Natural compounds proposed for the management of non-alcoholic fatty liver disease.

Although non-alcoholic fatty liver disease (NAFLD) presents as an intricate condition characterized by a growing prevalence, the often-recommended lifestyle interventions mostly lack high-level evidence of efficacy and there are currently no effective drugs proposed for this indication. The present review delves into NAFLD pathology, its diverse underlying physiopathological mechanisms and the available in vitro, in vivo, and clinical evidence regarding the use of natural compounds for its management, through three pivotal targets (oxidative stress, cellular inflammation, and insulin resistance). The promising perspectives that natural compounds offer for NAFLD management underscore the need for additional clinical and lifestyle intervention trials. Encouraging further research will contribute to establishing more robust evidence and practical recommendations tailored to patients with varying NAFLD grades.

Microfluidic Diffusion Sizing Applied to the Study of Natural Products and Extracts That Modulate the SARS-CoV-2 Spike RBD/ACE2 Interaction.

The interaction between SARS-CoV-2 spike RBD and ACE2 proteins is a crucial step for host cell infection by the virus. Without it, the entire virion entrance mechanism is compromised. The aim of this study was to evaluate the capacity of various natural product classes, including flavonoids, anthraquinones, saponins, ivermectin, chloroquine, and erythromycin, to modulate this interaction. To accomplish this, we applied a recently developed a microfluidic diffusional sizing (MDS) technique that allows us to probe protein-protein interactions via measurements of the hydrodynamic radius (Rh) and dissociation constant (KD); the evolution of Rh is monitored in the presence of increasing concentrations of the partner protein (ACE2); and the KD is determined through a binding curve experimental design. In a second time, with the protein partners present in equimolar amounts, the Rh of the protein complex was measured in the presence of different natural products. Five of the nine natural products/extracts tested were found to modulate the formation of the protein complex. A methanol extract of Chenopodium quinoa Willd bitter seed husks (50 µg/mL; bisdesmoside saponins) and the flavonoid naringenin (1 µM) were particularly effective. This rapid selection of effective modulators will allow us to better understand agents that may prevent SARS-CoV-2 infection.

Smallholders' knowledge about healing goat gastrointestinal parasite infections with wild plants in southern DR Congo.

Gastrointestinal parasite (GIP) infections control has an important role to play in increasing livestock production from a limited natural resource base and to improve animal health and welfare. This study aimed to collect indigenous knowledge and identify wild plants locally used by goat smallholders of three territories of Haut-Katanga province for treating signs of gastrointestinal parasitism. Ethnoveterinary surveys were conducted by semi-structured interviews and a bibliographic screening of the biological activities relating to cited plants was carried out. Our interviews showed that ethnosemantic diagnoses of GIP diseases are based on signs. Eighty-seven informants reported that 27 plant species from 15 families, dominated by Fabaceae (29.6%) and Lamiaceae (18.5%) were commonly used in their goats treatment. Among these plants, five species with palmately compound leaves were considerably more used. From those, we noted a substitution of Vitex congolensis De Wild. and T. Durand (Lamiaceae) by Oldfieldia dactylophylla (Welw. Ex Oliv.) J. Leonard (Picrodendraceae) and of Vitex mombassae Vatke by Vitex madiensis Oliv. Subsp. Milanjiensis (Britten) F. White. Roots (46.9%), leaves (28.0%) and seeds (12.5%) were the most frequently used plant organs, and maceration is applied for most of the medicinal preparations (62.2%). Recipes were administered by oral route, for GIP 1) prevention (33.3%), by macerating the ground plant material in drinking water for 2 weeks at the start of each season (dry and rainy); and 2) treatment (66.7%). According to the literature, some of these plants have few or no studies investigating their anthelmintic activity. The cited plants are worth investigating further as they could constitute an effective alternative strategy in maintaining animal productivity. Studies on the biological activity of these plants can also provide indications of promising leads for extracts that could be developed into commercial standardized medications.

Assessment of the Cytotoxicity, Mutagenicity, and Genotoxicity of Two Traditional Chinese Herbs: Aristolochia baetica and Magnolia officinalis.

