Chromosome-scale assembly of the Moringa oleifera Lam. genome uncovers polyploid history and evolution of secondary metabolism pathways through tandem duplication.

The African Orphan Crops Consortium (AOCC) selected the highly nutritious, fast growing and drought tolerant tree crop moringa (Moringa oleifera Lam.) as one of the first of 101 plant species to have its genome sequenced and a first draft assembly was published in 2019. Given the extensive uses and culture of moringa, often referred to as the multipurpose tree, we generated a significantly improved new version of the genome based on long-read sequencing into 14 pseudochromosomes equivalent to n = 14 haploid chromosomes. We leveraged this nearly complete version of the moringa genome to investigate main drivers of gene family and genome evolution that may be at the origin of relevant biological innovations including agronomical favorable traits. Our results reveal that moringa has not undergone any additional whole-genome duplication (WGD) or polyploidy event beyond the gamma WGD shared by all core eudicots. Moringa duplicates retained following that ancient gamma events are also enriched for functions commonly considered as dosage balance sensitive. Furthermore, tandem duplications seem to have played a prominent role in the evolution of specific secondary metabolism pathways including those involved in the biosynthesis of bioactive glucosinolate, flavonoid, and alkaloid compounds as well as of defense response pathways and might, at least partially, explain the outstanding phenotypic plasticity attributed to this species. This study provides a genetic roadmap to guide future breeding programs in moringa, especially those aimed at improving secondary metabolism related traits.

The impact of Moringa oleifera leaf supplementation on human and animal nutrition, growth, and milk production: A systematic review.

Childhood undernutrition contributes to up to 45% of deaths in children under age 5. Moringa oleifera (moringa) leaves are nutrient dense and promote breastmilk production. We performed a systematic review assessing the impact of moringa leaf supplementation in humans and animals on the outcomes of iron, vitamin A status, the measures of growth, and/or breastmilk production. Our inclusion/exclusion criteria were as follows; inclusion: quantitative primary data assessing the effect of moringa leaf supplementation on humans or animals including any of the outcomes defined earlier with no exclusion for geography, age, or language. Exclusion: full text not available. Our search yielded 148 unique studies; among them, 33 were included (seven human studies and 26 animal studies). Quality assessment by Effective Public Health Practice Project guidelines was strong for one study and moderate for all other studies. In humans, moringa at higher (14-30 g/day) not lower (<10 g/day) doses improved hemoglobin (Hgb) in children with iron deficiency anemia, improved Hgb and vitamin A status in postmenopausal women, and increased BMI in HIV+ underweight adults. Moringa (0.5 g/day) also increased breastmilk volumes. In animals, moringa increased milk production in two of three studies, inconsistently affected growth, and had no effect on iron status. Evidence on moringa supplementation's utility is limited but promising. Larger, more rigorous trials are needed to characterize its impact.

Investigating the Impact of Moringa oleifera Supplemented to Kenyan Breastfeeding Mothers on Maternal and Infant Health: A Cluster Randomized Single-Blinded Controlled Pilot Trial Protocol.

Undernutrition contributes to up to 45% of deaths globally in children <5 years, with an optimal time for intervention before 24 months of age. Breastmilk microbiome helps establish the infant intestinal microbiome and impacts infant intestinal and nutritional health. Inadequacies in breastmilk composition such as low vitamin A contribute to infant nutrient deficiencies. Changes in milk fatty acid composition (reduced saturated and increased unsaturated fatty acids) may reduce susceptibility to enteric infection and increase protective intestinal bacteria. Moringa oleifera leaves (moringa) provide high nutrient concentrations (including protein, iron, vitamin A) and increase milk production; this may enhance breastmilk quantity and quality and improve infant health. Objective: To investigate the role of moringa supplementation to improve maternal and infant nutritional and intestinal health via changes in maternal milk quantity and quality. Methods: Fifty mother-infant pairs exclusively breastfeeding will be enrolled in a single-blinded randomized controlled trial in Kombewa County Hospital and Chulaimbo SubCounty Hospital, Kisumu, Kenya. Intervention: Dietary Supplementation of 20 g of Moringa oleifera leaf powder divided twice daily in corn porridge consumed daily for 3 months while control comparator will receive porridge daily for 3 months. Outcomes: Change in infant growth and maternal milk output (primary); maternal and infant vitamin A and iron status, changes in infant and maternal intestinal health (secondary). Participating Centers: Pamoja Community Based Organization, Kombewa Sub-County Hospital, and Chulaimbo Sub-County Hospital.

Moringa Isothiocyanate-rich Seed Extract Delays the Onset of Diabetes in UC Davis Type-2 Diabetes Mellitus Rats.

