Caulerpa lentillifera (CL) is a green seaweed, and its edible part represents added value as a functional ingredient. CL was dried and extracted for the determination of its active compounds and the evaluation of its biological activities. The major constituents of CL extract (CLE), including tannic acid, catechin, rutin, and isoquercetin, exhibited beneficial effects, such as antioxidant activity, anti-diabetic activity, immunomodulatory effects, and anti-cancer activities in in vitro and in vivo models. Whether CLE has an anti-inflammatory effect and immune response remains unclear. Methods: This study examined the effect of CLE on the inflammatory status and immune response of lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and the mechanisms involved therein. RAW264.7 cells were treated with different concentrations of CLE (0.1–1000 µg/mL) with or without LPS (1 µg/mL) for 24 h. Expression and production of the inflammatory cytokines, enzymes, and mediators were evaluated. Results: CLE suppressed expression and production of the pro-inflammatory cytokines IL-6 and TNF-α. Moreover, CLE inhibited expression and secretion of the inflammatory enzyme COX-2 and the mediators PGE2 and NO. CLE also reduced DNA damage. Furthermore, CLE stimulated the immune response by modulating the cell cycle regulators p27, p53, cyclin D2, and cyclin E2. Conclusions: CLE inhibits inflammatory responses in LPS-activated macrophages by downregulating inflammatory cytokines and mediators. Furthermore, CLE has an immunomodulatory effect by modulating cell cycle regulators.Background:  Inflammation is a biological response to harmful stimuli including pathogens, toxic compounds, and irritants. Pro-inflammatory cytokines are produced predominantly by activated macrophages during inflammation . Abundanp53 (p53), the cyclin-dependent kinase inhibitor p27 (p27), cyclin D2, and cyclin E2) are involved in the immune response. The p53 gene and protein are part of the innate immune system, and play a key role in defense against infectious diseases [The immune response is the defense mechanism of the immune system against bacteria, viruses, and substances that appear foreign and harmful. Previous studies reported that cell cycle regulators (tumor protein diseases . MoreoveCaulerpa lentillifera (CL) is a green seaweed, known as sea grapes or green caviar seaweed [ seaweed . It is d seaweed . In addi seaweed ,10,11,12 seaweed . HoweverThe anti-inflammatory effect of CLE in RAW264.7 cells was determined using an ELISA assay. As shown in To further clarify the anti-inflammatory effect of CLE, expression of pro-inflammatory cytokine genes, including IL-6, IL-1β, and TNF-α, was also investigated using real-time PCR. Pro-inflammatory cytokine gene expression was significantly reduced in CLE-treated cells compared with control cells . These dAs shown in The inhibitory effect of CLE on DNA damage was evaluated. RAW264.7 cells treated with CLE with or without LPS for 24 h were stained with Hoechst 33342, a fluorescent stain for labeling DNA. The results showed that LPS induced morphologic changes in the DNA, which presented as nuclear fragmentation, chromatin condensation, and apoptotic body formation, compared with the control cells B. IntereIn addition, the effect of CLE on DNA damage was further confirmed by an ELISA assay. As shown in The effect of CLE on immune response enhancement was further investigated using qPCR. As shown in Caulerpa lentillifera extract (CLE) and identify their mechanisms in RAW 264.7 cells. The current study clearly showed that CLE exhibited anti-inflammatory effects in LPS-stimulated RAW264.7 cells by decreasing IL6 and TNF-α production and expression of the related genes. Consistently, previous studies found that the major constituent in CLE, tannic acid, suppressed IL-6 and IL-1β production and also reduced TNF-α protein expression [Inflammation is a response of the immune system that can be stimulated by several factors, including pathogens, damaged cells, and toxic compounds. Dysregulation of the inflammatory response is associated with the development of sepsis. Inflammation is a major cause of increased morbidity and mortality in intensive care units . Inflammpression . Accordipression . Thus, tDuring inflammation, iNOS enzymes produce high levels of NO, which has been determined to be a cytotoxic molecule. In addition, prostaglandin G2 is converted into prostaglandin H2 by COX enzymes and then activated prostaglandin synthases to generate PGE2, a principal mediator of inflammation. Moreover, pro-inflammatory cytokines are important mediators in the transcription of iNOS and COX-2 genes ,18,19. TV. angularis seeds inhibited supercoiled plasmid DNA damage [Inflammation produces reactive oxygen and nitrogen species (RONS), which can stimulate tissue repair and regeneration; however, the chemicals from this process can also induce DNA damage. This study showed that LPS induced DNA damage by nuclear fragmentation, chromatin condensation, and apoptotic body formation. Nevertheless, CLE reversed DNA damage, similar to NSAIDs. Consistently, complex polyphenols and tannins from wine decreased DNA oxidative damage in mucosal cells in rat colon . PreviouA damage . SimilarA damage .Anemarrhena asphodeloides extract activated the immune response by extending the cell cycle S-phase, inhibiting p27 and elevating cyclin D2 and cyclin E2 gene expression in RAW264.7 cells [Cell proliferation leads to an exponential increase in cell numbers, resulting in an increased tissue growth rate. Some of the important cell proliferation regulators are the D cyclins , which regulate cell cycle progression. Furthermore, during the late G1 stage, E cyclins (cyclins E1 and E2) become upregulated and activate cyclin-dependent kinase (CDK)-2, resulting in phosphorylation of various cell cycle-related proteins . In addiDulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco . β-Nicotinamide adenine dinucleotide phosphate; ,5-diphenyltetra-zolium bromide (MTT); lipopolysaccharides were purchased from Merck . ELISA kits were obtained from BioLegend . All other chemicals with high purity were obtained from commercial sources.Low-grade CL was obtained from Phetchaburi Coastal Fisheries Research and Development Center, Phetchaburi Province, Thailand. A voucher specimen  .3 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin in a humidified atmosphere containing 5% CO2. RAW264.7 cells were seeded in 12- and 96-well plates at a density of 2.0 × 105 cells/mL and grown for 3 days for further experiments.Murine RAW264.7 cells were purchased from the American Type Culture Collection (ATCC) . Cells in the 2nd–22nd passages were grown in Dulbecco’s modified Eagle’s Medium (DMEM)  containing 3.7 g/L NaHCOThe viability of RAW264.7 cells was determined using a 3--2,5-diphenyltetra-zolium bromide (MTT) assay. Cells were seeded at 2.0 × 105 cells/mL in 96-well plates. The cells were incubated in a culture medium with or without various concentrations of CLE (0.1–1000 µg/mL) or celecoxib (CX), a non-steroidal anti-inflammatory drug (NSAID), at 3.8 µg/mL for 24 h at 37 °C. After exposure, the cells were washed with PBS, and free DMEM containing a MTT reagent was added and incubated for the next 4 h at 37 °C. Finally, the MTT solution was aspirated and DMSO was added to each well and incubated for another 30 min at 37 °C. The viability of RAW264.7 cells was measured at a wavelength of 570 nm using a SynergyTM HT microplate reader . The sample detected at a wavelength of 680 nm was also used as a reference.g for 10 min at 4 °C. The supernatant was collected for determining the IL-6, IL-1β, TNF-α, NO, and PGE2 concentrations using commercial kits, according to the manufacturer’s instructions . The levels of inflammatory cytokines were measured at a wavelength of 540 nm using a SynergyTM HT microplate reader .The cells were seeded in 12-well plates at 2.0 × 105 cells/mL and cultured for 3 days at 37 °C. On the day of the experiment, 1 µg/mL of LPS or CX at 3.8 µg/mL was added to the culture medium with or without various concentrations of CLE (0.1–1000 µg/mL) for 24 h at 37 °C. The treated cells were then taken for the next assay and centrifuged at 2000× g for 20 min at 4 °C. The supernatant was subsequently collected for measurement of the nitric oxide concentration. The nitric oxide level was measured at a wavelength of 540 nm using a SynergyTM HT microplate reader .The cells were plated in a 12-well plate at 2.0 × 105 cells/mL and cultured for 3 days at 37 °C. On the day of the experiment, culture media with or without various concentrations of CLE (0.1–1000 µg/mL) or CX at 3.8 µg/mL, with or without 1 µg/mL of LPS, were added for 24 h at 37 °C. The treated cells were then taken for the next assay and centrifuged at 10,000× 2. The culture medium was removed, then the cells were supplemented with various concentrations of CLE (0.1–1000 µg/mL) or CX at 3.8 µg/mL, with or without 1 µg/mL of LPS, for 24 h at 37 °C. The treated cells were then taken for the next assay and centrifuged at 10,000× g for 20 min at 4 °C. The supernatant was subsequently collected for measurement of the 8-OHdG concentration using commercial kits, according to the manufacturer’s instructions .To further identify the effect of CLE on DNA damage, the effect of CLE on 8-hydroxy-2′- deoxyguanosine (8-OHdG), a DNA damage marker, was investigated using an ELISA assay. RAW264.7 cells were plated in 12-well plates (2 × 105 cells/mL) and incubated for 24 h at 37°C in a humidified atmosphere with 5% CO2. The treated cells were fixed with 4% paraformaldehyde for 10 min and then treated with Hoechst 33,342 staining at 5 µg/mL for 10 min. The cells were then washed twice with PBS and observed under a Nikon Eclipse Ni-U fluorescent microscope (Nikon).To confirm the effect of CLE on DNA damage, the cells were seeded in 8-well cell culture slides for 3 days. RAW264.7 cells were supplemented with various concentrations of CLE (0.1–1000 µg/mL) or CX at 3.8 µg/mL, with or without 1 µg/mL of LPS, for 24 h at 37 °C in a humidified atmosphere with 5% COTotal RNA was extracted and purified from RAW264.7 cells using TRIzol reagent , according to the manufacturer’s instructions. First-strand cDNA was obtained using a SensiFAST cDNA synthesis kit , and qPCR was performed using SYBR Real-Time PCR Master Mix  on a CFX Touch real-time PCR system . Forward and reverse primers were purchased from Macrogen  and used at a final concentration of 0.4 µM. The specific primer sets for mouse TNF-α, IL-1β, IL-6, COX-2, p53, p27, cyclin D2, cyclin E2, and GAPDH that were used for the qRT-PCR are presented in p < 0.05.Data are expressed as the mean ± S.E.M. Statistical differences were assessed using one-way ANOVA followed by Dunnett’s test. Statistical analyses were conducted using the Statistical Package for the Social Sciences version 23 . Differences were considered significant at CLE contains tannin and catechin, and it improves inflammatory status by inhibiting the gene expression and production of pro-inflammatory cytokines and their mediators, and also by suppressing apoptotic body formation and DNA damage. CLE also enhances the immune response by modulating cell cycle regulators.