Herbal remedies used in traditional medicine often contain several compounds combined in order to potentiate their own intrinsic properties. However, herbs can sometimes cause serious health troubles. In Belgium, patients who developed severe aristolochic acid nephropathy ingested slimming pills containing root extracts of an Aristolochia species, as well as the bark of Magnolia officinalis. The goal of the study was to evaluate, on a human renal cell line, Aristolochia and Magnolia extracts for their cytotoxicity by a resazurin cell viability assay, and their genotoxicity by immunodetection and quantification of the phosphorylated histone γ-H2AX. The present study also sought to assess the mutagenicity of these extracts, employing an OECD recognized test, the Ames test, using four Salmonella typhimurium strains with and without a microsomial fraction. Based on our results, it has been demonstrated that the Aristolochia-Magnolia combination (aqueous extracts) was more genotoxic to human kidney cells, and that this combination (aqueous and methanolic extracts) was more cytotoxic to human kidney cells after 24 and 48 h. Interestingly, it has also been shown that the Aristolochia-Magnolia combination (aqueous extracts) was mutagenic with a TA98 Salmonella typhimurium strain in the presence of a microsomial liver S9 fraction. This mutagenic effect appears to be dose-dependent.

Endogenous Synthesis of Tetrahydroisoquinoline Derivatives from Dietary Factors: Neurotoxicity Assessment on a 3D Neurosphere Culture.

Tetrahydroisoquinoline (THIQ) alkaloids and their derivatives have a structural similarity to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a well-known neurotoxin. THIQs seem to present a broad range of actions in the brain, critically dependent on their catechol moieties and metabolism. These properties make it reasonable to assume that an acute or chronic exposure to some THIQs might lead to neurodegenerative diseases including essential tremor (ET). We developed a method to search for precursor carbonyl compounds produced during the Maillard reaction in overcooked meats to study their reactivity with endogenous amines and identify the reaction products. Then, we predicted in silico their pharmacokinetic and toxicological properties toward the central nervous system. Finally, their possible neurological effects on a novel in vitro 3D neurosphere model were assessed. The obtained data indicate that meat is an alkaloid precursor, and we identified the alkaloid 1-benzyl-1,2,3,4-tetrahydroisoquinoline-6,7-diol (1-benz-6,7-diol THIQ) as the condensation product of phenylacetaldehyde with dopamine; in silico study of 1-benz-6,7-diol-THIQ reveals modulation of dopamine receptor D1 and D2; and in vitro study of 1-benz-6,7-diol-THIQ for cytotoxicity and oxidative stress induction does not show any difference after 24 h contact for all tested concentrations. To conclude, our in vitro data do not support an eventual neurotoxic effect for 1-benz-6,7-diol-THIQ.

Perceptions and use of traditional African medicine in Lubumbashi, Haut-Katanga province (DR Congo): A cross-sectional study.

In the Democratic Republic of Congo, the desire of the Ministry of Health to integrate Traditional African Medicine into the Official Health System remains limited by the lack of reliable data on several aspects of this medicine. This study aims to determine the perceptions of the Lubumbashi population towards Traditional African Medicine and the contexts of recourse to these therapeutic modalities. We conducted semi-structured interviews of population samples in each of the 7 Lubumbashi municipalities, which were semi-randomly selected in proportions to each population size, from January to June 2017 and from February to July 2018. A total of 4278 people (average age, 32.1 ± 10.4 years; 36.5% of women) have been surveyed. Among them, 75.8% define "Traditional African Medicine" as "herbal-based treatments"; 79.4% have resorted to traditional medicine, for several pathologies and social problems. This medicine was preferred for efficiency, speed of healing and low cost, as well as the presence of the diseases against which it would be the only one used. Most, (52.1%) have started with Conventional Medicine, then resorted to Traditional African Medicine, 34.7% started directly with Traditional African Medicine, while 13.2% combined these two medicines. From those who have resorted to Traditional African Medicine (n = 3396), 55% declare no concern towards traditional medicine, while 42.5% fear looseness, overdose, intoxication, and lack of hygiene; from those who have not resorted to Traditional African Medicine (n = 882), 78% are fearful of fear looseness, witchcraft, and fetishism. Traditional African Medicine remains an important health care resource for the Lubumbashi people. It is essential that decision-makers consider the importance of this health sector for the population, but also the reported fears and hindrances, and work towards the regulation, sanitization, and control of this medicine to ensure its safe use.

