RESUMO
Cyclotides are plant-derived disulfide-rich cyclic peptides that have a natural function in plant defense and potential for use as agricultural pesticides. Because of their highly constrained topology, they are highly resistant to thermal, chemical, or enzymatic degradation. However, the stability of cyclotides at alkaline pH for incubation times of longer than a few days is poorly studied but important since these conditions could be encountered in the environment, during storage or field application as insecticides. In this study, kalata B1 (kB1), the prototypical cyclotide, was engineered to improve its long-term stability and retain its insecticidal activity via point mutations. We found that substituting either Asn29 or Gly1 to lysine or leucine increased the stability of kB1 by twofold when incubated in an alkaline buffer (pH = 9.0) for 7 days, while retaining its insecticidal activity. In addition, when Gly1 was replaced with lysine or leucine, the mutants could be cyclized using an asparaginyl endopeptidase, in vitro with a yield of â¼90% within 5 min. These results demonstrate the potential to manufacture kB1 mutants with increased stability and insecticidal activity recombinantly or in planta. Overall, the discovery of mutants of kB1 that have enhanced stability could be useful in leading to longer term activity in the field as bioinsecticides.
Assuntos
Ciclotídeos , Inseticidas , Oldenlandia , Ciclotídeos/genética , Ciclotídeos/farmacologia , Ciclotídeos/química , Inseticidas/química , Inseticidas/farmacologia , Leucina , Lisina/genética , Mutagênese , Proteínas de Plantas/metabolismo , Oldenlandia/química , Estabilidade Proteica , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacosRESUMO
The Hedyotis diffusa-Scutellaria officinalis pair (HD-SB) has therapeutic effects on a variety of cancers. Our study was to explore the mechanism of HD-SB in the treatment of hepatocellular carcinoma (HCC). A total of 217 active ingredients of HD-SB and 1196 HCC-related targets were reserved from the TCMSP and the SwissTarget Prediction database, and we got 63 intersection targets from GeneCards. We used a Venn diagram, and Cytoscape found that the three core ingredients were quercetin, luteolin, and baicalein. The PPI analysis showed that the core targets were TP53, CDK2, XPO1, and APP. Molecular docking results showed that these core ingredients had good binding potential with the core targets. HD-SB acts simultaneously on various HCC-related signaling pathways, including proteoglycans in cancer and the P53 signaling pathway. In vitro experiments confirmed that HD-SB can inhibit HepG2 cell proliferation by increasing TP53 and APP levels and decreasing XPO1 and CDK2 levels. This study analyzed active ingredients, core targets, and central mechanisms of HD-SB in the treatment of HCC. It reveals the role of HD-SB in targeting the P53 signaling pathway in the treatment of HCC. We hope that our research could provide a new perspective to the therapy of HCC and find new anticancer drugs.
Assuntos
Carcinoma Hepatocelular , Hedyotis , Neoplasias Hepáticas , Oldenlandia , Scutellaria , Carcinoma Hepatocelular/tratamento farmacológico , Simulação de Acoplamento Molecular , Proteína Supressora de Tumor p53 , Neoplasias Hepáticas/tratamento farmacológico , Veículos FarmacêuticosRESUMO
The Oldenlandia genus comprises approximately 240 species of plants, yet only a limited number of these have been investigated for their chemical composition and medicinal properties. These species contain a wide range of compounds such as iridoids, anthraquinones, triterpenes, phytosterols, flavonoids, anthocyanidins, vitamins, essential oils, phenolic acids, and coumarins. These diverse phytochemical profiles underscore the pharmacological potential of Oldenlandia plants for various medical purposes. Among other chemical constituents, ursolic acid stands out as the most important active compound in Oldenlandia, owing to its proven anticancer, anti-inflammatory, antimicrobial, and hepatoprotective properties. The evaluation of Oldenlandia's pharmacological prospects indicates that the holistic utilization of the entire plant yields the most significant effects. Oldenlandia diffusa showcases anticancer and anti-inflammatory capabilities attributed to its varying constituents. Across a broad spectrum of pharmacological capacities, anticancer research predominates, constituting the majority of medical uses. Oldenlandia diffusa emerges as a standout for its remarkable anticancer effects against diverse malignancies. Antioxidant applications follow, with O. corymbosa demonstrating potent antioxidant properties alongside O. umbellata and O. diffusa. Subsequent priority lies in anti-inflammatory studies, wherein O. diffusa exhibits noteworthy efficacy, trailed by O. corymbosa also takes the lead in antimicrobial activity, with O. umbellata as a strong contender. Additional investigation is essential to ascertain the relative significance of these species in various pharmacological applications. This comprehensive assessment underscores the multifaceted potential of Oldenlandia as a versatile herbal resource, offering diverse pharmacological capacities. The call for sustained exploration and research remains essential to unlock the full extent of Oldenlandia's medicinal benefits.
