentration. FICI was calculated as follows:FICI values were interpreted as follows: FICI < 0.5, synergistic; 0.5 < FICI < 1, synergistic to additive; 1 < FICI < 4, indifferent; FICI > 4, antagonistic.B. cinerea spores inoculation. We compared tomato plants after treatment using distilled water (control), wuyiencin at 60 μg/mL, ε-PL at 1500 μg/mL, and wuyiencin at 60 µg/mL + ε-Pl at 1500 µg/mL. The leaves were collected at 0, 6, 12, 24, and 48 h post-treatment. One gram of leaf sample was ground in liquid nitrogen, homogenized in 5 mL of 0.02 mol/L phosphate buffer (pH 6.8) at 0–4 °C, and centrifuged at 8000× g at 0–4 °C for 20 min. The supernatant comprising the enzyme solution was stored at 4 °C for subsequent analyses.To assess the responses of the plant antioxidant enzyme system, different bio-fungicides and distilled water were applied 3 days prior to 240).CAT activity was determined using the method of García et al. . with sl2 and 0.3 mL of 0.02 mmol/L riboflavin; pH 7.8).SOD activity was determined using the method of Pokora et al. , with slPOD activity was determined using the method described by Wang et al. , with sl290).PAL activity was determined using the method described by Li et al. , with slp < 0.05 was considered to indicate a significant difference.Analysis of variance (ANOVA) in SPSS 13.0  was used for the statistical analyses of the data. Fisher’s protected least significant difference (LSD) tests were used to compare the significant differences between the means. Physalospora piricola, Rhizopus stolonifer, Botrytis cinerea, Fulvia fulva, Sclerotinia sclerotiorum, Gibberella sanbinetti, Colletotrichum lagenarium, Phytophthora infestans, Valsa mali, Alteraria alternata, Helminthosporium maydis, Phomopsis asparagi) were used to estimate ε-PL’s antifungal activity. ε-PL displayed an obvious inhibitory effect on the mycelia growth of various pathogenic fungi in a concentration-dependent manner   .B. cinerea significantly; therefore, we tested the inhibitory effect of ε-PL combined with wuyiencin. The inhibitory effect increased as the concentrations of ε-PL and wuyiencin increased. The inhibition rate was markedly higher following the combined treatment compared with ε-PL treatment alone  were selected to further observe the effects of treatment on nsparent a. The mynsparent c. The mynsparent b. The mynsparent d.B. cinerea, and the inhibition rate increased with increasing concentration. When ε-PL was used alone, the conidial inhibition rate reached 58.82% at 200 μg/mL, and when wuyiencin was used alone, the conidial inhibition rate reached 76.47% at 60 μg/mL. When 60 μg/mL wuyiencin and 200 μg/mL ε-PL were combined, the conidial inhibition rate reached 100%  showed changes in the quantity, morphology, and spore production of mycelial (Compared with the control group (treated with distilled water), mycelial . Treatmemycelial d, followmycelial b,c.The mycelia in the control group were well stretched, smooth, and full; uniform in thickness; smooth in line; and tapered at the growth point e. The myB.cinerea was 0.46 < 0.5, showing synergistic effect, due to the fact that synergy is attributed to any interaction with the FIC below 0.5. A promising combination of ε-PL and wuyiencin was considered to be used as biological fungicide products because of their powerful synergy against B.cinerea.The FICI method was chosen to determine the combined action of ε-PL and wuyiencin, since it is widely used in synergy studies. The FICI values obtained for each test isolate at 72 h are shown in Considering the very efficient inhibition by the combined treatment (60 μg/mL wuyiencin + 1500 μg/mL ε-PL), we speculated the addition of wuyiencin would activate systemic resistance-related enzymes ; therefore, the activities of these antioxidant enzyme activity were examined.The CAT activity increased initially, peaked at 12 h, and decreased in the infected leaves in the control and treatment groups. The CAT activity was highest in combined treatment group, being 2.58 times higher than that in the control group at 24 h a.2O2 production by the increased SOD activity in the combined treatment group.The same trend occurred for POD activity in the control group and single 60 µg/mL wuyiencin treatment, both of which showed gradually increased activity. In the single ε-PL and combined treatment (1500 µg/mL ε-PL + 60 µg/mL wuyiencin) the POD activity showed a similar gradual increase in all treatments up to 12 h, peaking at 12 h, and then declining. In the combined treatment group, POD activity was highest at 12 h, being 3.66 times the activity in the control group b. This iThe treatments resulted in different increases in SOD activity after 0 h compared