Rationale exists for combining immune checkpoint inhibitors and PARP inhibitors (PARPi), and results of clinical trials in ovarian cancer are promising, but data in other cancers are limited.Efficacy and safety of PARPi/anti-PD-1 in advanced solid tumors were retrospectively analyzed. The efficacy measures included objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS) and overall survival (OS).P = 0.008), and LDH≥250U/L was negatively correlated with lowered DCR (P = 0.018), while lymphocyte number, ECOG and LDH significantly influenced both PFS and OS. We found that the possible resistant mechanisms were sarcomatous degeneration and secondary mutation, including BRCA2 truncation mutation, A2M, JAK1,T790M, KEAP1 and mTOR mutation. 37.5% patients had ≥grade 3 adverse events.This retrospective study included data from 40 patients. The ORR was 27.5% , with a DCR of 85.0% . Except four patients in first-line treatment (three with PR and one with SD), the ORR of ≥second-line treatment, non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) was 22.2%, 23.1% and 28.6%, and the DCR was 83.3%, 84.6% and 71.4%, separately. The median PFS of all patients, ≥second-line treatment, NSCLC and SCLC was 4.6 m, 4.2 m, 4.5 m and 3.7 m. The median OS was 9.4 m, 11.4 m, 12.7 m and 5.4 m, respectively. Multivariable analysis revealed that BRCA1/2 mutation was positively correlated with ORR  contains supplementary material, which is available to authorized users. In recent years, immune checkpoint inhibitors (ICIs) targeting PD-1/PD-L1 have achieved substantial advancements and been commonly used to treat different solid tumors. However, only a small portion of populations derive benefit, such as patients with high expression of programmed death-ligand 1 (PD-L1), high tumor mutation burden (TMB) and microsatellite instability , elimina+/CD8+ T cells which were activated by the stimulator of interferon genes (STING) pathway and recruited to tumors by tumor-specific neoantigens when combined with ICIs , and patients with baseline lymphocyte ≥0.8 × 109/l had a better mPFS  of 40 patients), fatigue (9 [22.5%]), neutropenia (9 [22.5%]), thrombopenia (8 [20%]), loss of appetite (8 [20%]), nausea and vomiting (8 [20%]). And compared with chemo- group, the incidence of myelosuppression in chemo+ group was higher . The ORd CXCL10 , 14, mayd CXCL10 .P = 0.022) but not mOS , which might due to small case number and different cancer types between BRCA+ and BRCA− group. The subgroup analysis of PARPi/ICIs combination in other studies showed longer mPFS for BRCA+ in TNBC cohort instead of ovarian cancer [P = 0.045), subgroup analysis by TMB showed no statistic difference , and there was no statistic difference of ORR between patients with ΔTMB > 0 and ΔTMB ≤ 0. We thought that small case number was a reason, and the time to collect blood samples should be adjusted. We also did PD-L1-based subgroup analysis of PFS and OS, although the survival curves separated and the mPFS and mOS of PD-L1≥1% group improved, still statistical difference was not found. Chemotherapy used to be considered to promote the response of ICIs to cancer [Survival analysis in our study showed longer mPFS but shorter mOS for BRCA+ when compared with BRCA−, however, statistic difference was seen only in mPFS Below is the link to the electronic supplementary material.


Although pentoxifylline (PTX) was identified as a competitive non-selective phosphodiesterase inhibitor, its pharmacological effect has not been clearly elucidated. The present study explored the effect of low dose 10 μg/mL PTX (therapeutic dose) compared to high dose 300 μg/mL PTX  in RAW 264.7 cells through immunoprecipitation-based high performance liquid chromatography (IP-HPLC), immunohistochemistry, and western blot. 10 μg/mL PTX increased the expression of proliferation , epigenetic modification , protein translation , RAS signaling , NFkB signaling , protection , survival , neuromuscular differentiation , osteoblastic differentiation , acute inflammation , innate immunity , cell-mediated immunity , while decreased the expression of ER stress , fibrosis , and chronic inflammation  versus the untreated controls. The activation of proliferation by 10 μg/mL PTX was also supported by the increase of cMyc-MAX heterodimer and β-catenin-TCF1 complex in double IP-HPLC. 10 μg/mL PTX enhanced FAS-mediated apoptosis but diminished p53-mediated apoptosis, and downregulated many angiogenesis proteins , but upregulated HIF1α, VEGFR2, and CMG2 reactively. Whereas, 300 μg/mL PTX consistently decreased proliferation, epigenetic modification, RAS and NFkB signaling, neuromuscular and osteoblastic differentiation, but increased apoptosis, ER stress, and fibrosis compared to 10 μg/mL PTX. These data suggest PTX has different biological effect on RWA 264.7 cells depending on the concentration of 10 μg/mL and 300 μg/mL PTX. The low dose 10 μg/mL PTX enhanced RAS/NFkB signaling, proliferation, differentiation, and inflammation, particularly, it stimulated neuromuscular and osteoblastic differentiation, innate immunity, and cell-mediated immunity, but attenuated ER stress, fibrosis, angiogenesis, and chronic inflammation, while the high dose 300 μg/mL PTX was found to alleviate the 10 μg/mL PTX-induced biological effects, resulted in the suppression of RAS/NFkB signaling, proliferation, neuromuscular and osteoblastic differentiation, and inflammation. Pentoxifylline (PTX), a xanthine derivative, is primarily used as an antiproteolytic agent to treat muscle pain in people with peripheral artery disease ,2 by actin vitro protein expression experiment. Although PTX has short half-life (0.39–0.84 h for the various doses and 0.96–1.61 h for the metabolites), its therapeutic dose for adult human is usually 400 mg , three times a day  polymerase 1 (PARP1) by 13.1% at 12 h, and cleaved PARP (c-PARP) by 21.5% at 12 h versus the untreated controls, while it increased the expression of murine double minute-2 homolog  by 8.9% at 12 h and minimally affected the expression of B cell lymphoma 2  (≤ 5%) .RAW 264.7 cells treated with 10 μg/mL PTX showed increases in the expression of FAS-mediated apoptosis proteins, that is, FAS ligand (FASL) by 20.5% at 48 h, FAS (CD95) by 10.1% at 12 h, FAS-associated protein with death domain (FADD) by 15.3% at 12 h, BH3 interacting-domain death agonist (BID) by 8.5% at 24 h, caspase 3 by 19.2% at 48 h, c-caspase 3 by 16.1% at 48 h, caspase 7 by 24.8% at 48 h, c-caspase 8 by 14% at 24 h, c-caspase 10 by 21.4% at 24 h versus the untreated controls, while the expression of FLICE-like inhibitory protein (FLIP) was decreased by 9.3% at 48 h .10 μg/mL PTX-treated RAW 264.7 cells showed increases in the expression of cellular stress protection- and antioxidant-related proteins versus the untreated controls, as follows: heat shock protein-70 (HSP70) by 17.2% at 12 h, Cu-Zn superoxide dismutase-1 (SOD1) by 28.2% at 48 h, glutathione S-transferase ω 1/2  by 30.3% at 48 h, nitric oxide synthases-1 (NOS1) by 6.4% at 12 h, LC3β by 11.6% at 48 h, sodium-dependent vitamin C transporter 2 (SVCT2) by 6.6% at 48 h, a transcription factor regulating the expression of antioxidant proteins NRF2 by 4.1% at 24 h, and energy expenditure hormone leptin by 13.1% at 24 h versus the untreated controls. Whereas PTX decreased the expression of cellular maintenance proteins: heme oxygenase-1 (HO1) by 12.1% at 48 h, small heat shock protein HSP27 by 14.2% at 48 h, HSP90α/β by 15.8% at 24 h, mTOR by 19.5% at 24 h, and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) by 12.1% at 24 h. The expressions of 5’ AMP-activated protein kinase α  was only minimally affected by PTX (≤ 5%) .10 μg/mL PTX-treated RAW 264.7 cells showed increases in the expression of survival and aging-related proteins, that is, pAKT1/2/3 by 6.5% at 24h, PKC by 8.6% at 24h, pPKC1α by 11.5% at 24h, SP-1 by 8.3% at 48h, a stress responsive deacetylase sirtuin 6 by 5.8% at 24 h, a master regulator of the regulatory pathway in the development and function of regulatory T cells FOXP3 by 7.4% at 12h, a type-I membrane protein related to β-glucuronidase Klotho by 7.1% at 12h, and PLCβ2 by 13.1% at 48h versus the untreated controls. Whereas PTX decreased the expression of telomere reverse transcriptase (TERT) by 10.4% at 12h, SP3 by 16.4% at 48h, sirtuin 1 by 9% at 24h, and FOXP4 by 11% at 48h. The expressions of sirtuin 7, FOXO1 (a transcription factor negatively regulating adipogenesis), and FOXO3 were only minimally affected by PTX (≤ 5%) .2+ release from ER) by 17.8% at 24 h. The expressions of GADD153 (C/EBP homologous protein (CHOP)) and p-GADD153 were only minimally affected by PTX (≤ 5%)  by 9.3% at 12 h, eIF2α and p-eIF2α  by 8.7% at 12 h and 5.7% at 48 h, respectively, activating transcription factor 6α (ATF6α) by 9% at 12 h, binding immunoglobulin protein  by 8.4% at 12 h, serine/threonine-protein kinase/endoribonuclease inositol-requiring enzyme 1 α (IRE1α) by 8.2% at 24 h, PGC-1α by 12.1% at 24 h, caveolin-1 by 8.3% at 48 h, and AIF by 10.9% at 48 h compared to the untreated controls. On the other hand, PTX upregulated the proteins contributing to ER-stress environment in cells; HSP70 by 17.2% at 12 h, p-eIF2AK3 by 6.7% at 48 h, ATF4 (cAMP-response element binding protein 2) by 8% at 24 h, AP1M1 (the medium chain of the trans-Golgi network clathrin-associated protein complex AP-1) by 12.9% at 48 h, calnexin (a chaperone for the protein folding in the membrane of the ER) by 8.5% at 12 h, LC3β (microtubule-associated proteins 1A/1B light chain 3B contributing to autophagosome biogenesis) by 11.6% at 48 h, and endothelin-1 (inducing CaX (≤ 5%) .10 μg/mL PTX was found to influence the expression of SHH/PTCH/GLI signaling proteins positively or negatively in RAW 264.7 cells. PTX upregulated the upstream proteins of SHH/PTCH/GLI signaling; sonic hedgehog (SHH) by 14.6% at 24 h, patched homolog 1  by 8.8% at 48 h, and CD44  by 9.8% at 48 h versus the untreated controls, while downregulated the downstream