Dryopteris ramosa (Hope) C. Chr. (plant family: Dryopteridaceae) is an ethnomedicinal plant of the Galliyat region of Pakistan. The aqueous fraction (AqF) of D. ramosa is being used by inhabitants of the Galliyat region of Pakistan to treat their gastrointestinal tract ailments, especially those caused by bacteria. The aims of the present study were as follows: (i) to justify the ethnomedicinal uses of the AqF of D. ramosa; (ii) to isolate a bioactive compound from the AqF of D. ramosa; and (iii) to evaluate the antibacterial and cytotoxic potential of the isolated compound. Column chromatography (CC) techniques were used for the isolation studies. Spectroscopic techniques  were used for structural elucidation. The agar-well diffusion method was used to evaluate the antibacterial potential of “i3CβDGP” against five bacterial strains, and compare it with the known antibiotic “Cefixime”. The brine shrimp lethality test (BSLT) was used for cytotoxic studies. The AqF of D. ramosa afforded “iriflophenone-3-C-β-D glucopyranoside (i3CβDGP)” when subjected to LH20 Sephadex, followed by MPLC silica gel60, and purified by preparative TLC. The “i3CβDGP” showed a strong potential  against Klebsiella pneumoniae, Staphylococcus aureus, and Escherichia coli, respectively. On the other hand, the least antibacterial potential was shown by “i3CβDGP” (MIC = 125 ± 7.2 µg/mL), against Bacillus subtilis, in comparison to Cefixime (MIC = 62.5 ± 7.2 µg/mL). The cytotoxicity of “i3CβDGP” was significantly low (LD50 = 10.037 ± 2.8 µg/mL) against Artemia salina nauplii. This study not only justified the ethnomedicinal use of D. ramosa, but also highlighted the importance of ethnomedicinal knowledge. Further studies on AqF and other fractions of D. ramosa are in progress.Ethnopharmacological approaches provide clues for the search of bioactive compounds.  Chemical compounds obtained from plants present a great structural diversity and enact important precursor compounds for the development of new restorative agents . The evoIn the recent era, for the search of bioactive natural constituents from plants, ethnobotanical and ethnopharmacological knowledge is being used as a starting point. A large number of pharmaceutical natural products have been obtained from the plants, on the basis of this empirical and traditional knowledge .Dryopteris ramosa (Hope) C. Chr. is a common fern plant  of Dryopteris ramosa has been used as gastrointestinal tonic [D. ramosa has been reported to have antimicrobial and anticancer potential [D. ramosa. The crude extract and various fractions of D. ramosa showed strong antioxidant potentials. The total phenolics and flavonoid contents were determined in a previous study [rn plant  of the Gal tonic , to cureal tonic , for theal tonic ,7. D. raotential . In the otential  has discus study .D. ramosa (leaves/fronds), because in traditional medicines, the polar fraction of D. ramosa is being used to cure gastrointestinal tract (GIT) disorders. Further, it is to validate the traditional use of the aqueous fraction of Dryopteris ramosa for GIT disorders. In this research, the isolation of a pure bioactive compound and its structure elucidation has been carried out by using chromatographic and spectroscopic techniques. Furthermore, in vitro bioactivities, including the antimicrobial and cytotoxic potential of a pure isolated compound from the AqF of D. ramosa, have been discussed. The bacteria that are commonly responsible for GIT disorders were used in this study. D. ramosa. Furthermore, this is the first report of the isolation of antibacterial phytochemicals from D. ramosa, to the best of our knowledge.In this study, we focused on the aqueous fraction of Dryopteris ramosa  were collected (2 kg fresh weight) from Galliyat region (near Patriata/New Murree) of Pakistan according to the “Guidelines in the Conservation on Medicinal Plants (1993)”. The plant was identified by an expert taxonomist (Dr. Rehmatullah Qureshi) at PMAS-Arid Agriculture University Rawalpindi, Pakistan, and voucher specimen (MI-934/15) was deposited in herbarium of Quaid-I-Azam University, Islamabad for future reference.Fronds of Collected leaves of selected plant were dried and powder was formed in a crushing mill. By using cold maceration technique, crude methanolic extract (CME) was formed. Extract was rotary evaporated and stored at 4 °C until further use.The crude methanolic extract (10 g) was subjected to the polarity-based solvent–solvent fractionation. Fractionation procedure of CME in the form of a schematic diagram is presented in D. ramosa was chromatographed over Sephadex LH20 eluted isocratic with methanol. A total of 20 elutions (50 mL of each) were collected, and based on TLC analysis they were pooled into four groups. On the account of MIC against bacterial strains, group 2 (139 mg) was subjected to CC over Sephadex LH20 (120 × 2 cm) eluted  with methanol. Fractions were pooled into two groups and group 2a (83.71 mg) was purified by MPLC over silica gel 60, 40–60 µm particle size, incurring 39.15 mg of pure compound. TLC analyses were carried out on silica gel 60 F254 plates developed in the mixture of n-butanol:acetic acid:water .Two grams of aqueous fraction (AqF) of 3PO4 and 1.5 mM Bu4NOH), flow rate 1 mL/min., injection volume 10 µL and linear gradient starting from 20% MeOH to 90% at 17 min to 100% at 20 min kept for 8 min, wavelength scanned between 200 nm and 600 nm.HPLC analyses were conducted on Agilent 1100 series with UV-DAD detector, Hypersil BDS-C18 column . The solvent system was methanol in aqueous buffer  with positive mode. Spectra range: 100–2500 1H NMR and 100.63 MHz for 13C NMR) equipped with a 5 mm observe broadband probe head (BBFO) with z-gradients at room temperature with standard Bruker pulse programs. Solvent: 0.6 mL of MeO-d4 (99.8% D). 1H NMR: 32 k complex data points and apodized with a Gaussian window function (lb = −0.3 Hz and gb = 0.3 Hz), 13C-jmod spectra with WALTZ16, 1H decoupling was acquired using 64 k data points. Signal-to-noise enhancement was achieved by multiplication of the FID with an exponential window function (lb = 1 Hz). All two-dimensional experiments  were performed with 1 k × 256 data points. Measurement temperature: 298 K ± 0.05 K. Residual CD3OD was used as internal standard for 1 H (δH 3.34) and CD3OD for 13 C (δC 49.0) measurements.Instrument: Bruker Avance II 400  and Staphylococcus aureus (ATCC 6538) while three Gram-negative bacteria were as follows: Klebsiella pneumoniae (ATCC 700603), Salmonella enterica subsp. Enterica serovar Setubal (ATCC 19196) and Escherichia coli (ATCC 25922).The isolated compound from the aqueous extract of Cefixime (antibiotic) was used as standard and positive control while Dimethyl sulfoxide (DMSO) was used as negative control.8 ± 0.5 CFU).Authentic strains of bacteria were cultivated in nutrient broth agar at 37 ± 0.3 °C for 24–48 h. This culture was maintained throughout the study in agar slanted suspension at 4 °C. Bacterial culture were suspended in 0.85% NaCl solution and adjusted to a turbidity 0.5 MacFarlan standard . Equal volumes of test compound (dilution) and nutrient broth were mixed in each test tube. Inoculum  was also added in each tube. The tubes were incubated aerobically at 37 °C for 24 h. Two control tubes for each dilution were maintained. One included test compound (each dilution) and growth medium while second tube contained growth medium, physiological saline and inoculum. MIC was determined as the lowest concentration of the compound permitting no visible growth (no turbidity) when compared with the control tubes [With some modifications, brine shrimp lethality test (BSLT) was carried out to evaluate the cytotoxic potential of i3βDGP as described by .Artemia salina) eggs were carried out in artificial sea water prepared from commercial sea salt 38 g/L and with aeration for 3 h using air pump. Artemia salina hatched after 24 h as shrimps nauplii and was ready for the assay. A source of light (lamp/light bulb) was used to attract hatched nauplii closer to the wall of container.Hatching of brine shrimp (v/v) to achieve the maximum tolerable concentration of working solution as described by [2Cr2O7) was used as positive control while well-aerated artificial sea water was used as negative control. The phototropic nauplii (24–36 h old) were collected with the help of pasture pipette and 20 nauplii were introduced into each concentration/vial and incubated for 24 h at room temperature. After 24 h of incubation, the dead and alive nauplii were counted in each tube and the percentage death of nauplii was determined by using following equation:Pure isolated compound (10 mg/mL) was dissolved in 100% DMSO as stock solution. Less than 1.25% DMSO was used to prepare each dilution . Analysis of variance (ANOVA) was used to determine the correlation and main effects of various concentrations of plant extract, fractions and isolated pure compound at After maceration, about 10 g of CME  was obtained. This CME afforded nHF = 3.6 g, CF = 1.1 g, EAF = 1.8 g, AqF = 2.9 g, and MF = 0.6 g .D. ramosa, 39.15 mg of pure compound was isolated, as stated in D. ramosa, the spectroscopic data are as follows:From the AqF of max = 298nm (band-I) and 210 nm (band-II). 1HNMR : 7.61 , 7.59 , 6.78 , 6.75 , 5.94 , 4.88 , 3.89 , 3.84 , 3.48 , 3.44 , and 3.35 . 