with that in the control. In the control and treatment groups, the SOD activity in the infected leaves increased rapidly within 12 h and then decreased. At 24 h, the SOD activity was slightly higher in the ε-PL and wuyiencin single treatment groups compared with those in the control group. Interestingly, in the combined treatment group, the SOD activity in the infected leaves increased suddenly and reached a maximum during 24–48 h. The SOD activity was significantly higher than that in the control group c.B. cinerea  treatment could further increase the PAL activity in plants, thus making tomato plants more resistant to  cinerea d.Sclerotinia scleroriorum, B. cinerea, and Alternaria alternate [Botrytis cinerea and Alternaria alternata by ε-PL  when the concentration of ε-PL was 2000 µg/mL. Thus, we concluded that ε-PL as a single agent could inhibit infection by plant fungal pathogens.ε-PL has been used widely and is recognized as a safe food preservative . ε-PL halternate ,26,27 wh by ε-PL . It is wlternata . It is qB. cinerea compared with treatment using either agent alone [S. albulus CK-15 secondary metabolite wuyiencin is used widely in agriculture to control fungal diseases, including tomato gray mold, strawberry powdery mildew, and cucumber downy mildew [Sclerotinia sclerotiorum growth and infection-related genes [B. cinerea. The inhibition of B. cinerea mycelial growth using ε-PL at 500 μg/mL and wuyiencin at 50 μg/mL alone were 71.15% and 88.10%, respectively. Combined treatment using ε-PL concentrations ranging from 200 to 500 μg/mL and wuyiencin at 50 μg/mL resulted in the inhibition rate ranging from 95.21% to 100%  suppressed the normal growth and development of B. cinerea spores. Moreover, the combination bio-fungicide induced B. cinerea resistance in tomatoes. The combined bio-fungicide comprising ε-PL and wuyiencin could be developed for use in crop protection. However, whether the combined treatment can control B. cinerea infection of tomato plants requires field tests. There were also many aspects for further study, including compatibility of the bio-fungicides, the feasibility of the industrial scale-up, improvement of the rain fastness, evaluation of possible phytotoxicity issues, and efficacy tests using pathosystems closer to real agricultural practice starting from experiments under greenhouse conditions.In conclusion, ε-PL had broad-spectrum fungicidal activity against various pathogenic fungi, reaching 100% for some fungi. This combined bio-fungicide (500 μg/mL ε-PL + 50 μg/mL wuyiencin) showed 100% inhibition of


Citrus × paradisi L.) extracts vary according to the extraction and hydrolysis methods used. The primary aim of this work was to maximize the yields of naringin and naringenin from various parts of fresh grapefruit fruits  using different extraction and hydrolysis methods. In addition, we aimed to evaluate the excipient—magnesium aluminometasilicate—and determine its influence on the qualitative composition of grapefruit extracts. Extracts were obtained by heat reflux extraction (HRE), ultrasound-assisted extraction with an ultrasonic homogenizer (UAE*), and ultrasound-assisted extraction with a bath (UAE). Ultrasound-assisted extraction using a bath (UAE) was modulated using acidic, thermal, and alkaline hydrolysis. The highest yield of naringin 8A (17.45 ± 0.872 mg/g) was obtained from an albedo sample under optimal conditions using ultrasound-assisted extraction; a high yield of naringenin 23-SHR (35.80 ± 1.79 µg/g) was produced using the heat reflux method from the segmental part. Meanwhile, ultrasonic combined with thermal hydrolysis significantly increased flavanone extraction from the albedo and segmental parts: naringin from sample 9-A (from 17.45 ± 0.872 mg/g to 25.05 ± 1.25 mg/g) and naringenin from sample 15-S (from 0 to 4.21 ± 0.55 µg/g). Additionally, magnesium aluminometasilicate demonstrated significant increases of naringenin from all treated grapefruit parts. To our knowledge, this is the first report of magnesium aluminometasilicate used as an adsorbent in flavanone extractions.While flavanones exist in a variety of chemical forms, their favorable health effects are most prominent in their free form—aglycones. Their concentrations in grapefruit ( Citrus × paradisi L. is an essential member of the Citrus genus in the Rutaceae family. Grapefruit is a delicious fruit used in the juice and food industry. However, around half of all fruit waste is discarded as a waste product, even though it includes a huge number of biologically active components with distinct health benefits. Therefore, numerous studies have been conducted to extract and identify biologically active components present