Garcinia kola and garcinoic acid suppress SARS-CoV-2 spike glycoprotein S1-induced hyper-inflammation in human PBMCs through inhibition of NF-κB activation.

Symptoms and complications associated with severe SARS-CoV-2 infection such as acute respiratory distress syndrome (ARDS) and organ damage have been linked to SARS-CoV-2 spike protein S1-induced increased production of pro-inflammatory cytokines by immune cells. In this study, the effects of an extract of Garcinia kola seeds and garcinoic acid were investigated in SARS-CoV-2 spike protein S1-stimulated human PBMCs. Results of ELISA experiments revealed that Garcinia kola extract (6.25, 12.5, and 25 µg/ml) and garcinoic acid (1.25, 2.5, and 5 µM) significantly reduced SARS-CoV-2 spike protein S1-induced secretion of TNFα, IL-6, IL-1ß, and IL-8 in PBMCs. In-cell western assays showed that pre-treatment with Garcinia kola extract and garcinoic acid reduced expressions of both phospho-p65 and phospho-IκBα proteins, as well as NF-κB DNA binding capacity and NF-κB-driven luciferase expression following stimulation of PBMCs with spike protein S1. Furthermore, pre-treatment of PBMCs with Garcinia kola extract prior to stimulation with SARS-CoV-2 spike protein S1 resulted in reduced damage to adjacent A549 lung epithelial cells. These results suggest that the seed of Garcinia kola and garcinoic acid are natural products which may possess pharmacological/therapeutic benefits in reducing cytokine storm in severe SARS-CoV-2 and other coronavirus infections.

Zanthoxylum zanthoxyloides inhibits lipopolysaccharide- and synthetic hemozoin-induced neuroinflammation in BV-2 microglia: roles of NF-κB transcription factor and NLRP3 inflammasome activation.

OBJECTIVES: The effects of a root extract of Zanthoxylum zanthoxyloides on neuroinflammation in BV-2 microglia stimulated with LPS and hemozoin were investigated. METHODS: ELISA, enzyme immunoassay and Griess assay were used to evaluate levels of cytokines, PGE2 and NO in culture supernatants, respectively. Microglia-mediated neurotoxicity was evaluated using a BV-2 microglia-HT-22 neuron transwell co-culture. KEY FINDINGS: Treatment with Z. zanthoxyloides caused reduced elevated levels of TNFα, IL-6, IL-1ß, NO and PGE2, while increasing the levels of IL-10. In addition, there were reduced levels of iNOS and COX-2 proteins. This was accompanied by a prevention of microglia-mediated damage to HT-22 mouse hippocampal neurons. Z. zanthoxyloides reduced elevated levels of phospho-IκB and phospho-p65, while preventing degradation of IκB protein and DNA binding of p65. Further mechanistic studies revealed that Z. zanthoxyloides reduced the levels of pro-IL-1ß and IL-1ß in hemozoin-activated BV-2 microglia. This was accompanied by a reduction in caspase-1 activity and NLRP3 protein expression. Bioassay-guided fractionation resulted in the isolation of skimmianine as an anti-inflammatory compound in Z. zanthoxyloides. CONCLUSION: This is the first report showing the inhibition of neuroinflammation in LPS- and hemozoin-activated BV-2 microglia by the root extract of Z. zanthoxyloides by targeting the activation of both NF-κB and NLRP3 inflammasome.

In vitro bioactivity of the fractions and isolated compound from Combretum elaeagnoides leaf extract against selected foodborne pathogens.

ETHNOPHARMACOLOGICAL RELEVANCE: Combretum species are used traditionally for the treatment of diarrhoea, hookworm, fever, inflammation, pain and infectious diseases. Infections are commonly caused by the intake of food contaminated with foodborne pathogens. These are a significant concern in the food industry owing to their ability to form biofilms and cause food spoilage, despite the availability of modern food preservation techniques. Combretum elaeagnoides Klotzsch (Combretaceae) is used in southern African traditional medicine against infections and diarrhoea. AIM OF THE STUDY: This study evaluated the antimicrobial ability of C. elaeagnoides leaf fractions and the isolated compound quercetin-3-O-rhamnoside against a panel of foodborne pathogens, and biofilms formed by them. The samples were also assessed for their antioxidant activity and cytotoxicity. MATERIALS AND METHODS: Fractions prepared from the methanol extract of the leaves, and a bioactive compound (quercetin-3-O-rhamnoside) isolated from the ethyl acetate fraction were investigated for activity against nine reference and clinical strains of foodborne pathogens. The microdilution method was used to determine the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the fractions and compound. The inhibition of biofilm formation and the crystal violet staining assays were used to determine the antibiofilm efficacy. The DPPH (2, 2-diphenyl-1-picrylhydrazyl) assay and the 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) electron reduction assay were used to determine the antioxidant potential of the fractions and compound. The cytotoxicity was assessed using the 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay against Vero African monkey kidney cells. RESULTS: The fractions were active against all tested organisms, with MIC values ranging from 0.03 to 1.25 mg/mL. The best MBC was 0.63 mg/mL. All the fractions and the purified compound inhibited biofilm formation of Staphylococcus aureus and Salmonella Typhimurium, with percentage inhibition values greater than 50% at 1 mg/mL. The compound had very promising antibiofilm activity against Escherichia coli 1 (ATCC 25922) with percentage inhibition of >150%. The compound and fractions had good radical scavenging potential against the DPPH and ABTS radicals. Quercetin-3-O-rhamnoside and the fractions were relatively non-cytotoxic. CONCLUSION: The ability of the fractions and compound to reduce and inhibit biofilm biomass and their promising antioxidant potential provide motivation to further investigate the use of plants to protect food products from contamination, as well as to treat infections characterized by bacterial biofilms.

