Secondary metabolites isolated from plant endophytic fungi have been getting more and more attention. Some secondary metabolites exhibit high biological activities, hence, they have potential to be used for promising lead compounds in drug discovery. In this review, a total of 134 journal articles (from 2017 to 2019) were reviewed and the chemical structures of 449 new metabolites, including polyketides, terpenoids, steroids and so on, were summarized. Besides, various biological activities and structure-activity relationship of some compounds were aslo described. Burkholderia gladioli pv. agaricicola (Bga) Yabuuchi has a promising antibacterial activity against the two microorganisms B. megaterium and E. coli . An–E 57–61 . Compounions -octane motif, while kadhenrischinins E–H (97–100) feature acyclopentane ring in a side chain rarely found in the family Schisandraceae  octane motif pyran-2(8H)-one (192), 4-methoxy-6-methyl-5-(2-oxobutyl)-2H-pyran-2-one (193), 6-(hydroxymethyl)-4-methoxy-5-(2-oxobutyl)-2H-pyran-2-one (194) respectively -5-methyl-4-oxo-2-methyl-4,5-dihydrofuran-3 carboxylate ((5S) cis-gregatin B) 195  188  and (R)-188  195 , (5R)-5-195 , one new compound aflaquinolone H 221  with pyranoindole moieties, amoenamide C 227  . In. InZiziplide 253  was obta benzoic acid (346) -1(4H)-2′-naphthodioxin-4-one  pyrazine-1,4-dione 390 , isolated as endophyte of the medicinal plant Vochysia divergens, revealed two new carboxamides, vochysiamides A 394 -5-chloro-4-hydroxy-6-methoxymellein 404 –4–hydroxymethyl–3,5–dimethyldihydro–2–furanone . O. OTalaro319  . Nine pr. A. ATalaro366  Bunge . The endnine 368  and myco368 -377  . The stuione 390  126]. A. ATalaro390 -7-c404 , Verticillium dahliae (ACCC 36916), Fusarium oxysporum (ACCC 37438), Alternaria solani (ACCC 36023), Fusarium gramineum (ACCC 36249), and Rhizoctonia solani (ACCC36124) obtained from Agricultural Culture Collection of China (ACCC). The antifungal assay displayed that 1 and 5 exhibited pronounced biological effects against F. oxysporum with MIC (minimum inhibitory concentration) value of 8 g/mL, whereas 5 can potently inhibited F. gramineum at concentration of 8 g/mL, compared with the positive control ketoconazole   thymidine assay using breast cancer (MCF-7) and colon cancer (COLO-205) cell lines. Doxorubicin (10 µg), was used as a positive control with ED10 µg/mL .363 was evaluated for its cytototoxicity against different cancer cell lines MOLT-4, A549, MDA-MB-231and MIA PaCa-2 by MTT assay. Interestingly, compound 363 showed considerable cytotoxic potential against the human leukaemia cancer cell line (MOLT-4) with IC50 value of 20 µmol/L, it was not as active as the positive control flavopiridol  [Compound  µmol/L) .365 was evaluated. In the cytotoxic assay, compound 365 displayed weak in vitro cytotoxicity against the THP-1 cell line, with IC50 value of 40.2 µM [Cytotoxicity against four tumor cell lines  of compound  40.2 µM .389 was evaluated in vitro towards ovarian (SK-OV-3), epidermoid (KB), malignantmelanoma (SK-MEL), human breast adenocarcinoma (MCF-7), colorectal adenocarcinoma (HCT-116), and ductal (BT-549) carcinomas. Doxorubicin (positive control) and DMSO (negative control) were used. It had selective and potent effect towards BT-549, MCF-7, SKOV-3, and HCT-116 cell lines with IC50s 0.09 ± 0.05, 21 ± 0.07, 1.23 ± 0.03, and 0.59 ± 0.01 µM, respectively, compared to doxorubicin . Fusarithioamide B (389) may provide a lead molecule for future developing of antitumor and antimicrobial agents [The cytotoxic effect of l agents .395 displayed low activity against human non-small cell lung A549 and human prostate PC3 cell lines  = 86.4 μM for 395, PC3: EC50 = 40.25 μM for 395. 1.5 mM hydrogen peroxide was used as positive control (100% dead cells), 0.1% dimethyl sulfoxide was used as negative control (100% live cells) [In the cancer cell line cytoxicity assays, compound e cells) .396–397 were evaluated for their cytotoxic activity against four human tumor cell lines  by the SRB (Sulforhodamine B) method. As a result, compounds 396, 397 showed weak inhibitory activities against the four tumor cell lines with IC50 values ranging from 30 to 100 µM . Cisplatin was used as a positive control with IC50 values of 3.39 ± 0.29 µM, 3.19 ± 0.12 µM, 2.42 ± 0.14 µM and 1.56 ± 0.08 µM against the four human tumor cell lines [Compounds 50 values against HL-60, A-549, SMMC-7721, MCF-7, and SW480 cell: 4.05 ± 0.11, 19.40 ± 0.71, 14.91 ± 0.36, 22.96 ± 0.58 and 23.15 ± 0.22 μM). Compound 447 demonstrated moderate cytotoxicity against HL-60, A-549, SMMC-7721, MCF-7, and SW480 cell with IC50 values of 15.80, 15.93, 19.42, 19.22, and 23.03 μM, respectively [The in vitro cytotoxicity assay was performed according to the MTS method in 96-well microplates. Five human tumor cell lines were used: human myeloid leukemia HL-60, human hepatocellular carcinoma SMMC-7721, lung cancer A-549, breast cancer MCF-7, and human colon cancer SW480, which were obtained from ATCC . Cisplatin was used as the positive control for the cancer cell lines  as a positive control. Compounds 7 and 249 showed moderate inhibitory activity against α-glucosidase with IC50 values of 188.7 µM and 178.5 µM, respectively. The results indicated that the configureuration at C-5 in compounds 6 and 7 might affect α-glucosidase inhibitory activity. Moreover, the methoxy group at C-15 in the lasiodiplodin derivatives decreased the activity . For co  was used as a positive control of inhibition ratio at 52.63% with the concentration of 0.333 μM [AChE inhibitory activities of the compound Chattonella marina, Heterosigma akashiwo, Karlodinium veneficum, and Prorocentrum donghaiense by 31–37 were assayed. The results showed that 32–34 were more active to C. marina, K. veneficum, and P. donghaiense than 31 and 35–37 . A structure-activity relationship analysis revealed that the phenyl group in 32–34 may contribute to their inhibitory ability, but the isomerization at C-9 and/or C-11 of 32–37 only has slight influences on their activities. K2Cr2O7 was used as positive control with IC50 values of 0.46, 0.98, 0.89 and 1.9 μg/mL, respectively [The inhibition of the marine phytoplankton ectively .38 were evaluated on the seedling growth of Arabidopsis thaliana, and 38 displayed an effect on the root growth but no remarkable inhibition of leaf growth in Arabidopsis thaliana [The biological effects of compound thaliana .50 = 2.0 μM) and methanol were used as positive and negative controls, respectively. 49 and 413 showed remarkable antioxidant activity with IC50 values of 2.50 and 5.75 μM respectively [The antioxidant activity was estimated by using adapted 2, 2′-diphenyl-b-picrylhydrazyl (DPPH) method. Ascorbic acid  [The biological activity properties of compounds 0.21 μM) .76 was assayed for agonistic effects on PXR. Compound 76 displayed the significant agonistic effect on PXR with EC50 value of 134.91 ± 2.01 nM [Nuclear transcription factor (PXR) can regulate a suite of genes involved in the metabolism, transport, and elimination of their substances, such as CYP3A4 and MRP, therefore, it is regarded as an important target to treat cholestatic liver disorders. So compound  2.01 nM .80 displayed brine shrimp inhibiting activities with IC50 value of 10.1 μmol/mL. The SDS (sodium dodecyl sulfate) was employed as positive control and its inhibiting ratio was 95% for brine shrimp and LC50 0.6 μmol/mL [Brine shrimp inhibiting assay was assayed. Compound  μmol/mL .82–92 were evaluated for their inhibitory activity against NO production in LPS-activated RAW 264.7 marcrophages, while indomethacin was used as a positive control. Compounds 89–91 exhibited inhibitory effects with IC50 values of 21, 24 and 16 μM, respectively, which are lower than that of the positive control indomethacin (IC50 = 38 ± 1 μM), while compound 85 exhibited moderate inhibition with an IC50 value of 42 μM. Preliminary structure–activity relationships revealed that the analogues with the S absolute configureuration at C-18  significantly enhanced the activity, as exemplified by compound 89 showing inhibition against NO production in RAW 264.7 marcrophage cells with an IC50 value of 21 μM, whereas compound 87 exerted less than 40% inhibition at 50 μM. In addition, all isolated compounds (82–92) were tested for their inhibitory activity of Mycobacterium tuberculosis protein tyrosine phosphatase B (MptpB). Compound 89 displayed inhibition with an IC50 value of 19 μM, comparable to the positive control . Compounds 83, 85, 86 and 90 showed moderate inhibitory activity of MptpB with IC50 values of 39 ± 2 μM, 42 ± 3 μM, 28 ± 1 μM and 35 ± 1 μM, respectively [Monitoring the NO level in LPS-activated cells has become a common approach for evaluating the potential anti-inflammatory activities of compounds. Isolates n IC50 =  ± 1 μM, 135–146 were evaluated for their inhibitory effects on the NO production in LPS-stimulated RAW264.7 microglial cells using Griess assay. Meanwhile, the effects of compounds 135–146 on cell proliferation/viability were measured using the MTT method. As a result, compounds 138, 139, 142, 143, 145 and 146 exhibited inhibitory activity against NO production with IC50 values in the range of 56.3–98.4 μM . Indomethacin was used as a positive control (IC50 = 33.6 ± 1.4 μM) [Compounds 168 was investigated by applying D. S. Kim’s method. Compound 168 exhibited inhibitory activities on ATP release of thrombin-activated platelets with IC50 value of 57.6 ± 3.2 μM. Staurosporine served as the positive control with IC50 value of 3.2 ± 0.6 μM [Measurement of ATP release of thrombin-activated platelets of the isolated compound ± 0.6 μM .Staphylococcus aureus DSM 1104 was tested in 96-well tissue microtiter plates. The compounds were tested in concentrations of up to 256 µg/mL. MeOH and cytochalasin B were used as negative and positive control, respectively. Minimum Inhibitory Concentration (MIC) value of 256 µg/mL was observed for metabolite 169 and it showed a weak inhibition of biofilm formation of 20.78% at 256 µg/mL [The inhibition of biofilm formation against 56 µg/mL .176–180 was performed. 1-deoxynojirimycin  was used as a positive control. In addition, The DPPH radical scavenging assay of these compounds was also conducted with 96-well plates using a revised method. The positive control was used by Vitamin C. Compounds 176–178 showed significant α-glucosidase inhibitory activity with IC50 values of 35.8 μM, 53.3 μM and 60.2 μM, respectively, compared to 62.8 μM for the positive control (1-deoxynojirimycin). Moreover, compound 179 exhibited radical scavenging activity against DPPH with EC50 value of 68.1 μM, the EC50 value of positive control ascorbic acid was 22.3 μM [A colorimetric α-glucosidase  assay of compounds  22.3 μM .200, 276, 344–346 were investigated for their capacity to inhibit biofilm formation in the reference strains of S. aureus, E. faecalis and E. coli. Aacetylquestinol 276, 345 and 200 were found to cause a significant reduction inbiofilm production by E. coli ATCC 25922 with the percentage of biofilm formation: 50.6 ± 17.6%, 23.7 ± 24.8% and 57.6 ± 8.1%, respectively. On the other hand, emodin 344 and 345 showed inhibition of biofilm production in S. aureus ATCC 25923 (21.1 ± 11.5% and 21.8 ± 18.9%). Interestingly, 345, which is the most effective in inhibiting biofilm formation in E. coli ATCC 25922, also caused nearly 80% reduction of the biofilm production in S. aureus ATCC 25923 [The tested compounds CC 25923 .207 was evaluated for its acetylcholinesterase (AChE) inhibitory activity using the Ellman colorimetric method, it showed weak AChE inhibitory activity with the inhibition ratio of 11.9% at the concentration of 50 μmol/mL [Compound  μmol/mL .210–211 were evaluated by measuring the inhibitory activity of nitric oxide (NO) production levels in the lipopolysaccharide (LPS)-induced RAW264.7 macrophage cells. 210–211 exhibited moderate inhibitory activities on NO production in LPS-stimulated RAW264.7 cells without cell cytotoxicities [The anti-inflammatory activities of the isolated compounds xicities .224–225 and the parent compound L  exhibited comparable activity with the positive control ibuprofen (EC50 = 19.4 ± 0.7 nM). The transformed products 224–225 and L all exhibited considerable neuroprotective activity in the invitro LPS-induced neuro-inflammation injury assay, suggesting that the hupA moiety shared by these compounds may be used as a lead structure for the development of neuroprotective drugs [The transformed products mpound L  were evave drugs .228. Compound 228 displayed remarkable insecticidal activities against first instar larvae of the cotton bollworm Helicoverpa armigera with mortality rates of 70.2%. Commercially-available matrine was used as positive control, causing 87.4% mortality rate under the same conditions. Acute cytotoxicity towards hatching rate, malformation and mortality of zebrafish embryos or larvae were also performed. Compounds 227 and 228 significantly decreased the hatching rate of zebrafish embryos, compound 228, used at concentrations of 5–100 μg/L, decreased the hatching rate of zebrafish embryos to below 20% [The artificial insect mixed drug method was used to determine the insecticidal activities of compound elow 20% .246 against lettuce seedlings (Lactuca sativa L.) was studied. Aqueous solutions of 246 ranging between 25 and 200 μg mL−1, were assayed for its effects on seed germination, root length, and shoot length of the lettuce. Compound 246 showed the most robust inhibitory effect on root growth. Compound 246 inhibited root growth by 50% at a concentration of 25 µg/mL. In addition, the highest concentration of 246 (200 µg/mL) strongly exerted an inhibitory effect on seed germination (90% inhibition) [The potential phytotoxicity of ibition) .256–257 were investigated for their inhibitory activities against the LPS-activated production of NO in RAW264.7 cells using the Griess assay with indomethacin as a positive control (IC50 = 37.5 ± 1.6 μM). The effects of compounds on cell proliferation/viability were determined using MTT method, and none of the test compounds exhibited cytotoxicity at their effective concentrations. Compounds 256 and 257 showed strong inhibitory effects on the production of NO, with IC50 values of 0.78 ± 0.06 and 1.26 ± 0.11 μM, respectively [Compounds ectively .258–261 were evaluated in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. 