Development of a Method for Producing oxLDL: Characterization of Their Effects on HPV-Positive Head and Neck Cancer Cells.

Cardiovascular diseases (CVD) and cancers are the two main causes of death worldwide. The initiation and progression of atherosclerosis is, in large part, caused by oxidized low-density lipoproteins (oxLDL); interestingly, oxLDL may also play a role in cancer cell metabolism and migration. As oxLDL are generally obtained by tedious ultracentrifugation procedures, "home-made" oxLDL were obtained by (i) applying a purification kit to isolate LDL and VLDL from human plasma; (ii) isolating LDL from VLDL by gel permeation chromatography (GPC); and (iii) oxidating LDL through CuSO4 incubation. On three HPV-positive head and neck cancer cells (HNCC) (93VU-147T, UM-SCC47, and UPCI-SCC154), cell migration was assessed using Boyden chambers, the Wnt/ß-catenin pathway was analyzed by Western Blotting, and the expression of two oxLDL receptors, LOX-1 and CD36, in response to oxLDL exposure, was analysed by immunofluorescence. Our data indicate: (a) a non-significant difference between reference and "home-made" oxLDL; (b) a decreased migration, parallel to an inhibition of the ß-catenin pathway; and (c) an increase of CD36 and LOX-1 expression in all HNCC. In conclusion, we successfully produced oxLDL. Our results demonstrate a decrease in HNCC migration after oxLDL exposure, and an increased expression of LOX-1 and CD36 associated with lipid uptake.

Effects of Chemopreventive Natural Compounds on the Accuracy of 8-oxo-7,8-dihydro-2'-deoxyguanosine Translesion Synthesis.

Translesion synthesis is a DNA damage tolerance mechanism that relies on a series of specialized DNA polymerases able to bypass a lesion on a DNA template strand during replication or post-repair synthesis. Specialized translesion synthesis DNA polymerases pursue replication by inserting a base opposite to this lesion, correctly or incorrectly depending on the lesion nature, involved DNA polymerase(s), sequence context, and still unknown factors. To measure the correct or mutagenic outcome of 8-oxo-7,8-dihydro-2'-deoxyguanosine bypass by translesion synthesis, a primer-extension assay was performed in vitro on a template DNA bearing this lesion in the presence of nuclear proteins extracted from human intestinal epithelial cells (FHs 74 Int cell line); the reaction products were analyzed by both denaturing capillary electrophoresis (to measure the yield of translesion elongation) and pyrosequencing (to determine the identity of the nucleotide inserted in front of the lesion). The influence of 14 natural polyphenols on the correct or mutagenic outcome of translesion synthesis through 8-oxo-7,8-dihydro-2'-deoxyguanosine was then evaluated in 2 experimental conditions by adding the polyphenol either (i) to the reaction mix during the primer extension assay; or (ii) to the culture medium, 24 h before cell harvest and nuclear proteins extraction. Most of the tested polyphenols significantly influenced the outcome of translesion synthesis, either through an error-free (apigenin, baicalein, sakuranetin, and myricetin) or a mutagenic pathway (epicatechin, chalcone, genistein, magnolol, and honokiol).

Toxicity and Anti-Proliferative Properties of Anisomeles indica Ethanol Extract on Cervical Cancer HeLa Cells and Zebrafish Embryos.

In this study, we showed that crude extract of Anisomeles indica (AI-EtE) expressed its toxicity to HeLa cells with an IC50 dose of 38.8 µg/mL and to zebrafish embryos with malformations, lethality and hatching inhibition at 72-hpf at doses higher than 75 µg/mL. More interestingly, flow cytometry revealed that AI-EtE significantly promoted the number of cells entering apoptotic. Accordingly, the transcript levels of BAX, CASPASE-8, and CASPASE-3 in the cells treated with AI-EtE at IC50 dose were 1.55-, 1.62-, and 2.45-fold higher than those in the control cells, respectively. Moreover, treatment with AI-EtE caused cell cycle arrest at the G1 phase in a p53-independent manner. Particularly, percentages of AI-EtE-treated cells in G1, S, G2/M were, respectively 85%, 6.7% and 6.4%; while percentages of control cells in G1, S, G2/M were 64%, 15% and 19%, respectively. Consistent with cell cycle arrest, the expressions of CDKN1A and CDNK2A in AI-EtE-treated cells were up-regulated 1.9- and 1.64-fold, respectively. Significantly, treatment with AI-EtE also decreased anchorage-independent growth of HeLa cells. In conclusion, we suggest that Anisomeles indica can be considered as a medicinal plant with a possible use against cervical cancer cells; however, the used dose should be carefully monitored, especially when applying to pregnant women.

