Overexpression of GmPAL Genes Enhances Soybean Resistance Against Heterodera glycines.

Soybean cyst nematode (Heterodera glycines, soybean cyst nematode [SCN]) disease adversely affects the yield of soybean and leads to billions of dollars in losses every year. To control the disease, it is necessary to study the resistance genes of the plant and their mechanisms. Isoflavonoids are secondary metabolites of the phenylalanine pathway, and they are synthesized in soybean. They are essential in plant response to biotic and abiotic stresses. In this study, we reported that phenylalanine ammonia-lyase (PAL) genes GmPALs involved in isoflavonoid biosynthesis, can positively regulate soybean resistance to SCN. Our previous study demonstrated that the expression of GmPAL genes in the resistant cultivar Huipizhi (HPZ) heidou are strongly induced by SCN. PAL is the rate-limiting enzyme that catalyzes the first step of phenylpropanoid metabolism, and it responds to biotic or abiotic stresses. Here, we demonstrate that the resistance of soybeans against SCN is suppressed by PAL inhibitor l-α-(aminooxy)-ß-phenylpropionic acid (L-AOPP) treatment. Overexpression of eight GmPAL genes caused diapause of nematodes in transgenic roots. In a petiole-feeding bioassay, we identified that two isoflavones, daidzein and genistein, could enhance resistance against SCN and suppress nematode development. This study thus reveals GmPAL-mediated resistance against SCN, information that has good application potential. The role of isoflavones in soybean resistance provides new information for the control of SCN. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The R2R3-MYB-SG7 transcription factor CaMYB39 orchestrates surface phenylpropanoid metabolism and pathogen resistance in chickpea.

Flavonoids are important plant pigments and defense compounds; understanding the transcriptional regulation of flavonoid biosynthesis may enable engineering crops with improved nutrition and stress tolerance. Here, we characterize R2R3-MYB domain subgroup 7 transcription factor CaMYB39, which regulates flavonol biosynthesis primarily in chickpea trichomes. CaMYB39 overexpression in chickpea was accompanied by a change in flux availability for the phenylpropanoid pathway, particularly flavonol biosynthesis. Lines overexpressing CaMYB39 showed higher isoflavonoid levels, suggesting its role in regulating isoflavonoid pathway. CaMYB39 transactivates the transcription of early flavonoid biosynthetic genes (EBG). FLAVONOL SYNTHASE2, an EBG, encodes an enzyme with higher substrate specificity for dihydrokaempferol than other dihydroflavonols explaining the preferential accumulation of kaempferol derivatives as prominent flavonols in chickpea. Interestingly, CaMYB39 overexpression increased trichome density and enhanced the accumulation of diverse flavonol derivatives in trichome-rich tissues. Moreover, CaMYB39 overexpression reduced reactive oxygen species levels and induced defense gene expression which aids in partially blocking the penetration efficiency of the fungal pathogen, Ascochyta rabiei, resulting in lesser symptoms, thus establishing its role against deadly Ascochyta blight (AB) disease. Overall, our study reports an instance where R2R3-MYB-SG7 member, CaMYB39, besides regulating flavonol biosynthesis, modulates diverse pathways like general phenylpropanoid, isoflavonoid, trichome density, and defense against necrotrophic fungal infection in chickpea.

Millettia isoflavonoids: a comprehensive review of structural diversity, extraction, isolation, and pharmacological properties.