Agathisflavone isolated from Anacardium occidentale suppresses SIRT1-mediated neuroinflammation in BV2 microglia and neurotoxicity in APPSwe-transfected SH-SY5Y cells.

Agathisflavone is a bioactive compound in Anacardium occidentale. In this study, we investigated inhibition neuroinflammation in BV2 microglia by agathisflavone. Neuroprotective activity of the compound was investigated in differentiated SH-SY5Y cells. Experiments in lipopolysaccharide (LPS)-activated BV2 microglia showed that pretreatment with agathisflavone (5-20 µM) produced significant reduction in the release of tumour necrosis factor-α, interleukin-6, interleukin-1ß, NO, and PGE2 from the cells. Immunoblotting experiments also revealed that agathisflavone reduced levels of iNOS and COX-2 protein. Further studies revealed that agathisflavone reduced neuroinflammation by targeting critical steps in NF-κB signalling in BV2 microglia. Treatment of SH-SY5Y cells with conditioned medium from LPS-activated BV2 microglia produced a significant reduction in neuronal viability. However, conditioned medium from BV2 cells that were stimulated with LPS in the presence of agathisflavone did not induce neurotoxicity. Agathisflavone also produced neuroprotection in APPSwe plasmid-transfected SH-SY5Y neurons. The compound further attenuated LPS-induced and APPSwe plasmid-induced reduction in SIRT1 in BV2 microglia and SH-SY5Y, respectively. In the presence of EX527, agathisflavone lost its anti-inflammatory and neuroprotective activities. Our results suggest that agathisflavone inhibits neuroinflammation in BV2 microglia by targeting NF-κB signalling pathway. The compound also reduces neurotoxicity through mechanisms that are possibly linked to SIRT1 in the microglia and neurons.

Antimicrobial and selected in vitro enzyme inhibitory effects of leaf extracts, flavonols and indole alkaloids isolated from Croton menyharthii.

Croton species are used in folk medicine in the management of infections, inflammation and oxidative stress-related diseases. In order to isolate, characterize and evaluate the bioactive constituents of Croton menyharthii Pax leaf extracts, repeated column fractionation of the ethyl acetate fraction from a 20% aqueous methanol crude extract afforded three flavonols identified by NMR (1D and 2D) spectroscopic methods as myricetrin-3-O-rhamnoside (myricetrin, 1), quercetin-3-O-rhamnoside (2) and quercetin (3) along with an indole alkaloid, (E)-N-(4-hydroxycinnamoyl)-5-hydroxytryptamine, [trans-N-(p-coumaroyl) serotonin, 4]. All the compounds are reported from the leaf extract of this plant for the first time. The crude extracts, four solvent fractions (hexane, DCM, ethyl acetate and butanol) and isolated compounds obtained from the leaves were evaluated for their inhibitory effects on selected bacteria, a fungus (Candida albicans), cyclooxygenase (COX-2), α-glucosidase and acetylcholinesterase (AChE). Amongst the compounds, quercetin (3) was the most active against Bacillus subtilis and Candida albicans while myricetrin-3-O-rhamnoside (1) and trans-N-(p-coumaroyl) serotonin (4) were the most active compounds against Escherichia coli, Klebsiella pneumonia and Staphylococcus aureus. The inhibitory activity of myricetrin-3-O-rhamnoside (1) against COX-2 was insignificant while that of the other three compounds 2-4 was low. The AChE inhibitory activity of the alkaloid, trans-N-(p-coumaroyl) serotonin was high, with a percentage inhibitory activity of 72.6% and an IC50 value of 15.0 µg/mL. The rest of the compounds only had moderate activity. Croton menyharthii leaf extracts and isolated compounds inhibit α-glucosidase at very low IC50 values compared to the synthetic drug acarbose. Structure activity relationship of the isolated flavonols 1-3 is briefly outlined. Compounds 1-4 and the leaf extracts exhibited a broad spectrum of activities. This validates the ethnomedicinal use of the plant in folk medicine.