in various citrus fruits to understand the relationship between their presence in diet and health benefits and reduced risk of disease [ disease ,2. Citru disease ,4.Previous research has found that flavonoid types in citrus fruits varied between species and cultivars , as did their contents and distribution in different fruit tissues  . This bioflavonoid can be obtained from naringin hydrolysis with naringinase when the glucose molecule is removed from the structure .d-glucosidase catalyzes the hydrolysis of prunin to glucose and naringenin [Naringin is hydrolyzed into rhamnose and prunin by the naringinase exhibiting α-l-rhamnosidase activity, and then β-ringenin . HydrolyConventional extraction methods are based on the use of chemical solvents and the heating of the sample to maximize the solubility of active substances and accelerate their transfer. The extraction yield depends on various factors, including the type, concentration, and amount of the solvent used; its treatment time; and temperature. According to Sarah Luisa Rodriguez De Luna, 2020, the extraction time is a parameter that needs to be optimized in each experiment . An incr2 extraction method or ultrasound-assisted extraction, are environmentally friendly, safe, and non-toxic and are promising alternatives to conventional extraction methods [Meanwhile, green and sustainable extraction techniques, such as supercritical CO methods ,23,24. Ultrasound-assisted extraction is widely used to extract biologically active compounds such as flavonoids, anthocyanins, and phenolic acids. According to Londono Londono et al. (2010), ultrasound-assisted extraction demonstrated a higher efficiency of flavonoid extraction from citrus peels in 60 min using methanol as a solvent than Soxhlet extraction with a more extensive solvent selection . The bioTransformation of flavanone glycosides to aglycone can be achieved using an extraction method combined with hydrolysis. Chemical (using bases and acids) or thermal (high temperatures) hydrolysis can increase aglycon content ,26.The previous research demonstrated that magnesium aluminometasilicate can work as an adsorbent to increase the solubility of bioflavonoids in extracts ,28.Indeed, this study aimed to maximize the yields of naringin and naringenin from various parts of fresh grapefruit fruits using different extraction and hydrolysis methods, as well as to evaluate how magnesium aluminometasilicate affects the qualitative content of grapefruit extracts. Citrus × paradisi L., variety Star Ruby, Italy, region—unknown) were collected from the local market in Mastaičiai, Kaunas district, Lithuania. The fruit were separated into the flavedo, albedo, and segmental parts, then chopped up with a food processor and frozen in a freezer (−18 ± 0.9 °C) until extraction. The parts of the fruit used in this study are shown in The grapefruit fruits  was used as excipient. Ethanol 96%  was used as a solvent for extraction. Magnesium aluminometasilicate (Neusilin®), and in the third experiment, ultrasound-assisted extraction with a bath was modulated with acidic, thermal, and alkaline hydrolysis. The operational conditions for each extraction method are shown in Three different extraction techniques were used. In the first experiment, extracts were obtained by heat reflux extraction , ultrasoHPLC-grade and analytical-grade reagents were used: hydrochloric acid, sodium hydroxide, methanol, acetonitrile ; standards of naringin and naringenin ; and ethanol (96%) .flavedo, albedo, or segmental parts) was mixed with the solvent (70% ethanol (v/v)) at 1:10 ratio in a 250 mL round-bottom flask and refluxed in a sand bath at 100 ± 2 °C for one hour. The mixture was left to cool down at room temperature, and then centrifuged with Sigma 3-18K centrifuge  for 10 min at RCP 1789× g, followed by the decantation of the supernatant. The extracts were filtered through PVDF syringe filters  prior to HPLC (high-performance liquid chromatography) analysis. All the extraction conditions are displayed in The frozen, raw material was defrosted at room temperature and allowed to warm up to 25 ± 2 °C. A sample of 1 ± 0.05 g of defrosted grapefruit  at a ratio of 1:10, and extraction time (10 or 30 min), with the processing temperature of 50 ± 2 °C or 70 ± 2 °C . The mixture was allowed to cool down at room temperature (20 ± 5 °C) and then centrifuged with Sigma 3-18K centrifuge  for 10 min at RCP 1789× g, followed by the decantation of the supernatant. Next, the extracts were filtered through PVDF syringe filters  before analyzing with HPLC (high-performance liquid chromatography). All the extraction conditions are displayed in Ultrasonic extraction was performed using an