Anti-inflammatory properties of the ethanol extract from Clerodendrum cyrtophyllum Turcz based on in vitro and in vivo studies.

ETHNOPHARMACOLOGICAL RELEVANCE: Clerodendrum cyrtophyllum Turcz, a plant belonging to the Verbenaceae family, has been used in traditional medicine for the treatment of various inflammatory diseases in many Asian countries. AIM OF THE STUDY: The study aimed to evaluate anti-inflammatory properties of the ethanol extract from Clerodendrum cyrthophyllum Turcz leaves (EE-CC) through in vitro and in vivo models. MATERIAL AND METHODS: Total phenolic and flavonoid contents in the extract were determined using colorimetric methods and HPTLC. In red blood cell membrane stabilization model, rat erythrocyte suspension was treated with crude ethanol extract at different concentrations, the hemoglobin content of the supernatant solution released by red blood hemolysis was estimated. We also evaluated the effects of the ethanol extract from this plant on the production of nitric oxide (NO), tumor necrosis factor alpha (TNF-α) in stimulated RAW 264.7 cells. In order to elucidate its anti-inflammatory molecular mechanisms, we further evaluated the effects of the EE-CC on the expression of the inflammatory genes in inflammation-induced zebrafish model by tail-cutting using qPCR analysis. RESULTS: Colorimetric methods and HPTLC revealed high phenolic and flavonoid contents in the extract. In the red blood cell membrane stabilization model, the amount of hemoglobin released by red blood hemolysis significantly decreased in the presence of EE-CC, demonstrating a strong membrane stabilizing activity. EE-CC did not cause any toxic effect on cell viability but strongly inhibited NO and TNF-ɑ release due to LPS induction. The association with EE-CC significantly reduced the expression of cox-2, pla2, c3a, il-1(il1fma), il-8 (cxcl8b.1), tnf-α, and nf-ƙb, while increased the expression of the anti-inflammatory cytokine il-10 gene in cut-tail induced inflammation of zebrafish model. CONCLUSIONS: Taken together, the results suggest that the raw ethanol extract from C. cyrtophyllum Turcz leaves presents potent anti-inflammatory activities and may be useful for the treatment of various inflammatory diseases.

Linear regression analysis of silychristin A, silybin A and silybin B contents in Silybum marianum.

Quantitative correlations between the contents of the flavonolignans silychristin A and silybins A/B provide biosynthetic clues that support a pathway in which one mesomeric form of a taxifolin radical is undergoing an oxidative coupling with a coniferyl alcohol radical. The flavonolignan content and patterns reported in the literature for 53 samples, representing populations of the Silybum marianum plant growing in different parts of the world, were subject to a meta-analysis. Linear regression analyses were carried out on these data sets, and a mathematical model was derived that predicts the content of silychristin A relative to the metabolomic pattern of its congeners. The validity of the model was verified by applying it to test samples. This approach could potentially become a tool to enhance the understanding of both the relative composition of the silymarin complex and the biosynthetic pathways that underlie its formation.

Chemotaxonomic and biosynthetic relationships between flavonolignans produced by Silybum marianum populations.