There are approximately 260 known species in the genus Millettia, many of which are used in traditional medicine to treat human and other animal ailments in various parts of the world. Being in the Leguminosae (Fabaceae) family, Millettia species are rich sources of isoflavonoids. In the past three decades alone, several isoflavonoids originating from Millettia have been isolated, and their pharmacological activities have been evaluated against major diseases, such as cancer, inflammation, and diabetes. Despite such extensive research, no recent and comprehensive review of the phytochemistry and pharmacology of Millettia isoflavonoids is available. Furthermore, the structural diversity of isoflavonoids in Millettia species has rarely been reported. In this review, we comprehensively summarized the structural diversity of Millettia isoflavonoids, the methods used for their extraction and isolation protocols, and their pharmacological properties. According to the literature, 154 structurally diverse isoflavonoids were isolated and reported from the various tissues of nine well-known Millettia species. Prenylated isoflavonoids and rotenoids were the most dominant subclasses of isoflavonoids reported. Other subclasses of reported isoflavonoids include isoflavans, aglycone isoflavones, glycosylated isoflavones, geranylated isoflavonoids, phenylcoumarins, pterocarpans and coumaronochromenes. Although some isolated molecules showed promising pharmacological properties, such as anticancer, anti-inflammatory, estrogenic, and antibacterial activities, others remained untested. In general, this review highlights the potential of Millettia isoflavonoids and could improve their utilization in drug discovery and medicinal use processes. Supplementary Information: The online version contains supplementary material available at 10.1007/s11101-022-09845-w.

Synthesis of amide derivatives of 3-aryl-3H-benzopyrans as osteogenic agent concomitant with anticancer activity.

The present study reports a series of 3-aryl-3H-benzopyran-based amide derivatives as osteogenic agents concomitant with anticancer activity. Six target compounds viz 22e, 22f, 23i, and 24b-d showed good osteogenic activity at 1 pM and 100 pM concentrations. One of the potential molecules, 24b, effectively induced ALP activity and mRNA expression of osteogenic marker genes at 1 pM and bone mineralization at 100 pM concentrations. These molecules also presented significant growth inhibition of osteosarcoma (MG63) and estrogen-dependent and -independent (MCF-7 and MDA-MB-231) breast cancer cells. The most active compound, 24b, inhibited the growth of all the cancer cells within the IC50 10.45-12.66 µM. The mechanistic studies about 24b showed that 24b induced apoptosis via activation of the Caspase-3 enzyme and inhibited cancer cell migration. In silico molecular docking performed for 24b revealed its interaction with estrogen receptor-ß (ER-ß) preferentially.

Protein Modelling Highlighted Key Catalytic Sites Involved in Position-Specific Glycosylation of Isoflavonoids.

Uridine diphosphate glycosyltransferases (UGTs) are known for promiscuity towards sugar acceptors, a valuable characteristic for host plants but not desirable for heterologous biosynthesis. UGTs characterized for the O-glycosylation of isoflavonoids have shown a variable efficiency, substrate preference, and OH site specificity. Thus, 22 UGTs with reported isoflavonoid O-glycosylation activity were analyzed and ranked for OH site specificity and catalysis efficiency. Multiple-sequence alignment (MSA) showed a 33.2% pairwise identity and 4.5% identical sites among selected UGTs. MSA and phylogenetic analysis highlighted a comparatively higher amino acid substitution rate in the N-terminal domain that likely led to a higher specificity for isoflavonoids. Based on the docking score, OH site specificity, and physical and chemical features of active sites, selected UGTs were divided into three groups. A significantly high pairwise identity (67.4%) and identical sites (31.7%) were seen for group 1 UGTs. The structural and chemical composition of active sites highlighted key amino acids that likely define substrate preference, OH site specificity, and glycosylation efficiency towards selected (iso)flavonoids. In conclusion, physical and chemical parameters of active sites likely control the position-specific glycosylation of isoflavonoids. The present study will help the heterologous biosynthesis of glycosylated isoflavonoids and protein engineering efforts to improve the substrate and site specificity of UGTs.

Combined Omics Approaches Reveal Distinct Mechanisms of Resistance and/or Susceptibility in Sugar Beet Double Haploid Genotypes at Early Stages of Beet Curly Top Virus Infection.