258–261 exhibited excellent inhibitory effects on the production of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and nitric oxide (NO) in LPS-induced macrophages with the IC50 values ranging from 16.21 ± 1.62 μM to 35.23 ± 3.32 μM, from 19.83 ± 1.82 μM to 42.57 ± 4.56 μM, from 16.78 ± 1.65 μM to 38.15 ± 3.67 μM, respectively, similar with the positive control indomethacin. Those results indicated that, terrusnolides A–D (258–261) might play a significant role as a lead compound in the study of anti-inflammatory agents. In addition, compounds 258–261 were also investigated for the inhibitory activities against BACE1 by M-2420 method and acetylcholin esterase (AchE) using Ellman’s method. Compound 260 exhibited weak AchE inhibitory activity with IC50 value of 32.56 ± 3.16 µM, compound 261 exhibited weak BACE1 inhibitory activity with IC50 value of 37.45 ± 4.56 µM. LY2811376 and Donepezil were used as the positive control in BACE1 and AchE inhibitory assay with IC50 values of 0.25 ± 0.04 µM and 0.05 ± 0.01 µM, respectively [In vitro anti-inflammatory effects of compounds ectively .284–286 were carried out. The results showed that compound 285 possessed significant inhibitory activity against IDO with IC50 value of 0.11 μM. Epacadostat, as the positive control, was one of the most potent IDO inhibitors with IC50 value of 0.05 μM. For compounds 284 and 286, they showed relatively strong inhibitory activity with IC50 values of 1.47 μM and 6.36 μM, respectively [The Indoleamine 2,3-dioxygenase (IDO) inhibitory activity assay of compounds ectively .288 was investigated for its effects on NF-κB pathway by reporter gene assay. The results showed that it could activate the NF-κB pathway with increments in the relative luciferase activity at a concentration of 50 μM [NF-κB has been considered as an attractive therapeutic target for the cancer research. Compound of 50 μM .295 and 296 were investigated by seed germination test on lettuce (Lactuca sativa L.) with 2,4-dichlorophenoxyacetic acid (0.3 µg/mL) as the positive control. Compounds 295 and 296 each inhibited the growth of both roots and hypocotyls at 30 µg/mL. Furthermore, 295 suppressed seed germination at 100 µg/mL [The phytotoxic activities of 00 µg/mL .302 were assayed by the spectrophotometric method developed by Ellman with modification. 302 showed weak AChE inhibitory activity (The percentage inhibition was at 20%~60% in 50 μM) [Acetylcholinesterase (AChE) inhibitory activities of the compound n 50 μM) .306–308 from Fusarium sp. was assessed in an attempt to explore their activity against 5-LOX. It is noteworthy that 306 displayed prominent 5-LOX inhibitory activity with IC50 value of 3.61 μM, compared to that of indomethacin (IC50 = 1.17 μM), while 307 and 308 had moderate activity with IC50 values of 7.01 μM and 4.79 μM, respectively [The 5-lipoxygenase (5-LOX) inhibitory potential of ectively .313 displayed moderate activities [α-Glucosidase inhibitory activity was performed in the 96-well plates and acarbose was used as the positive compound. In the inhibitory assay against α-glucosidase, compound tivities .314–317 were evaluated as inhibitory activities against lipopolysaccharide (LPS) induced nitric oxide (NO) production in RAW264.7 cell lines. Compound 317 showed the most NO inhibitory effects, with the inhibition of 17.4% NO production in LPS stimulated RAW264.7 cells at 10 μM. At the same concentration, compound 315 significantly inhibited the NO production, with 11.2% inhibitory rate. Compound 314 showed weak NO inhibitory effects at 10 μM, with inhibitory rates of 6.5%. At the same concentration, quercetin, the positive control, inhibited NO production to 12.9% [The anti-inflammatory activities of selected isolated 4 compounds to 12.9% .331 was investigated. It displayed strong antioxidant activity with EC50 value of 1.08 mg/mL on superoxide anion racdicals. Ascorbic acid (Vc) was used as positive control with EC50 value of 0.33 mg/mL [The Superoxide anion radical scavenging activity of compound 33 mg/mL .333 and 334 were subjected to motility inhibitory and zoosporicidal activity tests against P. capsici (Phomopsis capsici). Compounds 333 and 334 showed more than 50% motility inhibitory activity (IC50) at a concentration of 50−100 μg/mL [Compounds 00 μg/mL .379–385 were assayed for their inhibitory activities against hCE 2. Loperamide was used as a positive control with IC50 value of 1.31 ± 0.09 µM. Compounds 379, and 383–385 displayed significant inhibitory activities against hCE 2 with IC50 values of 10.43 ± 0.51, 6.69 ± 0.85, 12.36 ± 1.27, 18.25 ± 1.78 µM, respectively [Human carboxylesterases (hCE 1 and hCE 2) are the important enzymes that hydrolyze chemicals with functional groups, such as a carboxylic acid ester and amide, and they are known to play vital roles in drug metabolism and insecticide detoxication. The isolated compounds ectively .386 were evaluated. The results demonstrated that bysspectin A 386 was a novel and highly selective inhibitor against hCE2 with the IC50 value of 2.01 µM. Docking simulation also demonstrated that active compound 386 created interaction with the Ser-288  of hCE2 via hydrogen bonding, revealing its highly selective inhibition toward hCE2 [The inhibitory effects on human carboxylesterases  of compound ard hCE2 .392–393 were also evaluated for growth inhibition activity against newly hatched larvae of H. armigera Hubner. Compounds 392 and 393 showed growth inhibition activities against newly hatched larvae of H. armigera Hubner with the IC50 values of 150 and 100 µg/mL, respectively. Azadirachtin was used as positive control with the IC50 value of 25 µg/mL [Compounds 25 µg/mL .403 was determined by DPPH assay and compared with the positive control BHT. Compound 403 showed moderate antioxidant activities with IC50 value of 120.1 ± 11.7 μg/mL [Antioxidant activity of the compound .7 μg/mL .406–407 were subjected for determination of the xanthine oxidase (XO) inhibitory activity using microtiter plate based NBT assay. Allopurinol was used as a positive control with IC50 value of 0.18 ± 0.02 µg/mL. 406 and 407 showed XO inhibitory activity with IC50 values of 2.81 ± 0.71 and 0.41 ± 0.1 µg/mL, respectively. The oxidized form of 406 also showed high XO inhibition with IC50 value of 0.35 ± 0.13 µg/mL [The new compounds 13 µg/mL .421 was tested for osteoclastic differentiation activity using murine macrophage derived RAW264.7 cells. 421 significantly increased the number of mature osteoclasts at the comparable levels to the positive control of kenpaullone, compared to the negative control (DMSO), suggesting that 421 activated a signaling pathway in osteoclastic differentiation [Compound ntiation .432 was carried out using a published protocol. The new compound (−)-dihydrovertinolide 432 exhibited phytotoxicity against lettuce seedlings at a concentration of 50 mg/L [Phtotoxicity assay against lettuce seedlings of compound  50 mg/L .436 showed significant inhibitory activity against NO production in LPS-induced RAW264.7 cells with an IC50 value of 1.9 μM. They were also evaluated for in vitro antidiabetic activities based on the inhibition of alpha-glucosidase, PTP1b, and XOD. Compounds 437 and 441 showed moderate inhibitory activities toward XOD and PTP1b, respectively, at 10 μM with inhibition rates of 67% and 76% [All new compounds were tested for in vitro anti-inflammatory activities against nitric oxide production in liposaccharide (LPS)-induced RAW264.7 cells, and dexamethasone was used as the positive control. Compound  and 76% .447 was tested for acetylcholinesterase (AChE) inhibitory activities using the Ellman method with tacrine as the positive control. The results revealed that compound 447 showed weak AChE inhibitory activity wth IC50 value of 23.85 ± 0.20 μM. Tacrine are the positive control used to estimate AChE inhibitory activity with IC50 value of 0.26 ± 0.02 μM [New compound  0.02 μM .All information about the new compounds are briefly summarized in the From 2017–2019, a total of 449 new secondary metabolites isolated from plant endophytic fungi using different culture method like common culture, co-culture with bacteria, addition of metal ions and so on, were summarized in this review. These compounds have a variety of unique structures, the difference in structure leads to various biological activities of these compounds. Some of these metabolites display significant antimicrobial effects, cytotoxic activities, antioxidant activities and other biological activities, which indicate that they have potential to be agents to treat some diseases. In this review, structure-activity relationships of some compounds were also reviewed.2 into fermentation medium of an endophytic fungus P. citrinum 46, two compounds were isolated. The results showed that adding Cu2+ into medium to activate silent fungal metabolic pathways can increase the discovery of new compounds.According to genome sequencing, a lot of microorganisms have the potential to produce secondary metabolites with novel structures. However, many fungal gene clusters may be silent under standard laboratory growth conditions. As a result, some pathways to yield secondary metabolites cannot be expressed. Therefore, activating these pathways means that we can get more novel compounds. The approach of microorganism co-culture, involving the cultivation of two or more microorganisms in the same lab environment can do a favour for us. Interestingly, 29 new compounds summarized above were obtained through co-culture of bacteria and fungi or two fungi. Besides, by adding CuClBecause the compounds mentioned above were isolated from endophytic fungi in different parts of different plants in different regions, they have a variety of structures and biological activities. In addition to anti-tumor and anti-microbial activities, some compounds also exhibit unique biological activities. Among them, 7 compounds showed weak to moderate AChE inhibitory activity. Some compounds exhibited moderate to potent α-glucosidase inhibitory activity compared with those of positive control. By using adapted 2,2′-diphenyl-b-picrylhydrazyl (DPPH) method, a few of compounds were found to show moderate to remarkable antioxidant activity. Some of them also showed weak to significant inhibitory activity against NO production in LPS-induced RAW264.7 cells. The biological activity properties of 18 compounds were evaluated for inhibitory activity against some enzymes like pancreatic lipase, the 5-lipoxygenase (5-LOX), the Indoleamine 2,3-dioxygenase (IDO), Mycobacterium tuberculosis protein tyrosine phosphatase B (MptpB), the xanthine oxidase (XO) and so on, they showed weak to high inhibition.Endophytic fungi isolated from different parts of plants are a huge treasure house on account of the discovery of novel secondary metabolites with biological activities and unique structures. Since the endophyte resources were discovered, more and more researches have been conducted on them. Just from my review article, the new secondary metabolites isolated from plant endophytes during the three years from 2017 to 2019 were counted. Among them, 38 articles were published in 2017, 136 new compounds were obtained; 39 articles were published in 2018, 117 new compounds were obtained; 57 articles were published in 2019, and 196 new compounds were obtained. It can be discovered that in the past three years, the research trend of plant endophytes and their metabolites have increased year by year. The more new compounds obtained, the greater the possibility of screening compounds with excellent biological activity. This is also an important significance for researchers to study plant endophytes. Through this review, i hope to arouse more people’s interest and attention in this field and screen out compounds with good biological activities to create a better life for mankind by utilizing endophytes resources.


