Polysaccharides from Basidiocarps of Cultivating Mushroom Pleurotus ostreatus: Isolation and Structural Characterization.

Oyster mushrooms are an interesting source of biologically active glucans and other polysaccharides. This work is devoted to the isolation and structural characterization of polysaccharides from basidiocarps of the cultivated oyster mushroom, Pleurotus ostreatus. Five polysaccharidic fractions were obtained by subsequent extraction with cold water, hot water and two subsequent extractions with 1 m sodium hydroxide. Branched partially methoxylated mannogalactan and slightly branched (1→6)-ß-d-glucan predominated in cold- and hot-water-soluble fractions, respectively. Alternatively, these polysaccharides were obtained by only hot water extraction and subsequent two-stage chromatographic separation. The alkali-soluble parts originating from the first alkali extraction were then fractionated by dissolution in dimethyl sulfoxide (DMSO). The polysaccharide insoluble in DMSO was identified as linear (1→3)-α-d-glucan, while branched (1→3)(1→6)-ß-d-glucans were found to be soluble in DMSO. The second alkaline extract contained the mentioned branched ß-d-glucan together with some proteins. Finally, the alkali insoluble part was a cell wall complex of chitin and ß-d-glucans.

Evaluation of IR and Raman spectroscopic markers of human collagens: Insides for indicating colorectal carcinogenesis.

Vibrational spectroscopic methods are widely used in the molecular diagnostics of carcinogenesis. Collagen, a component of connective tissue, plays a special role as a biochemical marker of pathological changes in tissues. The vibrational bands of collagens are very promising to distinguish between normal colon tissue, benign and malignant colon polyps. Differences in these bands indicate changes in the amount, structure, conformation and the ratio between the individual structural forms (subtypes) of this protein. The screening of specific collagen markers of colorectal carcinogenesis was carried out based on the FTIR and Raman (λex 785 nm) spectra of colon tissue samples and purified human collagens. It was found that individual types of human collagens showed significant differences in their vibrational spectra, and specific spectral markers were found for them. These collagen bands were assigned to specific vibrations in the polypeptide backbone, amino acid side chains and carbohydrate moieties. The corresponding spectral regions for colon tissues and colon polyps were investigated for the contribution of collagen vibrations. Mentioned spectral differences in collagen spectroscopic markers could be of interest for early ex vivo diagnosis of colorectal carcinoma if combine vibrational spectroscopy and colonoscopy.

Hydrocolloids from the Mushroom Auricularia heimuer: Composition and Properties.

The ear- to shell-shaped fruiting bodies of the genus Auricularia are widely used as food and in traditional medicinal remedies. This study was primarily focused on the composition, properties and potential use of the gel-forming extract from Auricularia heimuer. The dried extract contained 50% soluble homo- and heteropolysaccharides, which were mainly composed of mannose and glucose, acetyl residues, glucuronic acid and a small amount of xylose, galactose, glucosamine, fucose, arabinose and rhamnose. The minerals observed in the extract included approximately 70% potassium followed by calcium. Among the fatty and amino acids, 60% unsaturated fatty acids and 35% essential amino acids could be calculated. At both acidic (pH 4) and alkaline (pH 10) conditions, the thickness of the 5 mg/mL extract did not change in a temperature range from -24 °C to room temperature, but decreased statistically significantly after storage at elevated temperature. At neutral pH, the studied extract demonstrated good thermal and storage stability, as well as a moisture retention capacity comparable to the high molecular weight sodium hyaluronate, a well-known moisturizer. Hydrocolloids that can be sustainably produced from Auricularia fruiting bodies offer great application potential in the food and cosmetic industries.

Perspectives in the Application of High, Medium, and Low Molecular Weight Oat ß-d-Glucans in Dietary Nutrition and Food Technology-A Short Overview.