Sugar beet is susceptible to Beet curly top virus (BCTV), which significantly reduces yield and sugar production in the semi-arid growing regions worldwide. Sources of genetic resistance to BCTV is limited and control depends upon insecticide seed treatments with neonicotinoids. Through double haploid production and genetic selection, BCTV resistant breeding lines have been developed. Using BCTV resistant (R) [KDH13; Line 13 and KDH4-9; Line 4] and susceptible (S) [KDH19-17; Line 19] lines, beet leafhopper mediated natural infection, mRNA/sRNA sequencing, and metabolite analyses, potential mechanisms of resistance against the virus and vector were identified. At early infection stages (2- and 6-days post inoculation), examples of differentially expressed genes highly up-regulated in the 'R' lines (vs. 'S') included EL10Ac5g10437 (inhibitor of trypsin and hageman factor), EL10Ac6g14635 (jasmonate-induced protein), EL10Ac3g06016 (ribosome related), EL10Ac2g02812 (probable prolyl 4-hydroxylase 10), etc. Pathway enrichment analysis showed differentially expressed genes were predominantly involved with peroxisome, amino acids metabolism, fatty acid degradation, amino/nucleotide sugar metabolism, etc. Metabolite analysis revealed significantly higher amounts of specific isoflavonoid O-glycosides, flavonoid 8-C glycosides, triterpenoid, and iridoid-O-glycosides in the leaves of the 'R' lines (vs. 'S'). These data suggest that a combination of transcriptional regulation and production of putative antiviral metabolites might contribute to BCTV resistance. In addition, genome divergence among BCTV strains differentially affects the production of small non-coding RNAs (sncRNAs) and small peptides which may potentially affect pathogenicity and disease symptom development.

The Activity of Red Nigerian Propolis and Some of Its Components against Trypanosoma brucei and Trypanosoma congolense.

Propolis is a resin that is gathered by bees from exudates produced by various plants. Its exact chemical composition depends on the plants available near the hive. Bees use propolis to coat the surfaces of the hive, where it acts as an anti-infective. Regardless of the chemical composition of propolis, it is always anti-protozoal, probably because protozoan parasites, particularly Lotmarium passim, are widespread in bee populations. The protozoa Trypanosoma brucei and T. congolense cause disease in humans and/or animals. The existing drugs for treating these diseases are old and resistance is an increasingly severe problem. The many types of propolis present a rich source of anti-trypanosomal compounds-from a material gathered by bees in an environmentally friendly way. In the current work, red Nigerian propolis from Rivers State, Nigeria was tested against T. brucei and T. congolense and found to be highly active (EC50 1.66 and 4.00 µg/mL, respectively). Four isoflavonoids, vestitol, neovestitol, 7-methylvestitol and medicarpin, were isolated from the propolis. The isolated compounds were also tested against T. brucei and T. congolense, and vestitol displayed the highest activity at 3.86 and 4.36 µg/mL, respectively. Activities against drug-resistant forms of T. brucei and T. congolense were similar to those against wild type.

Radical Scavenging Capability and Mechanism of Three Isoflavonoids Extracted from Radix Astragali: A Theoretical Study.

As a valuable traditional Chinese herbal medicine, Radix Astragali has attracted much attention due to its extensive pharmacological activities. In this study, density functional theory (DFT) was used thermodynamically and kinetically in detail to predict the antioxidant activity and reaction mechanisms involved in the free radical scavenging reactions of three representative isoflavonoids (formononetin, calycosin, and calycosin-7-glucoside) extracted from Radix Astragali. Three main mechanisms, including hydrogen atom transfer (HAT), proton transfer after electron transfer (SET-PT), and sequential proton loss electron transfer (SPLET) were examined by calculating the thermodynamic parameters. It was found that HAT is the predominant mechanism in the gas phase, while SPLET is supported in the solvent environment. The isoflavonoids' order of antioxidant activity was estimated as: calycosin > calycosin-7-glucoside > formononetin. For the calycosin compound, the result revealed the feasibility of double HAT mechanisms, which involve the formation of stable benzodioxazole with significantly reduced energy in the second H+/e- reaction. In addition, the potential energy profiles and kinetic calculations show that the reaction of •OH into the 3'-OH site of calycosin has a lower energy barrier (7.2 kcal/mol) and higher rate constant (4.55 × 109 M-1 s-1) compared with other reactions in the gas phase.

