Seed coat metabolite profiling of cowpea (Vigna unguiculata L. Walp.) accessions from Ghana using UPLC-PDA-QTOF-MS and chemometrics

Abstract

Cowpea (Vigna unguiculata L. Walp.) is an important grain legume in Africa exhibiting high morpho-genetic diversity. However, not much information exists on the phytochemical profiles of its hulls. This study explored the metabolite profiles of seed-coats from thirteen cowpea accessions of varying phenotypes using UPLC-QTOF-MS and chemometric analysis. A total of 34 secondary metabolites were identified, which comprised phenolic acids, flavonoids, anthocyanins, sphingolipids and fatty acids. Quantification of selected phenolic compounds revealed marked variations among the cowpea accessions. The chemical profiles of the test accessions were distinguished by multivariate analysis, and the results revealed a marked influence of seed-coat pigmentation on the observed differences in their metabolite profiles. Moreover, delphinidin (traces to 2257.6 µg/g), catechin glucoside (traces to 2840.6 µg/g), catechin (traces to 2089.2 µg/g) and epicatechin (26.3 to 3222.7 µg/g) contributed to the segregation amongst the studied samples. The concentrations of the discriminant metabolites were greater in the dark seeded cowpeas compared to their lighter seeded counterparts. The findings represent a useful contribution to the literature on cowpea seed coat metabolites, and also reveal their potential for use in the development of food and pharmaceutical products.     

Effect of paclobutrazol on water stress amelioration through antioxidants and free radical scavenging enzymes in Arachis hypogaea L

An investigation was carried out to find out the extent of changes occurred in groundnut (Arachis hypogaea L.) cultivars in response to paclobutrazol (PBZ) treatment under water deficit stress. Two groundnut cultivars namely ICG 221 and ICG 476 were used for the study. Individual treatment with PBZ and drought stress showed an increase in ascorbic acid, -tocopherol and reduced glutathione, superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) activities. PBZ with drought stressed plants maintained higher levels of antioxidant and scavenging enzymes. Significant differences were observed between cultivars and treatments. These results suggests that the adverse effects of water stress can be minimized by the application of PBZ by increasing the antioxidant levels and activities of scavenging enzymes such as SOD, APX and CAT. The Cv. ICG 221 appears to be more tolerant to water stress than the ICG 476.     

Changes in antioxidant metabolism of Vigna unguiculata (L.) Walp. by propiconazole under water deficit stress

In the present study, a pot culture experiment was conducted to estimate the ameliorating effect of propiconazole (PCZ) on drought stress in cowpea (Vigna unguiculata (L.) Walp.) plants. From 30 days after sowing (DAS), the plants were subjected to 3, 6 and 9 days interval drought (DID) stress and drought stress with PCZ at 15 and 15 mg l(-1) PCZ alone and 1 day interval irrigation was kept as control. The plant samples were collected on 34 DAS (3 DID), 37 DAS (6 DID) and 40 DAS (9 DID). The plants were separated into root, stem and leaf for estimating the antioxidant contents and activities of antioxidant enzymes. Individual and combined drought stress and PCZ treatments increased ascorbic acid (AA), alpha-tocopherol (alpha-toc) contents, superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and polyphenol oxidase (PPO) activities when compared to control. The PCZ treatment mitigated the adverse effects of drought stress by increasing the antioxidant potentials and thereby paved the way for overcoming drought stress in V. unguiculata plants.     

Assessing genotypic variability of cowpea (Vigna unguiculata [L.] Walp.) to current and projected ultraviolet-B radiation