Isolation and characterization of antimicrobial constituents of Searsia chirindensis L. (Anacardiaceae) leaf extracts.

ETHNOPHARMACOLOGICAL RELEVANCE: Searsia chirindensis is used in South African traditional medicine for management of bacterial infections such as diarrhoea. Aim of the study was to examine the phytochemical composition from the leaves of Searsia chirindensis that is responsible for the ethnomedicinal use of this plant. MATERIALS AND METHODS: The crude extract (80% methanol) was extracted sequentially with dichloromethane (DCM), ethyl acetate (EtOAc) and n-butanol. The extracts and isolated compounds were tested for their antibacterial activity against Gram-negative (Campylobacter jejuni, Escherichia coli and Shigella flexneri) and Gram-positive (Staphylococcus aureus) bacterial strains using the microdilution method. Bioguided fractionation of EtOAc fraction afforded five phenolic compounds. Structural elucidation was carried out using NMR (1D and 2D) spectroscopic analyses. RESULTS: Of the three fractions obtained from the crude extract, EtOAc was the most active and its fractionation afforded methyl gallate (1), and four flavonol glycosides: myricetin-3-O-arabinopyranoside (2), myricetrin-3-O-rhamnoside (3), kaempferol-3-O-rhamnoside (4) and quercetin-3-O-arabinofuranoside (5). These compounds are reported from Searsia chirindensis for the first time. All the compounds showed good antibacterial activity against all bacterial strains tested. Their minimum inhibitory concentrations ranged from 30 to 250 µg/mL. CONCLUSIONS: Antibacterial activity demonstrated by the extracts and isolated compounds provides credence to the ethnomedicinal use of Searsia chirindensis against diarrhoea.

Potential antiradical and alpha-glucosidase inhibitors from Ecklonia maxima (Osbeck) Papenfuss.

Alpha-glucosidase inhibitors play a potential role in the treatment of type 2 diabetes by delaying glucose absorption in the small intestine. Ecklonia maxima, a brown alga which grows abundantly on the west coast of South Africa, is used to produce alginate, animal feed, nutritional supplements and fertilizer. The crude aqueous methanol extract, four solvent fractions and three phlorotannins: 1,3,5-trihydroxybenezene (phloroglucinol) (1), dibenzo [1,4] dioxine-2,4,7,9-tetraol (2) and hexahydroxyphenoxydibenzo [1,4] dioxine (eckol) (3) isolated from E. maxima were evaluated for antiradical and alpha-glucosidase inhibitory activities. All the phlorotannins tested had strong antioxidant activities on DPPH free radicals with EC50 values ranging from 0.008 to 0.128µM. Compounds 2 and 3 demonstrated stronger antioxidant activity and an alpha-glucosidase inhibitory property than positive controls. These results suggest that E. maxima could be a natural source of potent antioxidants and alpha-glucosidase inhibitors. This study could facilitate effective utilization of E. maxima as an oral antidiabetic drug or functional food ingredient with a promising role in the formulation of medicines and nutrition supplements.

Anti-oxidative and cholinesterase inhibitory effects of leaf extracts and their isolated compounds from two closely related Croton species.

A comparative evaluation of the antioxidant and acetylcholinesterase inhibitory activity of the leaf extracts of Croton gratissimus and Croton zambesicus (subgratissimus) and compounds isolated from the extracts was carried out to determine their potential and suitability or otherwise as a substitute for each other in the management of oxidative and neurodegenerative conditions. Different antioxidant assays (DPPH, FRAP, ß-carotene-linoleic and the lipid peroxidation models) and the microplate assay for acetylcholinesterase (AChE) inhibition were carried out separately to study the activities of the crude leaf extracts and four solvent fractions from each of the two Croton species. Bioassay guided fractionation was used to target antioxidant constituents of the crude extracts and ethyl acetate fractions of 20% aqueous methanol extract of C. gratissimus on silica gel and Sephadex LH-20 columns resulted in the isolation of kaempferol-3-O-ß-6''(p-coumaroyl) glucopyranoside (tiliroside, 2), apigenin-6-C-glucoside (isovitexin, 3) and kampferol (4). The extract of C. zambesicus yielded quercetin-3-O-ß-6''(p-coumaroyl) glucopyranoside-3'-methyl ether (helichrysoside- 3'-methyl ether, 1), kaempferol-3-O-ß-6''(p-coumaroyl) glucopyranoside (tiliroside, 2) and apigenin-6-C-glucoside (isovitexin, 3). Three of the isolated compounds and their different combinations were also included in the bioassays. In all the assays performed, the antioxidant capacity and AChE inhibitory effects of C. zambesicus extracts were weaker than those of C. gratissimus. This suggests that C. gratissimus may not be substituted by C. zambesicus, despite the similarity in some of their constituents. Generally, the combinations made from the isolated compounds showed better activities in most of the assays compared to the individual isolated compounds. This suggests mechanisms such as synergism and/or additive effects to be taking place. This study established low, moderate and high antioxidant activities as well as AChE inhibitory effects by the crude extracts, fractions, compounds and compound combinations. This means some of the extracts, isolated compounds and compound combinations could be useful in the management of neurodegenerative conditions and serve as sources of natural neurodegenerative agents.