Autophagy flux is the rate at which cytoplasmic components are degraded through the entire autophagy pathway and is often measured by monitoring the clearance rate of autophagosomes. The specific means by which autophagy targets specific cargo has recently gained major attention due to the role of autophagy in human pathologies, where specific proteinaceous cargo is insufficiently recruited to the autophagosome compartment, albeit functional autophagy activity. In this context, the dynamic interplay between receptor proteins such as p62/Sequestosome-1 and neighbour of BRCA1 gene 1 (NBR1) has gained attention. However, the extent of receptor protein recruitment and subsequent clearance alongside autophagosomes under different autophagy activities remains unclear. Here, we dissect the concentration-dependent and temporal impact of rapamycin and spermidine exposure on receptor recruitment, clearance and autophagosome turnover over time, employing micropatterning. Our results reveal a distinct autophagy activity response profile, where the extent of autophagosome and receptor co-localisation does not involve the total pool of either entities and does not operate in similar fashion. These results suggest that autophagosome turnover and specific cargo clearance are distinct entities with inherent properties, distinctively contributing towards total functional autophagy activity. These findings are of significance for future studies where disease specific protein aggregates require clearance to preserve cellular proteostasis and viability and highlight the need of discerning and better tuning autophagy machinery activity and cargo clearance. Macroautophagy is a major intracellular degradation pathway critical in protein removal and the maintenance of cellular homeostasis . Its dysThree variants of autophagy exist, of which macroautophagy is the most characterised and best explored. Macroautophagy, hereafter referred to as autophagy, is primarily activated during starvation conditions and is particularly involved in cytoplasmic bulk degradation targeting mainly long-lived proteins. In doing so, this process generates metabolic substrates and maintains the cell’s energetic state ,17. BasaGiven the crucial role of autophagy activity in controlling neuronal fate through enhanced clearance of particular protein cargo, this relationship deserves urgent attention. The nature of the relationship between autophagosome turnover and receptor clearance is currently largely unclear. It is also unknown to what extent mTOR-dependent or -independent autophagy induction may govern such change in the autophagosome pool, autophagosome turnover and receptor clearance. In light of these complexities, we aimed to examine the relationship between autophagosome turnover and selective receptor recruitment and clearance, focusing on the two predominant cargo receptors, p62 and NBR1. By employing rapamycin  and sper2 at 37 °C. MEFs were sub-cultured using trypsin  to detach adherent cells from flasks. After trypsinisation, DMEM was added in a 2:1 ratio and cells were collected into either a 15 mL  or a 50 mL Falcon tube  depending on the size of the original culture flask. Cells were centrifuged at 1500 rpm for three minutes at room temperature . Media was discarded and cells were resuspended in fresh DMEM. Cells were re-seeded into either T25 , T75  or T175  culturing flasks or onto in-house fabricated fibronectin patterned coverslips for micropatterning purposes  (p < 0.05)]. Additionally, LAMP2a protein levels following H Rapa at 24 h −Baf were also shown to be significantly higher than abundance at 2 h −Baf [1.15 ± 0.11 (p < 0.05)], and at 24 h +Baf these were significantly higher than at 2 h +Baf [1.60 ± 0.29 (p < 0.05)]. These data indicate that H Rapa induces a slow, but gradual, increase in the overall LAMP2a protein abundance. To our surprise, in this model system, p62 and NBR1 protein levels remained largely unchanged. Although p62 abundance was increased in Con +Baf [2.72 ± 0.65 (p < 0.05)] compared to the Con, demonstrating effective basal clearance, there was no other condition during which Rapa induced a change in p62 abundance (p < 0.05)] following L Rapa treatment was noted, which returned to basal levels at 8 h.LC3-II is recruited to phagophores and is a key component of mature autophagosomes. It is therefore a widely used marker to assess their relative abundance. Following H and L Rapa treatments, a significant increase in LC3-II abundance was observed at all given time points compared to the control (Con), except at 24 h following L Rapa exposure both in the presence (+Baf) and absence (−Baf) of Baf  A. Whilst< 0.05)] B. Indeedbundance C. Similabundance D. Howevep < 0.05)] (p < 0.05)]. However, this increase was relatively short lived as levels returned to basal at 24 h +Baf [4.38 ± 1.37 (p < 0.05)]. Taken together, both H Sper and L Sper concentrations elicited a robust autophagosome turnover, which was maintained over 24 h. LAMP2a protein levels remained relatively unchanged following H Sper (p < 0.05)] compared to both Con +Baf and 2 h +Baf . To our surprise, a robust response in receptor protein abundance was observed, especially in cells treated with H Sper (p < 0.05)] at both 8 h −Baf and 24 h −Baf  (p < 0.05)] as well as 24 h −Baf and 24 h +Baf . Additionally, both 8 h −Baf and 24 h −Baf treatment groups displayed significantly higher NBR1 protein levels than cells at 2 h −Baf [1.27 ± 0.04 (p < 0.05)], suggesting a slow increase in NBR1 that reaches significance after 8 h, but not reflective of any flux. Additionally, an initially increased abundance of NBR1 at 2 h −Baf was detected, subsided at 8 h −Baf and 24 h −Baf.Following H Sper treatment LC3-II protein abundance significantly increased compared to Con at 2 h +Baf and 24 h +Baf  A. Althoug H Sper B. Howeveh H Sper C. H Sper< 0.05)] C, indica< 0.05)] D. Here, The interaction between autophagosomes and their movement towards lysosomes has been previously established , includip < 0.05)] and absence of Baf  were significantly higher than at 24 h −Baf and 24 h +Baf . Although p62 levels suggested the presence of a turnover under basal autophagy flux conditions, since counts of Con +Baf [88.08 ± 9.50 (p < 0.05)] were significantly higher than in Con, there was no enhanced p62 clearance at any other time point for the duration of the experiment. Only cells at 2 h −Baf and 8 h −Baf  showed p62 puncta significantly higher than the Con (p < 0.05)] compared to Con, indicating an increase in the recruitment of p62 to the autophagosome (p < 0.05)], demonstrating a gradual change in the recruitment of p62 over time following H Rapa exposure. Similarly, results revealed the presence of a basal NBR1 turnover as its levels at Con +Baf [168.17 ± 28.34 (p < 0.05)] were significantly higher than those at Con (p < 0.05)], albeit NBR1 puncta gradually decreased at 24 h −Baf [8.75 ± 3.31 (p < 0.05)] compared to puncta at 2 h −Baf. The GFP-LC3/NBR1 co-localised puncta significantly increased compared to the Con +Baf at both 2 h +Baf and 8 h +Baf , suggesting effective receptor recruitment (p < 0.05)]. Indeed, increased NBR1 flux at 2 h −Baf was observed, which was lost after this time point. Of note, the overall total number of NBR1 puncta co-localising with GFP-LC3 was by far smaller than the number of p62 puncta co-localising with GFP-LC3 ] at both 2 h and 8 h  (p < 0.05)] and 2 h +Baf as well as between 8 h −Baf [78.46 ± 14.60 (p < 0.05)] and 8 h +Baf, suggesting a prolonged period of enhanced autophagy machinery turnover. It is also worth noting that, unlike in the H Rapa treatment group, GFP-LC3 puncta counts were significantly higher than those in Con at 24 h −Baf [102.79 ± 11.07 (p < 0.05)]. Although p62 puncta counts were significantly increased at 2 h −Baf [92.33 ± 15.25 (p < 0.05)], an increase in their clearance was only observed at 8 h  (p < 0.05)] and 8 h  (p < 0.05)] (GFP-LC3 puncta counts were significantly increased compared to their +Baf treated counterparts [64.13 ± 9.07 (< 0.05)] A. Additi< 0.05)] B. Howeve< 0.05)] C,F, sugg< 0.05)] D,E. Addi< 0.05)] G.p < 0.05)] and 8 h  (p < 0.05)] (p < 0.05)] having been significantly higher than in the Con. A return to basal receptor clearance levels was observed at 24 h of treatment . GFP-LC3/p62 co-localised puncta mirrored these results (p < 0.05)] and 8 h  of treatment intervention (p < 0.05)] (p < 0.05)], suggesting robust recruitment to the autophagosome membrane. This supports previous work by Kirkin et al.  A, demons< 0.05)] B. Howeve results C,G. NBR1rvention D. Howeve< 0.05)] E,G, brinn et al.  demonstrp < 0.05)] and 8 h  following L Sper treatment (p < 0.05)] relative to both 2 h +Baf and 8 h +Baf, suggesting diminishment of the overall autophagosome pool size after 24 h of L Sper treatment. Strikingly, p62 puncta were effectively cleared at 8 h  as well as 24 h , with a greater receptor clearance rate at the 8 h time point (p < 0.05)] and 8 h  (p < 0.05)] compared to control levels and the 2 h −Baf group [134.50 ± 23.58 (p < 0.05)] at 24 h −Baf [266.92 ± 71.82 (p < 0.05)]. Taken together, by using quantitative fluorescence-based single cell analysis, a tremendous increase in both sensitivity and accuracy of the measurements was achieved, evident through the discernment between the total receptor puncta count and their extent of recruitment and co-localisation with GFP-LC3 positive structures.Effective autophagosome turnover was detected at 2 h  C,F, supp< 0.05)] D. HoweveAutophagy is a crucial role player in the maintenance of cellular homeostasis, in particular proteostasis . The selOur results reveal that treatment with rapamycin and spermidine at different concentrations resulted in unique and dynamic changes in the autophagy system. Both interventions impacted and changed the expression profiles of key autophagy-related proteins in a distinct manner. Firstly, rapamycin induced a significant increase in LC3-II protein abundance greater than the control both in the presence and absence of Baf at multiple time points A, while Although it is known that both rapamycin and spermidine induce an increase in autophagosome and receptor puncta counts ,53, how In the present study, both rapamycin and spermidine significantly increased the number of autophagosomes and the abundance of cargo receptors at distinct concentrations. We highlight that, when effective autophagosome turnover is present, there is typically, but not always, effective cargo receptor clearance that follows. However, such effective clearance of receptor-associated autophagosomes engages typically one or the other receptor, not both concomitantly. It should be noted that the number of receptors that co-localised with autophagosomes was far less than expected, given their total cytoplasmic abundance. Indeed, a large reserve pool of non-recruited receptor protein clusters remained in the cytoplasm, demonstrating reserve capacity of the cell to fully engage in receptor recruitment upon favourable stimulation. It may also suggest that substantial alternative receptor functions be held . Indeed,NBR1 has been shown to play several roles within the cell to maintain cell survival ,63,64,65