ultrasonic bath  (frequency of 38 kHz). A sample of 1 ± 0.05 g of defrosted albedo, flavedo, or segmental parts) were defrosted at room temperature before being mixed with the 70% ethanol solvent (v/v) at a 1:5 ratio in a 100 mL chemical beaker and extracted for 1, 3, and 5 min at a temperature of from 33.2 to 40 ± 2 °C. Next, the mixture was centrifuged with Sigma 3-18K centrifuge at room temperature (25 ± 5 °C)  for 10 min at RCF 3382× g, followed by the decantation of the supernatant. Then, the extracts were filtered through PVDF syringe filters  before analyzing with HPLC (high-performance liquid chromatography). All the extraction conditions are displayed in Ultrasound-assisted extraction was performed using an UP-250 ultrasonic homogenizer . Firstly, 5 ± 0.25 g of samples (v/v) or purification water was used as the solvent at a ratio of 1:10, and the excipient was added to the extraction mixture. The excipient concentration was 1% (w/v). Magnesium aluminometasilicate (g) was based on ethanol quantity. The samples were centrifuged for 10 min at RCF 1789× g, followed by decantation of the supernatant, and then the extracts were filtered through PVDF syringe filters (pore size 0.22 µm) before HPLC analysis. Sample preparation conditions are listed in Samples were modified with magnesium aluminometasilicate. The extracts were made under the same conditions as previously listed . Again, v/v) was used as a solvent (ratio of 1:10), and the pH was adjusted with 2 M HCl to pH 2.5. After that, the extracts were sonicated using the UAE method at 50 ± 2 °C for 20 min. Next, the hydrolyzed extracts were allowed to cool down to 25 ± 2 °C and adjusted to pH 8 by adding an aqueous solution of 2 M NaOH while stirring. Finally, the neutralized extracts were centrifuged for 10 min at RCF 1789× g and filtered through PVDF syringe filters (pore size 0.22 µm) before HPLC analysis.The applied modified acidic hydrolysis method was based on Keun Young Min et al.’s 2014 studies . Firstlyflavedo, albedo, or segmental parts in 10 mL of 70% v/v ethanol solvent for a duration of 20 min at a temperature of 50 ± 2 °C . Thermal hydrolysis was completed by transferring the extract to a 250 mL round-bottom flask and refluxing in a sand bath at 100 ± 2 °C for 1 h. After cooling, the mixture was centrifuged for 10 min at 1789× g using a Sigma 3-18K centrifuge , followed by decantation of the supernatant. Before HPLC analysis, the extracts were filtered through PVDF syringe filters . The parameters of sample preparation are listed in Firstly, we used an ultrasonic bath (frequency of 38 kHz)  to macerate 1 ± 0.05 g of v/v) (ratio of 1:10). Next, the pH was adjusted with 2 M NaOH to pH 10 . Next, the extracts were sonicated using the UAE method at 50 ± 2 °C for 20 min . The parameters of sample preparation are listed in The applied modified alkaline hydrolysis method was based on Liuting Zhu’s 2020 studies. Firstly, 1 ± 0.05 g of raw material was macerated with 70% ethanol  using a hydro distillation technique. The procedure was as follows. Firstly, 44.5 ± 0.5 g of peels was placed in a round-bottom flask with 500 mL of distilled water and connected to a Clevenger’s distillation unit. Then, the essential oil was extracted by hydro distillation for 120 min. Next, the obtained essential oil, which was collected in a Clevenger’s receiver, was separated. Finally, the essential oil was stored in a glass bottle refrigerated at −4 °C until we determined the yield. Each extraction was performed three times under the same conditions. The yield of oil from the grapefruit peels’ flavedo and albedo parts (Y) obtained in each extraction was calculated by the formula:Essential oil was extracted from the grapefruit peels  was used in the study. In addition, a chromatographic column ACE C18 (250 mm × 4.6 mm) with a sorbent particle size of 5 μm was used to separate the biologically active compounds.The following are the procedure details of HPLC method. The tested compounds were separated using gradient elution. Then, 10 µL of each extract was injected and analyzed at 280 nm. Eluent A: acetonitrile; eluent B: water at a rate of 1 mL/min. Gradient elution: 0.0 min, 10% A; 5 min, 20% A; 25 min, 40% A; 30 min, 100% A; 35 min, 100% A; 36 min, 10% A. The temperature of the column was 25 °C. The peaks were identified by comparing their UV-vis spectra and retention times to those of authentic reference standards. The samples were analyzed twice. The chromatograms of naringenin and naringin standards are shown in 2 > 0.999, confirming the linearity of the concentration ranges.The quantification and validation followed the methodical revision of natural products presented by Wolfender (2009) . StandarThe method sensitivity was evaluated by determining the limit of detection (LOD) and quantitation (LOQ). LOD and LOQ were calculated as the concentrations that gave signal-to-noise ratios of 3 to 10, respectively.2 of the calibration curve , and the peak areas were used for quantification, A standard mixture of naringin and naringenin was used for intra-day and inter-day precision testing. The method precision was demonstrated by performing five replicate non-consecutive injections of the usual mix on the same day on four different days. The results are reported in terms of RSD. In this study, standards (naringin and naringenin) were analyzed, and their retention time and spectra were compared with the prepared extracts. The linearity was determined by estimating the correlation coefficient Ron curve  (naringip < 0.05. The data are presented as the mean and standard deviation (SD). Statistical analysis was performed with SPSS 20.0 . One-way ANOVA was used to analyze the differences between extractions. In addition, post hoc comparisons of the means were conducted according to Tukey’s HSD test. The means of the compared samples were considered significantly different when v/v) were used for the extraction. Some of the samples were modified with magnesium aluminometasilicate.The extracts from fresh fruit materials were obtained using the UAE, HER, and UAE* methods . Two difThe yield of naringin and naringenin was determined using HPLC-PDA.flavedo, albedo, and segmental) of fresh Citrus × paradisi L. fruit. The yields of naringin and naringenin obtained using different extraction methods are shown in Naringin was obtained from all extracts of different parts  as a solvent with ultrasonic time of 30 min at 50 ± 2 °C, resulting in 8-A (17.45 ± 0.87 mg/g). In contrast, the lowest amount of naringin was obtained from the segmental part using 50% ethanol (v/v) with ultrasonic time of 30 min at 50 ± 2°C—17-S (4.31 ± 0.96 mg/g) (p < 0.05). Meanwhile, naringenin was found only in albedo, and its highest concentration was detected using 70% ethanol (v/v) for 30 min at 50 ± 2 °C, 12-A (4.63 ± 0.23 µg/g).Using the UAE extraction method, the highest yield of naringin was obtained from the v/v)) was analyzed, it was discovered that 70% (v/v) ethanol produced better results in some cases. For example, samples taken from flavedo parts had increased naringin from 7-A (14.79 ± 0.73 mg/g) to 9-A (17.39 ± 1.10 mg/g), and the albedo part had increased naringenin from 7-A (3.36 ± 0.16 µg/g) to 9-A (4.57 ± 0.22 µg/g) (50% and 70% (v/v) ethanol, respectively) (p < 0.05).When the effect of ethanol concentration (50% or 70% (v/v) ethanol showed that an increase in the sonication time statistically significantly increased flavanone yield from the flavedo part, 14.79 ± 0.73 mg/g to 17.45 ± 0.87 mg/g and 3.36 ± 0.168 µg/g to 3.55 ± 0.17 µg/g . The differences between samples 7-A and 8-A  and segmental parts 23-SHR (35.80 ± 1.77 µg/g).When compared to the UAE method, the HRE extraction method produced a significant yield, with naringenin qualitatively doubled in extracts from the v/v) ethanol in a temperature range of 33.5 to 40 ± 2 °C; the highest amount of naringenin detected was from the sample 28-SUX2 (7.40 ± 0.37 µg/g).In the case of the UAE* method, the maximum yield was obtained when naringenin was released from the segmental part using 70%  (p > 0.05). However, given that the findings were not statistically significant, they are not included in Unfortunately, the supercritical COv/v) ethanol as a solvent (flavedo part (from 5.59 ± 0.279 mg/g to 6.25 ± 0.312 mg/g), doubled yield from the albedo part (from 17.39 ± 0.869 mg/g to 25.05 ±1.25 mg/g), and from the segmental part (from 5.26 ± 0.263 mg/g to 11.07 ± 0.55 mg/g)  (p < 0.05 when compared to extraction without hydrolysis). Statistically significant results are shown in The extracted sample was prepared using the UAE method with acidic, alkaline, and thermal hydrolysis and 70% ( solvent . Using U55 mg/g) . The higWenbin Chen et al. (2017) demonstrated that the complete hydrolysis of the flavonoid glycosides was achieved only by refluxing at 80 ± 2 °C . This teUsing excipients as adsorbent may improve the solubility of certain active substances in drugs that have poor water solubility ,32. Therflavedo (samples 21-FHR—2.92 ± 0.503 µg/g). There was also a statistically significant increase of naringenin yield in samples from segmental parts using the UAE method: 13-S (4.07 ± 0.203 µg/g); 14-S (5.11 ± 0.257 µg/g); 15-S (2.66 ± 0.133 µg/g); 16-S (2.83 ± 0.141 µg/g); 17-S (6.78 ± 0.34 µg/g); 18-S (3.75 ± 0.19 µg/g). The operational conditions for each extraction method are shown in The use of magnesium