Assuntos
Anti-Infecciosos , Oldenlandia , Oldenlandia/química , Antioxidantes , Iridoides , Compostos Fitoquímicos , Anti-Inflamatórios , Extratos Vegetais/farmacologiaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Osteoarthritis (OA) is a type of joint disorder that is marked by the gradual breakdown of cartilage and persistent inflammation of the synovial membrane, and is a leading cause of disability among elderly people worldwide. Oldenlandia diffusa (OD) is a member of the Rubiaceae family, and various researches have revealed that it possesses antioxidant, anti-inflammatory, and anti-tumor properties. Extracts of Oldenlandia diffusa is commonly used in traditional oriental medicine to treat various illnesses, including inflammation and cancer. AIM OF THE STUDY: This study is aimed at investigating the anti-inflammatory and anti-apoptosis effects of OD and its potential mechanisms on IL-1ß-induced mouse chondrocytes, as well as its characteristics in a mouse osteoarthritis model. MATERIALS AND METHODS: In this study, the key targets and potential pathways of OD were determined through network pharmacology analysis and molecular docking. The potential mechanism of OD in osteoarthritis was verified by in vitro and in vivo studies. RESULTS: The results of network pharmacology showed that Bax, Bcl2, CASP3, and JUN are key candidate targets of OD for the treatment of osteoarthritis. There is a strong correlation between apoptosis and both OA and OD. Additionally, molecular docking results show that ß-sitosterol in OD can strongly bind with CASP3 and PTGS2. In vitro experiments showed that OD pretreatment inhibited the expression of pro-inflammatory factors induced by IL-1ß, such as COX2, iNOS, IL-6, TNF-α, and PGE2. Furthermore, OD reversed IL-1ß-mediated degradation of collagen II and aggrecan within the extracellular matrix (ECM). The protective effect of OD can be attributed to its inhibition of the MAPK pathway and inhibition of chondrocyte apoptosis. Additionally, it was found that OD can alleviate cartilage degradation in a mouse model of knee osteoarthritis. CONCLUSION: Our study showed that ß-sitosterol, one of the active components of OD, could alleviate the inflammation and cartilage degeneration of OA by inhibiting chondrocyte apoptosis and MAPK pathway.
Assuntos
Oldenlandia , Osteoartrite , Camundongos , Animais , Condrócitos , Caspase 3/metabolismo , Simulação de Acoplamento Molecular , Osteoartrite/tratamento farmacológico , Osteoartrite/patologia , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Anti-Inflamatórios/metabolismoRESUMO
Plants accumulate a vast array of secondary metabolites, which constitute a natural resource for pharmaceuticals. Oldenlandia corymbosa belongs to the Rubiaceae family, and has been used in traditional medicine to treat different diseases, including cancer. However, the active metabolites of the plant, their biosynthetic pathway and mode of action in cancer are unknown. To fill these gaps, we exposed this plant to eight different stress conditions and combined different omics data capturing gene expression, metabolic profiles, and anti-cancer activity. Our results show that O. corymbosa extracts are active against breast cancer cell lines and that ursolic acid is responsible for this activity. Moreover, we assembled a high-quality genome and uncovered two genes involved in the biosynthesis of ursolic acid. Finally, we also revealed that ursolic acid causes mitotic catastrophe in cancer cells and identified three high-confidence protein binding targets by Cellular Thermal Shift Assay (CETSA) and reverse docking. Altogether, these results constitute a valuable resource to further characterize the biosynthesis of active metabolites in the Oldenlandia group, while the mode of action of ursolic acid will allow us to further develop this valuable compound.