Aspergillus falconensis, isolated from sediment collected from the Canyon at Dahab, Red Sea, yielded two new chlorinated azaphilones, falconensins O and P (1 and 2) in addition to four known azaphilone derivatives (3−6) following fermentation of the fungus on solid rice medium containing 3.5% NaCl. Replacing NaCl with 3.5% NaBr induced accumulation of three additional new azaphilones, falconensins Q−S (7−9) including two brominated derivatives (7 and 8) together with three known analogues (10−12). The structures of the new compounds were elucidated by 1D and 2D NMR spectroscopy and HRESIMS data as well as by comparison with the literature. The absolute configuration of the azaphilone derivatives was established based on single-crystal X-ray diffraction analysis of 5, comparison of NMR data and optical rotations as well as on biogenetic considerations. Compounds 1, 3−9, and 11 showed NF-κB inhibitory activity against the triple negative breast cancer cell line MDA-MB-231 with IC50 values ranging from 11.9 to 72.0 µM.The marine-derived fungus  Aspergillus sp., was the lead structure for the putative anticancer drug plinabulin, which has entered Phase III of clinical trials against non-small cell lung cancer + .Falconensin P (2): Yellow amorphous powder; c 0.2, MeOH); UV (MeOH) λmax 329 and 216 nm; 1H and 13C NMR data, m/z 513.1073 [M + H]+ .Falconensin Q (7): Yellow oil; c 0.2, MeOH); UV (MeOH) λmax 354 and 205 nm; 1H and 13C NMR data, m/z 479.0702 [M + H]+ . Falconensin R (8): Yellow oil; c 0.2, MeOH); UV (MeOH) λmax 353 and 206 nm; 1H and 13C NMR data, m/z 493.0853 [M + H]+ .Falconensin S (9): Yellow oil; c 0.2, MeOH); UV (MeOH) λmax 355 and 201 nm; 1H and 13C NMR data, m/z 443.1700 [M + H]+ .5 was measured with a Bruker Kappa APEX2 CCD diffractometer with a microfocus tube and Cu-Kα radiation (λ = 1.54178 Å). APEX2 was used for data collection, SAINT for cell refinement and data reduction [2 using SHELXL-2016/6 [2, and 0.98 Å for CH) and refined using riding models , with Uiso(H) = 1.2 Ueq, and 1.5 Ueq(CH3). The absolute structure configuration of 5 was solved using anomalous dispersion from Cu-Kα, resulting in a Flack parameter of 0.016(5) using Parsons quotient method. All graphics were drawn using DIAMOND [Crystals were obtained by solvent evaporation (MeOH). Data Collection: compound eduction , and SADeduction . The streduction , and refL-2016/6 . The hyd DIAMOND . The str2 atmosphere at constant 37 °C. The metastatic breast cancer cell line, MDA-MB-231, was purchased from the European Collection of Authenticated Cell Cultures (ECACC) . Sub-culturing was performed in RPMI 1640 medium (#21875-034) supplemented with 15% (v/v) fetal calf serum (FCS) and 1% (v/v) penicillin-streptomycin (pen-strep) . The MDA-MB-231 cell line stably expressing a plasmid with the NF-κB response element and the gene sequence for the firefly-luciferase protein (NF-κB- MDA-MB-231) was purchased from Signosis . For selection, 100 µg/mL hygromycin B  was applied in high glucose DMEM (#41966-029) supplemented with 10% (v/v) FCS, 1% (v/v) pen-strep. Starvation medium for the NF-κB inhibition assay contained 1% (v/v) FCS, 1% (v/v) pen-strep, and 100 µg/mL hygromycin B. Cell detachment occurred by trypsinization in 0.25% trypsin-EDTA and cell counting was performed at 1:1 (v/v) dilution in Erythrosin B  using the LUNA II automated cell counter (BioCat). Compounds 1, 4, 5, 7, 8, and 9 were dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 10 mM, whereas the compounds 3, 6, 11 were dissolved at 20 mM in DMSO. Further dilutions in cell culture medium contained maximal 2% DMSO.Culture medium and supplements were purchased from Gibco . Cell plates were obtained from Greiner bio-one . Cells were grown and incubated in a humidified 5% CO4 NFκB-MDA-MB-231 cells were seeded in total 100 µL medium per well in a 96-well plate . On the next day, medium was exchanged, and triplicates were pre-incubated for 20 min without (negative control) or with compounds in total 100 µL starvation medium. Final concentration of the compounds ranged a twofold serial dilution starting from 400 µM  or 200 µM  to 0.78 µM. To activate NF-κB signaling, untreated cells (positive control) or compound treated cells were subsequently stimulated for 24 h with 20 ng/mL TNFα . Finally, cell lysis and measurement were done according to the manufacturer’s instruction of the Luciferase Assay System . Injection of equal volume of luciferase substrate with 10 s integration time and subsequent luminescence measurement was performed using the Spark® microplate reader .3 × 10® Well vario pipetting robot , 18 µL of the MDA-MB-231 cell suspension (2.8 × 105 cells/mL) were seeded per well in a 384-well plate  and incubated for 24 h. The final concentration of the compounds ranged in twofold serial dilution steps starting from 400 µM  or 200 µM  to 0.78 µM. After 24 h compound stimulation in quadruples, cell lysis and measurement were done as prescribed in the manufacturer’s instruction of the CellTiter-Glo® Luminescent Cell Viability Assay . In short, it was applied equal volume of the CellTiter-Glo® reagent and luminescence was measured using the Spark® microplate reader (TECAN).Using the CyBio50) values were determined by nonlinear regression analysis based on the dose–response inhibition calculation “log(inhibitor) vs. response–variable slope (four parameters)” without curve fitting.Data of the NF-κB inhibitory assay and cell viability assay represent at least three individual experiments and were analyzed using GraphPad Prism . For the NF-κB inhibitory assay, data below the relative light unit (RLU) of the negative control were excluded for further analysis. Half maximal inhibitory concentration (IC


Pseudomonas balearica RAD-17, was identified and showed efficient inorganic nitrogen removal ability. The average NO3−-N, NO2−-N, and total ammonium nitrogen (TAN) removal rate  in a batch test was 6.22 mg/(L∙h), 6.30 mg/(L∙h), and 1.56 mg/(L∙h), respectively. Meanwhile, optimal incubate conditions were obtained through single factor experiments. For nitrogen removal pathways, the transcriptional results proved that respiratory nitrate reductases encoded by napA, which was primarily performed in aerobic denitrification and cell assimilation, were conducted by gluS and gluD genes for ammonium metabolism. In addition, adding the strain RAD-17 into actual wastewater showed obvious higher denitrification performance than in the no inoculum group (84.22% vs. 22.54%), and the maximum cell abundance achieved 28.5 ± 4.5% in a ratio of total cell numbers. Overall, the efficient nitrogen removal performance plus strong environmental fitness makes the strain RAD-17 a potential alternative for RAS (recirculating aquaculture system) effluent treatment.An aerobic denitrification strain,  Biological nitrogen removal is crucial for wastewater treatment, and the heterotrophic denitrification is the most selective method due to its high efficiency and flexibility . During The drawback of the biodegradable polymer-based denitrification is the relative high cost in media when compared with liquid carbon . On the Diaphorobacter polyhydroxybutyrativorans strain SL-205 enabled rapid reactor startup and improved nitrate removal performance when compared with active sludge inoculation in an anoxic solid-phase denitrification reactor [An interesting alternative to promote the denitrification performance is the supplement of functional bacteria through bioaugmentation technology . In a pr reactor . This in reactor . TherefonarG gene encoded for the nitrate reductase is sensitive to oxygen presence, which could block the sequential energy and electron transfer under aerobic condition [napFDAGHBC family while napA was responsible for synthesis of the catalytic subunit for electron delivery from NADH+ to nitrate aerobically [napA gene, which is located in the periplasm, was primarily infiltrated by nitrate and oxygen as compared to the narG gene located in plasma membrane [napA gene make these aerobic denitrifying strains use nitrate over oxygen preferentially [Pseudomonas stutzeri YZN-001 [Acinetobacter sp. HA2 [Pseudomonas stutzeri T13 [Marinobacter hydrocarbonoclasticus RAD-2 [Pseudomonas stutzeri C3 [Traditional denitrification or the similar reduction process only appeared under anoxic conditions ,14, as tondition . Recentlondition . The maiobically . Under amembrane . Therefoentially . Potentientially . Until n YZN-001 , Acineto sp. HA2 , Pseudomzeri T13 , Marinobus RAD-2 , Pseudomtzeri C3 , etc. Intzeri C3 ,24,25,26Pseudomonas balearica RAD-17, was isolated for a long-term PBS-supported denitrification reactor for RAS effluent treatment, demonstrated in a previous study [napA, nirS, norB, and nosZ) and glutamic biosynthesis genes (gluD and gluS) that are potentially related with ammonium assimilation. In addition, the strain’s bioaugmentation performance was also evaluated by adding it into actual RAS effluent. To the best of our knowledge, this is the first report of a functional strain with efficient aerobic nitrogen removal ability in the Pseudomonas balearica species. Overall, the results might provide new insight in aerobic denitrifying microbial resources and potential alternatives for enhancing nitrate-removal performance for RAS practice. In this study, a novel aerobic denitrifying halotolerant strain, us study . The 16Sw/v) agar. The DM (denitrification media) was prepared in the ratio of 2.0 g/L sodium acetate, 2.0 g/L KNO3 (or NaNO2), 0.2 g/L MgSO4·7H2O, 1.0 g/L K2HPO4, and 1.0% (v/v) trace-element solution for aerobic denitrification performance evaluation. The HNM (heterotrophic nitrification media) was prepared as follows: 2.0 g/L sodium acetate, 0.3 g/L NH4Cl, 0.2 g/L MgSO4·7H2O, 6.7 g/L Na2HPO4, 1.0 g/L KH2PO4, and 1.0% (v/v) trace-element solution for ammonium-nitrogen removal evaluation. The trace-element solution contained 50.0 g/L EDTA, 2.2 g/L ZnSO4, 5.5 g/L CaCl2, 5.06 g/L MnCl2·4H2O, 5.0 g/L FeSO4·7H2O, 1.1 g/L (NH4)6Mo7O2·4H2O, 1.57 g/L CuSO4·5H2O, and 1.61 g/L CoCl2·6H2O. In addition, the amounts of nitrogen and carbon in DM or HNM can also change according to the experimental setting. The initial pH of all media was set to 7.2 and then autoclaved for 20 min at 121 °C.The culture media used in this study were according to our previous study . The LB 4+-N and 150 mg/L NO3−-N and showed average TAN and nitrate removal rates of 47.35 ± 15.62 g NH4+–N·m−3·d−1) and 0.64 ± 0.14 kg NO3−-N·m−3·d−1with no obvious nitrite accumulation [Pseudomonas balearica, named RAD-17, was isolated. The genomic DNA of the RAD-17 strain was extracted using a DNA extraction kit . The 16S rRNA amplified product was sequenced by the Zhejiang Institute of Microbiology . Phylogenetic relationships of the strain RAD-17 with other denitrifying bacteria were constructed using the molecular evolutionary genetics analysis software . In addition, the purified strain RAD-17 was also stored in a 30% glycerol solution at −80 °C for following experiments.The RAD-17 strain was screened from a long-term PBS based denitrifying reactor, which operated under alternant aerobic/anoxic conditions in our previous study . The rea3−-N (around 300 mg/L) or NO2−-N (around 300 mg/L) was tested in DM media, which contained KNO3 or NaNO2, respectively. For heterotrophic ammonium removal, sole nitrogen source of NH4Cl (around TAN 80 mg/L) was carried out in similar operation in HNM media. For process, 3% (v/v) seed suspension was inoculated in 250 mL Erlenmeyer flasks and cultured for 48 h under aerobic condition at 25 °C and 150 rpm, respectively. Meanwhile, the cell-growth  and nitrogen concentrations were measured every 4 h.The inorganic nitrogen removal performance of the strain RAD-17 was evaluated on DM or HNM media. For aerobic denitrification capacity, a sole nitrogen source of NO3−-N concentration of 300 mg/L, C/N ratio 10, NaCl concentration 25‰, temperature 25 °C, rotation 150 rpm, and 3% inoculation (v/v). On C/N ratio test, the C/N ratios were set to 2, 5, 10, 15, and 20. On salinity test, the NaCl concentrations were set at 0‰, 2.5‰, 5‰, 15‰, and 25‰. On carbon sources, fructose, sodium acetate, lactin, glucose, and sodium citrate were tested. On revolution test, the speeds were set to 0 rpm, 50 rpm, 100 rpm, 150 rpm, and 200 rpm. On temperature test, 5 °C, 15 °C, 25 °C, and 40 °C were used. All tests were conducted in triplicate and non-seeded