aluminometasilicate during extraction significantly affected the release of naringenin in all samples. There was a statistically significant improvement in naringenin self-efficacy in the HRE from the flavedo part, with a yield of 2.38 ± 0.119 µg/g. However, given that the findings were not statistically significant, they are not included in Using the UAE technique , naringenin was detected in samples from the v/v as a solvent, sonication time 30 min at 50 ± 2 C°) from the albedo part 8-A (17.45 ± 0.872 mg/g); meanwhile, the highest yield of naringenin was obtained by heat reflux extraction method from the segmental part with 70% of ethanol (v/v) 23-SHR (35.80 ± 1.79 µg/g). Significant results of naringenin yield in terms of ultrasound-assisted extraction using an ultrasonic homogenizer were obtained using extracts from the segmental part—in a short time (3 min), the quantity of naringenin increased up to 28-SUX2 (7.40 ± 0.37 µg/g) compared with UAE method ; 9-A (25.05 ± 1.25 mg/g). The acidic and thermal hydrolysis influenced the amount of aglycon naringenin from the segmental part and demonstrated better quantification when compared with extraction without hydrolysis, (0 µg/g–1.12 ± 0.056 µg/g–4.21 ± 0.21 µg/g)  .flavedo, albedo, and segmental parts, using 50% or 70% of ethanol (v/v). Meanwhile, when purified water was used as the solvent, naringenin was detected in a small amount from the flavedo part (2.38 ± 0.119 µg/g). Thus, we could achieve the same quantity of aglycone with a lower concentration of solvent (ethanol) when using additional adsorbent magnesium aluminometasilicate. However, the quantity of naringin was reduced by 15% in extracts from different parts of the fruits (The magnesium aluminometasilicate, which was used as an adsorbent to increase flavanone yields from different fruits parts, increased the amount of naringenin in all samples from the e fruits .After reviewing the studies, we found that flavanone solubility may be increased in the aqueous solvent or a lower concentration of ethanol by adding additional agents, such as cyclodextrin, to improve the release and stability of the active compounds.Citrus × paradisi L. albedo and segmental parts using the UAE and HRE methods, so these parts are a promising material for further research and can be used to develop novel pharmaceutical products.The majority of the active compounds were extracted from the


Objective: To explore the impact of artemisinin (ARS) on myocardial ischemia-reperfusion (I/R) injury and the underlying mechanism.Methods: Myocardial I/R rat model and cell model were used in this study. The cell viability, morphological changes, apoptosis, and oxidative stress were evaluated in cardiomyocytes H9c2 cells in vitro by using cell counting kit-8, microscope, flow cytometry, and commercial kits. High throughput sequencing is used to identify molecular targets of ARS on myocardial I/R injury, and then the gene-gene interaction network was constructed. MiR-29b-3p, hemicentin 1 (HMCN1), and apoptosis-related genes were tested by qRT-PCR and Western blotting. In the myocardial I/R rat model, echocardiography, (Triphenyl tetrazolium chloride) TTC staining, Hematoxylin-eosin (H&E) staining, Masson Trichrome staining, and TUNEL staining are applied to evaluate the protective effect of ARS on the myocardial injury.Results:In vitro, we demonstrated that ARS alleviated H2O2-induced myocardial I/R injury, manifested by increased H9c2 viability, decreased pathological changes, apoptosis, and oxidative stress biomarker ROS, LDH, and CK-MB. Then, sequencing analysis revealed that miR-29b-3p/HMCN1 was the target of ARS for myocardial I/R injury. Notably, rescue experiments indicated that ARS inhibited myocardial I/R injury through targeted regulation miR-29b-3p/HMCN1. In vivo, we confirmed that ARS reduced myocardial injury, fibrosis, and apoptosis via modulation of miR-29b-3p/HMCN1.Conclusion: This study demonstrated the functional role of the ARS/miR-29b-3p/HMCN1 axis in alleviating myocardial I/R injury, which provided a new direction for myocardial I/R injury therapy. Globally, the morbidity and mortality of cardiovascular diseases remain high . Most ofArtemisinin (ARS), a sesquiterpene endoperoxide, is mainly derived from Artemisia annua . ARS hasvia modulating the miR-29b-3p/HMCN1 axis.In this study, we aimed to investigate the impacts of ARS on myocardial I/R injury, and its underlying mechanism. We found that ARS could be deemed as a potential agent for managing myocardial I/R A total of 50 male Sprague Dawley (SD) rats (250–300 g) were obtained from Shanghai Animal Laboratory Centre. SD rats were housed in a specific pathogen free animal room at room temperature