For centuries human civilization has cultivated oats, and now they are consumed in various forms of food, from instant breakfasts to beverages. They are a nutrient-rich food containing linear mixed-linkage (1 → 3) (1 → 4)-ß-d-glucans, which are relatively well soluble in water and responsible for various biological effects: the regulation of the blood cholesterol level, as well as being anti-inflammatory, prebiotic, antioxidant, and tumor-preventing. Numerous studies, especially in the last two decades, highlight the differences in the biological properties of the oat ß-d-glucan fractions of low, medium, and high molecular weight. These fractions differ in their features due to variations in bioavailability related to the rheological properties of these polysaccharides, and their association with food matrices, purity, and mode of preparation or modification. There is strong evidence that, under different conditions, the molecular weight may determine the potency of oat-extracted ß-d-glucans. In this review, we intend to give a concise overview of the properties and studies of the biological activities of oat ß-d-glucan preparations depending on their molecular weight and how they represent a prospective ingredient of functional food with the potential to prevent or modulate various pathological conditions.

Mid-Infrared Spectroscopic Study of Cultivating Medicinal Fungi Ganoderma: Composition, Development, and Strain Variability of Basidiocarps.

Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy was proposed for rapid, versatile, and non-invasive screening of Ganoderma basidiocarps to assess their potential for specific applications. Fifteen species and strains of this fungus were selected for analysis, and fine sections at different parts of young and mature basidiocarps were obtained. The spectra of fungal samples showed significant differences interpreted in terms of biochemical composition using characteristic bands of proteins, polysaccharides, lipids, and triterpenoids. Obviously, for the transverse sections in trama, especially in the basal part, the most intense bands at 950-1200 cm-1 corresponded to polysaccharide vibrations, while for the superficial sections, the bands of carbonyl and aliphatic groups of triterpenoids at 1310-1470, 1550-1740, and 2850-2980 cm-1 predominated. The pilei, especially hymenium tubes, apparently contained more proteins than the bases and stipes, as evidenced by the intense bands of amide vibrations at 1648 and 1545-1550 cm-1. The specificity of the Ganoderma basidiocarp is a densely pigmented surface layer rich in triterpenoids, as proved by ATR-FTIR spectroscopy. The spectral differences corresponding to the specificity of the triterpenoid composition may indicate the prospects of individual strains and species of this genus for cultivation and further use in food, cosmetics, or medicine.

Water-extractable polysaccharide fraction PNE-P1 from Pinus koraiensis pine nut: Structural features and immunostimulatory activity.

The polysaccharide fraction PNE-P1 was isolated from hot water extract (PNE) of the defatted meal of pine nuts (Pinus koraiensis) using DEAE-cellulose column chromatography. This fraction had three components of molecular masses 1251, 616, and 303 g/mol consisting mainly of arabinose, xylose, and galacturonic acid at a molar ratio of 2:1.6:1. Structural analysis with FTIR/Raman, methylation and GC-MS, and NMR revealed that PNE-P1 is a cell wall polysaccharide complex including arabinan, heteroxylan, homogalacturonan (HM) and rhamnogalacturonan I (RG-I) parts. Being nontoxic to RAW 264.7 macrophages in the concentration range of 10-200 µg/mL, PNE-P1 promoted proliferation of these cells, significantly induced the secretion of proinflammatory cytokines (TNF-α and IL-6) and chemokines (RANTES and MIP-1α) and enhanced the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and nitric oxide (NO). PNE-P1 also markedly induced macrophage-mediated phagocytosis of apoptotic Jurkat T cells. These results demonstrate that pine nuts Pinus koraiensis contain a complex of water-soluble plant cell wall polysaccharides, which can stimulate innate immunity by potentiating macrophage function.

Modification of chitosan-methylcellulose composite films with meso-tetrakis(4-sulfonatophenyl)porphyrin.

Polysaccharide films containing chitosan, methylcellulose, and a mixture of these polysaccharides in various ratios were prepared and modified with meso-tetrakis(4-sulfonatophenyl)porphyrin in an aqueous medium at pH 7. The modified films were compared with the initial films using spectroscopic methods and microscopic imaging. Electronic (UV-vis absorption, electronic circular dichroism (ECD)) and vibrational (FTIR and Raman) spectra showed that the porphyrin macrocycles had a strong affinity toward chitosan and did not interact with the methylcellulose. The total porphyrin uptake depended on the chitosan: methylcellulose ratio and pure methylcellulose films did not retain porphyrin macrocycles. ECD measurements detected the presence of optically active porphyrin species bound to the films. SEM and AFM images confirmed that the porphyrin macrocycles caused structural changes on the film surface and within the film layer.