Assuntos
Oldenlandia , Oldenlandia/química , Transcriptoma , Metabolômica , Genômica , Ácido UrsólicoRESUMO
OBJECTIVE: To investigate the anti-invasion efficacy of the ethanol extract of Oldenlandia diffusa Will. (EEOD) on a three-dimensional (3D) human malignant glioma (MG) cell invasion and perfusion model based on microfluidic chip culture and the possible mechanism of action of Oldenlandia diffusa Will. (OD). METHODS: The comprehensive pharmacodynamic analysis method in this study was based on microfluidic chip 3D cell perfusion culture technology, and the action mechanism of Chinese medicine (CM) on human MG cells was investigated through network pharmacology analysis. First, the components of EEOD were analyzed by ultraperformance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). Then, cell viability and apoptosis were assessed to determine the optimum concentration of EEOD for invasion experiments, and two-dimensional (2D) migration and invasion abilities of U87 and U251 MG cells were evaluated using scratch wound and Transwell assays. The possible mechanism underlying the effects of EEOD on glioma was analyzed through a network pharmacology approach. RESULTS: Thirty-five compounds of EEOD were detected by UPLC-Q-TOF/MS. EEOD suppressed the viability of MG cells, promoted their apoptosis, and inhibited their migratory and invasive potentials (all P<0.05). Network pharmacology analysis showed that OD inhibited the invasion of MG cells by directly regulating MAPK and Wnt pathways through MAPK, EGFR, MYC, GSK3B, and other targets. The anti-invasion effect of OD was also found to be related to the indirect regulation of microtubule cytoskeleton organization. CONCLUSIONS: ]EEOD could inhibit the invasion of human MG cells, and the anti-invasion mechanism of OD might be regulating MAPK and Wnt signaling pathways and microtubule cytoskeleton organization.
Assuntos
Medicamentos de Ervas Chinesas , Glioma , Oldenlandia , Humanos , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêutico , Glioma/tratamento farmacológico , Microfluídica , Farmacologia em Rede , Oldenlandia/química , Extratos Vegetais/farmacologiaRESUMO
OBJECTIVE: To evaluate the apoptosis and cycle arrest effects of Oldenlandia diffusa flavonoids on human gastric cancer cells, determine the action mechanisms in association with the mitochondrial dependent signal transduction pathway that controls production of reactive oxygen species (ROS), and evaluate the pharmacodynamics of a mouse xenotransplantation model to provide a reference for the use of flavonoids in prevention and treatment of gastric cancer. METHODS: Flavonoids were extracted by an enzymatic-ultrasonic assisted method and purified with D-101 resin. Bioactive components were characterized by high-performance liquid chromatography. Cell lines MKN-45, AGS, and GES-1 were treated with different concentrations of flavonoids (64, 96, 128, 160 µg/mL). The effect of flavonoids on cell viability was evaluated by MTT method, and cell nuclear morphology was observed by Hoechst staining. The apoptosis rate and cell cycle phases were measured by flow cytometry, the production of ROS was detected by laser confocal microscope, the mitochondrial membrane potential (MMP) were observed by fluorescence microscope, and the expression of apoptotic proteins related to activation of mitochondrial pathway were measured by immunoblotting. MKN-45 cells were transplanted into BALB/c nude mice to establish a xenograft tumor model. Hematoxylin and eosin staining was used to reveal the subcutaneous tumor tissue. The tumor volume and tumor weight were measured, the expression levels of proliferation markers proliferating cell nuclear antigen (PCNA) and Ki-67 were detected by immunohistochemistry, and the expression levels of CA72-4 were measured by enzyme linked immunosorbent assay. RESULTS: Oldenlandia diffusa flavonoids inhibited proliferation of MKN-45 and AGS human gastric cancer cells, arrested the cell cycle in G1/S phase, induced accumulation of ROS in the process of apoptosis, and altered MMP. In addition, flavonoids increased Apaf-1, Cleaved-Caspase-3, and Bax, and decreased Cyclin A, Cdk2, Bcl-2, Pro-Caspase-9, and Mitochondrial Cytochrome C (P<0.05). The MKN-45 cell mouse xenotransplantation model further clarified the growth inhibitory effect of flavonoids towards tumors. The expression levels of PCNA and Ki-67 decreased in each flavonoid dose group, the expression level of CA72-4 decreased (P<0.05). CONCLUSION: Flavonoids derived from Oldenlandia diffusa can inhibit proliferation and induce apoptosis of human gastric cancer cells by activating the mitochondrial controlled signal transduction pathway.