of 25°C and relative humidity of 50%–60% in the First Affiliated Hospital of Xiamen University. All the animal experiments were conducted according to the Principles of Laboratory Animal Care and the Use of Laboratory Animals. The ethics committee of The First Affiliated Hospital of Xiamen University approved the study. SD rats were randomly divided into five groups with 10 rats in each group, Control group, I/R group (Model group), ARS group, ARS + miR-29b-3p inhibiror, and ARS + miR-29b-3p inhibitor + si-HMCN1 group.The rats were first anesthetized with pentobarbital sodium (70 mg/kg). Then, the thoracotomy was performed to expose the heart. Next, the left coronary artery (LCA) was ligated and bound with 6/0 nylon monofilament for 30 min to induce ischemia. After occlusion for 30 min, the nylon monofilament was released, and reperfusion was performed for 2 h to obtain the I/R injury model. After 3 days, the treatment groups were intraperitoneally injected with artemisinin (10 mg/kg) or miR-29b-3p inhibitor (20 mg/kg) or si-HMCN1 (20 mg/kg), and model rats or control group were given normal saline for 10 consecutive days.2O2 or H2O2 plus ARS (Low (5 μM), Medium (20 μM), and High (40 μM) concentration) or miR-29b-3p inhibitor (20 nM) or si-HMCN1 (20 nM) for 12 h (The rat cardiomyocyte H9c2 cells (American Type Culture Collection) was cultured in DMEM in a favorable environment. To induce I/R injury and investigate the role of ARS on I/R, H9c2 cells were treated with 100 μM Hfor 12 h .The H9c2 were inoculated onto 96-well plates and treated accordingly. The CCK-8 solution  was added and then incubated for 2 h. Finally, the activity of each group of cells was determined at a 450 nm optical density.2O2, ARS, and H2O2 + ARS, respectively. After culturing for 24 h, the cell morphology was observed under a inverted phase contrast microscope .In short, H9c2 cells were treated with HFirst, H9c2 was suspended in ice PBS and then incubated in buffer solution with FITC-labeled Annexin-V. Next, PI was added and stored on ice in the dark. Finally, the samples were immediately tested on the Flow cytometer .Protein isolation was carried out using cell lysis buffer and their concentrations were measured. Proteins were separated by 15% SDS-PAGE and then transferred onto PVDF membranes . Blocked with 5% non-fat dry milk, the membranes were subjected to incubation with anti-Cleaved-caspase-3, anti-Cleaved-caspase-9, anti-HMCN1, and β-actin , followed by secondary antibody. Finally, ECL reagents were adopted to visualize the protein.© reagent (Invitrogen), TaqMan miRNA reverse transcription kit, and SYBR Green were adopted to extract sample RNA, synthesize cDNA and perform qRT-PCR, respectively. The primer sequences for this experiment were shown below: miR-29b-3p (NR_031837.1) sense, 5′-TAGCACCATTTGAAATCAGTGTT-3′ and antisense, 5′-CTCTACAGCTATATTGCCAGCCAC-3′; HMCN1 (XM_039090461.1) sense, 5′-GCCATCAGTGCCACCGAATA-3′, and antisense 5′-AGACATGCTAGGGGTAGGGG-3′; β-actin (V01217.1) sense, 5′-GCAAATGCTTCTAGGCGGAC-3′, and antisense, 5′-GCTCCCCACACCCAGTAGAA-3′.TRIZOLvia the CK-MB isoenzyme assay kit and LDH Cytotoxicity Assay Kit , respectively. Besides, the concentrations of interleukin (IL)-6 and TNF-ɑ were assessed using ELISA kits .To detect ROS-induced cell injury, the treated H9c2 were co-cultured with Dichloro-dihydro-fluorescein diacetate  and lactate dehydrogenase (LDH) were detected For the transcriptome libraries, the paired-end sequencing (125-bp reads) was executed. For QC, adapter sequences, the number of fuzzy bases (Ns), the 5′ and 3′ end low-quality sequences, and reads with quality scores below 20 were trimmed. After QC, clean reads were aligned to the reference genome (Rno6.0), compressed and sorted using SamTools, and counted with reference genome annotation (ENSEMBL) using FeatureCounts to obtain the original reads of the known genes. The DERNAs  < 0.05 and biological coefficient variation (BCV) = 0.1 were then identified by the “edgeR.”For the small RNA library, the single-end sequencing (50 bp) was executed. For raw sequencing reads, the quality was checked using FastQC. Briefly, adapter sequences and Ns were discarded. The average base quality (>15) was not less than 40%, and the length of the trimmed reads was between 17–35 nt. After QC, clean reads were aligned to the reference (Rno6.0), compressed and sorted using SamTools, and then counted with reference genome annotation (miRBase) using FeatureCounts to obtain the original reads of the known genes. “EdgeR” was used to identify the DE genes with BCV = 0.1 and FDR < 0.05.The intersection of DERNAs  of the two comparison groups were used to construct the network. LncBase v2, RNAInter, and