Flavonolignans constitute an important class of plant secondary metabolites formed by oxidative coupling of one flavonoid and one phenylpropanoid moiety. The standardized flavonolignan-rich extract prepared from the fruits of Silybum marianum is known as silymarin and has long been used medicinally, prominently as an antihepatotoxic and as a chemopreventive agent. Principal component analysis of the variation in flavonolignan content in S. marianum samples collected from different locations in Egypt revealed biosynthetic relationships between the flavonolignans. Silybin A, silybin B, and silychristin are positively correlated as are silydianin, isosilychristin, and isosilybin B. The detection of silyamandin in the extracts of S. marianum correlates with isosilychristin and silydianin content. The positive correlation between silydianin, isosilychristin, and silyamandin was demonstrated using quantitative 1H nuclear magnetic resonance spectroscopy (qHNMR). These correlations can be interpreted as evidence for the involvement of a flavonoid radical in the biosynthesis of the flavonolignans in S. marianum. The predominance of silybins A & B over isosilybin A & B in the silybin-rich samples is discussed in light of the relative stabilities of their respective radical flavonoid biosynthetic intermediates.

In vitro genotoxicity tests point to an unexpected and harmful effect of a Magnolia and Aristolochia association.

ETHNOPHARMACOLOGICAL RELEVANCE: in the 1990s, a Belgian cohort of more than 100 patients reported cases of Aristolochic Acid Nephropathy (AAN). This progressive renal and interstitial fibrosis, frequently associated with urothelial malignancies, was consecutive to the Chinese-herbs based slimming capsules intake where a plant Stephania tetrandra S. Moore was replaced by a highly genotoxic Aristolochia species. 70% of the Belgian patients evolved into end-stage renal disease, requiring dialysis or renal transplantation. Furthermore the prevalence of upper urinary tract carcinoma was found alarmingly high in these patients. The Aristolochia adulteration was blamed for the intoxication cases and, to the best of our knowledge, the prescription itself has not been further investigated. AIM OF THE STUDY: This work proposes to evaluate the in vitro cytotoxicity and genotoxicity of Aristolochia and Magnolia traditional aqueous decoctions and their association. MATERIALS AND METHODS: The cytotoxicity of extracts has been assessed by a MTT cell proliferation assay and the genotoxicity by measuring the presence of γ-H2AX, a phosphorylated histone associated with DNA damages. RESULTS: Treating cells for 24h with a mixture 1:1 of Magnolia officinalis and Aristolochia baetica decoctions led to an increase in the production of γ-H2AX. CONCLUSIONS: This genotoxic potentiation warrants further studies but may lead to an explanatory factor for the "Chinese herb nephropathy" cases.

Organocatalysis paradigm revisited: are metal-free catalysts really harmless?

Catalysts are commonly used in polymer synthesis. Traditionally, catalysts used to be metallic compounds but some studies have pointed out their toxicity for human health and environment, and the removal of metal impurities from synthetic polymer is quite expensive. Organocatalysts have been intensively synthesized and are now widely used in ring-opening polymerization (ROP) reactions to address these issues. However, for most of them, there is not any evidence of their safety. The present study attempts to assess whether well-established organo-based ROP catalysts used for the preparation of FDA-approved polyesters may present a certain level of cytotoxicity. In vitro toxicity is evaluated using a methyl-thiazol-tetrazolium cytotoxicity assay on two cell models (FHs74Int and HepaRG). Among the investigated organocatalysts, only functionalized thiourea shows an important cytotoxicity on both cell models. 4-Dimethylaminopyridine (DMAP), 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD), and meta-(trimethylammonio)phenolate betaine (m-BE) show cytotoxicity against HepaRG cell line only at a high concentration.

Pyrosequencing for the quantitative assessment of 8-oxodG bypass DNA synthesis.

Translesion synthesis (TLS) with specialized DNA polymerases allows dealing with a base lesion on the template strand during DNA replication; a base is inserted opposite the lesion, correctly or incorrectly, depending on the lesion, the involved DNA polymerase(s) and the sequence context. The major oxidized DNA base 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG) is highly mutagenic due to its ability to pair with either cytosine or adenine during DNA synthesis, depending on its conformation and involved DNA polymerases. To measure the correct or mutagenic outcome of lesion bypass, an original quantitative pyrosequencing method was developed and analytically validated. The method was applied to the study of DNA synthesis fidelity through an 8-oxodG or an undamaged guanine. After an in vitro primer-extension through 8-oxodG in the presence of the four deoxynucleotides triphosphates and a total nuclear protein extract, obtained from normal human intestinal epithelial cells (FHs 74 Int cell line), the reaction products were amplified by polymerase chain reaction and analyzed by pyrosequencing to measure nucleotides inserted opposite the lesion. The 8-oxodG bypass fidelity of FHs 74 Int cells nuclear extract is about 85.3%. We calculated within-day and total precisions for both 8-oxodG (2.8% and 2.8%, respectively) and undamaged templates (1.0% and 1.1%, respectively). We also demonstrated that only cytosine is incorporated opposite a normal guanine and that both cytosine and adenine can be incorporated opposite an 8-oxodG lesion. The proposed method is straightforward, fast, reproducible and easily adaptable to other sequences and lesions. It thus has a wide range of applications in the biological field, notably to elucidate TLS mechanisms and modulators.