Assuntos
Oldenlandia , Neoplasias Gástricas , Humanos , Animais , Camundongos , Oldenlandia/metabolismo , Antígeno Nuclear de Célula em Proliferação , Flavonoides/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Camundongos Nus , Antígeno Ki-67 , Linhagem Celular Tumoral , Apoptose , Extratos Vegetais/farmacologia , Caspases , Proliferação de CélulasRESUMO
Oldenlandia diffusa is an important Chinese traditional medicine with various biological activities such as anti-tumor, anti-inflammatory, anti-oxidant, antibacterial, neuroprotective and hepatoprotective effects. During our course of finding novel compounds from O. diffusa, two new alternariol derivatives named 9-O-(trans-p-coumaroyl)-alternariol (1), 9-O-(trans-caffeoyl)-alternariol (2), together with six known compounds (3-8) were isolated. Their structures were established on the basis of spectroscopic and physicochemical analysis. All isolates were evaluated for in vitro cytotoxic activities on MCF-7, HepG2, A549 and A2780 cancer cells. As a result, new compounds 1 and 2 exhibited potent cytotoxic activities on A2780 cancer cells with IC50 values of 3.1 and 9.4 µM, respectively. And a conclusion was deduced that the p-coumaroyl or caffeoyl moiety could greatly increased the cytotoxic activity of alternariol on cancer cells.
Assuntos
Oldenlandia , Neoplasias Ovarianas , Humanos , Feminino , Linhagem Celular Tumoral , Oldenlandia/químicaRESUMO
The pathogenesis of gastric cancer is a multistage process that involves glucose metabolism, inflammation, oxidative damage, angiogenesis, autophagy, and apoptosis. Moreover, microRNA-340 (miR340) also plays a vital role in tumorigenesis and the biology of gastric cancer as an epigenetic factor. It seems that the use of ketogenic diets (KDs) and plant extracts that have antitumor, anti-inflammatory, and antioxidant properties can be good treatment options to cure gastric cancer. The aim of this study was to investigate the role of miR-340 on pathways involved in the pathogenesis of gastric cancer and the improving effects of the KD, Oldenlandia diffusa extract (ODE), and curcumin in the animal model of gastric cancer. One hundred and ten male Wistar rats were divided into control and treatment groups. The expression of miR-340 along with genes involved in inflammation, oxidative damage, angiogenesis, and apoptosis were assessed. The results showed that the KD and different doses of curcumin and ODE in a dose-dependent behavior could induce apoptosis and the expression of the Akt/mTORC1 pathway and inhibit inflammation, oxidative damage, and angiogenesis in the gastric tissue of rats with cancer. In addition, there was no significant difference between cancer groups receiving ODE and curcumin. These results also showed that consumption of KD could significantly increase the efficacy of ODE and curcumin which may be due to increasing miR-340 expression. The results of this study suggested well that the KD along with conventional therapies in traditional medicine can be a useful solution for the prevention and treatment of gastric cancer. PRACTICAL APPLICATIONS: Gastric cancer is the third leading cause of cancer death, and genetic and epigenetic factors, including miR-340, are involved in its pathogenesis. However, the use of ketogenic diets (KDs) and plant products such as curcumin and Oldenlandia diffusa extract (ODE) can play an effective role in inhibiting tumorigenesis in some cancers. Our results showed that the KD and different doses of curcumin and ODE could induce apoptosis and the expression of the Akt/mTORC1 pathway and inhibit inflammation, oxidative damage, and angiogenesis in the gastric tissue. Moreover, the KD could significantly increase the efficacy of ODE and curcumin which may be due to an increase in miR-340 expression. These findings provide novel perceptions about the mechanisms of the KD, curcumin, and ODE to cure gastric cancer. It suggested that the KD as adjunctive therapy along with conventional therapies in traditional medicine could be considered a useful solution to prevent and treat gastric cancer.