proteins of SHH/PTCH/GLI signaling; GLI1 by 12.9% at 24 h, EpCAM  by 3.1% at 48 h, and BCL2  by 4.6% at 12 h.On the other hand, PTX downregulated Notch/Jagged signaling proteins; Notch1 ) by 5.3% at 48 h, Jagged2 (ligand for Notch) by 6.1% at 24 h, and VEGF-A  by 6.7% at 48 h. The expressions of Notch upstream signaling proteins, HIF1α (hypoxia-inducible factors 1α) and Wnt1 were compensatory increased by 13% and 13% at 24 h, respectively .10 μg/mL PTX was found to influence the expression of cytodifferentiation proteins positively or negatively in RAW 264.7 cells. PTX upregulated some cytodifferentiation proteins, that is, α-actin by 12% at 24 h, transglutaminase 2 (TGase 2) by 15.3% at 48 h, protein kinase C , p-PKC1α(11.5% at 24 h), focal adhesion kinase , A-kinase anchoring proteins 13 , calmodulin  by 8.2% at 48 h, calnexin by 8.5% at 12 h, cystatin A (a thiol protease inhibitor) by 6.9% at 12 h, SOX9 (SRY-related HMG-box) by 9.6% at 48 h, AP-1 complex subunit mu-1  by 12.9% at 48 h), phosphoinositide-specific phospholipase C β2 (PLCβ2) by 13.1% at 48 h, leucine-rich repeat-containing G-protein coupled receptor 4 (LGR4) by 6.4%, E-cadherin by 17.4% at 48 h, and VE-cadherin  by 12.9%at 12 h, while downregulated other cytodifferentiation proteins, that is, p63 by 9% at 24 h, vimentin by 14.5% at 24 h, TGase 4 by 11% at 24 h, hexokinases 2 (HK2) by 10.9% at 12 h, Krox-25 by 7.1% at 24 h, DLX2  gene expressed in the head and limbs of the developing fruit fly) by 17.3% at 24 h, laminin α5 by 14.2% at 48 h, pancreatic lipase by 5.4% at 48 h, and prostate-specific antigen ) by 16.9% at 48 h. On the other hand, the expressions of cysteine-rich protein that participates in cytoskeletal remodeling (CRIP1) by 16.3% at 12 h), TBX22 (T-box transcription factor), and epithelial cell adhesion molecule (EpCAM) were only minimally affected by PTX (≤ 5%) .10 μg/mL PTX was found to have positive influence on the expression of neuromuscular differentiation proteins in RAW 264.7 cells. PTX upregulated some neuromuscular differentiation proteins, that is, neuron specific γ enolase (NSEγ) by 13.6% at 24 h, glial fibrillary acidic protein (GFAP) by 9.5% at 12h, myosin heavy chain 2 (MYH2) by 28.6% at 24 h, desmin by 12.2% at 12h, calmodulin (CaM) by 8.2% at 48 h, calnexin by 8.5% at 12 h, cystatin A by 6.9% at 12 h, AP-1 complex subunit mu-1 (AP1M1) by 12.9% at 48 h, focal adhesion kinase  by 6.3% at 48 h, SHH by 14.6 at 24 h, β-catenin by 18.5% at 24 h, Wnt1 by 13% at 24 h, TGase 2 by 15.3% at 48 h, and α-smooth muscle actin (α-SMA) by 30.3% at 12 h, while downregulated other neuromuscular differentiation proteins, that is, S-100 by 13.8% at 48 h, unconventional myosin-1a (membrane binding class I myosin), 10.9% at 12 h, neurofibromin 1 (NF1) by 6.3% at 12 h. The expressions of neurokinin 1 receptor  and cysteine-rich protein 1 (CRIP1) were only minimally affected by PTX (≤ 5%) .10 μg/mL PTX was found to decrease the expression of fibrosis-inducing proteins; FGF1 by 5.5% at 12 h, FGF2 by 10.3% at 12 h, TGF-β1 by 12.5% at 12 h, CTGF by 17.7% at 12 h, collagen 3A1 by 13.7% at 48 h, collagen 4 by 14.6% at 24 h, collagen 5A by 6.9% at 24 h, laminin α5 by 14.2% at 48 h, integrin β1 by 8.7% at 12 h, plasminogen activator inhibitor-1 (PAI1) by 8.7% at 12 h, α1-antitrypsin by 7% at 24 h, elafin  by 5.2% at 48 h, and also to increase anti-fibrosis proteins; plasminogen by 15.6% at 48 h, integrin α2 by 16% at 24 h, integrin α5 by 12.2% at 48 h, and capillary morphogenesis gene 2 (CMG2) by 13.5% at 12 h. On the other hand, the expression of endothelin-1, a key role of vascular homeostasis, was reactively upregulated by 17.8% at 24 h. And the expressions of FGF7 and platelet-derived growth factor A (PDGF-A) were only minimally affected by PTX (≤ 5%) .10 μg/mL PTX was found to influence the expression of oncogenesis proteins positively or negatively in RAW 264.7 cells. PTX decreased the expression of tumor suppressor proteins; breast cancer type 1 susceptibility protein (BRCA1) by 8.4% at 12 h, breast cancer type 2 susceptibility protein (BRCA2) by 18.6% at 24 h, neurofibromin 1  by 6.3% at 12 h, ataxia telangiectasia caused by mutations  by 13.9% at 24 h, maspin  by 9.5% at 24 h, deleted in malignant brain tumors 1 protein  by 18% at 24 h, methyl-CpG-binding domain protein 4  by 6.7% at 12 h, p53 by 17.5% at 24 h, retinoblastoma protein (Rb1) by 9.8% at 48 h, but increased the expression of PTCH1  by 10.1% at 24 h. On the other hand, PTX increased the expression of oncogenic proteins; carcinoembryonic antigen (CEA) by 9.6% at 12 h, 14-3-3 θ proteins (a phosphoserine binding protein that regulates Cdc25C) by 10.9% at 48 h, survivin (a negative regulator of apoptosis) by 11.9% at 48 h, mucin 4 (an anti-adhesive glycoprotein that contributes to tumor development and metastasis) by 15.6% at 24 h, Yes-associated protein 1  by 10.3% at 12 h, but decreased the expression of mucin 1  by 21.2% at 48 h. the expression of phosphatase and tensin homolog  and PIM1 (proto-oncogene serine/threonine-protein kinase) were only minimally affected by PTX (≤ 5%) .10 μg/mL PTX reduced the expression of major angiogenesis proteins in RAW 264.7 cells; angiogenin by 5.3% at 12 h, vascular endothelial growth factor A (VEGF-A) by 6.7% at 48 h), VEGF-D by 15.6% at 24 h, von Willebrand factor (vWF) by 7.7% at 12 h, Fms-related tyrosine kinase 4 (FLT4) by 12.4% at 48 h, lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) by 10.9% at 48 h, fibroblast growth factor-2 (FGF2) by 10.3% at 12 h, CD106 (vascular cell adhesion molecule-1 (VCAM-1) by 15.5% at 12 h, matrix metalloprotease-2 (MMP2), 14.9% at 24 h, MMP-10 (9.4% at 12 h), plasminogen activator inhibitor-1 (PAI1) by 8.7% at 12 h, tumor-specific endothelial marker 8 (TEM8) by 12.3% at 12 h, CD54 (intercellular adhesion molecule 1 (ICAM-1)) by 12.6% at 12 h, and CD56 ) versus the untreated controls, while it compensatory elevated the expression of wound healing-related proteins, VEGF-C  by 10.7%10 μg/mL PTX-treated RAW 264.7 cells showed increase in the expression of osteogenesis proteins, that is, bone morphogenetic protein-2 , BMP3 (a negative regulator for bone density by antagonizing other BMPs) by 7.9% at 24 h, BMP4 by 21.3% at 24 h, BMPR1B (bone morphogenetic protein receptor type-1B) by 15.6% at 12 h, BMPR2 (bone morphogenetic protein receptor type 2) by 24.4% at 48 h, osteocalcin by 8.2% at 24 h, osteopontin by 18.4% at 48 h, osteonectin by 14.3% at 12 h, mammalian Runt-related transcription factor 2  by 21.3% at 24 h, osterix  by 13.3% at 48 h, alkaline phosphatase (ALP) by 13.1% at 48 h, osteoprotegerin (OPG) by 16% at 24 h, versican (abundant in the woven bone matrix) by 16.9% at 24 h, DMP1 by 19% at 24 h, SMAD4 by 14.9% AT 48 H, and activating transcription factor 4 (ATF4) by 8% at 24 h, while decreased the expression of aggrecan (a large chondroitin sulfate proteoglycan) by 7.4% at 12 h, receptor activator of nuclear factor kappa-Β ligand  by 6.6% at 12 h, TGF-β1 by 12.5% at 12 h, connective tissue growth factor  by 17.7% at 12 h, cathepsin K  by 9.2%PTX was found to downregulate TGF-β1, TGF-β2, TGF-β3, SMAD2/3, HGFα, Met, but reactively upregulate SMAD4, while it stimulated RAS signaling by upregulating KRAS, HRAS, pAKT1/2/3, PI3K, Rab1, ERK1, and pERK1 and downregulating JAK2, p-JNK1, and MEKK1, and simultaneously elevated NFkB signaling by upregulating NFkB, GADD45, p38, p-p38, and MDR, and downregulating p-mTOR and PGC1α.PTX increased the expression of acute inflammatory proteins , cell-mediated immunity proteins , and innate immunity proteins  were downregulated by 10 μg/mL PTX, while HIF1α, CMG2, VEGF-C, VEGFR2, and p-VEGFR2 were upregulated. On the other hand, PTX consistently increased the expression of osteogenesis proteins, BMP2, BMPR1B, osterix, RUNX2, osteocalcin, osteonectin, versican, and ALP, but decreased RANKL expression .300 μg/mL PTX reduced the expression of proliferation-related proteins in RAW 264.7 cells compared to 10 μg/mL PTX, that is, Ki-67 by 8.5% at 48 h, CDK4 by 9.9% at 12 h, cyclin D2 by 16.6% at 12 h, E2F1 by 14.8% at 24 h, cMyc by 6.4% at 12 h, MAX by 5.1% at 12 h, and MAD1 by 8.6% at 48 h, but increased the expression of p-Rb1 by 2.7% at 24 h. 10 μg/mL PTX stimulated the proliferation of RAW 264.7 cells by upregulating Ki-67, CDK4, cyclin D2, E2F1, cMyc, MAX, and downregulating p-Rb1, while 10 μg/mL PTX showed anti-proliferative effect on RAW 264.7 cells by downregulating Ki-67, cyclin D2, E2F1, and MAX compared to the untreated controls .300 μg/mL PTX reduced the expression of RAS/NFkB signaling proteins in RAW 264.7 cells compared to 10 μg/mL PTX, that is, KRAS by 11.3% at 12 h, pAKT1/2/3 by 11.3% at 12 h, p-ERK1 by 14.7% at 24 h, NFkB by 15.4% at 24 h, p-p38 by 6.9% at 24 h, GADD153 by 10.8% at 48 h, and p-PKC1α by 5.8% at 24 h. However, the expression of pAKT1/2/3 and p-PKC1α by 300 μg/mL PTX were increased to 102% at 48h and 106.9% at 12 h, respectively, compared to those by 10 μg/mL PTX. 10 μg/mL PTX enhanced RAS/NFkB signaling by upregulating KRAS, pAKT1/2/3, p-ERK1, NFkB, p-p38, GADD153, and p-PKC1α compared to the untreated controls, while 300 μg/mL PTX attenuated RAS/NFkB signaling by downregulating pAKT1/2/3, p-ERK1, NFkB, p-p38, and GADD153 .300 μg/mL PTX markedly decreased the expression of inflammation-associated proteins in RAW 264.7 cells compared to 10 μg/mL PTX, that is, TNFα by 11% at 12 h, CD4 by 21.8% at 24 h, CD80 by 6.9% at 24 h, M-CSF by 15% at 48 h, CXCR4 by 7.1% at 24 h, MMP1 by 19.3% at 48 h, and lactoferrin by 15.7% at 24 h, but increased the expression of an inflammation suppressor TGF-β1 by 23.5% at 48 h. It was evident 10 μg/mL PTX stimulated the inflammatory reaction of RAW 264.7 cells by upregulating TNFα, IL-6, CD4, CD80, M-CSF, CXCR4,MMP1, lactoferrin, and downregulating TGF-β1 compared to the untreated controls, while 300 μg/mL PTX showed anti-inflammatory effect on cells by downregulating TNFα, IL-6, CD4, M-CSF, MMP1, lactoferrin, and upregulating TGF-β1 .300 μg/mL PTX was found