Effects of chemopreventive natural products on non-homologous end-joining DNA double-strand break repair.

Double-strand breaks (DSBs) may result from endogenous (e.g., reactive oxygen species, variable (diversity) joining, meiotic exchanges, collapsed replication forks, nucleases) or exogenous (e.g., ionizing radiation, chemotherapeutic agents, radiomimetic compounds) events. DSBs disrupt the integrity of DNA and failed or improper DSBs repair may lead to genomic instability and, eventually, mutations, cancer, or cell death. Non-homologous end-joining (NHEJ) is the major pathway used by higher eukaryotic cells to repair these lesions. Given the complexity of NHEJ and the number of proteins and cofactors involved, secondary metabolites from medicinal or food plants might interfere with the process, activating or inhibiting repair. Twelve natural products, arbutin, curcumin, indole-3-carbinol, and nine flavonoids (apigenin, baicalein, chalcone, epicatechin, genistein, myricetin, naringenin, quercetin, sakuranetin) were chosen for their postulated roles in cancer chemoprevention and/or treatment. The effects of these compounds on NHEJ were investigated with an in vitro protocol based on plasmid substrates. Plasmids were linearized by a restriction enzyme, generating cohesive ends, or by a combination of enzymes, generating incompatible ends; plasmids were then incubated with a nuclear extract prepared from normal human small-intestinal cells (FHS 74 Int), either treated with these natural products or untreated (controls). The NHEJ repair complex from nuclear extracts ligates linearized plasmids, resulting in plasmid oligomers that can be separated and quantified by on-chip microelectrophoresis. Some compounds (chalcone, epicatechin, myricetin, sakuranetin and arbutin) clearly activated NHEJ, whereas others (apigenin, baicalein and curcumin) significantly reduced the repair rate of both types of plasmid substrates. Although this in vitro protocol is only partly representative of the in vivo situation, the natural products appear to interfere with NHEJ repair and warrant further investigation.

Measurement of translesion synthesis by fluorescent capillary electrophoresis: 7,8-dihydro-8-oxodeoxyguanosine bypass modulation by natural products.

Translesion synthesis (TLS) relies on a series of specialized DNA polymerases able to insert a base either correctly or incorrectly opposite a lesion on a DNA template strand during replication or post-repair synthesis. To measure the correct or mutagenic outcome of 7,8-dihydro-8-oxodeoxyguanosine (8-oxodG) bypass by TLS DNA polymerases, a capillary electrophoresis (CE) method with fluorescent label has been developed. Two oligonucleotides were designed and hybridized: (i) a 72-mer oligonucleotide framing one 8-oxodG at position 40 and (ii) the 39-mer oligonucleotide complementary to the first strand from the 3' end to the lesion and labeled at the 5' end with a fluorochrome. After incubation with FHs 74 Int human intestinal epithelial cell nuclear proteins, in the presence of either deoxyadenosine triphosphate (dATP) or deoxycytidine triphosphate (dCTP), and denaturation, the resulting elongated oligomers were analyzed by fluorescent capillary electrophoresis. This primer extension assay was then validated in terms of linearity (linear range=0.5-2.5 nM), detectability (limits of detection and quantification=0.023 and 0.091 nM, respectively), and precision (total precisions=8.1% and 3.7% for dATP and dCTP, respectively, n=9). The addition of some natural phytochemicals to the reaction mix significantly influences the outcome of TLS either in an error-free way or in a mutagenic way.