Assuntos
Curcumina , Dieta Cetogênica , MicroRNAs , Oldenlandia , Neoplasias Gástricas , Animais , Ratos , Curcumina/farmacologia , Extratos Vegetais/farmacologia , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genética , Proteínas Proto-Oncogênicas c-akt , Ratos Wistar , Apoptose , Estresse Oxidativo , MicroRNAs/genética , MicroRNAs/farmacologia , Inflamação , Carcinogênese , AutofagiaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Hedyotis diffusa Willd, also named Scleromitrion diffusum (Willd.) R.J. Wang, is one medical herb, which has been traditionally used by the She nationality in China. And H. diffusa represents a beneficial effect on Systemic lupus erythematosus (SLE) treatment in clinic. AIM OF THE STUDY: The underlying mechanisms of the protective effects of H. diffusa on SLE remain unclear. In this study, we treated MRL/lpr mice with H. diffusa water extract (HDW) to assess its therapeutic effects and verified its regulating signalling pathway through cytological experiments. MATERIALS AND METHODS: In the present study, the constituents of HDW were analysed through ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and SCIEX OS software. The protective activity and underlying mechanisms were studied in a MRL/lpr lupus mouse model. The blood cells, autoantibodies, metabolites and the cytokines in serum were identified with a hematology analyzer, specific ELISA kit, GC/MS system and cytometric assays. The histological and immunohistochemical analysis were engaged in the morphologic, and the expression and translocation of the crucial protein observation. The dual luciferase reporter assay was applied to identifying the regulative activity of HDW. The transcription and translation expression of the protein was studied by real-time PCR and Western blot assays. The network pharmacology analysis was employed to predict the IL-6/STAT3 pathway regulators and the screen the STAT3 inhibitors in HDW. RESULTS: The results revealed the capability of HDW to attenuate the production of autoantibodies, secretion of inflammatory cytokines (IL-6 and IFN-γ), and suppressed the IgG and C3 deposition, the development of glomerular lesions in MRL/lpr mice. Serum metabolomics study showed the improvement in serum metabolites, especially aminoacyl-tRNA biosynthesis, by HDW. IL-6 was clarified to be highly associated with the significantly changed metabolites in network analysis. We further demonstrated the effects of HDW on the IL-6/STAT3 pathway in vivo and in vitro. CONCLUSIONS: This study suggested that HDW exerts a therapeutic effect in SLE model mice by suppressing the IL-6/STAT3 pathway.
Assuntos
Hedyotis , Lúpus Eritematoso Sistêmico , Oldenlandia , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Autoanticorpos , Citocinas , Hedyotis/química , Interleucina-6 , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Camundongos , Camundongos Endogâmicos MRL lpr , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Fator de Transcrição STAT3RESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Hedyotis diffusa is a traditional ethnomedicinal plant in local communities in northeastern Asia and used to treat inflammation, nervous breakdown, among others. In recent years, it has been applied in the treatment of Alzheimer's disease (AD), while the specific chemical components responsible for the activity remain need to be explored. AIM OF THE STUDY: To prepare, screen and identify the potential anti-AD active components from Hedyotis diffusa. MATERIALS AND METHODS: The acetylcholinesterase (AChE) inhibitory activity of four different extracts of Hedyotis diffusa were initially assessed using a spectrophotometric Ellman's method. A more accurate LC-MS/MS screening method combining functional enzyme assay and affinity ultrafiltration (AU) screening assay was developed and applied for the screening of natural compound inhibitors of AChE from Hedyotis diffusa. The binding mode was further investigated between protein and ligands via molecular docking. Subsequently, CL4176, a transgenic nematode model for AD, was used for activity validation of one of these components. RESULTS: N-butanol extract of Hedyotis diffusa (NHD) appeared significant inhibitory activities on AChE, were chosen to delve deeper. Five bioactive components targeting AChE were screened out and identified using AU coupled to liquid chromatography-mass spectrometry. Molecular docking technique further confirmed the results of the screening assay. Finally, quercetin-3-O-sophoroside (QS) was confirmed as a potent anti-AD agent by in vivo experiments in C. elegans. CONCLUSION: This study explores a new idea for screening anti-AD active components from traditional medicine. The findings provide a molecular structure and bioactivity basis for future potential applications of Hedyotis diffusa in medical industries.
Assuntos
Hedyotis , Oldenlandia , Acetilcolinesterase , Animais , Caenorhabditis elegans , Cromatografia Líquida , Hedyotis/química , Simulação de Acoplamento Molecular , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Espectrometria de Massas em Tandem/métodos , UltrafiltraçãoRESUMO
During our continual investigations on Oldenlandia diffusa (Willd.) Roxb., two new iridoid glucosides, diffusosides C (1) and D (2), were isolated and their structures were elucidated through cumulative analysis of NMR and HRESIMS spectroscopic data as well as computational studies. Both isolated compounds displayed no obvious neuroprotective activity on H2O2-induced injury PC12 cells at 50 µM in vitro.