13CNMR : δ 198.89 (C-7), 163.42 (C-6), 162.90 (C-11), 161.44 (C-4), 161.07 (C-2), 133.26 (C-8), 132.82 (C-9), 132.82 (C-13), 115.50 (C-10), 115.50 (C-12), 107.17 (C-1), 104.67 (C-5), 94.61 (C-3), 82.55 (C-5’), 79.93 (C-3’), 76.61 (C-1’), 73.74 (C-2’), 71.46 (C-4’), and 62.48 (C-6’). EIMS ESI-TOF (+) = 409 (M + H) m/z, 246 m/z (M-163 + H), and other fragments include the following: 392 m/z (M-17 + H), 288 m/z (M-OH-105 + H), 126 m/z (M-163-121 + H). Molecular weight: 408 amu, and molecular formula: C19H20O10.UV–Vis spectroscopy: detector: DAD; RT: 6.467 min; λ D. ramosa and Cefixime (antibiotic/control), against different strains of bacteria, is presented in The antibacterial potential of the isolated compound (i3βDGP) from the AqF of 2Cr2O7) was used as a control. The mean percentage lethality against each concentration is presented in 50 (in µg/mL) was calculated by the regression equation. A low LD50 (µg/mL) value means that the compound is cytotoxic. LD50 of i3βDGP is 10.037 ± 2.89 µg/mL (The cytotoxicity of the isolated compound (i3βDGP) and Cefixime was evaluated through BSLT. Potassium dichromate  [j = 9.88) is coupled with the proton of H-2/. Similarly, H-13  was coupled with H-12 , and H-10  with H-9 . The heteronuclear multiple-bond correlation (HMBC) experiment was developed to assist in the identification (correlation) of proton nuclei with carbon nuclei that are separated by more than one bond. The HMBC plot of “i3CβDGP” was given in j-CH correlation was noticed between H-9 (δ 7.59 ppm), C-7 (δ 198.89 ppm), and C-11 (δ 162.90 ppm), as well as H-10 (δ 6.75 ppm), C-12 (δ 115.50 ppm), and C-8 (δ 133.26 ppm). Abundant 3-j-CH long-range correlation signals were observed with cross peak H-1/(δ 4.88 ppm) to C-3/(δ 79.93 ppm), C-5/(δ 82.55 ppm), C-6 (δ 163.42 ppm), and C-4 (δ 161.44 ppm). The TOF-ESI/MS (Dryopteris (and so to D. ramosa), to the best of our knowledge.In the quest of effective antibacterial compounds, the AqF of ey bush) . The 2D F-ESI/MS b showed -8 δ 133. ppm. AbuF-ESI/MS a. This iKlebsiella pneumoniae (MIC = 31.1 ± 7.2 µg/mL), while the same compound has shown minimum antibacterial potential against Bacillus subtilis (MIC = 125 ± 7.2 µg/mL). However, it was notable from Staphylococcus aureus, Klebsiella pneumoniae, and Escherichia coli, but for Salmonella enterica and Bacillus subtilis, the MIC of “i3CβDGP” was greater than Cefixime  against a selected strain of bacteria that is responsible for GIT disorders in humans is presented in Cefixime . This inD. ramosa ranges between 66 ± 0.7 mm (S. aureus) and 10 ± 0.7 mm (E. coli). The MIC for the CME against B. subtilis was the highest (250 ± 7.2 µg/mL), while it was the lowest against K. pneumoniae (62.5 ± 7.2 µg/mL). The antibacterial activity of crude extract was less as compared to isolated pure compound (i3CβDGP). This might be due to the synergetic effects between compounds. A similar observation was reported by [Terminalia laxiflora was less potent to P. aeruginosa than punicalagin . A recent study was carried out by [Dryopteris ramosa. According to them, “the water fraction produced a 12 mm inhibition zone against MRSA-10, as compared to 10 mm given by the standard and 6 mm zone against S. aureus. The crude extract was the most active, amongst all the fractions, against P. aeruginosa, where it exhibited a zone of 13 mm while the standard produced a zone of 11 mm. DMSO being the negative control did not inhibit the zones. The MIC was calculated for all the extracts. According to the results, the lowest MIC was shown by the crude extract (16 μg/mL) against P. aeruginosa, as compared to the standard Cefixime, with a MIC of 32 μg/mL. Similarly, against MRSA-10, the methanolic and water fraction have a MIC of 32 μg/mL, as compared to the MIC of Cefixime (64 μg/mL).The inhibition zone and the MIC (µg/mL) were compared and presented in orted by . They fod out by . They pr2Cr2O7). The results (50 = 10.037 ± 2.89 µg/mL). This is a positive property, in the sense that it may be used as a future antibiotic, without causing significant cytotoxic adverse effects on healthy human cells in human GIT or in the body. Toxicity evaluation of pure compounds or plant extracts is important, prior to their further development and commercialization. BSLT is an easy, bench-top and cost-effective preliminary test to evaluate cytotoxicity. This bioassay is being used by many researchers to determine the cytotoxicity of plant extracts and pure isolated compounds [The isolated pure compound “i3CβDGP” was subjected to BSLT to evaluate its cytotoxicity, and to compare it with the cytotoxicity of “cefixime” and potassium dichromate  ailments by the local inhabitants of the Gallyat region of Pakistan. We have successfully isolated the potential antibacterial compound “iriflophenone-3-C-β D glucopyranoside (i3CβDGP)” from the AqF of D. ramosa. “i3CβDGP” did not show significant cytotoxicity in BSLT, but “i3CβDGP” showed strong antibacterial potential against the tested strain of bacteria. This study not only justified the use of the AqF of D. ramosa, by the inhabitants of the Gallyat region of Pakistan, but also highlighted the importance of ethnomedicinal knowledge. Further studies on the AqF and other fractions of D. ramosa are in progress in our laboratory.The aqueous fraction (AqF) of


Notholirion thomsonianum for the management diabetes mellitus.Diabetes mellitus is a common disease effecting the lifestyles of majority world population. In this research work, we have embarked the potential role of crude extracts and isolated compounds of N. thomsonianum were initially evaluated for α-glucosidase, α-amylase and antioxidant activities. The compounds were isolated from the activity based potent solvent fraction. The structures of isolated compounds were confirmed with NMR and MS analyses. The isolated compounds were tested for α-glucosidase, α-amylase, protein tyrosine phosphatase 1B (PTP1B) and DPPH activities. The molecular docking studies were carried out to find the binding interactions of isolated compounds for α-glucosidase, α-amylase and PTP1B.The crude extracts of N. thomsonianum against different in-vitro targets. Among all, Nt.EtAc was observed a potent fraction among all giving IC50 values of 67, 70, < 0.1, 89 and 16 μg/mL against α-glucosidase, α-amylase, DPPH, ABTS and H2O2 respectively. Three compounds  were isolated from Nt.EtAc of N. thomsonianum. The isolated compounds Nt01, Nt02 and Nt03 exhibited IC50 values of 58.93, 114.93 and 19.54 μM against α-glucosidase, while 56.25, 96.54 and 24.39 μM against α-amylase respectively. Comparatively, the standard acarbose observed IC50 values were 10.60 and 12.71 μM against α-glucosidase, α-amylase respectively. In PTP1B assay, the compounds Nt01, Nt02 and Nt03 demonstrated IC50 values of 12.96, 36.22 and 3.57 μM in comparison to the standard ursolic acid (IC50 of 3.63 μM). The isolated compounds also gave overwhelming results in DPPH assay. Molecular docking based binding interactions for α-glucosidase, α-amylase and PTP1B were also encouraging.Initially, we screened out crude extracts and subfractions of N. thomsonianum is potential medicinal plant for the treatment of hyperglycemia. Overall, Nt.EtAc was dominant fraction in all in-vitro activities. Three compounds Nt01, Nt02 and Nt03 were isolated from ethyl acetate fraction. The Nt03 specifically was most potent in all in-vitro assays. The molecular docking studies supported our in-vitro results. It is concluded that N. thomsonianum is a rich source of bioactive antidiabetic compounds which can be further extended to in-vivo based experiments.In the light of current results, it is obvious that The online version contains supplementary material available at 10.1186/s12906-021-03443-7. DM is ahe blood . The orghe blood , 7. Varihe blood , 9. Howeα-glucosidase inhibitors (AGIs) like acarbose and voglibose in microorganisms, and nojirimycin and 1-deoxynojirimycin has been reported from plants [Natural products have long been and still are the source of treatment and prevention of different disorders including diabetes . Naturalm plants . Recentlm plants . SeveralAerobic life utilizes oxygen to metabolize food substrates which are rich in carbon and hydrogen to gain energy necessary for life activities. During the oxidation process, the oxygen molecule itself turns into reduced state and forms intermediates. During physiological metabolism, reactive oxygen species (ROS) are constantly created which are counter-balanced by cellular antioxidant protective mechanisms . These RNotholirion thomsonianum  Stapf belong to Liliaceae family. It is traditionally used in the treatment of a variety diseases including tumors, digestive and microbial mediated disorders [N. thomsonianum.isorders . We receisorders , 19. TheAll the chemicals and drugs used in this research were purchased from the local vendors of Sigma company.Notholirion thomsonianum was collected from different regions of Swat KP, Pakistan and was identified by Dr. Nasrullah, Plant Taxonomist at the department of Botany University of Malakand, Pakistan. A sample from the plant was pressed and deposited in herbarium of University of Malakand, Pakistan, under voucher specimen H.UOM.BG.106. As per our previous mentioned procedure, we did extraction and fractionation of the plant’s sample [s sample .α-Glucosidase inhibitory potentials of the samples from plant extracts and isolated compounds (Nt01 – Nt03) were analyzed using the method reported [α-glucosidase inhibitor, was used as standard drug. In 0.10 M of phosphate buffer having pH of 6.90, the α-glucosidase enzyme solution was prepared (0.50 U/ mL). The enzyme solution contains the mentioned amount of α-glucosidase and phosphate buffer  was added to complete the reaction. The same solution without the enzyme was used as a blank. The absorbances were recorded at 405 nm. All the experiments were repeated in triplicate. The percent inhibitions and subsequent IC50  were calculated accordingly.Yeast reported . Acarbosα-amylase activities on crude extracts and isolated compounds (Nt01 – Nt03) of N. thomsonianum were performed as per the standard procedure [The potential rocedure . The amy50 values were recorded accordingly.The PTP1B activity was performed only on the isolated compounds Nt01 – Nt03 using the standard procedure . Buffer 50 values of samples and standard acarbose were calculated as per the protocols.DPPH free radicals scavenging ability of the plant’s samples and isolated compounds (Nt01 – Nt03) were evaluated following previously reported procedures . Plant s50 values of samples and standard ascorbic acid were calculated as per the standard procedure [Plant crude samples were also evaluated against ABTS free radicals using standard protocol . In thisrocedure .N. thomsonianum. A solution of H2O2 (2 mM) was prepared in buffer solution of phosphate . In a test tube, 0.10 mL of crude extract was measured, and the volume was diluted to 0.40 mL by adding the phosphate buffer. Then 0.60 mL solution of H2O2 was added to it and was mixed properly. The absorbance of the crude sample and standard were recorded on 230 nm. The same procedure was repeated for all the crude samples. The percent inhibitions and IC50 values were calculated as per the reported procedure [This method was used to determine the antioxidant potentials of crude extracts from rocedure .50) were calculated via Microsoft Excel program. In free radicals’ assays including DPPH, ABTS and H2O2, the IC50s were calculated using same method [Samples’ concentrations that inhibited substrate hydrolysis by 50% . Preparation of downloaded enzymes such as determination of binding sites, energy minimization and 3-D protonation was performed by previously reported methods [We performed molecular docking simulations using Molecular Operating Environment software (MOE 2016). Three-dimensional structure of  methods , 27, 28.n-hexane with proper shaking in a separating funnel. The separating funnel was put in a stand and the two layers were given the time to separate. The organic layer (n-hexane) was separated, and