Mechanisms of anti-inflammatory property of Anacardium occidentale stem bark: inhibition of NF-κB and MAPK signalling in the microglia.

ETHNOPHARMACOLOGICAL RELEVANCE: Anacardium occidentale is used in traditional African medicine for the treatment of arthritis, fever, aches, pains, and inflammation of the extremities. AIM OF THE STUDY: In this study, we investigated the molecular mechanisms responsible for anti-inflammatory effects of a stem bark extract of A. occidentale (ANE) in LPS-stimulated microglia. MATERIALS AND METHODS: Nitric oxide (NO), prostaglandin E(2) and cytokine (TNFα and IL-6) production were evaluated in supernatants from LPS-stimulated BV-2 cells. Cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and microsomal prostaglandin E2 synthase (mPGES-1) protein expressions in rat primary microglia were measured using western blot. The effects of ANE on NF-κB activation and nuclear translocation were evaluated in the luciferase reporter gene assay and ELISA, while ability of ANE to influence IκB phosphorylation was determined using ELISA specific for phospho-IκB. The involvement of MAPK phosphorylation in the anti-inflammatory actions of ANE was evaluated using specific ELISA for phospho-p38, phospho-p42/44 and phospho-JNK. The MTT assay was used to determine the effect of ANE on BV-2 microglia viability. RESULTS: ANE (25-100 µg/ml) produced significant (p<0.05) reduction in the production of NO, PGE(2), TNFα and IL-6 in BV-2 microglia stimulated with LPS for 24h. Pre-treatment with ANE caused a significant (p<0.05) inhibition of COX-2, iNOS and mPGES-1 protein expressions in the rat primary microglia. Further experiments showed that ANE inhibited COX-2 and iNOS protein expression via IκB-mediated nuclear translocation and transactivation of NF-κB. Our studies also revealed that ANE produced significant (p<0.05) and dose-dependent inhibition of p38, p42/44 and JNK MAPK phosphorylation in LPS-activated BV-2 microglia. CONCLUSIONS: We conclude that ANE has an anti-inflammatory property related to inhibition of inflammation-associated cytokine production as well as iNOS and COX-2 gene expression by blocking NF-κB and MAPK pathways in the microglia. It is also suggested that mPGES-1 inhibition contributes to the effect of ANE on PGE(2) production in the microglia.

Bridelia ferruginea Produces Antineuroinflammatory Activity through Inhibition of Nuclear Factor-kappa B and p38 MAPK Signalling.

Bridelia ferruginea is commonly used in traditional African medicine (TAM) for treating various inflammatory conditions. Extracts from the plant have been shown to exhibit anti-inflammatory property in a number of in vivo models. In this study the influence of B. ferruginea (BFE) on the production of PGE(2), nitrite, and proinflammatory cytokines from LPS-stimulated BV-2 microglia was investigated. The effects of BFE on cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) protein expressions were evaluated in LPS-activated rat primary microglia. The roles of NF-κB and MAPK signalling in the actions of BFE were also investigated. BFE (25-200 µg) inhibited the production of PGE(2), nitrite, tumour necrosis factor-α (TNFα), and interleukin-6 (IL-6) as well as COX-2 and iNOS protein expressions in LPS-activated microglial cells. Further studies to elucidate the mechanism of anti-inflammatory action of BFE revealed interference with nuclear translocation of NF-κBp65 through mechanisms involving inhibition of IκB degradation. BFE prevented phosphorylation of p38, but not p42/44 or JNK MAPK. It is suggested that Bridelia ferruginea produces anti-inflammatory action through mechanisms involving p38 MAPK and NF-κB signalling.