Assuntos
Medicamentos de Ervas Chinesas , Oldenlandia , Medicamentos de Ervas Chinesas/química , Peróxido de Hidrogênio , Glucosídeos Iridoides , Oldenlandia/químicaRESUMO
A uni-molecular layer of lipids at air-water interface mimicking one of the leaflets of the cellular membrane provides a simple model to understand the interaction of any foreign molecules with the membrane. Here, the interactions of protein Kalata B1 (KB1) of cyclotide family with the phospholipids 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DPPG), and 1,2-distearoyl-sn-glycero-3-ethylphosphocholine chloride salt (DSEPC) have been investigated. The addition of KB1 induces a change in pressure of the lipid monolayers. The characteristic time of the change in pressure is found to be dependent on the electrostatic nature of the lipid. Even though the protein is weakly surface active, it is capable of modifying the phase behavior and elastic properties of lipid monolayers with differences in their strength and nature making the layers more floppy. The KB1-lipid interaction has been quantified by calculating the excess Gibb's free energy of interaction and the 1-anilino-8-naphthalenesulfonate (ANS) binding studies. The interaction with zwitterionic DPPC and negatively charged DPPG lipids are found to be thermodynamically favorable whereas the protein shows a weaker response to positively charged DSEPC lipid. Therefore, the long ranged electrostatic is the initial driving force for the KB1 to recognize and subsequently attach to a cellular membrane. Thereafter, the hydrophobic region of the protein may penetrate into the hydrophobic core of the membrane via specific amino acid residues.
Assuntos
Ciclotídeos/química , Bicamadas Lipídicas/química , 1,2-Dipalmitoilfosfatidilcolina/química , Naftalenossulfonato de Anilina/química , Elasticidade , Simulação de Dinâmica Molecular , Oldenlandia/química , Fosfatidilgliceróis/química , Eletricidade EstáticaRESUMO
Herbicide resistance is a worldwide problem in weed control. This prompts researchers to look for new modes of action to slow down the evolution of herbicide-resistant weeds. This research aims to determine the herbicidal action of thiazolo[3,2-a]pyrimidines derivatives, which are well known as antihypertensive drugs. The phytotoxic effects of ten compounds were investigated using leaf disc discoloration test and seed germination bioassay. At concentrations of 125 to 250 mg/L, the 5-(3-Fluoro-phenyl)-7-methyl-5H-thiazolo[3,2-a]pyrimidine-6-carboxylic acid ethyl ester (c) was highly active against Oldenlandia verticillata and Eleusine indica. At application rates of 1.25 to 2.5 kg ai/ha, formulated c demonstrated selective post-emergence and pre-emergence herbicidal activity against O. verticillata, E. indica and Cyperus iria. In the crop tolerance test, formulated c outperformed the commercial herbicide diuron, with aerobic Oryza sativa being the most tolerant, followed by Zea mays, and Brassica rapa. The addition of calcium chloride partially nullified compound c's inhibitory effects on weed shoot growth, indicating that it has potential as a calcium channel blocker. Compound c acted by triggering electrolyte leakage without affecting photosystem II. These findings imply that c could be explored further as a template for developing new herbicides with novel modes of action.
Assuntos
Anti-Hipertensivos/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Herbicidas/farmacologia , Eleusine/efeitos dos fármacos , Meio Ambiente , Oldenlandia/efeitos dos fármacos , Oryza/efeitos dos fármacos , Complexo de Proteína do Fotossistema II/efeitos dos fármacos , Zea mays/efeitos dos fármacosRESUMO
Red blood cells (RBCs) can serve as vascular carriers for drugs, proteins, peptides, and nanoparticles. Human RBCs remain in the circulation for â¼120 days, are biocompatible, and are immunologically largely inert. RBCs are cleared by the reticuloendothelial system and can induce immune tolerance to foreign components attached to the RBC surface. RBC conjugates have been pursued in clinical trials to treat cancers and autoimmune diseases and to correct genetic disorders. Still, most methods used to modify RBCs require multiple steps, are resource-intensive and time-consuming, and increase the risk of inflicting damage to the RBCs. Here, we describe direct conjugation of peptides and proteins onto the surface of RBCs in a single step, catalyzed by a highly efficient, recombinant asparaginyl ligase under mild, physiological conditions. In mice, the modified RBCs remain intact in the circulation, display a normal circulatory half-life, and retain their immune tolerance-inducing properties, as shown for protection against an accelerated model for type 1 diabetes. We conjugated different nanobodies to RBCs with retention of their binding properties, and these modified RBCs can target cancer cells in vitro. This approach provides an appealing alternative to current methods of RBC engineering. It provides ready access to more complex RBC constructs and highlights the general utility of asparaginyl ligases for the modification of native cell surfaces.