Chemical Composition and Rheological Properties of Seed Mucilages of Various Yellow- and Brown-Seeded Flax (Linum usitatissimum L.) Cultivars.

When seeds sown in the soil become wet, their hulls secrete viscous matter that can retain water and thus support germination. Flaxseed mucilage (FSM) is an example of such a material and is attractive for food, cosmetic, and pharmaceutical applications due to its suitable rheological properties. FSM consists mainly of two polysaccharides, namely, arabinoxylan and rhamnogalacturonan I, and it also contains some proteins, minerals, and phenolic compounds. The genotype and the year of the flax harvest can significantly affect the composition and functional properties of FSM. In this work, FSM samples were isolated from flax seeds of different cultivars and harvest years, and their structural and rheological properties were compared using statistical methods. The samples showed significant variability in composition and rheological properties depending on the cultivar and storage time. It was found that the ratio of two polysaccharide fractions and the contribution of less-prevalent proteins are important factors determining the rheological parameters of FSM, characterizing the shear-thinning, thixotropic, and dynamic viscoelastic behavior of this material in aqueous solutions. The yield strength and the hysteresis loop were found to be associated with the contribution of the pectin fraction, which included homogalacturonan and rhamnogalacturonan I. In contrast, the shear-thinning and especially the dynamic viscoelastic properties depended on the arabinoxylan content. Proteins also affected the viscoelastic properties and maintained the elastic component of FSM in the solution. The above structural and rheological characteristics should be taken into account when considering effective applications for this material.

Polysaccharides from Basidiocarps of the Polypore Fungus Ganoderma resinaceum: Isolation and Structure.

In this study, we focused on the isolation and structural characterization of polysaccharides from a basidiocarp of polypore fungus Ganoderma resinaceum. Polysaccharide fractions were obtained by successive extractions with cold water at room temperature (20 °C), hot water under reflux (100 °C), and a solution of 1 mol L-1 sodium hydroxide. The purity of all fractions was controlled mainly by Fourier transform infrared (FTIR) spectroscopy, and their composition and structure were characterized by organic elemental analysis; neutral sugar and methylation analyses by gas chromatography equipped with flame ionization detector (GC/FID) and mass spectrometry detector (GC/MS), respectively; and by correlation nuclear magnetic resonance (NMR) spectroscopy. The aqueous extracts contained two main polysaccharides identified as a branched O-2-ß-d-mannosyl-(1→6)-α-d-galactan and a highly branched (1→3)(1→4)(1→6)-ß-d-glucan. Mannogalactan predominated in the cold water extract, and ß-d-glucan was the main product of the hot water extract. The hot water soluble fraction was further separated by preparative anion exchange chromatography into three sub-fractions; two of them were identified as branched ß-d-glucans with a structure similar to the corresponding polysaccharide of the original fraction. The alkaline extract contained a linear (1→3)-α-d-glucan and a weakly branched (1→3)-ß-d-glucan having terminal ß-d-glucosyl residues attached to O-6 of the backbone. The insoluble part after all extractions was identified as a polysaccharide complex containing chitin and ß-d-glucans.

Sulfated Glucuronorhamnoxylan from Capsosiphon fulvescens Ameliorates Osteoporotic Bone Resorption via Inhibition of Osteoclastic Cell Differentiation and Function In Vitro and In Vivo.