the same procedure was repeated three times to get enough amount of n-hexane fraction. The n-hexane layer was subjected to rotary evaporator. The dried n-hexane fraction (Nt.Hex) was stored and labelled properly. The same solvent fractionation procedure was performed by using different solvent of increasing the polarity. So, the next fractions we got were of chloroform (Nt.Chf), ethyl acetate (Nt.EtAc), and the final layer obtained was that of aqueous layer (Nt.Aq).The plant materials were extracted with our previous reported method. After the extraction process, the methanolic extract was subjected to rotary evaporator for removal of solvent. The dried methanolic extract (Nt.Cr about 0.30 Kg) of the plant was suspended in 0.50 L of distilled water and was added 0.50 L of n-hexane. Afterwards, the polarity of eluent was gradually increased by adding a small amount of polar modifier (ethyl acetate solvent). There was a gradual increase of 2% in polarity of solvent system. At the end, the polarity was increased to 1:1 ratio of n-hexane/ethyl acetate. Semi-purified phytochemicals were collected and dried. Three of the semi-purified products were then individually loaded onto small pre-packed silica gel column. Each of the small column was also eluted with n-hexane/ethyl acetate. Starting from low polarity and gradually increasing the polarity, we were able to get the purified products (Nt01 – Nt03) as visualized on a TLC plate.Based on the overall potency in all in-vitro assays, the ethyl acetate (Nt.EtAc) was selected for the isolation of bioactive compounds. Initially, the Nt.EtAc was loaded on the top of a pre-packed silica gel gravity column. The column was initially eluted with pure J) of 8.49 Hz. The two methyl groups attached to a double bond gave two singlets at 2.00 and 2.11. The methyl group attached at para position to the benzene ring appeared at chemical shift of 2.27. The single vinylic hydrogen gave a singlet at 5.98. The three aliphatic protons gave a multiplet in the range of 2.93–3.28. The aromatic proton gave the typical splitting pattern of a para substitution by giving two consecutive doublets. The doublets appeared at 7.07 and 7.13 with J values of 8.05 and 7.98 Hz respectively. The carbon NMR was also in agreement with the compound’s structure. There were clear signals for aliphatic and aromatic/vinylic carbons. The carbonyl carbon appeared above chemical shift of 200. The MS and 1H NMR data of compound Nt02 is shown in Figure SJ value of 10.66 Hz. The high J value of the doublet shows a trans-confirmation. Similarly, the second vinylic H – atom also gave a multiplet at 6.43–6.29. The three methylene (−CH2-) appeared in the form multiplets between chemical shift 2.13 and 1.28. The methyl group gave a distinct triplet at 0.97 with J value of 7.23 Hz. The MS and 1H NMR data of compound Nt03 is shown in Figure SJ value of 6.83 Hz. The most downfield signal was that from the phenolic H – atom, which gave a broad singlet at 4.21.The structures of the isolated compounds were determined with NMR and MS analyses as shown in Fig. α-glucosidase and α-amylase inhibitory activities of the crude extracts and different solvent extracts of N. thomsonianum are summarized in Table α-glucosidase inhibition assay of N. thomsonianum, Nt.EtAc was found most active causing 68.57 ± 1.26, 61.89 ± 1.70, 57.77 ± 1.01, 53.61 ± 2.31 and 45.33 ± 0.66% inhibitions at concentrations of 1000, 500, 250, 125 and 62.50 μg/mL respectively attaining IC50 of 67 μg/mL. This was followed by Nt. Cr exhibiting 64.78 ± 0.92, 54.11 ± 0.07, 51.55 ± 0.41, 47.55 ± 0.67 and 40.00 ± 0.57% inhibitions at concentrations of 1000, 500, 250, 125 and 62.50 μg/mL respectively with IC50 of 185 μg/mL. The other tested samples from N. thomsonianum were less effective in an order of Nt. Chf > Nt. Hex > Nt. Aq with IC50 values of 394, 435 and 614 μg/mL respectively. In comparison, the observed/calculated IC50 value of the standard drug acarbose was 25 μg/mL using the same concentrations.The N. thomsonianum exhibited the alpha amylase activity as shown in Table 50 of 70 μg/mL. In comparison, the standard acarbose gave IC50 value of 13 μg/mL. The other fractions like Nt. Cr, Nt. Hex, Nt. Chf and Nt. Aq exhibited IC50 values of 180, 240, 450 and 950 μg/mL respectively.In almost a similar pattern, different extracts from N. thomsonianum using DPPH, ABTS and H2O2 free radicals are tabulated in Table The results of antioxidant assays of extracts of N. thomsonianum, Nt.EtAc exhibited highest activity causing 89.96 ± 0.55, 85.93 ± 0.30, 81.40 ± 0.65, 77.46 ± 0.50 and 72.10 ± 0.65% inhibitions at concentrations of 1000, 500, 250, 125 and 62.5 μg/mL respectively gaining IC50 < 0.1 μg/mL. Nt. Chf was comparatively active with 88.43 ± 1.26, 83.83 ± 0.66, 77.93 ± 0.90, 72.26 ± 0.77 and 67.10 ± 0.95% inhibitions at concentrations of 1000, 500, 250, 125 and 62.5 μg/mL respectively, IC50 10 μg/mL. Comparatively, standard drug ascorbic acid showed 92.55 ± 0.35, 87.84 ± 0.26, 81.33 ± 0.88, 76.54 ± 0.54 and 72.67 ± 0.19% inhibitions at the same concentrations gaining IC50 of < 0.10 μg/mL.In DPPH assay of 50 of 72 μg/mL. In this assay, the ethyl acetate fraction (Nt.EtAc) was observed to be less potent than chloroform fraction giving IC50 value of 89 μg/mL. The standard drug IC50 against the ABTS free radicals was also < 0.1 μg/mL.In ABTS free radicals scavenging assay, Nt. Chf exhibited highest activity of 74.70 ± 0.55, 66.20 ± 0.95, 62.00 ± 0.57, 55.93 ± 0.44, and 48.53 ± 0.40% inhibitions at concentrations of 1000, 500, 250, 125 and 62.50 μg/mL respectively attaining IC2O2 as shown in Table 50 values of crude extracts were 16 (Nt.EtAc), 79 (Nt.Chf), 221 (Nt.Cr), 788 (Nt.Hex) and 1403 (Nt.Aq) μg/mL. the observed and calculated IC50 of standard ascorbic acid was 9.87 μg/mL.Likewise majority of our observations, the Nt.EtAc was also dominant in scavenging the free radicals from HN. thomsonianum were evaluated for their in-vitro inhibitory potentials against the α-glucosidase enzyme as shown in Table 50) are provided in μg/mL as well as in μM (micromole) based on the molecular weight of each tested compound. Among the isolated compounds, Nt03 showed highest activity with IC50 value of 19.54 μM in comparison to the standard drug acarbose (IC50 of 10.60 μM). The calculated IC50 values of compounds Nt01 and Nt02 were 58.93 and 114.93 μM respectively.The isolated compounds (Nt01 – Nt03) from α-amylase enzyme as shown in Table 50 value of 24.39 μM . In comparison, the IC50 value given by acarbose was 8.20 μg/mL.Our isolated compounds (Nt01 – Nt03) were also good to moderate in inhibiting the 50 values in PTP1B target, we can claim that our compounds, specifically Nt03 is an excellent inhibitor in comparison to the standard ursolic acid. The IC50 value given by compound Nt03 was 3.57 μM in comparison to the standard drug (ursolic acid IC50 was 3.63 μM). Similarly, the compounds Nt01 and Nt02 exhibited IC50 values of 12.96 and 36.22 μM respectively.Besides the two targets , we also tested our compounds for protein tyrosine phosphatase 1B as shown in Table 50 value exhibited by the ascorbic acid was 1.78 μM . In comparison to the standard drug, our compounds exhibited IC50 values of 4.70 (Nt01), 41.53 (Nt02) and 2.75 μg/mL (Nt03).We also determined the antioxidant potentials of isolated compounds with DPPH free radical scavenging method Table . The obsα-glucosidase is shown in Figure SDocking studies on our previously reported homology model was carried out by using Molecular operating Environment MOE 2016) software package. Three-dimensional (3-D) plots of identified compounds (Nt01- Nt03) into the binding site of homology modelled α-glucosidase are shown in Fig. 016 softwα-amylase was performed by using MOE software. Crystal structure of human pancreatic α-amylase with accession code 4 W93 was obtained from PDB [α-amylase is shown in Figure SDocking studies on Human pancreatic from PDB . 3-D intDocking studies on Protein tyrosine phosphatase 1B (PTP1B) was performed by using MOE software. PTP1B in complex with catalytic inhibitor was retrieved from protein data Bank (PDB ID = 1NNY). 3-D interaction plots of all the six identified compounds are shown in Fig. Natural products are still believed as a potential source for the discovery of new drugs and play a vital role in the drug development programs –32. NumeN. thomsonianum using α-glucosidase and α-amylase targets. We also supplemented the activities with antioxidant potentials of the crude extracts using free radicals’ methods of DPPH, ABTS and H2O2. Based on the initial in-vitro screenings of the methanolic extract and subfractions, we observed the ethyl acetate fraction as the potent one. We subjected this fraction to isolation of bioactive compounds and were able to purify three compounds (Nt01 – Nt03). The antidiabetic potentials of the isolated compounds were tested using in-vitro targets of α-glucosidase, α-amylase, protein tyrosine phosphatase 1B and DPPH. Based on our result, we can claim that our isolated compounds, specifically Nt03 can be lead multitarget antidiabetic agent.Diabetes mellitus (DM) is a common metabolic disorder, characterized by an abnormal postprandial raise in blood glucose level. Control of postprandial hyperglycemia is believed as an important tool in the management of DM. Parallelly, the antioxidant-based drug therapies are commonly used for the mitigation and treatment of multiple diseases including atherosclerosis, stroke, cancer, Alzheimer’s disease and diabetes . In liviα-glucosidase, α-amylase, and protein tyrosine phosphatase 1B. The molecular docking studies revealed encouraging bind interactions with the target proteins for our compounds.The molecular docking is a powerful dry lab approach to find out the interactions and binding energies of a drug molecule in a drug target . This inN. thomsonianum as a potential plant for the management of diabetic through multitarget approach. In the initial in-vitro screening of the crude samples from the plant, we observed that ethyl acetate fraction is overall the potent in inhibiting all the tested targets. The bio-guided based approach for the isolation of compounds enables us to purify three compounds (Nt01 – Nt03). We also tested the isolated compounds α-glucosidase, α-amylase, protein tyrosine phosphatase 1B and DPPH in-vitro targets. Moreover, we also find out the binding interactions with the target proteins using molecular docking approach. The in-vitro and encouraging binding in-silico results revealed that N. thomsonianum is a potential source of bioactive compounds as emerging multitarget antidiabetic drugs.It is concluded from our results, that we have explored Additional file 1.