The Effect of Genistein on Anastomotic Healing in Intestinal Ischemia/Reperfusion Injury.

INTRODUCTION: Genistein is a natural isoflavonoid and has several pharmacological effects, such as antioxidant, antitumor activity, and improvement of glucose metabolism. The safety of intestinal anastomosis after ischemia-reperfusion (I/R) injury is a critical issue for surgeons. This experimental study aimed to investigate the effects of genistein on anastomotic healing after intestinal I/R injury. METHODS: A total of 36 male Wistar Albino rats were divided into four groups: control, I/R, genistein, and genistein + I/R. The control group received segmental ileal resection and ileoileal anastomosis. The I/R group received resection + anastomosis after intestinal I/R. The genistein group was administered subcutaneous injection of 1 mg/kg genistein 12 h and 1 h before the procedure and received ileal resection + anastomosis. The genistein + I/R group received I/R + ileal resection + anastomosis after genistein injection. Anastomotic bursting pressure, hydroxyproline, superoxide dismutase, and glutathione peroxidase levels and histopathological wound healing scores of all rats were measured on postoperative day 5. RESULTS: The anastomotic bursting pressure was significantly higher in the genistein and genistein + I/R groups (P < 0.001). Genistein increased the hydroxyproline concentration and the superoxide dismutase and glutathione peroxidase levels in the intestinal anastomosis (P < 0.001). In histopathological assessment, the mean wound healing score was significantly higher in the genistein group than in the other groups (P < 0.001). CONCLUSIONS: Genistein, with its anti-inflammatory and antioxidant properties, shows protective effects against increased oxidative stress after intestinal I/R injury and contributes positively to intestinal anastomotic healing.

In vitro efficacy of isoflavonoids and terpenes against Leishmania (Leishmania) infantum and L. amazonensis.

The main form of control of leishmaniasis is the treatment, however various side effects and poor efficacy are associated with presently available drugs. The investigation of bioactive natural products for new antileishmanial drugs is a valid approach. The present study reports the in vitro efficacy of natural isoflavonoids and terpenes against Leishmania infantum and L. amazonensis and their cytotoxicity against HepG2 cells. L. infantum and L. amazonensis promastigotes were exposed to the terpenes kaurenoic acid, xylopic acid, and (-)-α-bisabolol and to the isoflavonoids (-)-duartin and (3R)-claussequinone for antileishmanial activity and to cytotoxicity to HepG2 cells. The most effective substance against both L. infantum and L. amazonensis species was (3R)-claussequinone (IC50 = 3.21 µg/mL and 2.47 µg/mL, respectively) that disclosed low cytotoxicity against HepG2 cells (CC50 = 387.79 µg/mL). The efficacy of (3R)-claussequinone against intracellular amastigotes of L. infantum and the externalization of phosphatidylserine in promastigotes of this isoflavanoid were investigated by infection of Raw 264.7 macrophages and marking with Annexin V-FITC and propidium Iodide for flow cytometry analysis. The results for amastigotes showed that (3R)-claussequinone was able to reduce the rate of infection with IC50 = 4.61 µg/mL and did not alter the externalization of phosphatidylserine. In conclusion it is presently reported, for the first time, the striking antileishmanial activity of (3R)-claussequinone against L. infantum and L. amazonensis associated to low cytotoxicity. Furthermore, these results suggest that (3R)-claussequinone is a new hit aiming to develop new therapeutic alternatives.

Genistein lowers fertility with pronounced effect in males: Meta-analyses on pre-clinical studies.

