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Purification of the immunoreactive substance in the peanut extract. (a) The immunoreactive substance was purified using a silica gel column chromatography (Ethyl acetate-Hexane 1:9 v/v), and analyzed using TLC. The samples shown in the figure are: (1) olive TAGs (control), (2) mixture of 1 and 3, and (3) the purified immunoreactive substance in the peanut extract. For the purified peanut extract, a major single spot appeared on the TLC, whose migration matched that of the olive (control) TAGs. (b) The immunoreactive activity (i.e., melanin formation) was recovered from the TLC spot. Each of the ten sections of silica layer as shown in the bottom panel was curved and re-extracted to examine the immunoreactivity. As shown in the chart (top panel), the immunoreactivity co-migrated with the spot. The y-axis of the top panel represents the OD value (492 nm) of silkworm hemolymph was measured 3 h after sample injection. The mean value and the standard deviation obtained from duplicate measurements are shown in the chart. (c) Fatty acid compositions were determined as described in “Materials and Methods”. Each combination of three fatty acid moieties were color-coded as demonstrated in the legend. For example, ‘TG 18:1_18:1_18:1’ represents a TAG with three oleic acid moieties, and ‘TG 16:0_18:1_18:1’ represents a TAG with a palmitic acid and two oleic acid moieties.
Source publication
In this study, we investigated immunoreactivity of peanut (Arachis hypogaea) oil using the silkworm (Bombyx mori) model. The peanut oil induced melanin formation when injected to the silkworm hemocoel. We then purified the active substance and identified the triacylglycerols (TAGs) as the responsible molecule for the melanin-forming effect of peanu...
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Peanut is a well-known lipid and protein source for nutrition and its oil is considered to be nutritious and healthy, owing to its fatty acid composition and the presence of natural bioactive components. Olive leafs are the major by-products of oil processing and its industry. The phenolic content of peanut oil was 78.15 and varied between 271.8 an...
Citations
... In insects, including silkworms, the immune response via hemolymph melanization and the Toll pathway is mediated by the same signaling pathway (17,19). Therefore, the melanization response in silkworms is an indicator of innate immune induction (20,21). Melanization in silkworm hemolymph by pathogens such as Porphyromonas gingivalis, Cutibacterium acnes, and Candida albicans is useful as an indicator of innate immunity (22)(23)(24). ...
Staphylococcus aureus, a Gram-positive bacterium, causes inflammatory skin diseases, such as atopic dermatitis, and serious systemic diseases, such as sepsis. In the skin and nasal environment, peptidoglycan (PGN)-degrading enzymes, including lysozyme and lysostaphin, affects S. aureus PGN. However, the effects of PGN-degrading enzymes on the acute innate immune-inducing activity of S. aureus have not yet been investigated. In this study, we demonstrated that PGN-degrading enzymes induce acute silkworm hemolymph melanization by S. aureus. Insoluble fractions of S. aureus treated with lysozyme, lysostaphin, or both enzymes, were prepared. Melanization of the silkworm hemolymph caused by the injection of these insoluble fractions was higher than that of S. aureus without enzyme treatment. These results suggest that structural changes in S. aureus PGN caused by PGN-degrading enzymes affect the acute innate immune response in silkworms.
... Glycerolipid is a class of lipids that exists in peanuts, which mainly includes triacylglycerol (TAG) and Diacylglycerol (DAG). TAGs were major storage lipids in seeds, which comprised glycerol and three fatty acid moieties [26]. TAGs served as important fatty acid reserves for carbohydrate and energy production during oilseed development [27]. ...
The effect of nitrogen-modified atmosphere storage (NS) on peanut lipid oxidation was investigated in this paper. Non-targeted lipidomics was employed to detect the lipid metabolites in peanuts with the aim of exploring the mechanism of lipid oxidation in peanuts under different storage conditions. The results showed that compared with conventional storage (CS), NS significantly (p < 0.05) delayed the increase in acid value, carbonyl value, and 2-thiobarbituric acid value and the decrease in vitamin E content. However, the storage time has a much greater effect on lipid oxidation than the oxygen level in the storage environment. Lipidomics analysis revealed that there were significant differences in metabolite changes between CS and NS. NS reduced the decline of most glycerophospholipids by regulating lipid metabolism in peanuts. NS maintained higher levels of Diacylglycerol (DAG), sulfoquinovosyl diacylglycerol (SQDG), lysophophatidylcholine (LPC), lysophosphatidylethanolamine (LPE) and phosphatidylinositol (PI) compared to CS. This work provided a basis for the application of NS technology to peanut storage.
... Immunomodulatory activity was measured using a silkwormbased assay as previously reported (Urai et al., 2017;Li et al., 2022). In this assay, we measure silkworm muscle contraction as an index of BmPP (insect cytokine) level. ...