to have different effect on the expression of oncogenesis proteins in RAW 264.7 cells from 10 μg/mL PTX. The expressions of tumor suppressor protein p53, anti-apoptotic protein BCL2, apoptosis trigger FASL, and TGF-β1 by 300 μg/mL PTX were higher by 7.9% at 24 h, 13.3% at 48 h, 5.5% at 24 h, and 23.5% at 48 h, respectively, than those by 10 μg/mL PTX, while the expressions of FAS-associated protein with death domain (FADD), apoptosis executioners c-caspase 8 and c-caspase 3 by 300 μg/mL PTX were lower by 7.3% at 12 h, 9.9% at 24 h, and 13.3% at 48 h, respectively, than those by 10 μg/mL PTX. 10 μg/mL PTX induced significant apoptosis effect on RAW 264.7 cells through FAS-mediated apoptosis by upregulating FAS, FADD, c-caspase 8, and c-caspase 3 compared to the untreated controls, while 300 μg/mL PTX slightly reduced the apoptosis effect by downregulating FADD, c-caspase 8, and c-caspase 3 compared to 10 μg/mL PTX .300 μg/mL PTX increased the expression of ER stress proteins in RAW 264.7 cells compared to 10 μg/mL PTX, that is, eIF2AK3 (PERK) by 14.9% at 12 h, p-eIF2α by 12.8% at 48 h, ATF6α by 13.7% at 48 h, BIP by 10.3% at 12 h. and IRE1α by 3.3% at 24 h. And 300 μg/mL PTX minimally affected the expression of GADD153 (CHOP) compared to 10 μg/mL PTX. It was found that 10 μg/mL PTX reduced ER stress by downregulating eIF2AK3, p-eIF2α, ATF6α, BIP, and IRE1α, while 100 μg/mL PTX alleviated the reduction of ER stress by upregulating eIF2AK3, p-eIF2α, ATF6α, and BIP compared to 10 μg/mL PTX, particularly, the expression of eIF2AK3, p-eIF2α, ATF6α, and BIP were increased by 300 μg/mL PTX to 106% at 48 h, 106.4% at 48 h, 105.8% at 48 h, an 104.4% at 24 h, respectively, versus the untreated controls .300 μg/mL PTX increased the expression of fibrosis proteins in RAW 264.7 cells compared to 10 μg/mL PTX, that is, FGF1 by 7.1% at 24 h, FGF2 by 8.3% at 12 h, CTGF by 15.6% at 12 h, collagen 3A1 by 29.1% at 24 h, laminin α5 by 28% at 48 h, and α1-antitrypsin by 21.6% at 24 h. 10 μg/mL PTX showed potent anti-fibrosis effect on RAW 264.7 cells by downregulating FGF1, FGF2, CTGF, collagen 3A1 and 5A, laminin α5, and α1-antitrypsin, while 300 μg/mL PTX showed no anti-fibrosis effect rather increased the expression of FGF1 (by 6.4% at 48 h), collagen 3A1 (14% at 24 h), collage 5A (4.6% at 24 h), and laminin α5 (9.8% at 48 h) compared to the untreated controls .300 μg/mL PTX decreased the expression of neuromuscular differentiation proteins in RAW 264.7 cells compared to 10 μg/mL PTX, that is, NSEγ by 7.6% at 48 h, GFAP by 5.2% at 12 h, MYH2 by 18.1% at 24 h, desmin by 8.5% at 24 h, and α-SMA by 21.5% at 12 h, but the expression of myosin 1a (membrane binding class I myosin) was increased by 11.3% at 12 h. 10 μg/mL PTX induced nerve differentiation by upregulating NSEγ and GFAP, and muscle differentiation by upregulating MYH2 , desmin, and α-SMA, while 300 μg/mL PTX only slightly increased the expression of NSEγ, GFAP, MYH2, desmin and α-SMA compared to the untreated controls .300 μg/mL PTX decreased the expression of osteogenesis proteins in RAW 264.7 cells compared to 10 μg/mL PTX, that is, BMP2 by 25.8% at 48 h, RUNX2 by 27.7% at 12 h, osterix by 8.7% at 24 h, osteocalcin by 5.2% at 24 h, osteopontin by 11.3% at 48 h, and osteonectin by 9.4% at 24 h. However, the expression ratios of OPG and RANKL, which are essential signaling molecules of RANKL/RANK/OPG system regulating osteoclast differentiation/activation and calcium release from the skeleton, were increased by 6.6% and 9.6% at 12 h, respectively. It was found 10 μg/mL PTX showed strong osteogenic effect on RAW 264.7 cells by upregulating BMP2, RUNX2, osterix, osteocalcin, osteopontin, osteonectin, and OPG, while 300 μg/mL PTX alleviated the osteogenic effect by downregulating RUNX2, BMP2, and osteocalcin compared to the untreated controls .The pharmacological effect of PTX was frequently investigated in different cell types including RAW 264.7 cells –22 and aIn the present study, RAW 264.7 cells, which are originally murine monocytes, were explored for PTX-induced protein expression changes by administrating with two different doses, 10 μg/mL PTX similar to the therapeutic dose in human, and 300 μg/mL PTX which was frequently used in cell and animal experiments. First of all, the 10 μg/mL PTX-induced effect was compared with the 300 μg/mL PTX-induced effect. However, in clinical application, even the therapeutic dose PTX, about 10 μg/mL, produces diverse side effects including belching, bloating, stomach discomfort or upset, nausea, vomiting, indigestion, dizziness, flushing, angina, palpitations, hypersensitivity, itchiness, rash, hives, bleeding, hallucinations, arrhythmias, and aseptic meningitis ,57. TherIn the global protein expression of RAW 264.7 cells by 10 μg/mL PTX, a competitive non-selective phosphodiesterase inhibitor which is known to raise intracellular cAMP and activate PKA, actually enhanced RAS signaling by upregulating AKAP13, pAKT1/2/3, PKC, p-PKC1α, KRAS, and HRAS, and subsequently activated histone/DNA demethylation and acetylation by upregulating KDMD4, HMGB1, and PCAF, and downregulating HDAC10, MBD4, DMAP1, DNMT1 and EZH2, and subsequently induced cellular proliferation by upregulating cMyc/MAX/MAD network proteins, Wnt/β-catenin signaling objective protein TCF1, proliferation activating proteins Ki-67, PCNA, PLK4, cyclin D2, and cdc25A in this study. On the other hand, the expression of CDK inhibitors, p14, p15/16, and p21 were compensatory upregulated. The double IP-HPLC to assess the amount of protein complex containing two different target proteins showed the increase of cMyc-MAX heterodimer  and β-caAlthough the expression of cMyc and MAX were decreased by 10 μg/mL PTX at 12, 24, and 48 h, the expression of MAD1 was increased in western blot and IP-HPLC, and the double IP-HPLC showed dominant increase of cMyc-MAX heterodimer and decrease of cMyc-MAD heterodimer versus the untreated controls . TherefoCell counting assay of 10 μg/mL PTX-treated cells showed the increase of cell number at 12, 24, and 48 h. Regarding proliferation-related proteins, ICC revealed strong positive reaction of Ki-67 at 12, 24, and 48 h compared to the untreated controls, and western blot showed strong bands of Ki-67, E2F1, Wnt1, and TCF1 at 12, 24, and 48 h. Taken together, it is evident that 10 μg/mL PTX enhances RAS signaling, and subsequently activates histone/DNA demethylation and acetylation, cMyc/MAX/MAD network, and Wnt/β-catenin signaling, and resulted in the proliferation of RAW 264.7 cells. Whereas 300 μg/mL PTX showed a trend to decrease the expression of proliferation-related proteins, Ki-67, cMyx, MAX, p-Rb1, E2F1, and cyclin D2 compared to 10 μg/mL PTX.The non-selective phosphodiesterase inhibitor, PTX specifically induced RAS signaling, and subsequently stimulated NFkB signal by upregulating PKC, p-PKC-1α, AKAP13, MDR, GADD45, p38, and p-p38, and eventually influenced on the expression of protection-, ER stress-, apoptosis-, and inflammatory proteins in RAW 264.7 cells. Regarding cellular protection, 10 μg/mL PTX upregulated antioxidant proteins, SOD1, GSTO1/2, and SVCT2, but downregulated NRF2 regulating antioxidant expression, PGC-1α regulating mitochondrial biogenesis, and AMPK1α that plays a role in cellular energy homeostasis. And 10 μg/mL PTX mitigated ER stress by downregulating eIF2AK3, p-eIF2AK3, eIF2α, p-eIF2α, GADD153, p-GADD153, BIP, IRE1α, and ATF6α. Therefore, it is suggested that 10 μg/mL PTX-treated in RAW 264.7 cells are relatively safe under control of cellular protection and homeostasis. And more, the 10 μg/mL PTX-induced RAS signal by cAMP accumulation activated epigenetic modification through histone/DNA demethylation or acetylation, and subsequently elevated protein translation, which eventually positively influenced on cell proliferation, differentiation, protection and survival of RAW 264.7 cells.10 μg/mL PTX-induced NFkB signal also influenced on the expression of inflammatory protein positively or negatively in RAW 264.7 cells. It was found some acute inflammatory proteins , innate immunity proteins , and cell-mediated immunity proteins  were upregulated, while some chronic inflammatory proteins including IL-12, CD68, CD106, lysozyme, cathepsin C and G, COX2, and α1-antitrypsin were downregulated. Therefore, it is suggested 10 μg/mL PTX-treated cells are tend to differentiate into M1 type macrophages, which are pro-inflammatory type and important for phagocytosis and secretion of pro-inflammatory cytokines and microbicidal molecules to defend against pathogens, such as bacteria, virus –62, etc.On the other hand, 300 μg/mL PTX consistently decreased the expression of inflammatory proteins, TNFα, IL-6, CD4, CD80, M-CSF, CXCR4, MMP1, and lactoferrin but compensatory increased the expression of anti-inflammatory protein TGF-β1 compared to 10 μg/mL PTX. And 300 μg/mL PTX increased the expression of anti-apoptosis protein, BCL2, but minimally affected the expression of tumor suppressor protein, p53, compared to 10 μg/mL PTX, whereas the apoptosis triggering TNF family protein, FASL, and the initiator caspase, c-caspase 8 were more upregulated by 300 μg/mL PTX than 10 μg/mL PTX. Therefore, it is suggested 300 μg/mL PTX-treated cells showed anti-inflammatory effect and FAS-mediated apoptosis.The 10 μg/mL PTX-treated RAW 264.7 cells showed crosstalk between activated RAS and NFkB signalings, and were progressed to cytodifferentiation by upregulating some growth factors and SHH/PTCH signaling. Particularly, it is evident that the 10 μg/mL PTX-treated RAW 264.7 cells have potentials of neuromuscular and osteoblastic differentiation, which are able to influence on objective adjacent cells –65. The In the present study, 300 μg/mL PTX consistently decreased the expression of proliferation-, RAS/NFkB signaling-, inflammation-, and osteogenesis-related proteins but apoptosis proteins compared to 10 μg/mL PTX, therefore, it is thought that the high dose 300 μg/mL PTX may somehow disturb