Genistein, an isoflavonoid, is found in a plethora of plant-based foods, and has been approved for use in various therapies. A couple of studies in adult men observed a negative correlation between genistein exposure and reproductive parameters. To assess the effects of genistein exposure on reproduction and fertility in males and females, we performed quantitative meta-analyses by pooling data from published studies on animals that assessed various reproductive parameters. Pooled analysis showed significant decreases in sperm count in males exposed to genistein during adulthood (Hedges's g = -2.51, p = 0.013) and in utero (Hedges's g = -0.861, p = 0.016) compared with controls. In males exposed to genistein in utero, serum testosterone levels decreased (Hedges's g = -6.301, p = 0.000) and luteinizing hormone (LH) (Hedges's g = 7.127, p = 0.000) and FSH (Hedges's g = 6.19, p = 0.000) levels increased in comparison with controls. In females, the number of corpora lutea (Hedges's g = -2.103, p = 0.019) and the litter size (Hedges's g = -1.773, p-value = 0.000) decreased; however, female reproductive hormones remained unaffected. These meta-analyses show that genistein has detrimental effects on male reproductive system and on the progression and sustenance of pregnancy, with more pronounced adverse impact in males, particularly when exposed in utero.

Can genistein be a potential agent against skin side effects associated with the treatment of breast cancer?

Despite significant medical advances, cancer treatment is still associated with a high risk of side effects. The treatment is usually invasive and devastating and it affects the overall immunity of the whole organism, including the condition of the skin. In recent years there has been a growing interest in isoflavonoids, due to their wide range of biological and pharmacological activity, especially estrogen-like. It gives a broad perspective of their use as active ingredients of preparations, which eliminate skin lesions associated with oncological treatment. This article is an overview describing preclinical and clinical observations on the basis of available literature. It discusses the influence of genistein on skin health in women after breast cancer treatment. The overview focuses on studies conducted with genistein in vitro or in vivo to demonstrate its effect on skin, and anticancer properties. We selected articles from the last 20 years, available in the PubMed and Google Scholar databases.

Inhibition of calcitriol inactivating enzyme CYP24A1 gene expression by flavonoids in hepatocellular carcinoma cells under normoxia and hypoxia.

A vast number of epidemiological, preclinical and in vitro experimental data strongly indicate the anticancer potential of calcitriol, the biologically active form of vitamin D. However, for the implementation of a vitamin D based cancer therapy the increased deactivation of calcitriol in cancer cells by overexpressed CYP24A1 hydroxylase should be suppressed. Inhibition of this enzyme expression or activity nowadays is considered as important aspect of anticancer therapeutic strategies. Herein, we investigated the impact of genistein, biochanin A, formonentin and kaempferol on the expression of the CYP24A1 gene induced by calcitriol in hepatocellular cancer cells Huh7 under normoxia (21%O2) or hypoxia (1%O2). We demonstrate that calcitriol induces CYP24A1 under normoxia and hypoxia, but this induction is significantly more potent under hypoxia, the typical microenvironment of solid tumors. In the presence of isoflavones genistein, biochanin A and formononetin, this induction is abrogated to the control levels under normoxia, while under hypoxia there is some differentiation in suppression efficacy between these compounds with genistein ≥ biochanin > formononetin. At the same time, kaempferol turned out to be completely ineffective in the suppression of CYP24A1 gene expression.

Anti-diabetic potential of Masclura tricuspidata leaves: Prenylated isoflavonoids with α-glucosidase inhibitory and anti-glycation activity.

Investigation of chemical constituents of Masclura tricuspidata leaves resulted in the isolation of 47 isoflavonoids possessing prenyl groups with different numbers and structures. Among them, sixteen compounds named cudracusisoflavones A-P (1-16) were first isolated from nature. The isoflavonoids isolated from M. tricuspidata leaves showed anti-diabetic effects as measured by inhibition on α-glucosidase activity and advanced glycation end-products (AGEs) formations. Especially, cudracusisoflavone L (12), a new compound, together with gancaonin M (27), erysenegalensein E (41) and millewanin G (44) showed strong α-glucosidase inhibition with IC50 values <10.0 µM. In addition, cudracusisoflavones A (1), D (4), M (13) and N (14), together with known prenylated isoflavonoids efficiently inhibited methylglyoxal (MGO)- or glyoxal (GO)-induced AGE formations. Structure activity relationship together with molecular docking analysis suggested the importance of hydroxy group and linear type of prenyl moiety for α-glucosidase inhibition. Conclusively, diverse prenylated isoflavonoids in M. tricuspidata leaves might ameliorate glycotoxicity-induced metabolic diseases.