samples were used as blank control.Single-factor experiments were also carried out to evaluate the effect of various conditions on the aerobic denitrification performance of the strain RAD-17, for optimized incubated conditions. The basal condition was determined as follows: NOnapA, nirS, norB, nosZ, and the ammonium incorporation genes gluD (NADP-specific glutamate dehydrogenase) and gluS (glutamate synthase) with RNA samples in 48-h experiments. All primers were designed by the genome sequence of the strain RAD-17 and are listed in The transcriptional level gene expression intensity of the strain RAD-17 on nitrogen removal processes was investigated to reveal the metabolic pathways. In this study, real-time quantitative PCR was conducted to amplify the denitrifying genes 3−-N concentration and the C/N ratio were adjusted at approximately 100 mg/L and 15 by adding KNO3 and sodium acetate, respectively. Then, 500 μL of the strain RAD-17 (OD600 = 1.0) solution was added into the experimental tanks and aerobically cultured for 140 h. Another group without inoculation operated as control. Air-pumps  were used for aerobic condition and temperature was set at 25 ± 1 °C in a thermostatic chamber. All treatments were carried out in triplicate.The strain RAD-17 was added into actual RAS effluent to evaluate its bioaugmentation performance. The experimental tanks had a total volume of 10 L. A total of 5 L was used for raw RAS wastewater. The NOnapA gene by the strain RAD-17 that is relative to the total 16S rDNA genes using absolute qPCR. The qPCR primers are listed in napA gene and the 16S rDNA gene. The average number of 16S rDNA copies per bacterial cell is 4.2, and the napA gene carries a single copy in the strain RAD-17 cell [During the experimental phase, 2 mL solutions were cultured on DM media to evaluate the potential denitrification strains by the most probable number (MPN) standard method. Here, the amount of CFU can partly reveal the potential denitrifying ability as DM is a specific media for the denitrifier . Meanwhi-17 cell . The dif2−-N, and NO3−-N concentrations were analyzed according to standard methods [600 value using a spectrophotometer at 600 nm . The morphology of the strain RAD-17 was observed by a scanning electron microscope (SEM) . The pH value was measured using a portable pH meter . DO was measured using a DO meter . Physiological and biochemical characteristics were tested using API 20NE kits , and test strips were checked after incubation for 24 h [v/v) seed suspension was inoculated in 250 mL Erlenmeyer flasks and cultured for 20 h under aerobic condition at 25 °C and 150 rpm, respectively. The incubated nitrate was set at around 30 mg/L. Then, the nitrogen balance can be calculated on the initial and final nitrogen concentration [The solution samples were filtered through a 0.45 μm filter membrane before analyzing. The TAN, NO methods . Bacterifor 24 h . NitrogePseudomonas balearica DSM 6083. The phylogenetic analysis (threshold 100%) further confirmed the identification of the RAD-17 strain as Pseudomonas balearica , no study on the denitrifying function was reported in this sub-lineage.For strain screening, more than 20 pure isolates were obtained from the DM medium, while one named RAD-17 showed the highest aerobic denitrification performance. The colonies of the RAD-17 strain were pale yellow, salient, semitransparent, circular in shape, and presented a moist surface on the LB medium. The RAD-17 strain was a gram-negative strain with a bacilliform sharp in size of around 0.3–0.4 µm in diameter and 0.8–1.6 µm in length, respectively . The 16Salearica . For nittzeri C3 , Pseudomzeri T13 , Pseudomri PCN-1 , Pseudomsii Y-11 , etc. Insii Y-11 . HoweverThe API 20NE tests were carried out for further identification of the physiological and biochemical characteristics of the strain RAD-17 . The RAD3−-N concentration decreasing from the initial 301.43 ± 1.06 mg/L to a final 2.94 ± 1.66 mg/L, which indicated a 99.02% removal efficiency  and aerobic  conditions, respectively. However, a decreased denitrification performance was observed in oxygen-limited conditions . In a previous study, several denitrifying strains, especially in the genus ectively . Since dnditions ,45. HowenapA, nirS, norB, and nosZ. The napA firstly showed a quicker response to nitrate than nirS, norB, and nosZ, which is consisted with the fact that this process is a sequence of electrons transfer [nirS and nosZ were present, which was consistent with the nitrite peak concentration after 16 h  regulation should be further clarified, and the relevant control methodologies still need further study.Pseudomonas balearica strain RAD-17 showed efficient nitrogen removal performance with average NO3−-N, NO2−-N, and TAN removal rates of 6.22 mg·L−1·h−1, 6.30 mg·L−1·h−1, and 1.56 mg·L−1·h−1, respectively. The transcriptional results proved that aerobic nitrogen metabolic pathways were performed in respiratory nitrate reductases  for nitrate removal, or cell assimilation (gluS and gluD) for ammonium utilization. In addition, the bioaugmentation performance by the strain RAD-17 achieved maximum cell abundance of 28.5 ± 4.5% in total environmental cell numbers, as well as obvious higher denitrification performance than in the no inoculum group (84.22% vs. 22.54%).An aerobic denitrification


S. litura moths, where chlorantraniliprole, flubendiamide, and emamectin benzoate was found to have the highest levels of toxicity . After exposure to the low lethal concentration LC50 of chlorantraniliprole, fecundity of the moths was substantially reduced. Egg-hatching was lower for LC20- and LC50-treated moth pairs than for untreated control pairs. Net reproductive rate (R0), intrinsic rate of increase (r), and finite rate of increase (λ) were significantly reduced in LC50♀ × LC50♂ cohorts. Larval mortality was significantly higher in subsequent generations in pairs of LC50-treated moths. Chlorantraniliprole, which was most toxic and had significant sublethal effects on moths, can be used as an alternative insecticide to methomyl in the attracticide for controlling S. litura moths, and the LC50 indicated a high potential for efficacy in the control S. litura through attract-and-kill schemes.The integrated use of plant-derived volatile attractants and synthetic insecticides in attract-and-kill programs is a useful tool for integrated pest management programs reducing pesticide input. Efficient alternative insecticides are critically needed to replace methomyl, which has been banned on cruciferous vegetables in China because it is also highly toxic to nontarget organisms. In the present study, among 15 commonly used insecticides were screened for toxicity against  Spodoptera litura Fab. , is a serious pest of many agricultural crops, such as soybean (Glycine max L.), maize (Zea mays L.), and vegetables and fruit trees in the southern and eastern regions of Asia [S. litura, and they usually target the larvae [The tobacco caterpillar,  of Asia ,2,3. Lose larvae .S. litura in China can be divided into three zones: the year-round breeding region, the overwintering region, and the summer breeding region [In China, the range of g region ,6,7. Dueg region ,9. ThereS. litura [Gossypium spp.), maize (Zea mays L.), and peanut (Arachis hypogaea L.) [The attract-and-kill strategy is a potential tactic in the management of agricultural pests that presupposes the intelligent combination of an attracting agent  and a killing agent  . Such an. litura . In Chingaea L.) ,14,15. Mgaea L.) ; howevergaea L.) . TherefoS. litura, is almost entirely concentrated on the larvae [S. litura. An attracticide must rapidly incapacitate and kill moths to prevent them from laying eggs before they die; thus, the concentration of an insecticide in the attracticide is important. Because all pesticides can elicit sublethal effects on pests [S. litura moths so that the dosage can be reduced in attractants.The extensive literature on the toxicity and effectiveness of various insecticides against e larvae ,19,20 beon pests ,22, we nS. litura moths to 15 common insecticides. Further, we investigated the sublethal effects of the most effective insecticide, chlorantraniliprole, on adults of S. litura and highlight that chlorantraniliprole at low doses would be effective in the attract-and kill against S. litura.Against this background, here we evaluated the susceptibility of S. litura moths were captured by a searchlight trap at the Langfang Experimental Station , Chinese Academy of Agricultural Sciences (CAAS), in Hebei Province, China. Then, they were stored in cages with mesh sides for egg collection. The larvae of S. litura were reared on an artificial diet [v/v sugar and 2% v/v vitamin complex for nutrition supplement. Adults of the third and fourth generations were used for bioassays. For experiments on sublethal effects of chlorantraniliprole on S. litura moths, the insects were sexed after pupation and placed separately in ventilated plastic cages for emergence. Moths were used within 24 h of emergence. ial diet ,24 underw/v, as indicated) of 15 insecticides were tested as follows: chlorantraniliprole (95.3%), cyhalothrin (95%), thiodicarb (95%), flubendiamide (98%), abamectin (97%), spinosad (90%), indoxacarb (94%), emamectin benzoate (92%), chlorfenapyr (94.5%), beta-cypermethrin (96.5%), fenpropathrin (92%), fenvalerate (96%), chlorpyrifos (98%), carbosulfan (90%). All insecticides were provided by the Institute for the Control of Agrochemicals (ICA), the Ministry of Agriculture (MOA), China. A spinetoram suspension concentrate (SC)  was obtained from Langfang, China .Insecticides were technical grade formulations (% −1) of spinetoram was prepared in distilled water, while all other stock solutions  of insecticides were diluted using dimethyl sulfoxide (DMSO). Each stock solution was diluted using a 10% v/v honey solution containing 0.1% v/v Tween-80 to the desired experimental concentrations . The 10% honey solution containing 1% DMSO and 0.1% Tween-80 was used as a blank control. To prevent insect contact with the insecticide solution, the cotton ball with insecticide or honey solution was placed on the bottom of a plastic cup . Next, a bottomless plastic cylinder that was 9.5 cm high  was wrapped with two layers of cotton gauze and placed inside this cup, maintaining ~0.8 mm between the gauze and soaked cotton ball. Five S. litura moths (3 days old) were randomly chosen and placed in each cylinder as one replicate; all bioassay treatments had five replications. All cylinders were maintained at 25 ± 1 °C, 60 ± 5% RH, and had a photoperiod of 14: 10 h (L: D). The mortality rate of moths in the cylinder was observed after 24 h. For sublethal effects of chlorantraniliprole to S. litura moths, LC20, LC50, and LC90 values were calculated.A 100 ml stock solution  and 10% honey solution was placed on the bottom of the plastic cup. To obtain enough live moths treated with different doses of chlorantraniliprole, 80 replicates  were subjected to the LC50 treatment, 60 replicates  to the LC20 treatment, and 60 replicates  to the control treatment. Five 50♀ × LC50♂, LC50♀ × CK♂, CK♀ × LC50♂, LC20♀ × LC20♂, LC20♀ × CK♂, CK♀ × LC20♂, and CK♀ × CK♂. Each pair was placed in a plastic cup , and 10% honey solution was provided as food for moths. The top of the plastic cup was covered with gauze for oviposition. Eggs on the gauze were counted, and new gauze was placed on the plastic daily. Egg-hatching was recorded for a random subset of approximately 150–200 eggs. For this purpose, sections of cotton gauze with S. litura eggs were placed in Petri dishes  and examined for seven consecutive days. Adult longevity was also recorded. One hundred replicates were used for each treatment. After 24 h, the female and male moths