Glycine max (L.) Merr.] with different K efficiency. Two K high-efficiency vegetable soybean genotypes (Line 19 and Line 20) and two K low-efficiency genotypes (Line 7 and Line 36) were investigated in low K and normal K conditions. The root of K high-efficiency genotypes had a higher K+ affinity associated with a higher maximum K+ uptake rate (Imax), but lower Michaelis constant for K+ absorption (Km) and lower compensation concentration for K+ uptake (Cmin). Seedlings of K high-efficiency genotypes also had higher root vigor [triphenyl tetrazolium chloride (TTC) reduction method] and greater absorbing activity (methylene blue method), especially in the low K condition. Furthermore, the root bleeding-sap rate of K high-efficiency genotypes in low K stress was 9.9–24.3% greater than that of normal K conditions, which was accompanied by a relatively higher K concentration of root bleeding-sap in contributing to K+ upward flux. The root of K high-efficiency vegetable soybean genotypes exhibited K+ high-affinity and driving advantages. Photosynthetic parameters of K high-efficiency vegetable soybean genotypes were less affected by low K stress. Low K stress decreased the net photosynthetic rate of K high-efficiency genotypes by 6.1–6.9%, while that of K low-efficiency genotypes decreased by 10.9–15.7%. The higher chlorophyll (Chl) a/b ratio with enhanced relative content of Chl a in response to low K stress might be an adapted mechanism for K high-efficiency genotypes to maintain photosynthetic capacity. Stronger root K affinity drivers associated with photosynthetic adaptability to low K stress are the key factors in determining the K high-efficiency of vegetable soybeans.Significant variations of potassium absorption and utilization exist in vegetable soybean. Pot and hydroponic experiments were carried out to examine the characteristics of root potassium (K) affinity-associated drivers and photosynthesis in vegetable soybean (edamame) [ Potassium application benefits vegetable soybean yield and quality , while tDifferences in the K efficiency of crops can be understood from two main aspects, such as (1) the difference in K uptake efficiency and (2) the difference in K utilization efficiency . The utiExcept for root morphology, K uptake kinetic parameters, root bleeding-sap, and root vigor are also demonstrated as effective parameters in evaluating K uptake efficiency . SufficiLess photosynthetic assimilates and reduced assimilate transport out of the leaves to the developing fruit greatly contribute to the negative consequences that deficiencies of K have on yield and quality production . GenotypHence, understanding how plants take up and use K is of scientific and practical importance. Research into this mechanism has become increasingly urgent as a result of major ecological and agricultural issues. In our previous studies, K high-efficiency vegetable soybean genotypes were demonstrated with a strong K uptake and redistribution ability ,b. HowevBased on previous K efficiency selection , two K h–1, total N 2.3 g kg–1, total P 1.3 g kg–1, total K 18.9 g kg–1, available N 159 mg kg–1, available P 57.0 mg kg–1, and available K 85 mg kg–1  and K2SO4 120 kg ha–1 (K120). Plants were randomly harvested at fourth-node (V4), full bloom (R2), beginning seed (R5), and full seed (R6) stages  in 2017. The soil used was a typical Mollisol (Black soil) with the following properties: soil pH 6.6, organic matter 28.9 g kg) stages  for root+ absorption kinetic parameters, root vigor, and root absorbing activity test. Seeds of the four selected genotypes were sterilized and germinated on moistened filter paper in a plant growth chamber at 60% humidity and 28°C, under a 16 h light, 8 h dark cycle for 3–4 days. After that, the seedlings were transferred into light-proof glass boxes (volume 15 cm3 × 20 cm3 × 20 cm3) with half-strength modified Hoagland nutrient solution, as described by + (0.5 and 3 mmol L–1). The K source was K2SO4. The initial pH value of the nutrient solution was 6, which was adjusted by 0.1 mol L–1 NaOH or 0.1 mol L–1 HCl. After transplanting, the samples were continuously cultured for 9 days. The nutrient solution was changed every 3 days and kept enough O2 by regular ventilation with an air pump. The pH value of the solution was adjusted to 6 when the nutrient solution was changed.A hydroponic experiment was used for K–1 per plant) = bleeding-sap weight/4 h.Collection of root bleeding-sap: root bleeding-sap collection was based on the method used by Where K upward fluxes = root bleeding-sap rate × K concentration, K concentration was determined by flame spectrophotometer  .+ absorption kinetic parameters: A measurement of a depletion curve of a single plant. Five replicates per genotype were conducted. After K deficiency and starvation for 48 h, samples under the treatment of K3 at 9 days were immersed in 0.2 mmol L–1 CaSO4 solutions three times, dried, and put into a 200 ml absorption solution. The composition of the absorption solution was 0.05 mmol L–1 potassium chloride (KCl) + 0.2 mmol L–1 CaSO4. The absorption solution was placed in a dark conical flask, the culture temperature was (25 ± 2)°C, and the light intensity was 210–260 mol m–2 s–1. Parameter calculation including the maximum K+ uptake rate (Imax), Michaelis constant for K+ absorption (Km), compensation concentration for K+ uptake (Cmin), and statistical analysis refers to the modified method of Cmin represents the concentration of K+ in the solution when the concentration remains constant. Ion consumption curve equation: Y = a + bX + cX2 (1), take the negative derivative of the equation: Y’ = b + 2cX (2). Hence, if X goes to 0, then Y’ = b = Km.KRoot vigor, as determined by enzymatic reduction assay of triphenyl tetrazolium chloride (TTC) was conducted according to the modified protocol of Root absorption activity was determined by methylene blue adsorption . First, –1); C′, the concentration of the solution after leaching (mg ml–1); 1, 2, 3 is the beaker number; V, Volume of the solution; When 1 mg methylene blue forms a monolayer, it covers an area of 1.1 m2  were determined at 10:00–12:00 am at V4, R2, R4 (full pod), R5, and R6 stages with a Li-6800  at 25°C, 60% relative humidity, 500 μmol molDetermination of chlorophyll (Chl) content  was condDifferences among treatments and genotypes were examined by ANOVA using SPSS 17, and the means were separated by the LSD test at the 5% level. Linear regression analysis between root bleeding-sap rate per root length, K upward fluxes rate per root length, K concentration of root bleeding-sap, and plant K concentration were conducted after Pearson correlation analysis using a two-tailed test in SPSS 17. The figures were created using SigmaPlot 12.+ kinetics absorption parameters of the four vegetable soybean genotypes in a hydroponic experiment. The maximum K+ uptake rate (Imax) in K high-efficiency genotypes was around 58.2–65.5 μmol g–1 min–1 FW, which was significantly higher than that of K low-efficiency genotypes around 42.4–54.1 μmol g–1 min–1 FW (P < 0.05). The compensation concentration for K+ uptake (Cmin) in K high-efficiency genotypes was 0.83–1.32 μmol L–1, which was lower than that of K low-efficiency genotypes (3.16–3.22 μmol L–1) (P <0.05). The Michaelis constant for K+ absorption (Km) is a parameter evaluating the affinity between root and K+. The greater the value, the smaller the affinity. Higher affinity was found in K high-efficiency vegetable soybean genotypes with a lower Km of 32.8–35.0 μmol L–1 than that of 48.6–49 μmol L–1 in K low-efficiency genotypes (P < 0.05).–1 root–1 in K0 and 1.6 ml h–1 root–1 in K120, respectively. Compared with K120 treatment, K0 treatment increased the average of the root bleeding-sap rate per plant over the four stages by 9.9–24.3% in K high-efficiency genotypes, but by –2.2–5.1% in K low-efficiency genotypes. On the other hand, the root bleeding-sap rate per root length in four genotypes was found higher at the V4–R2 stage, dropped down at the R5–R6 stage. Interestingly, K high-efficiency genotypes had a higher root bleeding-sap rate per root length compared with K low-efficiency genotypes at the beginning seed stage (P < 0.05). At this time, root bleeding-sap rate per root length ranged 0.09–0.13 ml h–1 cm–1 in K0 and 0.10–0.15 ml h–1 cm–1 in K120 treatment, while in K low-efficiency genotypes kept around 0.05 ml h–1 cm–1 in K0 and 0.05–0.07 ml h–1 cm–1 in K120 treatment.As shown in P < 0.05). The K flux rate per root length of K high-efficiency genotypes was 19.4–32.4 μg h–1 cm–1 in K0 and 24.1–37.2 μg h–1 cm–1 in K120 treatment, that of 7.6–8.7 μg h–1 cm–1 in K0 and 9–15.2 μg h–1 cm–1 in K120 treatment in K low-efficiency genotypes. Meanwhile, K concentration of root bleeding-sap in K high-efficiency genotypes reached 51.1–63.2 μg ml–1 in K0 and 60.2–64 μg ml–1 in K120 treatment at the beginning seed stage, which was significantly higher than that of K low-efficiency genotypes with 41.1–48 μg ml–1 in K0 and 46.1–52.4 μg ml–1 in K120 treatment (P < 0.05).Higher K upward fluxes were found in K120 treatment over the four genotypes, which was accompanied consistently by a higher concentration of K in the root bleeding-sap . The K fP < 0.05)  .P < 0.05). Although no consistent trend was observed in K low-efficiency genotypes, low K stress decreased root vigor by 21% in Line 36 (P < 0.05). The highest root vigor of 516 μg g–1 FW h–1 was found in Line 20 under low K conditions.The root vigor was tested in a hydroponic experiment using the measurement of respiratory activity with triphenyl tetrazolium chloride (TTC) . At the P < 0.05). In contrast, an opposite trend was found in K low-efficiency genotypes. Under low K stress, the actively absorbing area decreased by 6.1–10.3% and the total absorbing area decreased by 6.6–15.7% (P > 0.05). Furthermore, K high-efficiency genotypes also had a higher ratio of total absorbing area to root volume, especially in Line 19 with 62.3 m2 cm–3 (K0.5) and 60.2 m2 cm–3 (K3). Low K stress increased the ratio of actively absorbing area to total absorbing area by 9.3–9.4% in K high-efficiency genotype (P < 0.05), but by 0.6–5% in K low-efficiency genotypes (P > 0.05).The root absorbing activity parameters are shown in 2 m–2 s–1 and 33.8 μmol CO2 m–2 s–1 under K0 and K120 treatments, respectively. Meanwhile, from full pod to full seed stage, the dramatic decline of the Pn was found in K low-efficiency genotypes. The Pn of Line 36 decreased by 87.2% in K0 and 86.2% in K120 treatment (P < 0.05), and that of Line 7 decreased by 54.3% in K0 and 59% in K120 treatment. Whereas the decline of the Pn in K high-efficiency genotypes was around 33.5–51.8% in K0 and 36.7–41.1% in K120 treatment, which was not as much as that of K low-efficiency