The current and projected terrestrial ultraviolet-B (UV-B) radiation affects growth and reproductive potential of many crops. Cowpea (Vigna unguiculata [L.] Walp.), mostly grown in tropical and sub-tropical regions may already be experiencing critical doses of UV-B radiation due to a thinner ozone column in those regions. Better understanding of genotypic variability to UV-B radiation is a prerequisite in developing genotypes tolerant to current and projected changes in UV-B radiation. An experiment was conducted in sunlit, controlled environment chambers to evaluate the sensitivity of cowpea genotypes to a range of UV-B radiation levels. Six cowpea genotypes [Prima, California Blackeye (CB)-5, CB-27, CB-46, Mississippi Pinkeye (MPE) and UCR-193], representing origin of different geographical locations, were grown at 30/22 degrees C day/night temperature from seeding to maturity. Four biologically effective ultraviolet-B radiation treatments of 0 (control), 5, 10, and 15 kJ m(-2)d(-1) were imposed from eight days after emergence to maturity. Significant genotypic variability was observed for UV-B responsiveness of eighteen plant attributes measured. The magnitude of the sensitivity to UV-B radiation also varied among cowpea genotypes. Plants from all genotypes grown in elevated UV-B radiation were significantly shorter in stem and flower lengths and exhibited lower seed yields compared to the plants grown under control conditions. Most of the vegetative parameters, in general, showed a positive response to UV-B, whereas the reproductive parameters exhibited a negative response showing the importance of reproductive characters in determining tolerance of cultivars to UV-B radiation. However, all cultivars, except MPE, behaved negatively to UV-B when a combined response index was derived across parameters and UV-B levels. Based on the combined total stress response index (C-TSRI) calculated as sum of individual vegetative, physiological and reproductive component responses over the UV-B treatments, the genotypes were classified as tolerant (MPE), intermediate (CB-5, CB-46 and UCR-193) and sensitive (CB-27 and Prima) to UV-B radiation. The differences in sensitivity among the cowpea genotypes emphasize the need for selecting or developing genotypes with tolerance to current and projected UV-B radiation.     

Protection of mitochondrial integrity from oxidative stress by selenium-containing glutathione transferase

The antioxidant activity of a novel artificial glutathione peroxidase-like enzyme, selenium-containing glutathione 5-transferase from Lucilia cuprina (seleno-LuGST1-1), was studied by using a ferrous sulfate/ascorbate-induced mitochondrial damage model system. Swelling of mitochondria, lipid peroxidation, and cytochrome-c oxidase activity were selected to evaluate the preservation of mitochondrial integrity in this system. Seleno-LuGST1-1 could effectively protect the mitochondria against oxidative damage in a dose-dependent manner and exhibited both higher catalytic activity and greater antioxidant ability than the classic mimic, 2-phenyl-1,2-benziososelenazol-3(2H)-one (Ebselen). This novel artificial biocatalyst therefore may have great protential for pharmacologic application in the treatment of reactive oxygen species-related diseases.     

Protein Fractionation of Cowpea (Vigna unguiculata (L.) Walp) Leaf, Flower and Seed by Capillary Electrophoresis and Its Potential for Variety Identification

The proteins of different faction of cowpea [Vigna unguiculata (L.) Walp] were fractionated by capillary electrophoresis (CE). The extracting solvent system was one of the most critical factors in the optimization exercise. To improve reproducibility, seed samples needed to be defatted with chloroform/methanol (V:V=2:1) as preferred prior to protein extraction. Proteins were extracted from seeds, leaves and flowers with 50% aqueous 1‐propanol and separated on a 50 (m×20 cm fused silica capillary column using a UV detector at 200 nm. Separation was conducted at constant voltage (10 kV, 40°C), using iminodiacetic acid (pH 2.5) containing 0.05% hydroxypropylmethylcellulose (HPMC) and 20% acetonitile. The results showed that proteins extracted from all fraction of cowpea were successfully separated by CE in less than 20 min. Seed extracts provided the greatest number of eluted protein peaks, followed by flower and leaf, respectively. The seed‐protein extracts provided unique CE patterns for different varieties; hence the seed was the tissue chosen as being most suitable for variety identification. As a result, an optimized procedure was developed to provide rapid identification of cowpea varieties, based on capillary electrophoregram patterns.     

Regulation of photosynthesis, fluorescence, stomatal conductance and water-use efficiency of cowpea (Vigna unguiculata [L.] Walp.) under drought

Drought is the major abiotic stress factor that causes extensive losses to agriculture production worldwide. The objective of this study was to evaluate the dynamics of photosynthesis and water-use efficiency parameters in 15 cowpea genotypes under well-watered and drought condition. Photosynthesis (A) and chlorophyll fluorescence (Fv'/Fm') declined linearly with decreasing soil water content whereas intrinsic water-use efficiency (WUE) increased under drought stress, suggesting stomatal regulation was a major limitation to photosynthesis. However, under increasing drought conditions, increase in ratio of intercellular CO(2) to ambient CO(2) concentrations along with reduced WUE showed the role of non-stomatal limitation of photosynthesis. The resistant nature of Fv'/Fm' and electron transport rate under drought appeared to be important mechanisms for photoinhibition protection under drought stress. Oxidative stress was apparent due to drought-induced reduction in total chlorophyll and carotenoid which was accompanied with increased leaf wax contents. The accumulation of proline appeared to be in response of drought injury rather than a drought tolerance mechanism. A clear separation based on the genotypes site of origin among the genotypes for drought tolerance could not be established when analyzed using principal component analysis. The identified genotypes and physiological traits from this study may be useful for genetic engineering and breeding programs integrating drought adaptation in cowpea.     