There is widespread interest in the immunostimulatory effects of food-derived substances that are expected to contribute to improving human health. Broccoli ( Brassica oleracea var. italica ) has been shown to contain potent polysaccharides for developing food materials with immunomodulatory function. In this study, immunostimulatory activity of previously uncharacterized broccoli polysaccharides was assessed using a silkworm ( Bombyx mori ) immunological assay (using muscle contraction as an index) across 10 commercial and 17 experimental cultivars of broccoli. For the commercial cultivars, there was a significant variation in specific activity (units/mg, measured by the silkworm-based immunological assay). For the experimental cultivars, a cultivar “winter dome” showed the highest activity. We further purified the immunostimulatory polysaccharides of “winter dome” by gel filtration. Using the silkworm-based assay, we found that the peak fraction (“Fraction II”) contained the active substance with higher specific activity than previously reported active substances (e.g., glucans). The substance appeared a heterogeneous molecular weight of more than 270 kDa. The major sugar composition of Fraction II was glucose, suggesting that glucose homopolymer is responsible for the activity. Further, Fraction II induced cytokine productions both in the silkworm (production of active B. mori paralytic peptide (BmPP) in vivo ) and mouse (production of TNFα in vitro ) immune system. These results suggest that glucose homopolymer of broccoli induces cytokines both in insect and mammalian immune systems, providing molecular insights to our understanding about how plant-derived substances interact with animal immune system.
... Long-term mammalian infection experiments using a large number of individuals, however, are problematic from the viewpoint of animal ethics [16]. Silkworms are useful laboratory animals for evaluating the pathogenic mechanisms of microorganisms that cause systemic infections and for assessing the activation of innate immunity [16][17][18][19]. Silkworms have several advantages in experiments using large numbers of individuals, and fewer ethical issues are associated with their use [20]. ...
... The phenoloxidases (PPO1 and PPO2)-deficient flies are sensitive to Staphylococcus aureus infection [28]. Therefore, hemolymph melanization in insects can be used as an indicator of innate immune system activation [17,19,29]. The induction of innate immunity by Porphyromonas gingivalis and Candida albicans was evaluated by silkworm hemolymph melanization [24,30]. ...
Cutibacterium acnes is a pathogenic bacterium that cause inflammatory diseases of the skin and intervertebral discs. The immune activation induced by C . acnes requires multiple cellular responses in the host. Silkworm, an invertebrate, generates melanin by phenoloxidase upon recognizing bacterial or fungal components. Therefore, the melanization reaction can be used as an indicator of innate immune activation. A silkworm infection model was developed for evaluating the virulence of C . acnes , but a system for evaluating the induction of innate immunity by C . acnes using melanization as an indicator has not yet been established. Here we demonstrated that C . acnes rapidly causes melanization of the silkworm hemolymph. On the other hand, Staphylococcus aureus , a gram-positive bacterium identical to C . acnes , does not cause immediate melanization. Even injection of heat-killed C . acnes cells caused melanization of the silkworm hemolymph. DNase, RNase, and protease treatment of the heat-treated C . acnes cells did not decrease the silkworm hemolymph melanization. Treatment with peptidoglycan-degrading enzymes, such as lysostaphin and lysozyme, however, decreased the induction of melanization by the heat-treated C . acnes cells. These findings suggest that silkworm hemolymph melanization may be a useful indicator to evaluate innate immune activation by C . acnes and that C . acnes peptidoglycans are involved in the induction of innate immunity in silkworms.
The relationship between insect pathogenic fungi and their insect hosts is a classic example of a co-evolutionary arms race between pathogen and target host: parasites evolve towards mechanisms that increase their advantage over the host, and the host increasingly strengthens its defenses. The present review summarizes the literature data describing the direct and indirect role of lipids as an important defense mechanism during fungal infection. Insect defense mechanisms comprise anatomical and physiological barriers, and cellular and humoral response mechanisms. The entomopathogenic fungi have the unique ability to digest the insect cuticle by producing hydrolytic enzymes with chitin-, lipo- and proteolytic activity; besides the oral tract, cuticle pays the way for fungal entry within the host. The key factor in insect resistance to fungal infection is the presence of certain types of lipids (free fatty acids, waxes or hydrocarbons) which can promote or inhibit fungal attachment to cuticle, and might also have antifungal activity. Lipids are considered as an important source of energy, and as triglycerides are stored in the fat body, a structure analogous to the liver and adipose tissue in vertebrates. In addition, the fat body plays a key role in innate humoral immunity by producing a range of bactericidal proteins and polypeptides, one of which is lysozyme. Energy derived from lipid metabolism is used by hemocytes to migrate to the site of fungal infection, and for phagocytosis, nodulation and encapsulation. One polyunsaturated fatty acid, arachidonic acid, is used in the synthesis of eicosanoids, which play several crucial roles in insect physiology and immunology. Apolipoprotein III is important compound with antifungal activity, which can modulate insect cellular response and is considered as important signal molecule.