from the different treatments were paired. The mating patterns were as follows: LC20 and LC50 concentrations were used to assess the sublethal effects of chlorantraniliprole on S. litura. To determine the LC20 and LC50 concentrations, we first generated a concentration–mortality regression line, then calculated the respective concentrations from the regression lines (see S. litura larvae were randomly sampled from each treatment (see The LCines see . Newly hment see  and placp < 0.05) using SPSS 13.0 software  [S. litura population parameters, including the net reproductive rate (R0), intrinsic rate of increase (r), finite rate of increase (λ), and mean generation time (T) were analyzed according to the age-stage, two-sex life table theory, using the program TWOSEX-MSChart [The median lethal concentrations, 95% confidence limits (CLs), and slope ± SE were calculated using probit analysis. One-way analysis of variance (ANOVA) was used to analyze the effects of lethal concentrations of chlorantraniliprole on female longevity, male longevity, fecundity, egg-hatching rate, developmental period, pupa weight, adult emergence, and larvae mortality, followed by Tukey’s honestly significant difference (HSD) test  . The sel-MSChart . Means, -MSChart . Before -MSChart .S. litura moths varied considerably, but mortality was consistently <5% in the control groups . The toxicity of chlorantraniliprole was the highest among the tested insecticides, with an LC50 value of 0.56 mg a.i. liter−1, while avermectin, spinetoram, spinosad, carbosulfan, and chlorfenapyr had the lowest toxicity, with LC50 values > 100 mg a.i. liter−1 . The longevity of male adults was reduced by the LC50 treatment, but not significantly compared with the control . Fecundity of female adults significantly decreased in the LC50♀ × LC50♂ cohorts compared with the control . Furthermore, the egg-hatching rate in the LC50♀ × LC50♂, LC50♀ × CK♂, CK♀ × LC50♂, and LC20♀ × LC20♂ cohorts was significantly lower than in the control treatments   .S. litura offspring, adult emergence for the LC50♀ × LC50♂ cohorts (65.50 ± 1.17%) was significantly lower than control treatments (85.09 ± 0.78%) . Larval mortality for the LC50♀ × LC50♂ (61.33 ± 1.76%) and CK♀ × LC50♂ cohorts (44.67 ± 4.06%) was significantly higher than for the control treatment (24.00 ± 2.00%)  (For < 0.001) .0R) was significantly reduced for LC50♀ × LC50♂ (2.44 ± 0.58) and LC50♀ × CK♂ (47.99 ± 17.23) cohorts compared to the control (137.43 ± 22.43). The intrinsic rate of increase (r) was the lowest for LC50♀ × LC50♂ cohorts (0.03 ± 0.01), followed by the LC50♀ × CK♂ (0.14 ± 0.02), CK♀ × LC50♂ (0.14 ± 0.01), LC20♀ × LC20♂ (0.15 ± 0.01), and CK♀ × LC20♂ cohorts (0.15 ± 0.01), which were all significantly lower than that of the control (0.18 ± 0.01). The finite rate of increase (λ) was the lowest for LC50♀ × LC50♂ cohorts (1.03 ± 0.01), followed by LC50♀ × CK♂ (1.15 ± 0.02), CK♀ × LC50♂ (1.15 ± 0.01), LC20♀ × LC20♂ (1.16 ± 0.01), and CK♀ × LC20♂ cohorts (1.16 ± 0.01), which were all significantly lower than that of the control (1.20 ± 0.01) (Net reproductive rate ( ± 0.01) . S. litura moths. In this study of the toxicity of 15 insecticides commonly used against S. litura moths, chlorantraniliprole had highest toxicity against S. litura moths. Therefore, chlorantraniliprole is a good candidate for replacing the hazardous methomyl in attracticide products. Chlorantraniliprole at LC20 or LC50 concentrations had sublethal effects, reducing egg-hatching and the longevity of female adults, and the LC50 concentration reduced fecundity. These negative effects may be related to the mechanism of action of chlorantraniliprole, which acts on the ryanodine receptors in insects and affects calcium homeostasis in the cell, leading to feeding cessation, lethargy, muscle paralysis, and ultimately, death of the insect [An attracticide, a combination of synthetic plant volatiles and an insecticide, is used to trap and control lepidopteran moths ,29,30. He insect .S. litura larvae having the ability to develop high resistance to chlorantraniliprole [50 of chlorantraniliprole against S. litura moths was found to be low; therefore, its high insecticidal toxicity and rapid efficiency against target pests make it a good candidate for controlling S. litura moths.The toxicity of chlorantraniliprole, flubendiamide, and emamectin benzoate was high against the moths, in accordance with their high toxicity against larvae ,20. Howeiliprole , furtherS. litura, the LC50 of chlorantraniliprole significantly reduced the fecundity of the LC50♀ × LC50♂ cohorts, as found for larvae of Platynota idaeusalis and Helicoverpa zea that fed on a diet containing a sublethal concentration of tebufenozide [20 and LC50 concentration of chlorantraniliprole were paired with untreated male and female moths. Whether the reduction in hatching rate was caused by a decrease in mating rate or the lack of hatching of fertilized eggs needs further study. Our results indicate that future use of this compound in attract-and-kill schemes may not be limited by S. litura adult abundance or immigration rates, because egg-hatching will drop sharply even when untreated immigrants mate with the resident, pesticide-tainted individuals. Fieldwork, however, is essential to validate these hypotheses and fine-tune pesticide delivery methods.In the current study with fenozide ,40. Somefenozide ,42; howerm), a measure of the ability of a population to increase exponentially in an unlimited environment, provides an effective summary of an insect’s life history traits [rm was lower for LC50♀ × LC50♂, LC50♀ × CK♂ (0.14 ± 0.02), CK♀ × LC50♂ (0.14 ± 0.01), LC20♀ × LC20♂ (0.15 ± 0.01), and CK♀ × LC20♂ cohorts after treatment with chlorantraniliprole compared with the controls, meaning a population increase would be delayed.The intrinsic rate of increase (y traits , which, y traits ,45,46. IS. litura moths. Thus, a low concentration will reduce S. litura fecundity and egg-hatching, and slow population growth. Hence, the inclusion of a low concentration of chlorantraniliprole in an attract-and-kill delivery scheme constitutes a highly desirable alternative to broad field-level applications or coating seeds with insecticide. Such an “attract-and-kill” measure will potentially enhance environmentally friendly pest management, and thus the IPM toolbox for controlling this global agricultural pest.In conclusion, the novel insecticide chlorantraniliprole had the highest toxicity and fastest activity among the insecticides tested against


Schinus molle (L.) is a dioecious plant of the Anacardiaceae family, originating in South America and currently widespread in many regions throughout the world. In this work leaf extracts and derived low-pressure column chromatography (LPCC) fractions of S. molle L. male and female plants were investigated for the antimicrobial activity. Leaf extracts were tested on microbes Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, Candida albicans and Bacillus subtilis. Furthermore, the extracts showing antimicrobial activity were fractionated by LPCC and the obtained fractions tested on the same microorganism strains. Positive fractions were investigated by gas-chromatography/mass spectrometry (GC-MS) and were seen to be rich in sesquiterpenes, sesquiterpenoids and other terpens. The obtained effects highlighted the antimicrobial properties of S. molle (L.) leaf compounds and revealed their importance as a source of bioactive molecules of potential pharmaceutical interest. To our knowledge, this is the first paper reporting investigations on the chemical composition of the extracts and derived positive fractions from Schinus molle (L.) plants grown in central Italy Over the decades, the extensive use of antibiotics, especially prophylaxis, has led to the development of resistant pathogens . DiseaseSchinus terebinthifolia Raddi) processing industry chain [Escherichia coli and Pseudomonas aeruginosa and gram-positive Staphylococcus aureus and Bacillus cereus [E. coli, Acinetobacter bohemicus, and Pseudomonas fluorescens) and gram-positive (B. cereus and Kocuria marina) bacteria [From this perspective, the literature of the last 20 years has grown in quantity and quality and many studies have been carried out to test the antimicrobial activity of extracts from plant matrices. Gomes and colleagues evaluated antibacterial activity against multidrug-resistant strains of hospital origin and standard strains using extracts exploiting a plant matrix of waste from the Brazilian pepper tree , known as “false pepper” or “pink pepper” due to the edible red/pink fruits whose flavour and fragrance are reminiscent of Piper nigrum (L.), is an evergreen plant belonging to the Anacardiaceae family. It is native to South America where it is known as the Peruvian pepper tree [per tree , whereasper tree ,14,15,16per tree ,14, antiper tree ,18, hypoper tree , anti-inper tree , and anaper tree  effects,per tree , acariciper tree ,24, repeper tree ,26,27,28Antibacterial activity has been investigated in extracts and essential oils from leaves, flowers, berries, and bark derived from this plant ,30,31,32S. molle (L.) fresh leaf extracts and they were shown to be mainly composed of sesquiterpene hydrocarbons and monoterpene hydrocarbons [In our search for new compounds of possible pharmaceutical interest, we previously carried out chemical investigations of male and female ocarbons . We founocarbons ,43,44,45S. molle (L.) grown in central Italy was evaluated on clinically relevant bacterial strains and the chemical composition of biological active fractions investigated.For the first time, in this work the antibacterial activity of extracts and their fractions of leaves from S. molle (L.) using four solvents with increasing polarity with this crescent order: petroleum ether, diethyl ether, acetone and distilled water.The investigation of antibacterial activity was carried out on female and male leaf extracts of S. molle (L.) leaf extracts was carried out using the disk diffusion test. Treatments with petroleum ether (SM♀L1) and diethyl ether (SM♀L2) on extracts from the S. molle (L.) female plant (S. molle (L.) male plant  assay of the tested extracts with a positive response in the disk diffusion test (DDT) recorded values between 25 µg/mL and 400 µg/mL. The DTT of the leaf extracts from the female S. molle (L.)