Protective role of exogenous nitric oxide against oxidative-stress induced by salt stress in barley (Hordeum vulgare)

To probe into the potential of relieving the oxidative damage of salt stress, we investigated the protective role of nitric oxide on barley under salt stress. Salt stress resulted in increased ion leakage, lipid peroxidation and protein oxidation in barley leaves. Simultaneous treatments of barley leaves with 50 microM sodium nitroprusside, a nitric oxide donor, alleviated the damage of salt stress, reflected by decreased ion leakage, and malendialdehyde (MDA), carbonyl, and hydrogen peroxide content in barley leaves. The presence of the nitric oxide donor increased the activities of superoxide dismutases (SOD), ascorbate peroxidases (APX), and catalases (CAT). Meantime, sodium nitroprusside addition increased accumulation of ferritin at the protein level, indicating that nitric oxide directly regulated ferritin accumulation. These results suggested that nitric oxide can effectively protect seedlings from salt stress damage by enhancing activities of antioxidant enzymes to quench the excessive reactive oxygen species caused by salt stress and inducing the increase of ferritin accumulation to chelate larger number of ferrous ion. Information from this study can be used to improve soil management practices for sustainable use of salt-affected soils in the future.     

Synthesis of keratine,silver, and flavonols nanocomposites to inhibit oxidative stress in pancreatic beta-cell (INS-1) and reduce intracellular reactive oxygen species production

Flavonols (FLA) from Vaccinium macrocarpon (V. macrocarpon) were identified using high-performance liquid chromatography coupled with mass spectrometry detection. Nanoparticles were prepared using highly crosslinked keratin (KER) from human hair and silver and entrapped with flavonols [KER + FLA + AgNPs]. Nanocomposites were characterized using UV–Vis spectroscopy, transmission electron microscopy (TEM), X-ray diffraction, zeta potential, and dynamic light scattering, and release profiles. The interactions between the capping agent and the silver core have been investigated using FTIR spectroscopy·H₂O₂ is a source of Reactive Oxygen Species (ROS) and acts as an activator of oxidative stress affecting NS-1 cells, and the protective effect of the nanocomposites were evaluated against H₂O₂-induced pancreatic β-cell damage. LC-MS/MS and HPLC analyses revealed the presence of 12 flavonols in V. macrocarpon plant extract. The cell apoptosis and proliferation, were evaluated by Hoechst 33342 staining, flow cytometry and Cell Counting Kit-8 respectively. Preincubation of the NS-1 cells with 250 µg/mL of H₂O₂ induced oxidative stress conditions that show pancreatic β-cell dysfunction, including ROS, cell death, mitochondrial function, antioxidant enzymes, and lipid peroxidation. Nevertheless, pretreatment with FLA and [KER + FLA + AgNPs] prevented mitochondria disruption, maintained cellular ATP levels, inhibited LDH release, intracellular ROS production, decreased lipid peroxidation, increased expression of antioxidant enzymes (CAT, SOD, and GPx) and GSH levels. These results indicate that nanocomposites could protect rat INS-1 pancreatic β-cell from H₂O₂-induced oxidative damage, apoptosis and proliferation by reducing the production of intracellular reactive oxygen species.     