miRWalk were adopted to predict the relationship between lncRNAs and miRNAs. MiRDB, DIANA microT_CDS, and TargetScan were adopted to predict the relationship between mRNAs and miRNAs. Then, the above predictions were integrated to establish a lncRNA-miRNA-mRNA network. Pearson correlation coefficients between DEmiRNAs and DEmRNAs were calculated to enhance the reliability.First, the rats were anesthetized with 3% isoflurane and placed supine on a rat plate. Then, the acupuncture needles were inserted into the limbs and connected to the leads. Finally, the Vevo2100 imaging system  was used to collect the two-dimensional cardiac ultrasound images of the rats. The catheter was inserted into the carotid artery for ultrasonic index detection and then advanced into the left ventricle. As a result, the interventricular septal thickness (IVS), left ventricular internal end-systolic diameter (LVIDs), and left ventricular posterior wall thickness (LVPW) were detected. In addition, the maximum rate of the rise of left ventricular pressure increase (+dp/dt max) and the maximum rate of the rise of left ventricular pressure decrease (−dp/dt max) were recorded for hemodynamic evaluation.After reperfusion, the hearts were excised, and the tissue sections were stained with 1% TTC for 25 min. Then, they were placed in 10% formaldehyde for 2 days. Finally, when the infarct area appeared white, the infarct size was then calculated.After the corresponding treatment, the myocardial tissues were fixed with formaldehyde (10%) for 24 h, tissues were placed in a 5% nitric acid decalcification solution for 3–5 days. After washing with water, routine dehydration, clearing, paraffin immersion, embedding, and sectioning, the tissue sections were stained with H&E. Finally, the pathological changes in myocardial tissues were observed under a microscope .First, slices of myocardial tissue were fixed in Bouin’s solution at 37°C. Then, after washing with distilled water, Mayer’s Hematoxylin and acidic Ponceau were stained for 10 min. Subsequently, the section was dissolved in a 1% phosphomolybdic acid aqueous solution, and after 10–15 min, the specimen was transferred to aniline blue and stained at 37°C for 20 min. Finally, the samples were rapidly dehydrated in 95% ethanol and then treated with hyaluronic acidification with dimethylbenzene.First, myocardial tissues were fixed with 4% formaldehyde and then washed in PBS containing proteinase K (20 μg/ml) at 37°C. Afterward, myocardial tissues were incubated overnight with the two  TUNEL reagents. Finally, TUNEL staining was observed under an optical microscope .post hoc test. A p < 0.05 was regarded as statistically different.SPSS21.0 was used to analyze the data in this study and data were presented as the mean ± SD. Each experiment was repeated ≥3 times. Differences between all multiple groups were compared using one- or two-way ANOVA and Tukey’s 2O2 produced cell cytotoxicity and reduced H9c2 cell viability compared to the Control group. However, when ARS was added, it significantly reduced the toxicity of H2O2 to cells in a concentration-dependent pattern. Therefore, we selected the high concentration of ARS with the most significant cytotoxic remission for subsequent analysis. Under the microscope, we observed that H2O2 caused damage to the H9c2 cells, while ARS significantly alleviated the pathological injury induced by H2O2 , there were 23 differential expressions of lncRNAs (DElncRNAs), with eight up-regulated and 15 down-regulated . There w2O2 inhibited the miR-29b-3p expression and promoted HMCN1 expression. After ARS intervention, the expression of the above two genes was significantly reversed  in H2O2 + ARS + miR-29b-3p inhibitor + si-HMCN1 group was also remarkably decreased   . SimilarSH-SY5Y) , and theSH-SY5Y) . ConsistSH-SY5Y) . In the SH-SY5Y) . ART cou5Y cells . Consistin vitro and in vivo studies confirmed that low expression of miR-29b-3p contributed to the deterioration of myocardial I/R injury, increased pathological change, fibrosis, apoptosis, and oxidative stress.MicroRNAs (miRNAs) can inhibit mRNA translation or activate mRNA degradation, thereby mediating gene silencing . MiRNAs Caenorhabditis elegans (Hemicentin 1 (HMCN1), also known as fibulin-6, was first discovered in  elegans . HMCN1 i elegans . Studies elegans , tumorig elegans , Fraser  elegans , and cel elegans  and glom elegans . Little In summary, we reported that ARS intervention reduced myocardial I/R injury. Furthermore, the observed beneficial effects of ARS are related to the miR-29b-3p/HMCN1 axis. The finding of this new mechanism will help develop new and better treatment options for patients with myocardial I/R injury.