Assuntos
Carbono-Nitrogênio Ligases/química , Membrana Eritrocítica/metabolismo , Peptídeos/química , Anticorpos de Domínio Único/química , Animais , Carbono-Nitrogênio Ligases/genética , Engenharia Celular , Linhagem Celular Tumoral , Cisteína Endopeptidases/genética , Diabetes Mellitus Experimental/prevenção & controle , Membrana Eritrocítica/química , Transfusão de Eritrócitos , Feminino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Mutação , Oldenlandia/enzimologia , Proteínas de Plantas/genéticaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Scutellaria barbata D.Don (SB) and Oldenlandia diffusa (Willd.) Roxb are commonly known as Ban Zhi Lian and Bai Hua She Cao in Chinese herbal medicines, respectively. As a pair of herbs, they have traditionally been used as ethnomedicines for clearing away heat and toxins, removing blood stasis, and promoting blood circulation, diuresis, and detumescence. AIM OF THE STUDY: The aim of the present study was to determine the active ingredients in SB and OD extracts and to investigate whether these extracts can inhibit the growth of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) cell lines (HepG2.2.15 and Hep3B) in vitro and in vivo, as well as to explore their mechanisms of action. MATERIALS AND METHODS: We determined the levels of total flavonoids, luteolin, and apigenin in SB and OD extracts via ultraviolet-visible spectrophotometry and high-performance liquid chromatography. The effects of SB and OD extracts on HBV-associated HCC cell growth were assessed by HepG2.2.15 and Hep3B cells phenotype and RNA sequencing of Hep3B cells in vitro, and xenograft models in vivo. RESULTS: The extracts of SB and OD contained total flavonoids. There were active ingredients of luteolin and apigenin in SB, but not in OD. The extracts of SB and OD significantly inhibited HCC growth, migration, invasion, and HBV activity in vitro and in vivo, as well as altered circRNA expression in Hep3B cells. Moreover, we constructed a circRNA-miRNA-mRNA co-expression network. CONCLUSIONS: The extracts of SB and OD may inhibit HCC cell growth and HBV activity in vitro and in vivo through altering circRNA-miRNA-gene expression and that the efficacies of these extracts may be related to the presence of luteolin and apigenin.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Oldenlandia/química , RNA Circular/metabolismo , Scutellaria/química , Animais , Apigenina/análise , Apoptose/efeitos dos fármacos , Proteínas Relacionadas à Autofagia/metabolismo , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/uso terapêutico , Flavonoides/análise , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Hepatite B/complicações , Humanos , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/patologia , Luteolina/análise , Camundongos Nus , RNA Circular/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
MAIN CONCLUSION: We demonstrate the production of a structurally correct cyclotide in rice suspension cells with co-expression of a ligase-type AEP, which unlocks monocotyledons as production platforms to produce cyclotides. Cyclotides are a class of backbone-cyclic plant peptides that harbor a cystine knot composed of three disulfide bonds. These structural features make cyclotides particularly stable, and thus they have attracted significant attention for their use in biotechnological applications such as drug design. Currently, chemical synthesis is the predominant strategy to produce cyclotides for research purposes. However, synthetic production becomes costly both economically and environmentally at large scale. Plants offer an attractive alternative to chemical synthesis because of their lower cost and environmental footprint. In this study, rice suspension cells were engineered to produce the prototypical cyclotide, kalata B1 (kB1), a cyclotide with insecticidal properties from the African plant Oldenlandia affinis. Engineered rice cells produced structurally validated kB1 at yields of 64.21 µg/g (DW), which was dependent on the co-expression of a peptide ligase-competent asparaginyl endopeptidase OaAEP1b from O. affinis. Without co-expression, kB1 was predominantly produced as linear peptide. Through HPLC-MS co-elution, reduction, alkylation, enzymatic digestion, and proton NMR analysis, kB1 produced in rice was shown to be structurally identical to native kB1. This study reports the first example of an engineered plant suspension cell culture with the required molecular machinery for efficient production and cyclisation of a heterologous cyclotide.