Moringa seeds have been used traditionally in the management of type 2 diabetes mellitus (T2DM) and contain potent bioactive isothiocyanates. This study evaluated the efficacy of an isothiocyanate-rich moringa seed extract in delaying the onset of T2DM in UC Davis T2DM rats, a well validated model which closely mimics T2DM in humans. Rats were separated into three groups; control, moringa seed extract at 0.4%, and a weight matched group. Rats were fed respective diets for 8 months, during which energy intake, body weight, the onset of diabetes circulating hormones, metabolites and markers of inflammation and liver function, and were monitored. The MS group had a significantly slower rate of diabetes onset p = 0.027), lower plasma glucose (p = 0.043), and lower HbA1c (p = 0.008) compared with CON animals. There were no significant differences in food intake and body weight between all groups. This study demonstrated MS can delay the onset of diabetes in the UC Davis T2DM rat model to a greater extent than moderate caloric restriction (by comparison to the WM group). The results support its documented traditional uses and a bioactive role of moringa isothiocyanates and suggest the potential efficacy for moringa supplementation for diabetes management in populations at risk for T2DM.

Isothiocyanate-enriched moringa seed extract alleviates ulcerative colitis symptoms in mice.

Moringa (Moringa oleifera Lam.) seed extract (MSE) has anti-inflammatory and antioxidant activities. We investigated the effects of MSE enriched in moringa isothiocyanate-1 (MIC-1), its putative bioactive, on ulcerative colitis (UC) and its anti-inflammatory/antioxidant mechanism likely mediated through Nrf2-signaling pathway. Dextran sulfate sodium (DSS)-induced acute (n = 8/group; 3% DSS for 5 d) and chronic (n = 6/group; cyclic rotations of 2.5% DSS/water for 30 d) UC was induced in mice that were assigned to 4 experimental groups: healthy control (water/vehicle), disease control (DSS/vehicle), MSE treatment (DSS/MSE), or 5-aminosalicyic acid (5-ASA) treatment (positive control; DSS/5-ASA). Following UC induction, water (vehicle), 150 mg/kg MSE, or 50 mg/kg 5-ASA were orally administered for 1 or 2 wks. Disease activity index (DAI), spleen/colon sizes, and colonic histopathology were measured. From colon and/or fecal samples, pro-inflammatory biomarkers, tight-junction proteins, and Nrf2-mediated enzymes were analyzed at protein and/or gene expression levels. Compared to disease control, MSE decreased DAI scores, and showed an increase in colon lengths and decrease in colon weight/length ratios in both UC models. MSE also reduced colonic inflammation/damage and histopathological scores (modestly) in acute UC. MSE decreased colonic secretions of pro-inflammatory keratinocyte-derived cytokine (KC), tumor necrosis factor (TNF)-α, nitric oxide (NO), and myeloperoxidase (MPO) in acute and chronic UC; reduced fecal lipocalin-2 in acute UC; downregulated gene expression of pro-inflammatory interleukin (IL)-1, IL-6, TNF-α, and inducible nitric oxide synthase (iNOS) in acute UC; upregulated expression of claudin-1 and ZO-1 in acute and chronic UC; and upregulated GSTP1, an Nrf2-mediated phase II detoxifying enzyme, in chronic UC. MSE was effective in mitigating UC symptoms and reducing UC-induced colonic pathologies, likely by suppressing pro-inflammatory biomarkers and increasing tight-junction proteins. This effect is consistent with Nrf2-mediated anti-inflammatory/antioxidant signaling pathway documented for other isothiocyanates similar to MIC-1. Therefore, MSE, enriched with MIC-1, may be useful in prevention and treatment of UC.

Miniaturized Cultivation of Microbiota for Antimalarial Drug Discovery.

The ongoing search for effective antiplasmodial agents remains essential in the fight against malaria worldwide. Emerging parasitic drug resistance places an urgent need to explore chemotherapies with novel structures and mechanisms of action. Natural products have historically provided effective antimalarial drug scaffolds. In an effort to search nature's chemical potential for antiplasmodial agents, unconventionally sourced organisms coupled with innovative cultivation techniques were utilized. Approximately 60,000 niche microbes from various habitats (slow-growing terrestrial fungi, Antarctic microbes, and mangrove endophytes) were cultivated on a small-scale, extracted, and used in high-throughput screening to determine antimalarial activity. About 1% of crude extracts were considered active and 6% partially active (≥ 67% inhibition at 5 and 50 µg/mL, respectively). Active extracts (685) were cultivated on a large-scale, fractionated, and screened for both antimalarial activity and cytotoxicity. High interest fractions (397) with an IC50 < 1.11 µg/mL were identified and subjected to chromatographic separation for compound characterization and dereplication. Identifying active compounds with nanomolar antimalarial activity coupled with a selectivity index tenfold higher was accomplished with two of the 52 compounds isolated. This microscale, high-throughput screening project for antiplasmodial agents is discussed in the context of current natural product drug discovery efforts.