the protein expressions and give a harmful effect on RAW 264.7 cells, and resulted in the increase of FAS-mediated apoptosis, whereas the low dose 10 μg/mL PTX showed characteristic protein expression of competitive non-selective phosphodiesterase inhibitor, which may be helpful for the investigation of PTX pharmacological effect in human.Contrary to the neuromuscular and osteoblastic differentiation in PTX-treated RAW 264.7 cells, 10 μg/mL PTX suppressed fibroblastic differentiation and attenuated collagen production by downregulating the fibrosis-inducing proteins, FGF1, FGF2, TGF-β1, CTGF, collagen 3A1, 4, and 5, laminin α5, integrin β1, α1-antitrypsin, and upregulating the fibrosis-inhibiting proteins, plasminogen, CMG2, integrin α2 and α5. On the other hand, 10 μg/mL PTX increased the expression of endothelin-1 having a key role of vascular homeostasis. In the global expression of 10 μg/mL PTX-treated RAW 264.7 cells, the anti-fibrotic protein expression is closely relevant to the reduction of chronic inflammation-associated M2 macrophage polarization by downregulating CD68, CD106, lysozyme, MMP9, and α1-antitrypsin, and the low level of ROS damage and ER stress after 10 μg/mL PTX treatment. Therefore, it is suggested that 10 μg/mL PTX inhibits M2 type macrophage polarization through RAS/NFkB/TNFα signaling , and redde novo angiogenesis for wound healing Reviewers' comments:Reviewer's Responses to Questions <font color="black"> Comments to the Author1. Is the manuscript technically sound, and do the data support the conclusions? </font> The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes********** <font color="black"> 2. Has the statistical analysis been performed appropriately and rigorously?  </font> Reviewer #1: N/AReviewer #2: Yes********** <font color="black"> 3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. </font> The Reviewer #1: YesReviewer #2: Yes********** <font color="black"> 4. Is the manuscript presented in an intelligible fashion and written in standard English? </font> PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes********** <font color="black"> 5. Review Comments to the Author </font> Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The "unbiased" large scale analysis of multiple signaling components at protein level adds new details to the mode of action, especially of the dose-dependent actions, of this old, but still used drug.The weak point of the paper is that the results are based on a single mouse macrophage cell line. Since the agent is nowadays rarely used in mouse models, the considerable effort of the authors might be better spent using human models, better mimicking the clinical situation. Although analogous mature human macrophage cell lines are not available, the experiments might at least be supplemented some experiments with primary human macrophages or in vitro "matured" human monocyte cell lines.The paper is still acceptable in its present form, however, the use of human primary cells or human cell lines would greatly increase its practical value.Reviewer #2: The present work suggested that PTX has different biological effects on RWA 264.7 cells depending on the concentration of 10 μg/mL and 300 μg/mL PTX. The low dose 10 μg/mL PTX enhanced RAS/NFkB signaling, proliferation, differentiation, and inflammation, particularly, it stimulated neuromuscular and osteoblastic differentiation, innate immunity, and cell-mediated immunity, but attenuated ER stress, fibrosis, angiogenesis, and chronic inflammation, 44 while the high dose 300 μg/mL PTX was found to alleviate the 10 μg/mL PTX-induced biological effects, resulted in the suppression of RAS/NFkB signaling, proliferation, neuromuscular and osteoblastic differentiation, and inflammation. However, I have one comment to the authors as following:?" ext-link-type="uri" xlink:type="simple">https://doi.org/10.3390/app11178273"?- Could the authors explains what is the difference between this work and the recently published paper "Appl. Sci. 2021, 11, 8273. ********** <font color="black"> 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). 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Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.Reviewer #1: YesReviewer #2: Yes________________________________________2. Has the statistical analysis been performed appropriately and rigorously?Reviewer #1: N/AReviewer #2: Yes________________________________________3. Have the authors made all data underlying the findings in their manuscript fully available?The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.Reviewer #1: YesReviewer #2: Yes________________________________________4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes________________________________________5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The "unbiased" large scale analysis of multiple signaling components at protein level adds new details to the mode of action, especially of the dose-dependent actions, of this old, but still used drug.The weak point of the paper is that the results are based on a single mouse macrophage cell line. Since the agent is nowadays rarely used in mouse models, the considerable effort of the authors might be better spent using human models, better mimicking the clinical situation. Although analogous mature human macrophage cell lines are not available, the experiments might at least be supplemented some experiments with primary human macrophages or in vitro "matured" human monocyte cell lines.The paper is still acceptable in its present form, however, the use of human primary cells or human cell lines would greatly increase its practical value.[Answer] PTX is an old and well-known drug, but its effect of non-selective phosphodiesterase inhibitor may give an important roles in the treatment of different diseases. Recently, PTX is frequently recommended as a wound healing drug by inhibiting fibrosis and stimulating osteogenesis to prevent severe complications of osteoradionecrosis in head and neck cancer patients.  Regarding the selection of human cell line, I am appreciate for your comment using primary human macrophages or in vitro "matured" human monocyte cell lines. It is really critical to use human therapeutic dose of PTX in human cell line, therefore, in the following experiment, we have a plan to perform by using human macrophages or Human Umbilical Vein Endothelial Cells (HUVECs). It is sorry to inform that we are unable to fulfill the reviewer’s comment at this time. Reviewer #2: The present work suggested that PTX has different biological effects on RWA 264.7 cells depending on the concentration of 10 μg/mL and 300 μg/mL PTX. The low dose 10 μg/mL PTX enhanced RAS/NFkB signaling, proliferation, differentiation, and inflammation, particularly, it stimulated neuromuscular and osteoblastic differentiation, innate immunity, and cell-mediated immunity, but attenuated ER stress, fibrosis, angiogenesis, and chronic inflammation, 44 while the high dose 300 μg/mL PTX was found to alleviate the 10 μg/mL PTX-induced biological effects, resulted in the suppression of RAS/NFkB signaling, proliferation, neuromuscular and osteoblastic differentiation, and inflammation. However, I have one comment to the authors as following:https://doi.org/10.3390/app11178273"?- Could the authors explains what is the difference between this work and the recently published paper "Appl. Sci. 2021, 11, 8273. [Answer] Dr. Kim, Soung Min, who is a corresponding author of the paper published in Appl. Sci. 2021, was cooperated with me for the PTX effect on RAW 264.7 cells. After the preliminary experiment, I found a great discrepancy between human therapeutic dose (10 μg/mL) of PTX and high dose (100 – 500 μg/mL) frequently used in many animal experiments. And then, Dr. Kim submitted the preliminary data to the Journal of Applied Science by inclining to the results reported in the literature in the absence of any discussion, therefore, I had retrieved my authorship from the paper. The present manuscript is a revised and upgraded one through careful analysis using different methods. We hope this manuscript contribute a little advance in the elucidation of PTX pharmacological effect. ________________________________________6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.Reviewer #1: NoReviewer #2: Nohttps://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, [Answer]All figure files have been examined and converted by PACE digital diagnostic tool.Besides the editor and reviewer’s comments, the present manuscript is added more IP-HPLC data using 6 antisera, including AXIN2, HMGB1, LGR4. Klotho, FOXO1, and FOXO3. Because the graph of protection proteins become much crowded and complicated, it is divided into two graphs of protection-related proteins and survival and aging-related proteins in Fig 13. And the diagram of global protein signaling pathway of PTX, Fig 19, is upgraded to demonstrate the panoramic scope of protein expression. The additional minor correction of mistyped letter or sentences is done and traceable in “Revised Manuscript with Track Changes-PTX-220302.docx”.Thank you very much for your kindness.AttachmentResponse to Reviewers-220302.docxSubmitted filename: Click here for additional data file. 7 Mar 2022Pentoxifylline-induced Protein Expression Change in RAW 264.7 Cells as Determined by Immunoprecipitation-based High Performance Liquid ChromatographyPONE-D-21-38689R1Dear Dr. Lee,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. 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For more information, please contact Kind regards,Michael SchubertAcademic EditorPLOS ONE 11 Mar 2022PONE-D-21-38689R1 Pentoxifylline-induced Protein Expression Change in RAW 264.7 Cells as Determined by Immunoprecipitation-based High Performance Liquid Chromatography Dear Dr. Lee:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. 