—SM♀L1, SM♀L2, SM♀L3 (acetone extract), SM♀L4 (water extract)—and the male—SM♂L1, SM♂L2, SM♂L3, SM♂L4—produced no effects on E. coli and P. aeruginosa. The extracts SM♀L3, SM♀L4, SM♂L3 and SM♂L4 had no effects on any of the strains used  were fractioned by the low-pressure column chromatography (LPCC) technique using solvents with increasing polarity in this order: petroleum ether 100%, petroleum ether/ethyl acetate 95%/5%, 90%/10%, 80%/20%, 70%/30% and 0%/100% and methanol 100%. The obtained fractions have been investigated by disk diffusion and microdilution tests. Concerning the size of the inhibition halo and the MIC values, as shown in S. molle (L.) leaf extracts that produced an inhibition halo equal to or greater than 10 mm were further investigated with the gas-chromatography/mass spectrometry. The identification of the components separated by GC/MS was carried out by comparing the mass spectra for each compound with that reported in mass spectrometry (MS) libraries and by calculating their linear retention indices (LRIs). S. molle (L.) leaves; 40 identified molecules are reported. S. molle (L.) leaves; 12 identified molecules are listed. The composition of the biologically active fractions obtained by LPCC from the male and female extracts in petroleum ether showed a particular abundance of sesquiterpenes and monoterpene hydrocarbons. The fractions obtained from the male and female diethyl ether extracts were rich in sesquiterpenes and alcohol terpenes; fatty acids, phenols, esters and hydrocarbons were also present.The fractions derived from female and male Plant secondary metabolites possess numerous biological activities and are useful in the treatment of human and animal health problems. Antibacterial activities have been well defined in different classes of natural compounds and, due to the high incidence of antibiotic resistance, the search for new antibiotics is ongoing.S. aureus showed an increase in the inhibition halo, from 10.3 ± 1.5 mm to 20 ± 1 mm, and a reduction in the MIC, from 400 µg/mL to 128 µg/mL, compared to the extract SM♀L1. The biological activity could be related to the presence of β-eudesmol (67.9%), together with cyclotetradecene (15.7%), γ- and α-eudesmol , dehydroxyisocalamendiol (3.4%) and elemol (2.7%). Salem and colleagues [S. molle (L.), which has a high percentage of β-eudesmol; this work supports the relationship between the chemical content found and the biological activity observed. Furthermore, the lowest MIC against S. aureus was obtained in the fraction SM♀L1-F4, containing elemol (38.1%), β-terpinene (33.8%)—known for its antibacterial activity against S. aureus [S. aureus led us to consider a synergism between the molecules present in the fraction and the antimicrobial activity of this strain.The antibacterial activity of the SM♀L1-F7 fraction against lleagues  demonstreudesmol 7.9%, togB. subtilis, with an increase in halo diameter from 14.3 ± 1.2 mm to 24.7 ± 0.6 mm for the disk diffusion test and an MIC reduced from 400 µg/mL to 8 µg/mL compared to extract SM♀L1. Similar values were also obtained from the fraction SM♀L1-F6 . The major constituent of these fractions was elemol (SM♀L1-F5 = 74.6% and SM♀L1-F6 = 71.6%). In the fraction SM♀L1-F5 α-, β- and γ-eudesmol  were present; these molecules are known for their antimicrobial effects [The fraction SM♀L1-F5 showed good inhibitory activity against  effects ,36. In dS. aureus showed an increase in antibacterial activity compared to the extract SM♀L2. The inhibition halo increased from 10 mm to 18 mm, while the MIC reduced from 400 µg/mL to 64 µg/mL. In this fraction, cyclotetradecene (77.6%) and 2,4-di-tert-butyl phenol  (22.4%) were identified. In particular, 2,4 DTBP is known to be an antifungal and cytotoxic molecule, as has been widely documented [The results of the SM♀L2-F4 fraction against cumented ,47,48. Acumented  also desC. albicans. A reduction in its inhibition halo was observed, from 14.3 ± 2.1 mm to 9.7 ± 0.6 mm, and a decrease was seen in the MIC from 50 µg/mL to 32 µg/mL.Among fractions obtained from the extract SM♀L2, only SM♀L2-F4 showed a modest activity against B. subtilis, fractionation produced better effects. The two fractions SM♀L2-F2 and SM♀L2-F3 were active, with inhibition halos of 25.7 ± 0.6 mm and 24.7 ± 1.2 mm and MICs of 1 µg/mL and 2 µg/mL, respectively. These values were the lowest recorded. The GC-MS analysis of SM♀L2-F2 revealed the presence of 3,7,11,15-tetramethyl-2-hexadecen-1-ol (86.9%), β-terpinene (5.8%), hexadecanoic acid (4.0%) and α-, β- and γ-eudesmol . SM♀L2-F3 consisted of only phytol, a stereoisomer of 3,7,11,15-tetramethyl-2-hexadecen-1-ol with a similar antimicrobial activity [Regarding activity . Both ofactivity .E. faecalis, with an increase in the inhibition halo from 8.3 ± 0.6 mm to 14.7 ± 1.2 mm and a reduction in the MIC from 200 µg/mL to 128 µg/mL.The fraction SM♀L2-F4 was the only one to show activity in response to B. subtilis was related exclusively to the disk diffusion test (not to the MIC). Indeed, the inhibition diameter reached 26.3 ± 0.6 mm, compared to 13.7 ± 0.6 mm for the SM♂L1 extract. This phenomenon could be due to the presence of molecules already known for their antibacterial activity, such as germacrene-D [As shown in acrene-D ,42, δ-caacrene-D , elixeneacrene-D ,54 and βacrene-D ,41.Piper nigrum (L.) fruits [S. aureus. In this study, the SM♂L1-F6 fraction showed a good antibacterial activity against this strain, with a halo of 16.7 ± 0.6 mm and an MIC of 8 µg/mL. In this fraction, isocalamendiol (84.8%), β-eudesmol (12.9%) and spathulenol (2.3%) were present. The antibacterial activity of β-eudesmol has been evaluated through the analysis of essential oils of Amazonian species of the genus Guatteriopsis [S. aureus was reported, with an MIC of over 1000 µg/mL. Our results supported the hypothesis of an antibacterial activity against S. aureus, exerted by isocalamendiol or by a synergistic action of the three molecules. The inhibitory activity against S. aureus could be associated with the high presence of β-eudesmol, also found in the fractions SM♂L1-F10 (91.7%) and SM♀L1-F7 (67.9%), which have a DDT = 15.3 ± 1.2 mm/MIC = 128 µg/mL and a DDT = 20 ± 1 mm/MIC = 128 µg/mL, respectively.As already reported for ) fruits , there ceriopsis  and a loInteresting effects were also recorded for SM♂L1-F7 . As revealed by GC-MS investigations, the fraction consisted of β-eudesmol (59.6%), spathulenol (14.2%), isocalamendiol (12.2%), squalene (7.0%) and isoaromadendrene epoxide (7.0%).S. aureus of isoaromadendrene, epoxide [Syzygium jambos L. (Alston), in which no antibacterial activity occurred during the testing of squalene.Some papers have reported the experimental data antibacterial activity against  epoxide . The ine epoxide  in a stuC. albicans, mainly expressed by the fraction SM♂L2-F6, with a similar DDT result  and enhanced MIC activity of 400 µg/mL to 64 µg/mL. As shown by GC-MS investigations, the fraction consisted of hexadecanoic acid. Biological activities of hexadecenoic acid reported in literature, such as larvicidal [B. subtilis, with an increase in the inhibition halo from 11.3 ± 1.5 mm in the total extract up to 28.3 ± 1.2 mm in the fraction SM♂L2-F6. This last fraction, in line with the cited literature, also showed an increase in antibacterial activity with the broth microdilution method, with an MIC reduced from 100 µg/mL to 16 µg/mL.Most of the fractions derived from SM♂L2 exhibited inhibitory activity against the fungus rvicidal , anti-inrvicidal  and antirvicidal ,36. In ttert-butyl- [The fraction SM♂L2-F7 showed the biggest inhibition halo (29.7 ± 0.6 mm) recorded, an activity in contrast with the increase in the MIC value from 100 µg/mL to 256 µg/mL. The composition of this fraction was more heterogeneous than the fraction SM♂L2-F6 . The comt-butyl- ,48,49, aAll the solvents used for chemical analysis, extraction and fractionation purposes were analytical grade . The silica gel  was from Macherey-Nagel . The microorganism media and reagents for antimicrobial tests were purchased from Sigma–Aldrich .S. molle (L.) plants were collected during the flowering period from the “Angelo Rambelli” botanical garden in Viterbo  and identified by Dr. Monica Fonck (Scientific Supervisor) with the number AS21  and AS22  in the Botanical Garden Catalogue. The leaves were washed with distilled water, air-dried in dark conditions at room temperature and stored at −80 °C until lyophilization. The frozen leaves were lyophilized for 3 days to eliminate traces of water, ground to obtain a powder  and stored at 4 °C. In order to perform serial extractions, the powder was put in a cellulose thimble and processed in a soxhlet apparatus. Three solvents with increasing polarity—petroleum ether (PE), diethyl ether (DE) and acetone (AC)—were used. After the acetone extraction, the solid residue was macerated in distilled water overnight and the aqueous fraction filtered through Whatman paper. The solvent was evaporated by a rotary vapor  and the dried residues put into glass vials and stored at 4 °C until use. The obtained extracts were named as SM♀L1 , SM♀L2 , SM♀L3 , SM♀L4 , SM♂L1 , SM♂L2 , SM♂L3  and SM♂L4 .The leaves of male and female All the obtained extracts—SM♀L1, SM♀L2 and SM♂L1, SM♂L2—were fractionated by silica gel in the solid phase and solvents with increasing polarity in the mobile phase. The silica gel was packed using petroleum ether and helped by vibrations to improve the packing of the stationary phase. A layer of sand was put on top of the silica to avoid the dispersion of the solid phase and the creation of air bubbles . The solThe active fractions were analyzed with a Perkin Elmer GC/MS Clarus 500. The gas chromatograph was equipped with a Stabilwax fused-silica capillary column  .m/z. The relative percentages for quantification of the components were calculated by the electronic integration of the GC-FID (Gas Chromatograph-Ionization Flame Detector) peak areas using the normalization method. The identification of the components separated by GC/MS was performed by comparing the mass spectra for each compound with that reported in the MS library search (Wiley and Nist 02). Furthermore, the linear retention indices (LRI) of each compound were calculated according to van den Dool and Kratz [n-alkanes  injected directly into the GC injector with the same operating conditions reported above. All the analyses were repeated twice. The percentage area values were obtained electronically from the GC-FID response without the use of an internal standard or correction factors.The analytical conditions were set as follows: the injector was at 280 °C; the oven temperature was programmed from 60 °C to 220 °C at a rate of 5 °C/min and held for 30 min; the carrier gas was helium at 1.0 mL/min. One microliter of methanol was added to the extracts and 2 µL of each was injected directly with a split ratio of 1:20. The Clarus 500 Mass Spectrometer single quadrupole operated in the electron impact (EI) mode at 70 eV. The mass range was from 30 to 450 nd Kratz , using aS. aureus ATCC 6538, Escherichia coli ATCC 25922, P. aeruginosa ATCC 9027, E. faecalis ATCC 29212, B. subtilis ATCC 6633 and C. albicans ATCC 10231.The antibacterial activity was tested using selected pathogens and commensal strains from the American Type Culture Collection (ATCC) : The strains were maintained at 4 °C on slants of Tryptic Soy Agar  and Malt Extract Agar for bacteria and molds, respectively. The bacterial and fungal strains were previously activated in Tryptic Soy Agar (TSA) at 37 °C/24 h and Sabouraud Dextrose Agar (SDA) at 25 °C/5 days.All the inocula were prepared with fresh cultures plated the day before the test.S. molle (L.) fractions were placed on TSA, having been previously seeded with the bacterial strain. The microorganisms were suspended in sterile Tryptic Soy broth with a turbidity from 0.5 to 1 McFarland (approximately 107–108 CFU/mL). S. molle (L.) fractions which had growth inhibitory activity were highlighted by the absence of microorganism growth around spots and the haloes were measured using a Vernier calliper rule and expressed in mm [Sterile disks (6 mm diameter) impregnated with ed in mm ,64. EachS. molle (L.) fractions were determined against all strains by means of a microwell dilution method [The MIC values of n method ,66. Dilun method . As a nen method .p values of ≤ 0.05 considered statistically significant.The results were expressed as means ± the standard deviation (SD). Data were analyzed with a one-way analysis of variance (ANOVA) using GraphPad Prism software , with S. molle (L.) leaf extracts and derived fractions from S. molle (L.) male and female plants. In most cases, the obtained fractions showed improved antimicrobial activity compared to the in toto extracts.In conclusion, our findings confirm the antimicrobial properties of The chemical investigations of positive fractions could suggest a possible correlation between molecules highlighted by GC-MS analysis and the antibacterial activity. Further studies are needed for a more detailed evaluation of the antibacterial properties of single identified molecules in extracts and their possible synergic effects.