genotypes.A pot experiment was conducted to determine photosynthetic parameters of two K efficiency vegetable soybean genotypes affected by K deficiency (K0) are shown in Potassium deficiency also decreased the stomatal conductance (Gs) in all genotypes, but K high-efficiency genotypes were less affected. The decrease of Gs by K deficiency was 7.5–7.8% in K high-efficiency genotypes, but 9.4–12.5% in K low-efficiency genotypes. Low K treatment had the most obvious effect on Gs at the beginning seed stage.2 concentration (Ci), especially in K low-efficiency genotypes. The increase of Ci by K deficiency was 7.3–7.9% in K low-efficiency genotypes, but 2–6% in K high-efficiency genotypes.As opposed to Pn and Gs, K deficiency increased the intercellular CO2O m–2 s–1 in K0 and 9–10.1 mmol H2O m–2 s–1 in K120 treatment, which was higher than that of 7.1–8.2 mmol H2O m–2 s–1 in K0 and 7.1–8.6 mmol H2O m–2 s–1 in K120 treatment in K low-efficiency genotypes. At the full seed stage, higher Tr was found in K high-efficiency genotypes with 4–4.5 mmol H2O m–2 s–1 in K0 and 4.2–4.7 mmol H2O m–2 s–1 in K120 treatment, but only 1–1.2 mmol H2O m–2 s–1 and 1.4 –1.6 mmol H2O m–2 s–1 in K low-efficiency genotypes.Potassium deficiency decreased the transpiration rate (Tr) of all genotypes. Across the five stages, Tr of K high-efficiency genotypes was around 8.8–9.8 mmol H–1 in K0 and 2.89–2.91 mg g–1 in K120 treatment, which was lower than that of K low-efficiency genotypes with 2.57–3.2 mg g–1 in K0 and 3.08–3.5 mg g–1 in K120 treatment. However, K deficiency increased the ratio of Chl a to Chl b, especially in K high-efficiency genotypes. The ratio of Chl a to Chl b was increased by 36.2–38.1% in K high-efficiency genotypes (P < 0.05), but only by 6–9.8% in K low-efficiency genotypes.Pot experiment indicated that K deficiency significantly decreased leaf chlorophyll content of the four vegetable soybean genotypes . The ave+ upward flux. In K high-efficiency cotton cultivars, higher root bleeding-sap and K upward fluxes could also be induced by low K stress (P < 0.05). This is another evidence demonstrating that the root of K high-efficiency genotypes has a stronger affinity for K+.Potassium high-efficiency vegetable soybean genotypes are more efficient in root architecture adjustment associated with higher specific root K uptake rate  to adapt to low K conditions, which ensures an adequate supply of K . Based oK stress . SuitablK stress . Due to K stress , increasKm) and compensation concentration for K+ uptake (Cmin), and a higher maximum K+ uptake rate (Imax) from K high-efficiency genotypes, compared with K low-efficiency genotypes. Lower Km and Cmin indicate higher affinity between the roots and K+ and stronger ability to use the low-concentration K+, while higher Imax ensures a faster K uptake rate (Potassium kinetic parameters and root activities are also important factors assessing K efficiency when plants suffering low K stress . The preake rate . The absake rate . The stuake rate . In the ake rate , which wake rate . Therefoake rate .Pn of K high-efficiency genotypes decreased by 6.1–6.9% and K low-efficiency genotypes by 10.9–15.7%. Many investigations also indicate that crops with high K efficiency should be more efficient in photosynthesis or that photosynthetic capacity is less affected by low K stress (Pn of K-efficient cotton cultivar Liaomian 18 is 19.4% higher than that of K-inefficient cultivar NuCOTN99B. Besides, photosynthetic parameters of Liaomian 18 were less affected by low K stress (Pn in K high-efficiency genotypes, but they have a higher HI and KHI [the relevant results have been published by Photosynthetic parameters affected by K levels are direct references to characterize the photosynthetic capacity of crops . Hence, K stress . For insK stress . However+ concentration of vegetable soybean, but K high-efficiency genotypes were less affected (Consistent with photosynthetic parameters, Chl content also reflects the photosynthetic activity in leaves . In the affected . This coIt is root uptake power that drives up nutrients transport, while photosynthetic capacity and assimilate redistribution capacity are the key to crop yield . In the + associated with root activity under low K stress is essential to promote root K absorption. The strong drivers, represented with higher root bleeding-sap rate and Tr induced by low K stress, ensure the upward flux of K+ and other essential nutrients as much as possible. The photosynthetic system of K high-efficiency vegetable soybean genotypes is less affected and reasonably regulated by low K stress to maintain photosynthates. Therefore, the ability to redistribute photosynthetic products seemed more important than photosynthetic capacity in K high-efficiency genotypes (The higher affinity of root to Kenotypes . OverallThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.XL and QZ designed the experiments, supervised the study, and revised the manuscript. CL and XW performed the research and wrote the manuscript. HC, BT, and YL helped in planting and data analysis. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Eupolyphaga sinensis Walker polysaccharide (ESPS) has been isolated and characterized, while its antiviral effect on HBV remained unclear. The anti-HBV activity of ESPS and its regulatory pathway were investigated in vitro and in vivo. The results showed that ESPS significantly inhibited the production of HBsAg, HBeAg, and HBV DNA in the supernatants of HepG2.2.15 in a dose-dependent manner; HBV RNA and core protein expression were also decreased by ESPS. The in vivo studies using HBV transgenic mice further revealed that ESPS (20 and 40 mg/kg/2 days) significantly reduced the levels HBsAg, HBeAg, and HBV DNA in the serum, as well as HBV DNA and HBV RNA in mice liver. In addition, ESPS activated the Toll-like receptor 4 (TLR4) pathway; elevated levels of IFN-β, TNF-α, and IL-6 in the serum were observed, indicating that the anti-HBV effect of ESPS was achieved by potentiating innate immunity function. In conclusion, our study shows that ESPS is a potential anti-HBV ingredient and is of great value in the development of new anti-HBV drugs.Hepatitis B virus (HBV) infection remains a major global threat to human health worldwide. Recently, the Chinese medicines with antiviral properties and low toxicity have been a concern. In our previous study,  Hepatitis B virus (HBV) infection remains a major and serious global health problem, and with 257 million chronically infected people worldwide, HBV infection often results in acute or chronic infection and contributes to a high risk of severe liver disease including liver failure, cirrhosis, hepatocellular carcinoma (HCC), or death . Due to Eupolyphaga sinensis Walker (ESW) belongs to the family Corydiidae (Blattodea), which is a kind of important insect and widely distributed in China , fucose (3.1%), arabinose (13.9%), xylose (9.3%), glucose (39.7%), and galactose (26.5%) . We applESPS was prepared in our laboratory . LPS andThe hepatoma cell line HepG2.2.15 was purchased from ATCC (USA), cultured in DMEM medium containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin and incubated in an atmosphere at 37°C and 5% carbon dioxide.Male C57BL/6J mice, weighing 21 ± 2 g, were purchased from Weitong Lihua Laboratory Animal Technology Co., Ltd. , which can freely access to food and water under the conditions of temperature (22–25°C), humidity (55–70%), and 12-h light/dark cycle according to the China Pharmaceutical University Animal Care and Use Guidelines .After 1-week adaptation, 8 μg pAAV-1.2HBV plasmid was injected into the tail vein of C57BL/6J mice by high-pressure hydrodynamic method within 5–8 s . Twenty-4 cells per well, cultured at 37°C with 5% CO2. The cytotoxicity of ESPS on HepG2.2.15 and Huh7 cells was evaluated by CCK8 method . The primers of forward and reverse sequences from 5′ to 3′ were respectively shown in −ΔΔCt.Then expression levels of the following genes were analyzed with quantitative real-time PCR (qPCR) assays using TB Premix Ex Taq in the CFX96 Real-time fluorescence PCR system  or 3TC (2.45 μg/ml) or LPS (1 μg/ml)for 6 days or 16 h. And then, HepG2.2.15 cells were lysed in Ripa  containing 1% PMSF  and 0.01% phosphatase inhibitor . The loading buffer was added into the homogenate, accounting for 20%. After boiling at 100°C for 10 min, the protein extracts were electrophoretically separated on 10% SDS-PAGE gel and then transferred to a polyvinylidene fluoride (PVDF) membrane at 340 mA for 90 min. Then, the membranes were blocked with 5% BSA  for 2 h, incubated with anti-TLR4 , anti-TLR2 , anti-p-P65 , anti-p65 , anti-IRF3 , anti-p-IRF3 , anti-HBc , anti-HBs , anti-HB pol , anti-HNF4α , and anti-β-actin  at 4°C overnight and probed with secondary antibody  conjugated with horseradish peroxidase at 37°C for 2 h. Finally, the bands of protein on the membrane were visualized by 30% acrylamide and DAB Immunohistochemistry Color Development Kit  on a Bio-Imaging system, and the band intensities of proteins were quantified using ImageJ and normalized to β-actin levels.HepG2.2.15 cells were seeded in a 6-well plate with 2 × 10The levels of HBsAg, HBeAg, and IFN-β in HepG2.2.15 cells and mice serum were determined using ELISA kits according to the manufacturer’s instructions.For immunohistochemical investigation , a portip-value <0.05 as statistically significant.All of the experimental data were presented as the mean ± SD. Also, statistical analysis was calculated using GraphPad Prism 8.0. Comparison between groups was performed using one-way ANOVA, while considering a in vitro (p < 0.001) (p < 0.01)  , while a < 0.01) . The int < 0.01) . Therefop > 0.05) (p < 0.05)  and 4 (TLR4) expression in HepG2.2.15 cells. The mRNA and protein levels of TLR2 were not changed ( > 0.05) , while t < 0.05)  after trvia enhancement of the interferon system in vitro.The contribution of host innate immunity on clearance of HBV has been explored, and antiviral mediators such as interferon (IFN) and other immunoglobulin factors are considered to be the first line of antiviral immunity . We wondNF-κB is an important regulator of inflammation, immunity, or apoptosis and other pathological processes . Activatp < 0.05; p < 0.01), respectively.The results showed that the level of phosphorylated NF-κB was significantly increased after 16-h treatment of ESPS , compared with negative control group, while the total level of NF-κB had no significant change. The positive control LPS also increased the phosphorylated level of NF-κB . As showp < 0.01) (p < 0.01) , which is a very important factor for HBV transcription . Thus, w < 0.01) . As expe < 0.01) . Therefoin vitro, compared with the PBS group, the level of serum HBsAg