Excessive osteoclast differentiation and/or bone resorptive function causes a gradual loss of bone, leading to the pathogenesis of bone diseases such as osteoporosis (OP). In this study, a sulfated glucuronorhamnoxylan polysaccharide (designated SPS-CF) of the green alga Capsosiphon fulvescens was evaluated for anti-osteoporotic activity using osteoclastic cells differentiated from RAW264.7 macrophages by receptor activator of NF-κB ligand (RANKL) treatment and ovariectomized (OVX) female mice as a postmenopausal OP model. With negligible cytotoxicity, SPS-CF (50 µg/mL) significantly suppressed tartrate-resistant acid phosphatase (TRAP) activity, actin ring formation, and expression of matrix metalloproteinase 9 (MMP-9), cathepsin K, TRAF6, p-Pyk2, c-Cbl, c-Src, gelsolin, carbonic anhydrase II (CA II), and integrin ß3, indicating that SPS-CF inhibits the differentiation and bone resorptive function of osteoclasts. Removal of sulfate groups from SPS-CF abolished its anti-osteoclastogenic activities, demonstrating that sulfate groups are critical for its activity. Oral administration of SPS-CF (400 mg/kg/day) to OVX mice significantly augmented the bone mineral density (BMD) and serum osteoprotegerin (OPG)/RANKL ratio. These results demonstrated that SPS-CF exerts significant anti-osteoporotic activity by dampening osteoclastogenesis and bone resorption via downregulation of TRAF6-c-Src-Pyk2-c-Cbl-gelsolin signaling and augmentation of serum OPG/RANKL ratios in OVX mice, suggesting that SPS-CF can be a novel anti-osteoporotic compound for treating postmenopausal OP.

Fractionation of the water insoluble part of the heterotrophic mutant green microalga Parachlorella kessleri HY1 (Chlorellaceae) biomass: Identification and structure of polysaccharides.

The water-insoluble part of Parachlorella kessleri HY1 biomass was subjected to the extraction of cell-wall polysaccharides using polar aprotic solvents (DMSO, LiCl/DMSO) and aqueous alkaline solutions (0.1, 1 and 4 mol·l-1 of NaOH). Proteins predominated in all the crude extracts and in the insoluble residues were partially removed by treatment with proteolytic enzymes (pepsin and pronase), and in some cases with the HCl/H2O2 reagent, yielding purified polysaccharide-enriched fractions. These treatments led to the solubilisation of some products in water. The composition and structure of isolated polysaccharides were characterised based on monosaccharide composition, glycosidic linkage and spectroscopic analyses. The DMSO extract contained mainly proteins, and polysaccharides were not detected. The water-soluble parts isolated from the LiCl/DMSO extract contained α-l-rhamnan, α-d-glucan and ß-d-glucogalactan; the water-insoluble part contained (1 â†’ 4)-ß-d-xylan, first isolated from the biomass of green microalgae. The alkali extracts contained polysaccharides of similar structure, and also water-insoluble (1 â†’ 4)-ß-d-mannan. The insoluble part after all extractions contained α-chitin as the main polysaccharide, which was confirmed by spectroscopic methods. All these polysaccharides can play a certain role in the cell wall structure of this microalga.

Antioxidant and Anti-Inflammatory Activity of Five Medicinal Mushrooms of the Genus Pleurotus.

Within the group of higher fungi, edible medicinal mushrooms have a long history of being used as food and in folk medicine. These species contain biologically active substances with many potential beneficial effects on human health. The Pleurotus genus is representative of medicinal mushrooms because Pleurotus ostreatus is one of the most commonly cultivated culinary mushrooms. In our study, we focused on lesser-known species in the genus Pleurotus and measured their antioxidant and anti-inflammatory activity. We prepared extracts of the mushrooms and analyzed them using HPLC-HRMS, GC-MS, and 1H-NMR. Significant differences in biological activities were found among the Pleurotus spp. extracts. A MeOH extract of P. flabellatus was the most active as a radical scavenger with the highest ORAC, while a chloroform extract had significant anti-inflammatory COX-2 activity. The 80% MeOH extract of P. flabellatus contained the highest amounts of ergosterol, ergothioneine, and mannitol. The 80% MeOH extract of P. ostreatus Florida was the most active in the NF-κB inhibition assay and had the highest content of ß-glucans (43.3% by dry weight). Given the antioxidant and anti-inflammatory properties of P. flabellatus, the potential therapeutic usefulness of this species is worth evaluating through in-depth investigations and confirmation by clinical trials.

Determination of phytic acid and inositolphosphates in barley.