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Odontotermes formosanus and obtained a variety of valuable natural products.Actinomycetes are important microbes, and they are very important for developing active substances for useful drugs. Actinomycetes are numerous inhabitants, and they are widely distributed in the nest of fungus-growing termites. Previously, we isolated and purified numerous actinomycetes from the combs of Odontotermes formosanus using medium-based cultures. Among the eight media tested, M7 and I-HV media were found suitable for isolating actinomycetes. Further, 84 actinomycetes, including 79 Streptomyces isolates, were isolated and purified from O. formosanus and its combs, which belong to four genera . Then, the type and quantity of actinomycetes were positively correlated with the activity range of termites. Twenty-two actinomycetes strains showed antimicrobial activities. Among them, the BYF18, BYF48, BYF70, and BYF106 strains exhibited antifungal activities against five pathogenic fungi, with zone of inhibition (ZOI) values ranging from 3 to 21 mm. Grincamycin N was isolated and purified from the metabolites of Streptomyces lannensis (BYF106), and it displayed antibacterial activities against Staphylococcus aureus (ZOI = 13.82 ± 0.52 mm) and Micrococcus tetragenus (ZOI = 17.6 ± 0.5 mm) .Here, we isolated and purified actinomycetes from fungus-growing termite O. formosanus are important sources of new active substances.Our results confirmed that the actinomycetes associated with The online version contains supplementary material available at 10.1186/s12866-022-02501-5. Microorganisms are important sources of natural products, and they have great potential for application in medical, chemical, and agricultural industries . For exaInsects are by far the most diverse and abundant animal clade on earth, in terms of both the number of species and biomass . They arOdontotermes formosanus (Blattaria: Termitidae) is abundantly found and is widely distributed in Asia and Africa +. The 1H NMR and 13C NMR data were as follows: 1H NMR  δ1.19 , 1.25 , 1.27 , 1.63 , 2.41 , 2.47 , 2.51 , 2.91 , 3.06 , 3.51 , 3.86 , 4.34 , 4.71 , 5.01 , 5.22 , 7.74 , 7.82 , 7.92 , 7.93 ; and 13C NMR  δ158.0 , 137.0 , 133.6 , 118.8 , 131.9 , 187.8 , 115.0 , 169.6 , 135.2 , 139.7 , 118.3 , 131.0 , 187.8 , 115.4 , 39.6 , 71.0 , 46.3 , 172.4 , 26.4 , 70.4 , 35.8 , 75.7 , 73.5 , 23.6 , 17.3 , 90.4 , 70.8 , 39.7 , 208.7 , 76.9 , 16.0 . These data were almost identical to those reported for fridamycin D (C31H32O12) [Compound BYF106-2 (fridamycin D) was obtained as a yellow powder, and its molecular formula, CS. aureus and M. tetragenus. The ZOI value against S. aureus was significantly higher for BYF106-1 than for BYF106-2 . The ZOI value against M. tetragenus was also significantly higher for BYF106-1 than for BYF106-2   in fungus combs [In the world of fungus-growing termites, all termites live in the stable environment of the nest, where there is a clear social division of labor. The king and queen are responsible for reproduction, soldiers protect the nest, and workers are responsible for collecting food, building the nest, and cultivating crops , which reflected their broad-spectrum antimicrobial activity. This result indicated that actinomycetes produce a variety of compounds, rather than a single compound, to antagonize specific pathogenic fungi. BYF106 and BYF48 strains also showed antifungal activity against four plant pathogenic fungi , indicating that actinomycetes associated with O. formosanus have great potential as sources of new active substances. The monomer compounds BYF106-1 (grincamycin N) and BYF106-2 (fridamycin D) produced by S. lannensis (BYF106) also showed inhibitory activity against two human pathogens. Thus, BYF106-1 also can be developed as an active substance.Antifungal tests showed that many O. formosanus, and the distribution of actinomycetes was affected by the termite’s role in the nest. Most of the isolated actinomycetes were Streptomyces, and many of them are known to have antimicrobial activity. Grincamycin N, a monomer compound with significant antibacterial activity, was isolated from the fermentation broth of BYF106. Our results highlight that actinomycetes associated with O. formosanus are good sources of new active compounds.We found that culturable actinomycetes were mainly distributed on the body surfaces of The samples were collected in sterile petri dishes from the Jiangyin City, Jiangsu Province, China .Alternaria solani, Botrytis cinerea, Fusarium graminearum, Curvularia clavata, Curvularia lunata, Colletotrichum graminicola, Fusarium oxysporum f. sp. cucumerinum, Fusarium oxysporum f. sp. vasinfectum, and Fusarium oxysporum f. sp. momordicae, and three pathogenic bacteria, Escherichia coli, Staphylococcus aureus, and Micrococcus tetragenus, were obtained from the Zhejiang Normal University, Jinhua City, Zhejiang Province, China .Nine different phytopathogenic fungi, 2PO4, 1.42 g of Na2HPO4, 8 g of NaCl, and 0.2 g of KCl in 1L of distilled water) to obtain rinse solutions. Then, they were dissected under sterile conditions to obtain the whole guts, which were ground using sterile mortars to produce gut slurries. In addition, fungus combs (4 g) were ground and treated using four separate methods to obtain slurries. The following four methods were used: (i) 1 g of fungus comb was heated at 120 °C for 1 h before adding the 10 mL PBS buffer (pH 7.4); (ii) 1 g of fungus comb was subjected to microwave heating  for 9 min before adding the 10 mL PBS buffer (pH 7.4); (iii), 1 g of fungus comb in 10 mL of 1.5% phenol was placed in a 30 °C water bath for 30 min; and (iv) 1 g of fungus comb in 10 mL of 0.05% SDS was placed in a 40 °C water bath for 20 min [In total, 20 workers, 20 soldiers, 1 king, and 1 queen were removed from a freshly excavated termite nest and were separately placed in 10.0-mL sterile micro-centrifuge tubes for processing. Next, they were washed with sterile phosphate-buffered saline (PBS) buffer (pH 7.4) . All PCR reactions were carried out on an ABI Veriti thermocycler (USA) and using Phusion® High-Fidelity PCR Master Mix (New England Biolabs) and the specific primer pair 27F (5′-TCCTCCGCTTATTGATATGC-3′)-1492R (5′-GGTTACCTTGTTACGACTT-3′).2O, 25 µL Premix Taq (LA Taq Version 2.0), 2.5 µL DNA template, 1 µL 27F, and 1 µL 1492R. PCR was performed under the following conditions: 98 °C for 3 min, followed by 32 cycles of 98 °C for 10 s, 56 °C for 30 s, and 72 °C for 90 s, followed by 72 °C for 7 min and 16 °C for 4 min. The same volume of 1 × loading buffer (containing SYBR green) was mixed with PCR amplicons and electrophoresis was conducted on 1.2% agarose gels for amplicon detection. The products were sent to the General Biosystems Limited Company  for sequencing, and the results were used for BLAST analysis and Ezbiocloud sequence alignment to construct the phylogenetic trees.The 50-µL PCR reaction mixture contained 20.5 µL ddHFor phylogenetic reconstructions, the nucleotide substitution model determined by the Bayesian Information Criterion (BIC) value calculated by MEGA X software. The substitution model was Tamura 3-parameter and gamma distributed (G) (T92 + G) with 1,000 bootstrap replicates. The sequences of the 16 s rRNA gene of all test actinomycetes isolates were deposited in GenBank (accession number: MT498694-778).A. solani, C. clavata, C. lunata, C. graminicola, and Fusarium oxysporum f. sp. cucumerinum as the test strains. The test pathogenic fungi were inoculated onto the centers of the potato dextrose agar (PDA) plate  were screened for their antimicrobial activities. We used nine different phytopathogenic fungi, The in vitro antifungal activities against phytopathogenic fungi were assayed using the growth rate method with slight modifications , 25. GenInhibition (%) = (1 − Da/Db) × 100.where Da represents the diameter of the growth zone in the experimental dish (mm) and Db represents the diameter of the growth zone in the control dish (mm).2Cl2/MeOH" mixtures with stably growing polarity  affording 16 parts (F1-F16). Further purification of part 2 by silica gave pure compound 1 (9.7 mg), and further purification of part 4 by silica gave pure compound 2 (5.6 mg).The cultural broth (20 L) was extracted with ethyl acetate (4 × 20 L). Ethyl acetate extracts were combined and concentrated by evaporation of menstruum in vacuo. Then the crude extract (10 g) was chromatographed on a silica gel (200–300 mesh) column eluting with CH1H NMR and 13C NMR spectra were measured in CDCl3 with a Bruker AVANCE-400  spectrometer, and the chemical shifts were obtained in δ (ppm) by referring to the solvent signal and tetramethylsilane (TMS) as internal standards [Structural identification of the compound 1 and compound 2 was made by mass and nuclear magnetic resonance (NMR) spectroscopies . The electrospray ionization mass spectrometry (ESI–MS) of the purified compound was recorded on a Mariner Mass 5304 instrument. tandards .E. coli, S. aureus and M. tetragenus) as the test strains. The disc diffusion method was applied to evaluate the antimicrobial activities of isolated metabolites. Filter paper disks with metabolite dissolved in Dimethyl Sulfoxide (DMSO) at a concentration (30 µg/filter paper) were added to the Luria Broth (LB) (Table SS. aureus and M. tetragenus) for 24 h. Filter papers with DMSO, gentamycin sulfate was set as positive control. The zone of inhibition (ZOI) was determined by measuring the distance from the center of the disk to the end of the clear zone, and the experimental data were recorded using the “cross method” [Purified metabolites were screened for their antimicrobial activity. We used three common pathogenic bacteria  Table S medium,  method” .All tests were performed in triplicate. Data are shown as mean values ± standard deviation.Data were analyzed using SPSS 19.0, and figures were drawn using GraphPad Prism 9.0 and MEGA X.Additional file 1:Table S1. Medium formula used in the experiment.Additional file 2:Table S2. List of all actinobacterial strains isolated from Odontotermes formosanus and combs.