Interactive Effects of Molybdenum,Zinc and Iron on the Grain Yield,Quality, and Nodulation of Cowpea (Vigna unguiculata (L.) Walp.) in North-Western India

Micronutrient deficiency is a major constraint for the growth, yield and nutritional quality of cowpea which results in nutritional disorders in humans. Micronutrients including molybdenum (Mo), iron (Fe) and zinc (Zn) play a pivotal role in crop nutrition, and their role in different metabolic processes in crops has been highlighted. In order to increase the nutritional quality of cowpea, a field experiment was conducted for two years in which the effect of Mo along with iron (Fe) and zinc (Zn) on productivity, nitrogen and micronutrient uptake, root length and the number of nodules in cowpea cultivation was investigated. It was found that the foliar application of Fe and Zn and their interaction with Mo application through seed priming as well as soil application displayed increased yield, nutrient concentration, uptake and growth parameters which helped to enhance the nutritional quality of cowpea for consumption by the human population. The results of the above experiments revealed that among all the treatments, the soil application of Mo combined with the foliar application of 0.5% each of FeSO₄·7H₂O and ZnSO₄·7H₂O (M₂F₃ treatment) enhanced the grain and stover yield of cowpea, exhibiting maximum values of 1402 and 6104.7 kg ha⁻¹, respectively. Again, the M₂F₃ treatment resulted in higher Zn, Fe and Mo concentrations in the grain (17.07, 109.3 and 30.26 mg kg⁻¹, respectively) and stover (17.99, 132.7 and 31.22 mg kg⁻¹, respectively) of cowpea. Uptake of Zn, Fe and Mo by the grain (25.23, 153.3 and 42.46 g ha⁻¹, respectively) as well as the stover (104.2, 809.9 and 190.6 g ha⁻¹, respectively) was found to be maximum for the M₂F₃ treatment. The root length (30.5 cm), number of nodules per plant (73.0) and N uptake in grain and stover (55.39 and 46.15 kg ha⁻¹) were also higher for this treatment. Overall, soil application of Mo along with the foliar application of FeSO₄·7H₂O (0.5%) and ZnSO₄·7H₂O (0.5%) significantly improved yield outcomes, concentration, uptake, root length, nodules plant⁻¹ and N uptake of cowpea to alleviate the micronutrient deficiency.     

Soil applied cobalt alters the nodulation, leg-haemoglobin content and antioxidant status of Glycine max (L.) Merr

The present investigation has been carried out to estimate the effect of cobalt on the nodulation leg-haemoglobin content and antioxidant enzyme activities of soybean plants. The experiments were conducted in earthen pots containing 3 kg of air dried soil. The inner surface of pots was lined with polythene sheet. Soybean plants were raised in soil amended with different concentration of cobalt (0, 50, 100, 150, 200 and 250 mg/kg). The root nodule formation, leg-haemoglobin contents and antioxidant enzyme activities were analysed at 15 days intervals, namely 15, 30, 45, 60 and 75 days after sowing (DAS). There found a significant increase in the total number of root nodules, leg-haemoglobin content and antioxidant enzyme activities at 50 mg/kg cobalt application in the soil in all the sampling days when compared to control. Further increase in the cobalt level (100–250 mg/kg) in the soil decreased root nodules formation as well as leg-haemoglobin content antioxidant enzyme activities of soybean plants in all the sampling days.     

Extraction of Phenolic and Flavonoid Compounds from Mung Bean (Vigna radiata L.) Seed Coat by Pressurized Liquid Extraction

Mung bean seed coat (MBC) is a by-product of the mung bean processing industry. It contains a large number of phenolic compounds with therapeutic anti-inflammatory, anti-diabetic and antioxidant properties. This research aimed to investigate the optimum conditions for phenolic and flavonoid extraction from MBC by pressurized liquid extraction (PLE). Response surface methodology (RSM) was used to study the effects of temperature (80–160 °C), pressure (1200–1800 psi) and ethanol concentration (5–95%) on total phenolic content (TPC), total flavonoid content (TFC) and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) scavenging activity (ABTS). Scale-up extraction was also performed. The optimum conditions for extraction were 160 °C, 1300 psi and 50% ethanol. Under optimum conditions, the TPC was 55.27 ± 1.14 mg gallic acid equivalent (GAE)/g MBC, TFC was 34.04 ± 0.72 mg catechin equivalent (CE)/g MBC and ABTS scavenging activity was 195.05 ± 2.29 mg trolox equivalent (TE)/g MBC. The TFC and ABTS scavenging activity of the extracts obtained at the pilot scale (10 L) was not significantly different from the laboratory scale, while TPC was significantly increased. The freeze-dried MBC extract contained vitexin and isovitexin 130.53 ± 17.89, 21.21 ± 3.22 mg/g extract, respectively. In conclusion, PLE was able to extract phenolics, flavonoids with ABTS scavenging activity from MBC with the prospect for future scale-up for food industry.     