Phytic acid (PA) and lower inositolphosphates (InsP(n) ) is the main storage form of phosphorus in grains or seeds. The content of PA and InsP(n) in different varieties of barley was analyzed by capillary isotachophoresis and online-coupled capillary isotachophoresis with CZE. The electrolytes (in demineralized water) for the isotachophoretic analysis consisted of 10 mM HCl, 14 mM glycylglycine, and 0.1% 2-hydroxyethylcellulose (leading) and 10 mM citric acid (terminating). The optimized electrolytes for the online coupling isotachophoresis with zone electrophoresis analysis were mixtures of 5 mM HCl, 7 mM glycylglycine, and 0.1% 2-hydroxyethylcellulose (leading), 20 mM citric acid, 10 mM glycylglycine, and 0.1% 2-hydroxyethylcellulose (background) and 10 mM citric acid (terminating). PA and all studied InsP(n) were separated within 25 min and detected by a conductivity detector. Simple sample preparation (acidic extraction), sufficient sensitivity, speed of analysis, and low running cost are important attributes of the electrophoretic methods. The method was used for the determination of PA and InsP(n) in barley varieties within an ongoing research project.

Screening of the Chemical Composition and Identification of Hyaluronic Acid in Food Supplements by Fractionation and Fourier-Transform Infrared Spectroscopy.

Hyaluronic acid, together with collagen, vitamins or plant extracts, is a part of many cosmetic and food preparations. For example, this polysaccharide is used in formulation of many food supplements due to its protective effects on human health. In this work, the screening of the chemical composition of three chosen dietary supplements (powder, tablets and capsules) containing hyaluronic acid was carried out using Fourier-transform infrared spectroscopy. Because of the low amount of analyte in all these samples, it was isolated or concentrated prior to the analysis using a suitable sequential fractionation protocol. Individual isolation procedures were established for each sample based on their declared composition. Firstly, the major components such as collagen or vitamins were removed to obtain polysaccharide fractions by the enzymatic treatment and/or washing out with the appropriate solvents. In some cases, the water insoluble part was removed from the rest dissolved in water. Then, hyaluronic acid was precipitated with copper(II) cations and thus separated from the other polysaccharides. Finally, the analyte was identified in the enriched fractions by the characteristic vibrational bands. The amount of hyaluronic acid in the purified fractions was determined in three ways: gravimetrically, spectrophotometrically, and using isotachophoresis. The combination of the appropriate preparative and analytical steps led to the successful evaluation of chemical composition, finding and quantification of hyaluronic acid in all the studied samples.

Spectroscopic Discrimination of Bee Pollen by Composition, Color, and Botanical Origin.

Bee pollen samples were discriminated using vibrational spectroscopic methods by connecting with botanical sources, composition, and color. SEM and light microscope images of bee pollen loads were obtained and used to assess the botanical origin. Fourier transform (FT) mid- and near-infrared (FT-MIR, FT-NIR), and FT-Raman spectra of bee pollen samples (a set of randomly chosen loads can be defined as an independent sample) were measured and processed by principal component analysis (PCA). The CIE L*a*b* color space parameters were calculated from the image analysis. FT-MIR, FT-NIR, and FT-Raman spectra showed marked sensitivity to bee pollen composition. In addition, FT-Raman spectra indicated plant pigments as chemical markers of botanical origin. Furthermore, the fractionation of bee pollen was also performed, and composition of the fractions was characterized as well. The combination of imaging, spectroscopic, and statistical methods is a potent tool for bee pollen discrimination and thus may evaluate the quality and composition of this bee-keeping product.

Preparation and characterization of hydrophobic and hydrophilic amidated derivatives of carboxymethyl chitosan and carboxymethyl ß-glucan.