decreased by about 35% at day 8 in the low-dose and high-dose group; the level of serum HBeAg decreased by about 30% at day 12 in the low-dose and high-dose groups (We further investigated the influence of ESPS on HBV replication in a hydrodynamic injection (HI) mouse model. After injection for 24 h, HBsAg-positive mice were randomly divided into four groups, including PBS (model group), low-dose group (20 mg/kg/2 days ESPS treatment group), high-dose group (40 mg/kg/2 days ESPS treatment group), and positive control group (30 mg/kg/2 days 3TC treatment group) . Consiste groups . In the e groups . Similare groups . In addie groups . Moreovein vitro showed that ESPS played an anti-HBV role through innate immune signaling pathway mediated by TLR4. As expected, as shown in The results in vivo and in vitro, and its antiviral activity has a time- and dose-dependent effect. The anti-HBV activity of ESPS may be associated with appropriate activation of TLR4 signaling pathway, which results in the enhancement of I interferon and pro-inflammatory factors. Therefore, the Eupolyphaga sinensis Walker–derived polysaccharide ESPS was valuable for further investigation as a novel innate immune response–associated anti-HBV agent.In this study, we found that ESPS exhibited significant inhibitory activity on the replication of HBV Cell surface receptors play a vital role in identifying invading pathogens and initiating innate immune responses. For example, during HBV infection, both HBV DNA and pgRNA can be recognized by the cyclic GMP-AMP synthase (cGAS) and retinoic acid inducible gene I (RIG-I) receptors to activate the innate immune response ; howeverin vitro was comparable with lamivudine. Our data suggested that ESPS promoted the activation of cellular interferon system, stimulated the host to improve the level of interferon, and played an antiviral role. In addition, the in vitro data showed that ESPS inhibited HNF4α expression through the TLR4/NF-κB/ERK axis, thereby downregulating the activity of HBV core promoter, which may also be one of the mechanisms of ESPS for against HBV (As a nucleoside analogue, lamivudine targets the DNA polymerase of HBV, but its long-term application can cause mutations in the HBV-YMDD gene sequence, leading to HBV resistance . Unlike inst HBV . Since mPolysaccharides are widely distributed in nature. A large number of experiments have proved that many biological polysaccharides have immune and anti-tumor effects . The prein vitro and in vivo via activation of the TLR4 signaling pathway. Our study provided a reference for ESPS as an anti-HBV agent.In summary, we concluded that ESPS inhibited HBV replication


Enterococcus durans strains with the aim to contribute to basic dairy microbiology but also for further application potential and strategy. The total enterococcal count in cheeses reached 3.93 CFU/g (log 10) ± 1.98 on average. Based on a MALDI-TOF mass spectrometry evaluation, the strains were allotted to the species E. durans . The strains were gelatinase and hemolysis-negative (γ-hemolysis) and were mostly susceptible to commercial antibiotics. Among the strains, E. durans ED26E/7 produced the highest value of lactase enzyme β-galactosidase (10 nmoL). ED26E/7 was absent of virulence factor genes such as Hyl , IS 16 element and gelatinase (GelE). To test safety, ED26E/7 did not cause mortality in Balb/c mice. Its partially purified bacteriocin substance showed the highest inhibition activity/bioactivity against Gram-positive indicator bacteria: the principal indicator Enterococcus avium EA5 , Staphylococcus aureus SA5 and listeriae . Moreover, 16 staphylococci (out of 22) were inhibited (100 AU/mL), and the growth of 36 (out of 51) enterococcal indicators was as well. After further technological tests, E. durans ED26E/7, with its bacteriocin substance, can be supposed as a promising additive to dairy products.Slovak ewe’s milk lump cheese is produced from unpasteurized ewe’s milk without any added culture. Because of the traditional processing and shaping by hand into a lump, this cheese was given the traditional specialty guaranteed (TSG) label. Up till now, there have existed only limited detailed studies of individual microbiota and their benefits in ewe’s milk lump cheese. Therefore, this study has been focused on the beneficial properties and safety of  Enterococcus are found in many food products; these foods are animal-derived, e.g., dairy products [Enterococcus faecium, E. faecalis, E. hirae and/or E. durans [p < 0.05), and also higher average daily weight gains were detected. Using Ent 2019 and its producing strain E. faecium EF2019 = CCM7420, a tendency to modulate the serum biochemistry parameters and to improve immunity, jejunal morphometry, weight gains, feed conversion and meat quality was reported [E. faecium [E. durans species have begun to be studied as beneficial microorganisms [E. durans is grouped in the E. faecium group/cluster based on gene similarity 16S rRNA analysis [E. durans strains are able to produce bacteriocins—durancins [E. durans L28-1 isolated from soil. It shows antibacterial activity against lactobacilli, lactococci, carnobacteria and Weissella spp. Durancin TW-49M was characterized as a novel non-pediocin-like Class II bacteriocin produced by E. durans QU49 from carrot [E. durans E204 from camel milk. Anti-listerial activity in ham due to bacteriocin produced by E. durans 152 from a floor drain sample was reported by Lihui Du et al. [Ewe’s milk and cheeses made from it have a high nutritive value, which has led to high demand for those products that is increasing worldwide ,2. The iproducts ,9,10. Th. durans ,8,9,10. . durans ,12; but . durans ,14,15 an. durans ,17. Lauk. durans  reported faecium ,17,18. Hanalysis . Similarurancins ,23,24. Ym carrot . El QuarE. durans strains isolated from ewe’s milk lump cheese and especially on beneficial properties  and safety of the strain E. durans ED26E/7  with the aim to contribute to basic dairy microbiology but also for its further application as an additive. The novelty of this study lies in the fact that E. durans from Slovak local ewe’s milk lump cheese has not been characterized up to now from this aspect. Therefore, knowing its beneficial potential brings a promising perspective for dairy processing, which indicates a practical aspect of the study.Slovak ewe’s milk lump cheese is a popular food article among the population in Slovakia. It is produced from unpasteurized ewe’s milk without any added culture. Ewe’s milk lump cheese should contain at least 47% dry matter and 52% fat in dry matter. Its processing was described in more detail in our previous study . The finThirty-eight (38) ewe’s milk lump cheeses were sampled. Cheeses were made from milk of the Valachian breed, the most represented sheep breed in Slovakia . This brThe processing of ewe’s milk to manufacture milk lump cheese has already been described in our previous study . BrieflyE. durans DSM 20633T DSM). Identical colonies evaluated by the MALDI-TOF score value were excluded. Identified strains were maintained on M-Enterococcus agar  for the next testing and also stored using the Microbank system .Cheese samples were treated by the standard microbiological method ; 10 g of each cheese sample in 90 mL of Ringer solution  was stirred using the Stomacher–Masticator (Spain) and diluted. The appropriate dilutions were plated on Slanetz-Bartley agar and M-Enterococcus agar . The plates were incubated at 37 °C for 48 h. Fifty-nine (59) presumed colonies were streaked on Brain heart agar  enriched with defibrinated sheep blood (5%) to check their purity. Pure colonies were submitted for identification using MALDI-TOF mass spectrometry based on protein “fingerprints“  (MALDI-TE. durans strains was performed based on consensus matrix of tests for identification of Enterococcus spp. as reported by Manero and Blanch [N-acetyl-glucosamine), hippurate hydrolysis, etc. Type strain E. durans ATCC 19432 was used as a control. Phenotypization of d Blanch , also inN-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase. Enzyme activities were evaluated according to the manufacturer’s recommendations (after 4 h of incubation at 37 °C). Color intensity values from 0 to 5 and relevant values in nanomoles (nmoL) were assigned for each reaction according to the color chart supplied with the kit.The following enzymes were tested using the API-ZYM system: alkalic phosphatase, esterase, esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, acidic phosphatase, naphtol-AS-B1-phosphohydrolase, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, Hemolysis was detected by streaking the cultures on MRS agar (Difco) supplemented with 5% of defibrinated sheep blood. Plates were incubated at 37 °C for 24–48 h under semi-anaerobic conditions. The presence or absence of clearing zones around the colonies was interpreted as β-hemolysis and negative γ-hemolysis, respectively .2 in 20% HCl). The loss of turbidity halos around colonies was then checked at 4 °C [Gelatinase is a proteolytic enzyme  that acts on a variety of substrates, such as insulin-beta chain, collagenous material in tissues, the vasoconstrictor endothelin-1, as well as sex-pheromones and their inhibitor peptide . Gelatin at 4 °C .E. faecium CCM4231 [E. faecalis strain ATCC 29212 was a positive control.The antibiotic phenotype was tested using two methods; the disk diffusion method , as well CCM4231 . AntibioE. durans strains, the most frequently detected genes for enterocins production were analyzed: Ent A, P, B, L50B [Ents genes were used according to Aymerich et al. [Ent P, L50B and B was 56 °C instead of 58 °C. The PCR product was visualized by 2% agarose electrophoresis (1 μg ethidium bromide). As positive controls, we used E. faecium EK13 = CCM7419 [E. faecium L50 [Ent L50B and B. Briefly, template (2 µL) was added to 8.75 µL of the reagent mixture, which contained 0.5 µL of each primer, 1 µL of (10 mmoL/L) dNTPs (Invitrogen) and water to a total volume of 50 µL. The sequences of the primer pairs used for PCR amplification of the Ents structural genes are as follows: Ent A, F5′-GGT ACC ACT CAT AGT GC AAA-3′, R 5′-CCC TGG AAT TGC TCC ACC TAA-3′; Ent P, F5′-GCT ACG CGT TCA TAT GGT AAT-3′, R5′-TCC TGC AAT ATT CTC TTT AGC-3′;Ent L50B, F5′-ATG GGA GCA ATC GCA AAA TTA-3′, R5′-TAG CCA TTT TTC AAT TTG ATC-3′; Ent B, F5′-CAA AAT GTA AAA GAA TTA AGA TCG-3′, R5′-AGA GTA TAC ATT TGC TAA CCC-3′. DNA (template) was extracted by the rapid alkaline lysis method [To detect structural enterocin genes in  B, L50B ,39. The h et al. , Casaus h et al.  and Cinth et al. ,43: 5 mi CCM7419  for Ent cium L50 ,43 for Es method .Ents genes possessing strains were tested for antimicrobial activity using the quantitative agar diffusion