Anti-Influenza a virus of a new oligosaccharide citric acid derivative isolated from Vigna angularis (ohwi et ohashi. var. Dainagon) Seeds

A new oligosaccharide citric acid derivative was isolated by hot aqueous extraction at 85?°C from Vigna angularis seeds, and it was purified using anion exchange chromatography DE-52. The structure was inferred by using UV, 1?D and 2?D NMR spectroscopy and HR-MALDI/MS, and identified as α-D-galactopyranosyl-(1→6)-α-D-galactopyranosyl-(1→6)-α-D-glucopyranosyle-(1→2)-β-D-fructofuranosyle citric acid. The isolated oligosaccharide citric acid derivative revealed promising anti-influenza A virus with 50% inhibition concentration (IC₅₀) of 0.162?nM/mL and 50% cytotoxic dose (CC₅₀) of >2.38?nM/mL.     

Physical, proximate, functional and pasting properties of flour produced from gamma irradiated cowpea (Vigna unguiculata, L. Walp)

Cowpeas are leguminous seeds widely produced and consumed in most developing countries of sub Saharan Africa. The aim of this study was to determine the physical, proximate, functional and pasting properties of flour obtained from gamma irradiated cowpea. Four cowpea cultivars were irradiated with gamma radiation at dose levels of 0.25, 0.5, 0.75, 1.0 and 1.5 kGy with the unirradiated cultivars serving as controls. The samples were hammer milled, sieved and stored at 4 °C for analysis. Physical, proximate, functional, pasting properties were determined using appropriate methods. In general, the irradiation dose applied to cowpea for insect control did not significantly affect the physical and proximate properties of the flour. However, significant increase (p<0.05) was achieved in paste bulk density, water and oil absorption capacities, foam capacities and least gelation concentrations of flour in general, which may be attributed to the irradiation. The radiation reduced the swelling power and water solubility index significantly. The peak temperature, peak viscosity and setback viscosity of the pastes were significantly (p<0.05) reduced while breakdown viscosity was significantly (p<0.05) increased by the radiation. It was established that the doses used on cowpea affected both the functional and pasting properties of the flour.     

The phenolic contents,antioxidant and anticholinesterase activity of section Amaracus (Gled.) Vogel and Anatolicon Ietsw. of Origanum L. species

Origanum boissieri Ietsw., O. saccatum P.H.Davis, O. solymicum P.H.Davis and O. ayliniae Dirmenci & T.Yazıcı belonging to sect. Amaracus (Gled.) Vogel, O. sipyleum L. and O. hypericifolium O.Schwarz & P.H.Davis belonging to sect. Anatolicon Ietsw. were analyzed for their chemical composition of essential oil and phenolic components. The essential oil compositions were analysed by using GC-MS and GC-FID. The phenolic contents of the chloroform, acetone, and methanol extracts were analyzed using LC-MS/MS. Antioxidant activities of the extracts were investigated by using three methods; DPPH free radical scavenging activity, β-carotene linoleic acid assays and CUPRAC assays. The essential oil compositions of the section Amaracus were found to be as carvacrol type (O. ayliniae, O. boissieri) and p-cymene type (O. saccatum, O. solymicum). In the section of Anatolicon, while O. sipyleum was found as γ-terpinene type, O. hypericifolium was carvacrol type. In the extracts, the most abundant components were determined as flavonoids, coumaric acids and derivatives. Especially rosmarinic acid and penduletin were detected in high amounts. Among the studied species, extracts of O. ayliniae showed quite good activity for all methods. The extracts from all species showed remarkable antioxidant activity. Inhibition capability of the extracts against acetyl and butyrylcholinesterase enzymes (AChE and BChE) were determined. The extracts were found as inactive against AChE. The moderate inhibition capacity observed against BChE.     

Terpenoids of Linaria alpina (L.) Mill. from Dolomites,Italy

This paper reports the first phytochemical analysis of Linaria alpina (L.) Mill., collected in Dolomites (Italy), a species characteristic of mountain environment. Besides aucubin (4), which is rare in the subgenus Antirrhineae of Plantaginaceae, mainly acidic compounds were found, i.e. oleanolic acid (1), ursolic acid (2) maslinic acid (3) and shikimic acid (5). The pentacyclic triterpenes of L. alpina resulted in relatively high content, whereas flavonoids resulted in low content.