Synthesis of new α-Aryl-α-tetralones and α-Fluoro-α-aryl-α-tetralones, preliminary antiproliferative evaluation on drug resistant cell lines and in silico prediction of ADMETox properties.

α-aryl-α-tetralones and α-fluoro-α-aryl-α-tetralones derivatives were synthesized by palladium catalyzed α-arylation reaction of α-tetralones and α-fluoro-α-tetralones, with bromoarenes in moderate to good yields. These compounds were evaluated for their in vitro anti-proliferative effects against human breast cancer and leukemia cell lines with diverse profiles of drug resistance. The most promising compounds, 3b, 3c, 8a and 8c, were effective on both neoplastic models. 3b and 8a induced higher toxicity on multidrug resistant cells and were able to avoid efflux by ABCB1 and ABCC1 transporters. Theoretical calculations of the physicochemical descriptors to predict ADMETox properties were favorable concerning Lipinski's rule of five, results that reflected on the low effects on non-tumor cells. Therefore, these compounds showed great potential for development of pharmaceutical agents against therapy refractory cancers.

Natural isoflavonoids in invasive cancer therapy: From bench to bedside.

Cancer is a public health problem worldwide, and one of the crucial steps within tumor progression is the invasion and metastasis of cancer cells, which are directly related to cancer-associated deaths in patients. Recognizing the molecular markers involved in invasion and metastasis is essential to find targeted therapies in cancer. Interestingly, about 50% of the discovered drugs used in chemotherapy have been obtained from natural sources such as plants, including isoflavonoids. Until now, most drugs are used in chemotherapy targeting proliferation and apoptosis-related molecules. Here, we review recent studies about the effect of isoflavonoids on molecular targets and signaling pathways related to invasion and metastasis in cancer cell cultures, in vivo assays, and clinical trials. This review also reports that glycitein, daidzein, and genistein are the isoflavonoids most studied in preclinical and clinical trials and displayed the most anticancer activity targeting invasion-related proteins such as MMP-2 and MMP-9 and also EMT-associated proteins. Therefore, the diversity of isoflavonoids is promising molecules to be used as chemotherapeutic in invasive cancer. In the future, more clinical trials are needed to validate the effectiveness of the various natural isoflavonoids in the treatment of invasive cancer.

Soybean (Glycine max) Is Able to Absorb, Metabolize and Accumulate Fenbendazole in All Organs Including Beans.

Although manure is an important source of minerals and organic compounds it represents a certain risk of spreading the veterinary drugs in the farmland and their permeation to human food. We tested the uptake of the anthelmintic drug fenbendazole (FBZ) by soybean, a common crop plant, from the soil and its biotransformation and accumulation in different soybean organs, including beans. Soybeans were cultivated in vitro or grown in a greenhouse in pots. FBZ was extensively metabolized in roots of in vitro seedlings, where sixteen metabolites were identified, and less in leaves, where only two metabolites were found. The soybeans in greenhouse absorbed FBZ by roots and translocated it to the leaves, pods, and beans. In roots, leaves, and pods two metabolites were identified. In beans, FBZ and one metabolite was found. FBZ exposure did not affect the plant fitness or yield, but reduced activities of some antioxidant enzymes and isoflavonoids content in the beans. In conclusion, manure or biosolids containing FBZ and its metabolites represent a significant risk of these pharmaceuticals entering food consumed by humans or animal feed. In addition, the presence of these drugs in plants can affect plant metabolism, including the production of isoflavonoids.

Evaluation of Firefly and Renilla Luciferase Inhibition in Reporter-Gene Assays: A Case of Isoflavonoids.