Isothiocyanate-rich Moringa oleifera extract reduces weight gain, insulin resistance, and hepatic gluconeogenesis in mice.

SCOPE: Moringa oleifera (moringa) is tropical plant traditionally used as an antidiabetic food. It produces structurally unique and chemically stable moringa isothiocyanates (MICs) that were evaluated for their therapeutic use in vivo. METHODS AND RESULTS: C57BL/6L mice fed very high fat diet (VHFD) supplemented with 5% moringa concentrate (MC, delivering 66 mg/kg/d of MICs) accumulated fat mass, had improved glucose tolerance and insulin signaling, and did not develop fatty liver disease compared to VHFD-fed mice. MC-fed group also had reduced plasma insulin, leptin, resistin, cholesterol, IL-1ß, TNFα, and lower hepatic glucose-6-phosphatase (G6P) expression. In hepatoma cells, MC and MICs at low micromolar concentrations inhibited gluconeogenesis and G6P expression. MICs and MC effects on lipolysis in vitro and on thermogenic and lipolytic genes in adipose tissue in vivo argued these are not likely primary targets for the anti-obesity and anti-diabetic effects observed. CONCLUSION: Data suggest that MICs are the main anti-obesity and anti-diabetic bioactives of MC, and that they exert their effects by inhibiting rate-limiting steps in liver gluconeogenesis resulting in direct or indirect increase in insulin signaling and sensitivity. These conclusions suggest that MC may be an effective dietary food for the prevention and treatment of obesity and type 2 diabetes.

Direct and indirect antioxidant activity of polyphenol- and isothiocyanate-enriched fractions from Moringa oleifera.

Moringa oleifera Lam. is a fast-growing, tropical tree with various edible parts used as nutritious food and traditional medicine. This study describes an efficient preparatory strategy to extract and fractionate moringa leaves by fast centrifugal partition chromatography (FCPC) to produce polyphenol and isothiocyanate (ITC) rich fractions. Characterization and further purification of these fractions showed that moringa polyphenols were potent direct antioxidants assayed by oxygen radical absorbance capacity (ORAC), whereas moringa ITCs were effective indirect antioxidants assayed by induction of NAD(P)H quinone oxidoreductase 1 (NQO1) activity in Hepa1c1c7 cells. In addition, purified 4-[(α-l-rhamnosyloxy)benzyl]isothiocyanate and 4-[(4'-O-acetyl-α-l-rhamnosyloxy)benzyl]isothiocyanate were further evaluated for their ORAC and NQO1 inducer potency in comparison with sulforaphane (SF). Both ITCs were as potent as SF in inducing NQO1 activity. These findings suggest that moringa leaves contain a potent mixture of direct and indirect antioxidants that can explain its various health-promoting effects.

Polyphenol-rich Rutgers Scarlet Lettuce improves glucose metabolism and liver lipid accumulation in diet-induced obese C57BL/6 mice.

OBJECTIVE: The aims of the following experiments were to characterize antidiabetic in vitro and in vivo activity of the polyphenol-rich aqueous extract of Rutgers Scarlet Lettuce (RSL). METHODS: RSL extract (RSLE) and isolated compounds were evaluated for inhibitory effects on glucose production as well as tumor necrosis factor alpha-dependent inhibition of insulin activity in H4IIE rat hepatoma cells. Additionally, high-fat diet-induced obese mice were treated with RSLE (100 or 300 mg/kg), metformin (250 mg/kg), or vehicle (water) for 28 d by oral administration and insulin and oral glucose tolerance tests were conducted. Tissues were harvested at the end of the study and evaluated for biochemical and physiological improvements in metabolic syndrome conditions. RESULTS: A polyphenol-rich RSLE, containing chlorogenic acid, cyanidin malonyl-glucoside, and quercetin malonyl-glucoside, was produced by simple boiling water extraction at pH 2.0. In vitro, RSLE and chlorogenic acid demonstrated dose-dependent inhibition of glucose production. In vivo, RSLE treatment improved glucose metabolism measured by oral glucose tolerance tests, but not insulin tolerance tests. RSLE treated groups had a lower ratio of liver weight to body weight as well as decreased total liver lipids compared with the control group after 28 d of treatment. No significant differences in plasma glucose, insulin, cholesterol, and triglycerides were observed with RSLE-treated groups compared with vehicle control. CONCLUSION: RSLE demonstrated antidiabetic effects in vitro and in vivo and may improve metabolic syndrome conditions of fatty liver and glucose metabolism.

Stable, water extractable isothiocyanates from Moringa oleifera leaves attenuate inflammation in vitro.