Plasmodium falciparum’s resistance to available antimalarial drugs highlights the need for the development of novel drugs. Pyrimidine de novo biosynthesis is a validated drug target for the prevention and treatment of malaria infection. P. falciparum dihydroorotate dehydrogenase (PfDHODH) catalyzes the oxidation of dihydroorotate to orotate and utilize ubiquinone as an electron acceptor in the fourth step of pyrimidine de novo biosynthesis. PfDHODH is targeted by the inhibitor DSM265, which binds to a hydrophobic pocket located at the N-terminus where ubiquinone binds, which is known to be structurally divergent from the mammalian orthologue. In this study, we screened 40,400 compounds from the Kyoto University chemical library against recombinant PfDHODH. These studies led to the identification of 3,4-dihydro-2H,6H-pyrimidobenzothiazin-6-imine and its derivatives as a new class of PfDHODH inhibitor. Moreover, the hit compounds identified in this study are selective for PfDHODH without inhibition of the human enzymes. Finally, this new scaffold of PfDHODH inhibitors showed growth inhibition activity against P. falciparum 3D7 with low toxicity to three human cell lines, providing a new starting point for antimalarial drug development. Significant morbidity and mortality caused by protozoan parasitic infection pose serious threats to global health. Among parasitic diseases, malaria is the most devastating, with approximately 409,000 deaths reported during 2019, 67%  of whom were children under 5 years old from sub-Saharan Africa benzothiazin-6-imine , which was used for screening 40,400 compounds from the Kyoto University chemical library. The quality of our screening was evaluated by calculating the following parameters: Z’-factor (0.88 ± 0.09), signal window , signal-to-noise ratio (S/N = 85.0 ± 26.0), signal-to-background (S/B = 115.3 ± 28.6), and negative coefficient of variation (CV = 2.38 ± 1.85%), all of which were excellent. After the initial screening at 4.5 µM, we identified 43 compounds that meet the hit criteria (>50% inhibition), for a hit rate of 0.11%  against PfDHODH. Substitution at R1 to other 2-membered rings, such as Compounds 2 to 7, did not drastically affect the IC50s against PfDHODH (IC50 ranging from 0.70 to 1.3 µM) as shown in 1  or a phenyl group at R2  maintained the activity towards PfDHODH. Dual substitutions with benzothiophene and tert-butyl groups at R1 and R3 , respectively, resulted in a 6-fold increase in the IC50 compared to 1 (11) resulted in an increased IC50 to 6.42 µM relative to 1. Substitutions of the 2-naphtyl group of 1 to a phenyl group (12) had little effect on IC50  or trifluoromethyl (29) substitutions at R1 showed the highest IC50s of 18.5 and 9.71 µM against PfDHODH, respectively. Other BIP derivatives with substitutions at R1 (30 to 35) or R2 (36) inhibited PfDHODH activity stronger than 28 (30 at R1 (37 to 39), R2 (40 and 41), or R3 (42 and 43) lowered the IC50 compared to 30 , the remaining hits did not inhibit the activities of mammalian complexes I-III and II-III, at least in the range tested (0.0007 to 22.7 µM) in this study.To investigate whether or not the hits obtained in this study also inhibit HsDHODH, a counter-assay was conducted using purified recombinant HsDHODH. As a result, none of the hits inhibited HsDHODH . Except P. falciparum 3D7 based on PfLDH/diaphorase assays. A total of 31 compounds displayed EC50s below 10 µM, while the other 12 compounds did not inhibit parasite growth. From the compounds exhibiting antimalarial activity, 19 compounds showed an EC50 between 1 and 10 µM, and 12 compounds were below 1 µM  in P. falciparum  (accessed on 10 May 2021). Our efforts led to the identification of a new class of PfDHODH inhibitors that share DPBI and DBIP moieties. Those derivatives have previously been reported to inhibit the proliferation of human immunodeficiency virus (HIV) [P. falciparum 3D7 and displayed low toxicity to mammalian normal and cancer cell lines.To discover selective PfDHODH inhibitors with a novel scaffold, we screened 40,400 compounds from Kyoto University chemical library. This library is open-access and composed of many original compounds and diverse scaffolds (us (HIV) ,40,41,42us (HIV) , and herP. falciparum-expressing Family 1A DHODHs, such as yDHODH, are known to be resistant to PfDHODH inhibitors [6, 13, 14, 19, 21, 30, and 43, which showed growth inhibition against 3D7 but were inactive against 3D7-yDHODH , liver-stage parasites, as well as in vivo models (P. berghei) will be required for the future development of the compounds described in this study.In this study, we have identified PfDHODH inhibitors with new chemical scaffolds that inhibited the growth of the E. coli, as recently reported [E. coli BL21 Star (DE3) strain, harboring pETSUMO/PfDHODH, was selected in Luria–Bertani agar plates supplemented with 50 µg/mL kanamycin (Sigma). A single colony was inoculated onto 360 mL of LB medium containing the same antibiotic (pre-culture) and cultured at 37 °C for 16 h with shaking at 200 rpm. The pre-culture (60 mL) was transferred to 600 mL of Terrific-Broth media supplemented with 50 µg/mL kanamycin in an Ultra Yield®® flask (Thomson), and then cultured at 37 °C with vigorous shaking at 200 rpm . Protein expression was induced by 25 µM isopropyl β-d-1-thiogalactopyranoside  when the optical density at 600 nm reached 0.6–0.8, and then cultured for 16 h at 20 °C.The codon-optimized PfDHODH gene, encoding amino acid residues 158 to 569 fused to a His6-SUMO tag in its N-terminus, was constructed and optimized for expression in reported . Brieflyg for 10 min, and then resuspended in lysis buffer containing 50 mM HEPES-NaOH (Dojindo) pH 7.6, 50 mM NaCl (Wako), 5 mM imidazole (Wako), 20% (v/v) glycerol (Wako), and 0.25 mM phenylmethylsulfonyl fluoride . Suspended cells were disrupted by a French press (Ohtake) at 180 MPa, followed by centrifugation at 30,000× g for 20 min to discard unbroken cells and debris. Triton X-100 (Roche) was mixed with a supernatant to a final concentration of 1% (w/v), stirred for 30 min, and then further centrifuged at 40,000 × g for 90 min. The supernatant was mixed with 3 mL of pre-equilibrated nickel-nitriloacetic acid  resin and kept overnight, rotating at 10 rpm. The protein–resin mixture was centrifuged at 1500× g for 15 min and the resin was suspended in a minimal volume of supernatant and loaded into a gravity-flow column. Next, the column was sequentially washed with 60 mL of buffer A  glycerol, 0.2 mM orotate), 60 mL of buffer A containing 0.05% (w/v) polyoxyethylene(9)dodecyl ether , and followed by 60 mL of buffer A containing 0.05% C12E9 and 20 mM imidazole. The bound protein was eluted with buffer A containing 0.05% C12E9 and 300 mM imidazole. The eluted fraction was concentrated using a centrifugal filter unit , Millipore) at 3500× g until the final volume reached 0.5 mL. The concentrated protein plus detergent solution was diluted with buffer A containing 0.05% (w/v) C12E9 until the imidazole concentration was reduced to 75 mM.The subsequent purification steps were performed at 4 °C. Cells were harvested by centrifugation at 5000× w/v) C12E9, and 0.2 mM orotate) and incubated for 14 h. Equilibrated Ni-NTA resin was added into the cleavage mix and then incubated for 2.5 h at 4 °C, followed by loading onto a gravity-flow column. The flow-through was collected and concentrated as described above. An equal volume of cold glycerol was added to the concentrated protein, FMN was added to a final concentration of 200 µM, and it was stored at −30 °C until use.The His6-SUMO tag was cleaved by SUMO protease at a 1:50 ratio relative to PfDHODH to a final volume of 40 mL in cleavage buffer  sodium dodecyl sulfate  was added to columns 23 and 24 as positive controls (100% inhibition). Next, 38 µL of assay mix  glycerol, 150 mM NaCl, 0.05% (v/v) Triton X-100, 15 µM decylubiquinone , 120 µM 2,6-dichlorophenolindophenol , and 20 nM PfDHODH) [®® (Eppendorf). The reaction was started by the addition of 5 µL of 1.8 mM L-DHO (Sigma) as the substrate and mixed as described above for 20 s. The absorbance at 600 nm was recorded using a SpectraMax®® Paradigm®® Multi-Mode Microplate Reader (Molecular Devices) before (t0) and after 20 min (t20) incubation at room temperature. The readings at t0 were subtracted from t20, and PfDHODH inhibition was calculated as the inhibition relative to the negative and positive controls in a single-point assay. Hits were defined as compounds inhibiting more than 50% of PfDHODH activity at 4.5 µM. The quality of the screening system was evaluated by calculating statistical parameters: Z′-factor, S/N, S/B, SW, and CV, as previously reported [50 values of the hit compounds were determined using the same assay system containing serial dilutions of each compound in triplicates  using GraphPad Prism 8.0 software .The screening of 40,400 compounds was conducted in 384-well plates by adapting the end-point assay reported before . First, PfDHODH)  were disreported ,49. The v/v) Triton X-100, 2 mM potassium cyanide , 60 µM dUQ, and 120 µM DCIP, with the reaction started by the addition of 200 µM L-DHO  in triplicate. For this assay, DMSO was used as a negative control and 10 µM of lapachol was used as a positive control (100% inhibition). The relative inhibition was calculated as described above.Selected hit compounds from PfDHODH screening were tested for potential inhibition against HsDHODH at 4.5 µM. The HsDHODH used in this study was purified as previously reported . The HsDc reductase (complexes I–III) and succinate-cytochrome c reductase (complexes II–III) activities were assayed in 384-well plates following an established protocol [c , and 15 µg/mL mitochondria. Plates containing 1 µL serial dilutions of PfDHODH inhibitors were prepared and mixed with 38 µL of the reaction mix, as described above. The reaction was started by the addition of 5 µL of 2.6 mM NADH, and the assay was performed as described for PfDHODH, but at 550 nm and with 15 min of incubation. Complex II–III activity was assayed similarly to complex I–III activity but with 100 µM cytochrome c and 25 µg/mL mitochondria, and the reaction was started by 10 mM succinate. For both assays, DMSO and 5 µM ascochlorin were used as negative and positive controls, respectively. Assays were performed in triplicate.Bovine heart mitochondria were prepared as previously described ,51,52. Tprotocol ,54,55,56P. falciparum 3D7 strain was cultured in 2% hematocrit type O human RBC in RPMI-1640 medium (Gibco) supplemented with 23.8 mM sodium bicarbonate (Wako), 50 mg/L hypoxanthine (Wako), 25 mg/L gentamycin (Sigma), and 0.5% (w/v) AlbuMAX®® II (Gibco) under a 5% O2, 5% CO2, and 90% N2 atmosphere at 37 °C. Parasites were synchronized with 5% (w/v) D-sorbitol (Wako).This assay