Assuntos
Biotecnologia , Ciclotídeos , Oldenlandia , Oryza , Biotecnologia/métodos , Ciclotídeos/biossíntese , Ciclotídeos/genética , Oldenlandia/genética , Oryza/enzimologia , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismoRESUMO
Cyclotides are a class of cyclic disulfide-rich peptides found in plants that have been adopted as a molecular scaffold for pharmaceutical applications due to their inherent stability and ability to penetrate cell membranes. For research purposes, they are usually produced and cyclized synthetically, but there are concerns around the cost and environmental impact of large-scale chemical synthesis. One strategy to improve this is to combine a recombinant production system with native enzyme-mediated cyclization. Asparaginyl endopeptidases (AEPs) are enzymes that can act as peptide ligases in certain plants to facilitate cyclotide maturation. One of these ligases, OaAEP1b, originates from the cyclotide-producing plant, Oldenlandia affinis, and can be produced recombinantly for use in vitro as an alternative to chemical cyclization of recombinant substrates. However, not all engineered cyclotides are compatible with AEP-mediated cyclization because new pharmaceutical epitopes often replace the most flexible region of the peptide, where the native cyclization site is located. Here we redesign a popular cyclotide grafting scaffold, MCoTI-II, to incorporate an AEP cyclization site located away from the usual grafting region. We demonstrate the incorporation of a bioactive peptide sequence in the most flexible region of MCoTI-II while maintaining AEP compatibility, where the two were previously mutually exclusive. We anticipate that our AEP-compatible scaffold, based on the most popular cyclotide for pharmaceutical applications, will be useful in designing bioactive cyclotides that are compatible with AEP-mediated cyclization and will therefore open up the possibility of larger scale enzyme-mediated production of recombinant or synthetic cyclotides alike.
Assuntos
Ciclotídeos/química , Cisteína Endopeptidases/química , Proteínas de Plantas/química , Sequência de Aminoácidos , Ciclização , Ciclotídeos/síntese química , Ciclotídeos/genética , Cisteína Endopeptidases/genética , Escherichia coli/genética , Oldenlandia/enzimologia , Proteínas de Plantas/síntese química , Proteínas de Plantas/genética , Engenharia de ProteínasRESUMO
Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled protein polymerization process is always a challenge. Here, we have developed an enzymatic methodology for constructing polymerized protein step by step in a rationally-controlled sequence. In this method, the C-terminus of a protein monomer is NGL for protein conjugation using OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases) 1) while the N-terminus was a cleavable TEV (tobacco etch virus) cleavage site plus an L (ENLYFQ/GL) for temporary N-terminal protecting. Consequently, OaAEP1 was able to add only one protein monomer at a time, and then the TEV protease cleaved the N-terminus between Q and G to expose the NH2-Gly-Leu. Then the unit is ready for next OaAEP1 ligation. The engineered polyprotein is examined by unfolding individual protein domain using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Therefore, this study provides a useful strategy for polyprotein engineering and immobilization.
Assuntos
Cisteína Endopeptidases/metabolismo , Proteínas Imobilizadas/metabolismo , Oldenlandia/enzimologia , Polimerização , Biossíntese de Proteínas , Imagem Individual de Molécula/métodos , Cisteína Endopeptidases/química , Endopeptidases/química , Endopeptidases/metabolismo , Microscopia de Força Atômica , Potyvirus/enzimologia , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Espectrofotometria UltravioletaRESUMO
G-quadruplexes (G4) are non-canonical nucleic acid structures with important implications in biology. Based on an α-helical fragment of the RHAU helicase that displays high specificity for parallel-stranded G-quadrplexes, herein we demonstrate its head-to-tail cyclization by a high-efficiency ligase. The cyclic peptide exhibits superior stability and binding affinity to a G-quadruplex, and can serve as an excellent investigational tool for chemical biology applications.