Moringa (Moringa oleifera Lam.) is an edible plant used as both a food and medicine throughout the tropics. A moringa concentrate (MC), made by extracting fresh leaves with water, utilized naturally occurring myrosinase to convert four moringa glucosinolates into moringa isothiocyanates. Optimum conditions maximizing MC yield, 4-[(α-L-rhamnosyloxy)benzyl]isothiocyanate, and 4-[(4'-O-acetyl-α-L-rhamnosyloxy)benzyl]isothiocyanate content were established (1:5 fresh leaf weight to water ratio at room temperature). The optimized MC contained 1.66% isothiocyanates and 3.82% total polyphenols. 4-[(4'-O-acetyl-α-L-rhamnosyloxy)benzyl]isothiocyanate exhibited 80% stability at 37°C for 30 days. MC, and both of the isothiocyanates described above significantly decreased gene expression and production of inflammatory markers in RAW macrophages. Specifically, both attenuated expression of iNOS and IL-1ß and production of nitric oxide and TNFα at 1 and 5 µM. These results suggest a potential for stable and concentrated moringa isothiocyanates, delivered in MC as a food-grade product, to alleviate low-grade inflammation associated with chronic diseases.

Screening mangrove endophytic fungi for antimalarial natural products.

We conducted a screening campaign to investigate fungi as a source for new antimalarial compounds. A subset of our fungal collection comprising Chinese mangrove endophytes provided over 5000 lipophilic extracts. We developed an accelerated discovery program based on small-scale cultivation for crude extract screening and a high-throughput malaria assay. Criteria for hits were developed and high priority hits were subjected to scale-up cultivation. Extracts from large scale cultivation were fractionated and these fractions subjected to both in vitro malaria and cytotoxicity screening. Criteria for advancing fractions to purification were developed, including the introduction of a selectivity index and by dereplication of known metabolites. From the Chinese mangrove endophytes, four new compounds (14-16, 18) were isolated including a new dimeric tetrahydroxanthone, dicerandrol D (14), which was found to display the most favorable bioactivity profile.

Anthelmintic screening of Sub-Saharan African plants used in traditional medicine.

AIM OF STUDY: This study screened for anthelmintic activity of plant species traditionally used in the treatment of intestinal parasites and their symptoms in Sub-Saharan Africa in an effort to confirm their local use and aid in the search for new compounds since resistance is a growing concern. MATERIALS AND METHODS: Aqueous and organic extracts of 33 plant parts from 17 plant species traditionally used in the treatment of intestinal infections in Sub-Saharan Africa were evaluated for their anthelmintic activity. This activity was assessed using a standard motility assay against a levamisole resistant strain of the nematode Caenorhabditis elegans. RESULTS AND CONCLUSIONS: Anthelmintic activity was confirmed in 12 plant species. Of these, eight showed strong evidence of activity (p<0.0001), one exhibited moderate evidence of activity (p<0.001), three demonstrated weak evidence of activity (p<0.05), and five plants showed no evidence of activity. The eight species with the strongest evidence of activity were Acacia polyacantha, Anogeissus leiocarpus, Bridelia micrantha, Cassia sieberiana, Combretum nigricans, Grewia bicolor, Strychnos spinosa and Ziziphus mucronata. In only two cases, Anogeissus leiocarpus and Cassia sieberiana, anthelmintic activity has been previously confirmed. The activity demonstrated against the levamisole resistant strain of Caenorhabditis elegans and the presence of molecules in these plants known or suspected of having a broad spectrum of activity provide support for further study of these plants and their compounds as possible treatments for parasitic worm infections.

Comparison of motility, recovery, and methyl-thiazolyl-tetrazolium reduction assays for use in screening plant products for anthelmintic activity.

The primary objective of these experiments was to compare the effectiveness of motility, recovery, and methyl-thiazolyl-tetrazolium (MTT) reduction assays for determining anthelmintic activity of plant extracts and purified compounds from these extracts. Caenorhabditis elegans was used as the test organism. High-performance liquid chromatography (HPLC) grade water and M9 medium were used as the solvents. Copper, a common metal pollutant, and the anthelmintic drug levamisole were used as reference compounds. Extracts from the West African plant Anogeissus leiocarpus, which is used to treat worm infections, as well as two active compounds found in this plant, gallic and gentisic acids, were included in this comparison. MTT assay results for viability of worms were significantly lower (p < 0.01) than motility and recovery assay results. However, both gallic acid and the plant extract, in the absence of worms, caused reduction of MTT. Worm survival for levamisole using M9 medium was significantly (p < 0.01) higher than for HPLC grade water for all three methods. On the other hand, gallic acid showed significant (p < 0.05) activity in M9 medium but no activity in HPLC grade water, whereas gentisic acid was effective in HPLC grade water but had no activity in M9 medium. Activity of the A. leiocarpus extract also varied with solvent. In conclusion, plant extracts can be screened using motility assays that include both HPLC grade water and M9 salts.