was conducted using human red blood cells (RBC) following the guidelines of the ethics committee of Nagasaki University (permission no. 19). Human RBC was obtained from the Japanese Red Cross Society. The v/v). Parasite growth was monitored by the diaphorase-coupled P. falciparum lactate dehydrogenase (PfLDH) assay, as previously described [®® Paradigm®® Multi-Mode Microplate Reader. The first and second columns contained 0.4% (v/v) DMSO as a negative control, and the 23rd and 24th columns had a mixture of 1 µM atovaquone and artemisinin as a positive control. The EC50 was calculated using GraphPad Prism 8.0 software from the mean of quadruplets values.Synchronized ring-stage parasites at 0.3% parasitemia and 2% hematocrit were cultured in a 384-well plate at 25 µL with test compounds at a final concentration in the range of 0.0003 to 10 µM with a fixed DMSO concentration of 0.4%  heat-inactivated fetal bovine serum (Gibco) and the cells were maintained at 37 °C under 5% CO2.Human dermal fibroblast cells  were cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 medium , human pancreatic carcinoma cells  were cultured in DMEM (Gibco), and human colorectal adenocarcinoma cells  were cultured in RPMI-1640 medium . The culture mediums were supplemented with 10%  DMSO in column 1 and wells containing only the culture medium in column 12 were used as the negative and positive controls, respectively. After 48 h of incubation, the cells were washed with PBS, and 99 µL of fresh media was replaced, followed by the addition of 10 µL of Cell Counting Kit-8 (Dojindo) to each well. After 3 h of incubation, the absorbance was measured at 450 nm using a SpectraMax®® Paradigm®® Multi-Mode microplate Reader. The calculation of cell viability in the test well was based on the absorbance of control wells according to the manufacturer’s protocol. The mean of quadruplet values was used to calculate the CC50 using GraphPad Prism 8.0 software.HDF, PANC-1, and DLD-1 were seeded at 2.5 × 10P. falciparum 3D7 strain expressing the cytosolic yeast DHODH (3D7-yDHODH) was prepared as follows. pHHyDHODH-GFP plasmid  was transfected to the P. falciparum 3D7 strain following the established transfection method [2, 10 mM K2HPO4/KH2PO4 pH 7.6, 25 mM HEPES-KOH pH 7.6, 2 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid , and 5 mM magnesium chloride (Wako)) was transfected into the red blood cells by the Gene Pulser Xcell (Bio-Rad). Percoll–sorbitol synchronized parasites were mixed with red blood cells containing plasmid and maintained under 5 nM WR99210 for 2 weeks. The growth of resistant parasites against DSM265 and atovaquone, as PfDHODH and complex III inhibitors respectively, was confirmed by PfLDH/diaphorase assay. A total of 10 µM PfDHODH inhibitors were tested against wild-type 3D7 and 3D7-yDHODH following the method described above in triplicate. The significance of difference between the inhibition of the two parasite strains was evaluated by Student’s t-test (The n method . Brieflys t-test .50 determination. We successfully identified new classes of PfDHODH inhibitor harboring DPBI and DBIP moieties with growth inhibition activity against the asexual stage of P. falciparum 3D7. As DHODH does not seem to be essential in sexual stages, future studies will focus on the activity of our compounds against the liver stage parasite. Moreover, the compounds identified in this study are selective for PfDHODH and displayed low toxicity in several human cell lines, providing a new starting point for future antimalarial drug development.In this work, 40,400 compounds from the Kyoto University chemical library were screened against PfDHODH. Several quality control parameters were calculated in every assay plate, indicating excellent performance of our screening. The reproducibility of inhibition was further confirmed by the dose-response assay and IC


Neorautanenia mitis, Hydnora abyssinica, and Senna surattensis are medicinal plants with a variety of traditional uses. In this study, we sought to isolate the bioactive compounds responsible for some of these activities, and to uncover their other potential medicinal properties.N. mitis and H. abyssinica, and the leaves of S. surattensis were prepared and their phytochemical components were isolated and purified using chromatographic methods. These extracts and their pure phytochemical components were evaluated in in-vitro models for their inhibitory activities against Plasmodium falciparum, Trypanosoma brucei rhodesiense, Mycobacterium tuberculosis, α-amylase (AA), and α-glucosidase (AG).The DCM and ethanol extracts of the roots of Plasmodium falciparum showing a high safety ratio (SR) and IC50 values of 0.40 ± 0.07 μM (SR - 108) and 0.74 ± 0.29 μM (SR - 133) against TM4/8.2 and K1CB1, respectively. While (−)-2-isopentenyl-3-hydroxy-8-9-methylenedioxypterocarpan showed the highest inhibitory activity against T. brucei rhodesiense with an IC50 value of 4.87 ± 0.49 μM (SR > 5.83). All crude extracts showed inhibitory activities against AA and AG, with three of the most active phytochemical components; rautandiol A, catechin, and dolineon, having only modest activities against AG with IC50 values of 0.28 mM, 0.36 mM and 0.66 mM, respectively.Rautandiol B had significant inhibitory activities against two strains of Plasmodium falciparum. The relatively higher inhibitory activities of the crude extracts against AG and AA over their isolated components could be due to the synergistic effects between their phytochemical components. These crude extracts could potentially serve as alternative inhibitors of AG and AA and as therapeutics for diabetes.These studies have led to the identification of lead compounds with potential for future drug development, including Rautandiol B, as a potential lead compound against The online version contains supplementary material available at 10.1186/s12906-021-03406-y. Neorautanenia mitis (A. Rich) Verdc. (Fabacae), Hydnora abyssinica A. Braun (Hydnoraceae), and Senna surattensis (Burm. f.) H. Irwin and Barneby (Fabaceae), were selected based on their promising preliminary screening results, they have shown various bioactivities and are traditionally used for the treatments of many disease conditions. The roots of N. mitis, are used for the treatment of bilharzia, syphilis, diarrhea, skin infection, dysmenorrhea and neuropsychiatric conditions. They are also used as an anticonvulsant, anti-malarial, fish poison, insecticide, and for killing bilharzias-carrying fresh water snails in many African countries  hypoxanthine incorporation assay [3H]-hypoxanthine solution was added and incubated for 20 h. The parasites were harvested onto a UniFilter-96 GF/B plate . The filters in the plates were air-dried, and then 25 μL of liquid scintillation fluid  was added. The radioactivity was then measured using a microplate scintillation counter . The IC50 values of the crude extracts/ pure compounds were determined.on assay , 46. BriT. brucei rhodesiense (STIB-900) was maintained in Minimal Essential Medium (MEM) with Earle’s salts supplemented with 3 g/L sodium bicarbonate, 4.5 g/L glucose, 25 mM HEPES, pH 7.3, 0.05 mM bathocuproinedisulfonic acid disodium salt, 1.5 mM L-cysteine, 1 mM hypoxanthine, 0.16 mM thymidine, 1 mM sodium pyruvate, 0.2 mM 2-mercaptoethanol, 1% MEM non-essential amino acid, and 15% heated fetal bovine serum, at 37 °C in a 5% CO2 incubator. To assay anti-Tbr activity, a modified reported protocol was used [4 T. brucei rhodesiense parasites in 175 μL culture media were incubated with 25 μL of varying concentrations of each compound in a 96-well plate under the same culture conditions. Following 72 h incubation, 20 μL Alamar Blue (a resazurin solution) was added in each well. The mixture was further incubated for 3 h. The fluorescence signals were measured by a spectrofluorometer at ex530/em585 nm. The results were read as concentration of each compound that exhibit 50% growth inhibition (IC50) from the dose-response curve established from the fluorescence signals at each concentration of compounds.was used , 48. BriMycobacterium tuberculosis H37Rv (ATCC 27294) were grown in Löwenstein–Jensen (LJ) medium agars (Biomedia Thailand cat. no. BMT B24420) in a biosafety level 3 facility at the Department of Microbiology, Faculty of Science, Mahidol University. Two–three loopfuls of each M. tuberculosis strain from the LJ medium were scratched and put into a tube containing 10 mL of Middlebrook 7H9 broth supplemented with 0.2% glycerol and 10% Middlebrook OADC. Clumps of colonies were dispersed using a loop. The bacterial suspensions were left undisturbed for 15 min before the upper parts were collected. Absorbance at OD600 was measured. Finally, bacterial suspensions were prepared to OD600 at 0.005. Anti-tuberculous activities were evaluated by the Microplate Alamar Blue Assays [M. tuberculosis (OD600 of 0.005) was added to each well. Bacterial growth control and compounds/crude extracts control wells contained the medium with either bacteria or the test sample, respectively, while the medium control wells were without both. All were done in triplicate. The plate was incubated in a 37 °C incubator for 5 days. Then 20 μL of alamar blue and 12.5 μL of 20% Tween80 were added to one well of each control and the plate was further incubated for 24 h to test bacterial growth by observing color change. A blue color in the well was interpreted as no bacterial growth while a pink color represented proper bacterial growth. Therefore, if the well was still blue, the additional control wells were tested daily until the color changed to pink. Then the reagents were added to the entire plate and incubated for 24 h. Minimum inhibitory concentration (MIC) was then read from the lowest concentration of samples that prevented the change from blue to pink. Isoniazid and rifampin were employed as controls.e Assays . The assquantitatively determined in a 96-well plastic plate according to the method described by Nguyen et al. [p-nitrophenyl α-D-glucopyranoside (Sigma) as the substrate and 0.25 units/mL of AG from S. cerevisiae (Sigma). The absorbance at 405 nm was measured using a microplate reader . Acarbose (5 mM) was used as the positive control.AGI values of extracts and pure phytochemicals were n et al. . The enz2 and 5 mM KI). Following color development, the absorbance at 580 nm was measured using a microplate reader  [AAI values were determined using a microplate-based starch–iodine assay. Assay reactions were initiated by adding 40 μL of starch (Sigma S-2630) solution (2.0 g/L) and 40 μL of enzyme in 0.1 M phosphate buffer at pH 7.0 to microplate wells. The enzyme was incubated with the samples for 5 min before adding the substrate. To minimize evaporative loss during incubation, a plastic mat was used to cover the