method [g (at laboratory temperature). Supernatants were concentrated 10-fold using Concentrator plus  to achieve a volume of 4 mL. Their activity was tested against the principal , and against Listeriae from different food products , L. innocua LMG13568  and Staphylococcus aureus SA5 (our strain isolated from mastitis milk). Bacteriocin activity was expressed in arbitrary units per mL, indicating the highest dilution of bacteriocin that can inhibit the growth of the indicator strain. Based on the results of inhibition activity, ED26E/7 was selected to prepare semi-purified bacteriocin (precipitate). Inhibition activity testing was performed in duplicate.n method . For thiproducts  ListeriaE. avium EA5 strain was measured. Therefore, partial purification was performed according to the protocol for durancin 28L [durancin gene was not tested). ED26E/7 was cultivated in 500 mL of MRS  at 37 °C for 18 h. Then the broth culture was centrifuged  for 30 min (min) at 4 °C. The pH of the supernatant was adjusted to 4.2 and filtrated using a 0.45 µm filter . The supernatant was precipitated with ammonium sulfate (80% saturation) at 4 °C overnight (18 h). After precipitation and centrifuging  for 30 min at 4 °C, the precipitate was re-suspended in the minimum volume of phosphate buffer (pH 6.5) and inhibition activity was tested against E. avium EA5. Inhibition activity reached 102,400 AU/mL. Moreover, other indicators were used: S. aureus SA5, L. innocua LMG13568, L. monocytogenes (12) from various food products, various staphylococci from ewe’s milk lump cheese and raw goat milk, E. faecium, E. faecalis, E. hirae from raw goat milk and meat products, E. thailandicus from beavers, and fecal strains E. mundtii from roe and red deers.At first, partial purification of the bacteriocin substance followed the protocol according to Mareková et al.  for Ent ncin 28L , there wE. durans ED26E/7, three specific genes for virulence factors were tested. PCR amplification with the primers and conditions used followed the protocols according to Kubašová et al. [GelE (gelatinase), hylEfm  and Is16 (element IS). The PCR products were separated by means of agarose gel electrophoresis  with 1 µL/mL content of ethidium bromide (Sigma-Aldrich) using 0.5× TAE buffer . PCR fragments were visualized with UV light. E. faecium P36  was the positive control. The PCRs were carried out in 25 µL volume with a mixture consisting of 1× reaction buffer, 0.2 mmoL/L of deoxynucleoside triphosphate, 3 mmoL MgCl2, 1 µmoL/L of each primer, 1 U of Taq DNA polymerase and 1.5 µL of the DNA template, with the cycling conditions as previously reported by Kubašová et al. [To control safety of á et al.  and Lauká et al. . The gená et al.  and Lauká et al. .E. durans ED26E/7 strain, pathogen-free eight-week-old male Balb/c mice  were used. Their weight was around 18–20 g. Mice maintenance conditions were the same as previously reported by Vargová et al. [n = 10) and Group 1 (n = 10). To differ the ED26E/7 strain from other enterococci, its rifampicin-resistant variant was prepared [per os daily at a dose of 109 CFU/mL in a total dose 100 µL. Counts of ED26E/7, as well as other enterococci, were enumerated after standard microbiological dilution of feces and jejunum  and plated on BHI agar enriched with rifampicin (100 µg), and M-Enterococcus agar. Counts were expressed in CFU/g (log10) ± SD. Sampling occurred at the start of the experiment (n = 20) and on days 7 and 30.For the in vivo safety control of á et al. . Mice weprepared . ED26E/7E. durans with evaluation scores from 1.781 up to 2.245 , as well as raffinose. The Voges-Proskauer test was positive; the hydrolysis of esculin showed a dubious reaction and hippurate hydrolysis as well.The total enterococcal count in the screened cheeses reached 3.93 CFU/g (log 10) ± 1.98 on average. Based on MALDI-TOF mass spectrometry evaluation, strains were allotted to the species to 2.245 . Among tE. durans strains produced the enzyme alkaline phosphatase at a slight amount, 5 nmoL, similar to lipase, leucine arylamidase, valine arylamidase, cystin arylamidase, α-chymotrypsin, trypsin and acid phosphatase. The enzyme β-glucuronidase was not produced (except for in ED24E/9), and α-mannosidase and α-fucosidase were produced in 5 nmoL or not produced. However, beneficial hydrolase, β-galactosidase was produced by ED26E/7 strain at the amount 10 nmoL; the other strains either produced 5 nmoL or did not produce this enzyme. Esterase, esterase lipase and naphtol-AS-Bi-phosphohydrolase were also produced in amounts of 5 or 10 nmoL. The amount of β-galactosidase was the highest in the ED26E/7 strain (10 nmoL).E. durans strains did not form hemolysis (γ-hemolysis). The gelatinase test was also negative. The strains were susceptible to all antibiotics using the disk diffusion method with inhibition zones ranging from 10 up to 28 mm except for resistance to clindamycin in one strain, novobiocin, streptomycin or kanamycin. However, E. durans ED26E/7 was susceptible to all antibiotics with an inhibition size ranging from 13 up to 28 mm. The antibiotic profile of this strain was also evaluated using an E-test. E. durans ED26E/7 was susceptible to penicillin (8 µg/mL), ampicillin (2 µg/mL), tetracycline (5 µg/mL), erythromycin and vancomycin (4 µg/mL). MIC breakpoints (µg/mL) for these antibiotics are: P, S ≤ 8/R ≥ 16; Amp, S ≤ 4/R ≥ 16; TCT, S ≤ 4/R ≥ 16;E, S ≤ 0.5/R ≥ 8; Van, S ≤ 4/R ≥ 32.All Ent A and Ent P genes were detected in tested E. durans strains. Ent P genes were found in the strains E. durans ED25E/6, ED26E/1, ED7E/9 and ED26E/7, and the Ent A gene was detected in the strain ED24E/9. ED24E/9 and ED26E/1 did not show inhibition activity against the indicators used. Inhibition activity of strain concentrates ED25E/6, ED7E/9 and ED26E/7 are shown in E. avium was inhibited by concentrates of the strains ED26E/7 and ED7E/9 with inhibition activities of 800 ± 28.2 and 100 ± 12.1 AU/mL. L. monocytogenes CCM4699 was inhibited by concentrates of all three ED strains (100 AU/mL). The other listeriae (five strains) were not inhibited by ED25E/6, and ED7E/9 concentrates; however, concentrate ED26E/7 inhibited the growth of three listeriae .E. coli. Fifty-one enterococcal species strains  were used and 22 staphylococcal species strains  from ewe’s milk lump cheeses and from raw goat milk. S. aureus SA5 (from mastitis) was inhibited with activity 25,600 ± 116.6 AU/mL , 12 strains of L. monocytogenes from different products  were inhibited with inhibition activity in the range from 100 up to 400 AU/mL  and 4 strains of E. hirae (3 from the meat products and 1 from raw milk) with activity 200–6400 AU/mL. Using 22 staphylococcal strains isolated from raw goat milk, only four strains were inhibited (100 AU/mL); 12 strains were from cheeses  (4.61 ± 0.14 CFU/g). It does not cause the mortality of mice.ED26E/7 did not possess genes for E. durans 41D was isolated from Hispanic style cheese. In this study, three out of five identified strains showed identification scores corresponding to secure genus identification/probable species identification ; the scores of the strains ED7E/9 and ED26E/1 corresponded with probable genus identification. However, phenotypic biochemical testing supported characteristics for the species E. durans [Enterococcal counts in ewe’s milk cheese were similar as, e.g., in fermented meat products , but a l. durans .E. durans tested. N-acetyl-β-glucosaminidase was negative or only 5 nmoL; that one enzyme is required to proliferate in vivo [E. durans strains is also promising from a safety aspect. Enterococci considered for industrial applications should be antibiotic-resistant or have virulence factor genes absent [E. faecalis, as the secreted protease can damage host tissue and thus allow bacterial migration and spread infection. However, ED26E/7 was hemolysis and gelatinase-negative. The bacteriocin-producing species strain E. durans was also isolated from important Egyptian cheese OSY-W [E. durans LAB18s isolated from Brazilian soft cheese with the antioxidant activity of its culture supernatant and intracellular extract. Further beneficial/desirable traits may include the production of antioxidants or the expression of pathogens antagonism [E. durans ED26E/7 strain in mice infected with Trichinella spiralis. Moreover, the respiratory burst of blood polymorphonuclear cells was stimulated, which can contribute to a decreased larval migration and destruction of muscle larvae, and therefore, reduced parasite burden in the host. The application of ED26E/7 caused a significant decrease in the number of muscle larvae of T. spiralis and showed the highest inhibition effect on female fecundity (94%) [L. monocytogenes Ohio, its initial count of 6.7 (log 10) CFU/g was decreased to 1.9 CFU/g in one week after enterocin addition. In “Bryndza“, bacterial reduction (expressed in order of magnitude) was noted after Enterocin CCM4231 application.To evaluate strains as safe, no antibiotic resistance and/or virulence factor genes can be present, or their phenotype has to be assessed, as well as no or slight production of damaging enzymes (disease markers). The production of intestinal disorder enzymes, such as β-glucuronidase, α-chymotrypsin or β-glucosidase, was slight in the  in vivo . The tes in vivo . It is ks absent . In genes absent . Furtherse OSY-W . Pieniz se OSY-W  describetagonism . Dvorožňtagonism  reportedty (94%) . Moreovety (94%) ). The imty (94%) . Howeverty (94%) . In our ty (94%) ,61. In SE. durans ED26E/7 was selected from ewe’s milk lump cheese. This gelatinase and hemolysis-negative (γ-hemolysis) strain, most susceptible to commercial antibiotics producing 10 nmoL lactase enzyme β-galactosidase, represents a promising additive for dairy products. ED26E/7 did not possess virulence factor genes, such as hyl , the IS16 element or GelE gene. Its partially purified bacteriocin substance inhibited the growth of Gram-positive indicator bacteria; the principal indicator E. avium EA5, S. aureus SA5 and the other 16 out of 22 staphylococci, listeriae, and also 36 out of 51 enterococcal indicators were inhibited. In the experimental Balb/c mice, ED26E/7 did not cause mortality.It can be summarized that bioactive E. durans ED26E/7 represents a promising beneficial strain for dairy products manufacturing as a natural, bacteriocin-producing adjunct, which can prolong the stability of the products (cheese) by influencing microbiota without negative influence on cheese quality, and with respect for the One Health concept strategy. This study also contributes new alternative approaches for safety assessment in food hygiene/technology and based on previous results, also in feed or food-producing animal production system.It can be concluded that