Ursolic acid (UA), a natural product, shows a broad spectrum of anticancer effects. However, the poor bioavailability and efficacy of UA limit its clinical application. We developed novel analogues of UA with enhanced antitumor activities by the extensive chemical modification of UA. We developed multiple compounds by structural modification of UA, and found that UA232 had stronger activity than UA. The effects of UA232 (0–50 μM) on inhibiting the proliferation of A549 and H460 cells were determined by CCK-8 for 24, 48, or 72 h. The proapoptotic effect of UA232 was analyzed by microscopy and flow cytometry, and the potential signal pathway affected by UA232 was further validated by Western blotting and flow cytometry.50 = 5.4–6.1 μM for A549 and 3.9–5.7 μM for H460 cells). UA232 could induce not only cell cycle arrest in the G0/G1 phase but also apoptosis in both A549 and H460 cells. The treatment of UA232 could lead to an increase of CHOP expression rather than an increase in Bax or caspase-8, indicating that the apoptosis induced by UA232 was correlated with the endoplasmic reticulum stress (ER stress) pathway. Treatment with the ER stress-specific inhibitor, 4-PBA, decreased the ability of UA232 to induce apoptosis in A549 and H460 cells. Compared with UA, UA232 showed a stronger ability to inhibit the proliferation of lung cancer cells (IC UA232 induced apoptosis through the ER stress pathway, and showed stronger growth-inhibitory effects in A549 and H460 cells compared to UA, which may be a potential anticancer drug to suppress the proliferation of lung cancer. Lung cancer is the leading cause of cancer-related deaths worldwide  is a pentacyclic triterpene complex that can be isolated from a variety of plants and fruits, such as rosemary, lavender, thyme, and apple peel  at 400 MHz. Chemical shifts were reported in ppm using CDCl3 solution with TMS as internal standards. Mass spectra (ESI-MS) were performed on Agilent 1200 HPLC-6310 liquid chromatography-mass spectrometer . All reagents and solvents were used as received from commercial sources without further purification.Reactions were monitored by thin-layer chromatography (TLC) using silica gel 60 UV254 pre-coated silica gel plates. The detection was using a UV lamp. Flash column chromatography was performed on 300–400 mesh silica gel. 2CO3 , and KI  in 50 mL DMF, the mixture was stirred at room temperature. 1, 4-Dibromo-butane  was added dropwise to the mixture in 0.5 h and the reaction was monitored by TLC. When reactant was consumed completely, the mixture was poured into 300 mL water with stirring for 0.5 h and the mixture was stirred at 80 °C for 0.5 h and then was extracted with 300 mL ethyl acetate. The organic layer was dried with anhydrous Na2SO4, and then evaporated the solvent. The residue was purified by silica gel column chromatography (hexane: ethyl acetate = 15:1→8:1) to obtain 5.31 g UA-2-3-0 as white foamy solid with the yield of 81.9%. mp: 91.2–93.0 °C; ESI-MS m/z 591.34, 593.33 [M + H]+, 1H-NMR  δ 5.24 , 4.02 , 1.75 , 1.67 , 1.64 , 1.62–1.57 , 1.54–1.45 , 1.40–1.27 , 1.06 , 0.99 , 0.85 , 0.82 , 0.73 .To a solution of ursolic acid , K2CO3  in 15 mL DMF, the mixture was stirred at 80 °C for 0.5 h and then piperazine  was added. The reaction was monitored by TLC. When UA-2-3-0 consumed completely, the mixture was cooled to room temperature and then poured into 100 mL water with stirring for 0.5 h. Filtrated and dissolved the precipitate with ethyl acetate, then dried with anhydrous Na2SO4, 0.82 g UA-2-3-2 was obtained as a white solid powder after evaporating the solvent under reduced pressure with the yield of 81%. mp: 124.1–124.9 °C; ESI-MS m/z 597.43 [M + H]+; 1H-NMR  δ 5.22 , 3.99 , 3.28 , 3.20 , 2.71 , 2.42 , 2.21 , 1.99 , 1.87 , 1.75 , 1.56 , 1.30 , 1.07 , 1.04 , 0.97 , 0.94 , 0.90 , 0.85 , 0.77 , 0.73 , 0.71 .To a solution of UA-2-3-0  and KThe purity of UA232 was more than >98%. UA was purchased from MedChemExpress . UA232 and UA were dissolved in dimethyl sulfoxide (DMSO). Crystal violet was purchased from Beyotime Biotechnology . Polyvinylidenedifluoride (PVDF) membrane was purchased from Millipore Bedford . Propidium iodide (PI), FITC-labeled Annexin V, and propidium iodide (PI) double-staining were purchased from KeyGen Biotech . Primary antibodies against cyclin D1, CDK4, Bax, Bcl-2, caspase-8, PARP1, and CHOP were purchased from Proteintech Group . Antibodies for PERK, p-PERK, eIF2α, and p-eIF2α were purchased from Bioss . β-actin and horseradish peroxidase (HRP)-conjugated secondary antibodies (goat-anti-mouse or goat-anti-rabbit) were obtained from ZSGB-BIO .2.Human non-small cell lung cancer cell lines H460, A549, and human embryonic kidney cell line HEK293T were obtained from the American Type Culture Collection (ATCC). The A549 and H460 cells were cultured in RPMI 1640 medium supplemented with 10% foetal bovine serum (FBS), 1% penicillin, and 1% streptomycin. The HEK293T cells were cultured in DMEM medium, other medium components were consistent with other cells. All the cells were incubated at 37 °C with 5% COA549, H460, or HEK293T cells were seeded in 96-well plates at a density of 3000 cells/well and cultured for 24 h. Treatment cells with different concentrations of UA232 or UA for 24, 48, or 72 h. Then 20 μL CCK8 was added to each well and incubated at 37 °C for 2 h. Optical density at 450 nm was measured using a TECAN Infinite 200 .A549 or H460 cells were seeded into 6-well plates at a density of 5000 cells/well and incubated for 24 h. Treatment cells with different concentrations of UA232 or UA for 48 h and then remove it with fresh medium. After 2 weeks of growth, the cells were fixed at room temperature with 4% paraformaldehyde for 30 min and stained with crystal purple for 15 min. The colonies were photographed by Sony digital camera.2 cell culture bottle at a density of 3 × 105 cells/bottle and incubated for 24 h. Treatment of cells with UA232 for 24, 48, or 72 h. The collected cells were fixed with pre-cooled 70% ethanol for 24 h, then washed with PBS twice and stained with PI working solution containing RNase at room temperature for 30 min. Then the samples were analyzed by FACScan flow cytometry . The distribution of the cell cycle was analyzed by ModFit LT software.A549 or H460 cells were seeded into 25 cm2 cell culture bottle at a density of 3 × 105 cells/bottle and incubated for 24 h. Cells were treated with different concentrations of UA232 or UA for 48 h, then collected and stained using an Annexin V-FITC/PI staining kit. Samples were analyzed by FACScan flow cytometry . Apoptosis rate analysis was done using ImageJ2 software.A549 or H460 cells were seeded into 25 cm2 cell culture bottle at a density of 3 × 105 cells/bottle and incubated for 24 h. Cells were treated with different concentrations of UA232 or UA for 48 h. Cells were collected and lysed in a RIPA buffer at 4 °C for 1 h. After centrifugation at 14,000 rpm for 20 min, the protein concentration of the supernatant was determined using a BCA Protein Assay Kit . Protein samples (40 μg) were electrophoresis on 10% SDS-polyacrylamide gel and then transferred to PVDF membrane. The membranes were incubated with 5% skimmed milk at room temperature for 1 h, then incubated overnight with a specific primary antibody for 4 °C. On the second day, the membrane was incubated with the corresponding HRP-conjugated secondary antibody  for 2 h. The target protein was detected by the chemiluminescence instrument. The grey value of the western blot was analyzed by ScnImage software.A549 or H460 cells were seeded into 25 cmp < 0.05 was considered statistically significant. Data were expressed as mean ± S.D.Statistical differences between groups were assessed by a one-way ANOVA using SPSS software 22.0. Supplemental Figure 1. Compared with UA, UA232 exhibited stronger cytotoxicity against H460 and A549 cells, while the cytotoxicity of UA232 to human normal cell HEK293T was similar to that of UA (50 values with UA232 for H460 and A549 cells at 24, 48, and 72 h. The inhibitory effect of UA232 on cell viability was significantly stronger than that of UA at each time point. As shown in In the early stage, we synthesized a series of compounds by structural modification of UA, and found that compared with UA, the novel compound UA232 has a more significant inhibitory effect on the cell viability of A549 and H460 cells. The chemical structures and characterization of UA232 were shown in at of UA . Table 1The effects of UA232 on the cell cycle regulation of H460 or A549 cells were further analyzed by flow cytometry. As shown in We further applied Annexin V-FITC/PI staining analysis to verify whether UA232 could induce apoptosis in H460 and A549 cells. As shown by the flow cytometry analyses in ER stress-mediated apoptosis is associated with PERK/eIF2α-dependent induction of the pro-apoptotic transcriptional factor CHOP (Ghosh et al. About 60% of approved anticancer drugs come from natural resources, so natural products play a leading role in drug discovery (Gupta et al. 50 of UA232 at 24, 48, and 72 h, indicating that UA232 could exert its antitumor effect more quickly. Due to the high activity of UA232 on lung cancer cells, the cellular effects and molecular mechanisms of UA232 on H460 and A549 cells were further studied for the first time. Colony formation assays suggest that the growth of H460 and A549 cells has been inhibited by UA232. Therefore, the effects of UA232 on the cell cycle distribution in H460 and A549 cells were further investigated. Flow cytometry indicated that UA232 arrested the growth of H460 and A549 cells at the G0/G1 phase. It has been shown that inhibition of the expression of cell cycle-related proteins CDK4 and cyclin D1 can lead to cell cycle arrest in the G0/G1 phase and inhibit cell proliferation (Kim et al. In the present study, UA232 potently inhibited cell viability and colony formation ability of H460 and A549 cells. Interestingly, there was no significant difference in ICThe results of cell morphology observation showed that UA232 could lead to vacuole-like changes in lung cancer cells and lead to cell death. The results of Annexin V-FITC/PI double staining further confirmed that UA232 could induce apoptosis of H460 and A549 cells, indicating that UA232 induced cell death by inducing apoptosis. Apoptosis is regulated by a variety of pathways, including the death receptor pathway, mitochondrial apoptosis pathway, and endoplasmic reticulum stress pathway (Li et al. CHOP is a marker of PERK branch activation in the ER stress pathway (Walter and Ron in vitro. UA232 inhibited cell proliferation by inducing the G0/G1 phase arrest, and induced cell apoptosis, which is correlated with the ER stress pathway, especially the activation of the PERK/eIF2α/CHOP axis. To sum up, the novel compound UA232 is a promising anticancer drug.UA232 is a novel UA analogue, which exhibits a potent antitumor effect