microplate. After 30 min of incubation at 50 °C, 20 μL of 1 M HCl was added to stop the enzymatic reaction, followed by the addition of 100 μL of iodine reagent  .S. surratensis were coded from its species name ‘cassia’ as CDCM and CEOH, respectively. While the DCM and ethanol extracts of H. abyssinica were coded from its local name ‘kaushe’ as KDCM and KEOH, respectively. And the DCM and ethanol extracts from N. mitis were coded from its local name ‘abargora’ as ABDCM and ABEOH, respectively. The color and appearances and percentage yields of the DCM and ethanol extracts from all three plants are reported in Table S. surrattensis-CAQ, H. abyssinica-KAQ and N. mitis-ABAQ) were described earlier [S. surrattensis and H. abyssinica respectively, compared to their DCM extracts. However, the ethanol extract of N. mitis had a lower yield of 1.0% while its DCM yield was 1.5%.The DCM and ethanol extracts from  earlier . Higher S. surrattensis, KEOH from H. abyssinica and ABDCM from N. mitis, was selected for phytochemical studies which involved the isolation and structural characterisation of the pure chemical constituents. This selection was based on their promising preliminary biological activities and TLC profiles.One extract from each plant, which were CEOH from 1) [2) [3), which was also isolated from KEOH [4) [Three known compounds where isolated from CEOH, they were; kempherol 1) , quercet1) [2) , and (+)rom KEOH  togetherKEOH [4)   , querce5–24 , neodulin (6), ferulic acid (7), ambonane (8), stigmasterole (9), pachyrrhizine (10), neotenone (11), 7-methoxy-3- chroman-4-one (12), 12a-hydroxydolineon (13), dolineon (14), (−)-2-isopentenyl-3-hydroxy-8-9-methylenedioxypterocarpan (15), nepseudin (16), neorautenol (17), isoneorautenol (18), (−)-2-hydroxypterocarpin (19), rotenone (20), 12a-hydroxyrotenone (21), dehydroneotenone (22), rautandiol A (23) and rautandiol B (24), as described previously [The phytochemical studies on ABDCM lead to the isolation of compounds eotenone , 7-methoP. falciparum giving IC50 values of 2.99 ± 0.59 μg/mL (SR - 0.9) and 2.67 ± 1.05 μg/mL (SR - 1.01) on TM4/8.2 and K1CB1, respectively. It also showed significant activity against T. brucei rhodesiense with an IC50 value of 3.04 ± 0.27 μg/mL. Compound 24 had significant inhibitory activities against both strains of P. falciparum with IC50 values of 0.40 ± 0.07 μM (SR - 108) and 0.74 ± 0.29 μM (SR - 133) against TM4/8.2 and K1CB1, respectively. Compounds 11, 14, 15, 20, and 21 also showed inhibitory activity against T. brucei rhodesiense, with the highest activity displayed by compound 15 having an IC50 value of 4.87 ± 0.49 μM (SR > 5.83). ABDCM and its compounds were not active against M. tuberculosis  and 8.44 ± 0.16 μM (SR- 4.52), respectively. None of the compounds from KEOH were active against T. brucei rhodesiense and neither the extracts nor the compounds isolated from CEOH or KEOH were active against M. tuberculosis , S. surrattensis  and H. abyssinica  show significantly higher percentage inhibitory activities than the positive control acarbose (71.3%) except the aqueous extract (ABAQ) from N. mitis, which gave a lower inhibitory activity of 45.4%. The highest inhibitory activity of 100% was from the DCM extract (ABDCM) from N. mitis.In this study, the crude extracts were screened for their AGI activities as shown in Fig. H. abyssinica (KEOH) had the highest inhibitory activity with IC50 values of 0.21 ± 0.09 μg/mL against AG and 0.06 ± 0.02 mg/mL against AA. Both IC50 values were lower than that of acarbose which had IC50 values of 438.5 ± 4.9 μg/mL and 0.42 ± 0.07 mg/mL for AG and AA, respectively.The most active extracts were further analysed for their AAI and AGI activities. The data in Fig. 50 value of 0.7 ± 0.06 μg/mL and 0.31 ± 0.01 mg/mL against AG and AA, respectively. Also, the inhibitory activity of the DCM (ABDCM) and ethanol (ABEOH) extracts of N. mitis against AG gave significant IC50 values of 26.8 ± 1.01 μg/mL and 6.20 ± 0.32 μg/mL, respectively.KAQ had an IC3, 14, and 23 showed significant percentage inhibitory activities of 97.5 ± 0.6%, 54.2 ± 8.9% and 83.9 ± 1.0% when compared to the positive control acarbose that showed 86.5 ± 0.4% inhibition  had modest IC50 values of 0.36 mM and 2.26 mM against AG and AA, respectively. Its inhibitory activity against AG was more than three times more potent than that of acarbose with an IC50 of 0.91 mM, however its potency against AA was three times less.The ICAA Table . Compoun14 and 23 from ABDCM also showed moderate inhibitory activities against AG with IC50 values of 0.66 mM and 0.28 mM, respectively. Although the IC50 values of their inhibitory effect against AA were not determined, they may have played a role in the inhibitory activity displayed by the extract ABDCM.Compounds S. surrattensis, collected from different locations, revealed the presence of quercetin, rutin, quercetin 3-O-glucoside 7-O- rhamnoside in the leaves [O-β-D-galactopyranoside, chrysophanol, physcion, kaempferide, quercetin were isolated from extracts of the pods [O-α-L-rhamnopyranosyl (1–6)-β-D-glucopyranoside, chrysophanol, physcion, stearic acid, β-sitosterol and β-sitosterol-β-D-glucoside were isolated from the stem [O-β-D-glucopyranosyl-(1–4)-O-α-L-arabinopyranoside, γ-sitosterolin and digitolutein [1 and 3 were isolated from this plant for the first time, the differences in the chemical constituents from the different parts of this plant may account for the variety of biological activities shown by this plant. Earlier reports of the phytochemical studies on the whole plant of H. abyssinica, found from different locations, revealed the presence of cirsiliol, trans 3,5-dihydroxy-4,7-dimethoxydihydroflavonol, vanillin, protocatechuic acid, catechin, stigmasterol, oleic acid, myristic acid and palmitric acid [The higher percentage yields obtained from the ethanol extracts can relate to the fact that both polar and semi-polar constituents of plant material can more readily dissolve in ethanol, leading to higher yields . Previoue leaves , while 5the pods . The barthe pods  while 8-the stem , 59. Thetolutein , 61. Ourric acid . While, ric acid . Our stuN. mitis have resulted in the isolation of neorautanone, 4-methoxyneoduline [6), pachyrrhizine (10), neotenone (11), 12a-hydroxydolineon (13), dolineon (14), rotenone (20), 12a-hydroxyrotenone (21), dehydroneotenone (22) [16) [23) and rautandiol B (24) [12) and nine known compounds including compounds 5, 7, 8, 9, 12, 15, 17, 18 and 19, which were isolated for the first time from N. mitis [Several other phytochemical studies on the roots of eoduline  12a-hydreoduline , neoraudeoduline , togetheeotenone , 12a-hyddolineon , rotenonone (22) , nepseud22) [16) , rautandl B (24) . Our eareotenone , 12a-hydP. falciparum, T. brucei rhodesiense, and M. tuberculosis, are causative agents for malaria, trypanosomiasis and tuberculosis, respectively. These diseases are considered as re-emerging and their reoccurrence is either as a result of a breakdown in public health measures, the appearance of new strains of the causative organism or drug resistance [P. falciparum and T. brucei rhodesiense, however, low safety ratios (SR) were observed in both strains of P. falciparum (TM4/8.2 and K1CB1) and this can be attributed to the earlier reports which suggested that the cytotoxicity of ABDCM may be caused by compounds 20 and 21, as they were both cytotoxic [24 against the two strains of P. falciparum can account for the same activity showed by the ABDCM extract from which it was isolated. This demonstrates its potential as an antimalarial agent due to its activity against the mosquito vector and its larva [24 was reported as non-cytotoxic on VERO and BHK21 cells [24 can be considered as a potential lead candidate for the development of a new anti-malaria drug.sistance , 41. Dueytotoxic , 15. Thets larva , 12. Imp21 cells . With thT. brucei rhodesiense, compounds 11, 14, 15, 20 and 21 are thought to be responsible for this activity, compound 15 gave the highest inhibitory activity and can serve as a lead candidate in the development of new therapy for trypanosomiasis. The inhibitory activity of ABDCM and its compounds against very important heamo-parasites like P. falciparum and T. brucei rhodesiense gives a lead for further investigations against other hemooparasites. Compounds 1 and 2 also gave moderate inhibitory activity against T. brucei rhodesiense and could be responsible for the same inhibitory activity shown by CEOH, from which these compounds were isolated. The inhibitory activities of the ABDCM and CEOH extracts against T. brucei rhodesiense are reported here for the first time based on our literature search.ABDCM also showed significant inhibitory activity against All of the crude extracts from the selected plants except ABAQ, showed significant inhibitory activities against AG and AA, indicating their potential usefulness in the treatment of type II diabetes, which is a condition in which the capacity of the body to produce sufficient insulin is gradually lost. It is a degenerative condition in which the body becomes resistant to the normal effects of insulin, due mainly to excessive glucose absorption from the gastrointestinal tract (GIT) , 64. TheH. abyssinica was reported for its antioxidant and antiglycation activities, and these activities are also thought to be helpful in the management of diabetes complications [S. surrattensis was previously screened for antidiabetic activity [3) was isolated from both extracts and it is thought to be partly responsible for this activity. Catechin (3) is known for its bioactivities and health benefits, studies have revealed its ability to treat the symptoms of diabetes and its complications through the modification of oxidative stress [The root crude extract of ications . The ethactivity . Both KEe stress , 65. TheP. falciparum, T. brucei rhodesiense, α-amylase (AA) and α-glucosidase (AG). This study has revealed some of the individual compounds responsible for these inhibitory activities, giving indications for possible lead compounds for the development of new therapies against diseases caused by these agents. The inhibitory activity of the N. mitis compound 24 (rautandiol B) against P. falciparum was identified for the first time. It was found to be relatively safe based on its high safety ratio and therefore further studies on this compound are highly recommended for the development of alternative antimalarial therapy. Our study also revealed the importance of screening already identified and useful medicinal plants, as an aid in the discovery of new bioactivities which may be useful in the future development of new alternative therapeutic drugs.The extracts from the three selected medicinal plants have shown significant inhibitory activities against Additional file 1.