Firefly luciferase is susceptible to inhibition and stabilization by compounds under investigation for biological activity and toxicity. This can lead to false-positive results in in vitro cell-based assays. However, firefly luciferase remains one of the most commonly used reporter genes. Here, we evaluated isoflavonoids for inhibition of firefly luciferase. These natural compounds are often studied using luciferase reporter-gene assays. We used a quantitative structure-activity relationship (QSAR) model to compare the results of in silico predictions with a newly developed in vitro assay that enables concomitant detection of inhibition of firefly and Renilla luciferases. The QSAR model predicted a moderate to high likelihood of firefly luciferase inhibition for all of the 11 isoflavonoids investigated, and the in vitro assays confirmed this for seven of them: daidzein, genistein, glycitein, prunetin, biochanin A, calycosin, and formononetin. In contrast, none of the 11 isoflavonoids inhibited Renilla luciferase. Molecular docking calculations indicated that isoflavonoids interact favorably with the D-luciferin binding pocket of firefly luciferase. These data demonstrate the importance of reporter-enzyme inhibition when studying the effects of such compounds and suggest that this in vitro assay can be used to exclude false-positives due to firefly or Renilla luciferase inhibition, and to thus define the most appropriate reporter gene.

In Silico Studies of Some Isoflavonoids as Potential Candidates against COVID-19 Targeting Human ACE2 (hACE2) and Viral Main Protease (Mpro).

The Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the "COVID-19" disease that has been declared by WHO as a global emergency. The pandemic, which emerged in China and widespread all over the world, has no specific treatment till now. The reported antiviral activities of isoflavonoids encouraged us to find out its in silico anti-SARS-CoV-2 activity. In this work, molecular docking studies were carried out to investigate the interaction of fifty-nine isoflavonoids against hACE2 and viral Mpro. Several other in silico studies including physicochemical properties, ADMET and toxicity have been preceded. The results revealed that the examined isoflavonoids bound perfectly the hACE-2 with free binding energies ranging from -24.02 to -39.33 kcal mol-1, compared to the co-crystallized ligand (-21.39 kcal mol-1). Furthermore, such compounds bound the Mpro with unique binding modes showing free binding energies ranging from -32.19 to -50.79 kcal mol-1, comparing to the co-crystallized ligand (binding energy = -62.84 kcal mol-1). Compounds 33 and 56 showed the most acceptable affinities against hACE2. Compounds 30 and 53 showed the best docking results against Mpro. In silico ADMET studies suggest that most compounds possess drug-likeness properties.

Modulation of LPS-induced inflammation in RAW264.7 murine cells by novel isoflavonoids from Millettia pulchra.

Millettia pulchra is a renowned anti-inflammatory herbal medicine in southeast provinces of China. However, the underlying anti-inflammation mechanism remained incompletely understood. Herein, four new isoflavones, pulvones A-D and eleven reported constituents were isolated from the stems of Millettia pulchra with their structures being elucidated by HRMS and NMR analysis. The anti-inflammatory activities of pulvones A and C were further evaluated due to the better inhibitory activity on nitric oxide production in LPS-stimulated RAW264.7 cells and no obvious cytotoxicity to RAW264.7 cells. Western blot showed that pulvones A significantly decreased the levels of iNOS and COX-2 proteins and pulvones C only decreased the level of iNOS protein. ELISA analysis demonstrated that pulvones A inhibited the production of both interleukin-6 (IL-6) and IL-1ß while pulvones C showed better suppression effect on IL-1ß production in LPS-stimulated RAW264.7 cells. Then, their potential inhibitory effects on NF-κB pathway were tested in LPS-stimulated RAW264.7 cells. Immunofluorescence and western blot assay showed that pulvones A and C reduced the nuclear translocation of NF-κB(p65) and interrupted IκB phosphorylation. The ADP-Glo™ kinase assay showed pulvones A and C could directedly inhibit the IKKß kinase activity with the inhibitory rate of 40%, which were also verified by docking study. Collectively, these results suggested that pulvones A and C's anti-inflammatory effects were relevant to the interruption of NF-κB activation by inhibiting IKKß kinase.