Chitosan and ß-glucan are substances that are very widely used in the pharmaceutical and food industries, medicine and other areas. These polysaccharides have immense significance in human metabolism. They are able to affect the levels of cholesterol and lipids. Chemical modification of these polysaccharides allows the support of these attributes. A main goal is to prepare a group of amide derivatives of carboxymethyl chitosan and carboxymethyl ß-glucan to obtain a new group of polysaccharide derivatives and increase the benefits and attributes of these polysaccharides. The aim of carboxymethylation and subsequent two-step amidation is to achieve a high degree of substitution in the prepared derivatives. The amidation consisted methyl esterification followed by amino-de-alkoxylation with amidation reagents (n-alkylamines, hydrazine and hydroxylamine). The purity and substitution degree of the prepared derivatives were monitored by vibration spectroscopic methods (FTIR and FT Raman) and organic elemental analysis. The main, which was due to the presence of a secondary component (chitin) of polysaccharides, was the calculation of the substitution degree, which was based on the organic elemental analysis in combination with FTIR spectroscopy. These analytic methods confirmed the preparation of the substituted N-alkylamides, hydrazide and hydroxamic acid of carboxymethyl chitosan and carboxymethyl ß-glucan.

Immunoactive polysaccharides produced by heterotrophic mutant of green microalga Parachlorella kessleri HY1 (Chlorellaceae).

Hot water extract from biomass of heterotrophic mutant green alga Parachlorella kessleri HY1 (Chlorellaceae) was deproteinised, and three polysaccharidic fractions were obtained by preparative chromatography. The low-molecular fraction (1.5 × 104g mol-1) was defined mainly as branched O-2-ß-xylo-(1→3)-ß-galactofuranan where xylose is partially methylated at O-4. Two high-molecular fractions (3.05 × 105 and 9.84 × 104g mol-1) were complex polysaccharides containing α-l-rhamnan and xylogalactofuranan parts in different ratios. The polysaccharides were well soluble in hot water and, upon cooling, tended to self-segregate. Immunomodulatory activities of the obtained fractions were preliminary tested using ELISA, FACS and ImmunoSpot kits. The polysaccharides increased the TNF-α production in melanoma bearing mice with much higher intensity than in healthy mice. This was in agreement with the FACS results on T and B cells indicating their possibly secondary activation by innate immunity cells.

Hydrogels based on low-methoxyl amidated citrus pectin and flaxseed gum formulated with tripeptide glycyl-l-histidyl-l-lysine improve the healing of experimental cutting wounds in rats.

Hydrogels based on natural and modified polysaccharides represent growing group of suitable matrices for the construction of effective wound healing materials. Bioactive tripeptide glycyl-l-histidyl-l-lysine and amino acid α-l-arginine are known to accelerate wound healing and skin repair. In this study, hydrogels based on low-methoxyl amidated citrus pectin or flaxseed gum were prepared and used for the transport of these healing agents to the experimental cutting wounds affected by extensive skin damage. Fourier-transform infrared spectroscopy, rheology, differential scanning calorimetry, scanning electron microscopy, swelling and release tests confirmed that these hydrogels differed in structure and physical properties. The cationic tripeptide was found to bind to carboxylic groups in LMA pectin, and the C3OH hydroxyl and ring oxygen O5 are involved in this interaction. The pectin hydrogel showed high viscosity and strong elastic properties, while the flaxseed gum hydrogel was characterised as a viscoelastic system of much lower viscosity. The former hydrogel released the drugs very slowly, while the latter hydrogel demonstrated zero order releasing kinetics optimal for drug delivery. In the in vivo wound healing testing on rats, both polysaccharide hydrogels improved the healing process mediated by the mentioned biomolecules. The tripeptide applied in the hydrogels showed significantly higher healing degree and lower healing time than in the control animals without treatment and when it was applied in an aqueous solution. Despite the absence of a synergistic effect, the mixture of the tripeptide and α-l-arginine in the hydrogels was also quite effective in wound healing. According to histological analysis, complete healing was achieved only when using the tripeptide in the flaxseed gum hydrogel. These observations might have an important prospect in clinical application of polysaccharide hydrogels.

pH-controlled self-assembling of meso-tetrakis(4-sulfonatophenyl)porphyrin-chitosan complexes.

Solid meso-tetrakis(4-sulfonatophenyl)porphyrin (TPPS(4))-chitosan supramolecular complexes were prepared by addition of porphyrin to an aqueous solution of chitosan at pH values. The precipitates obtained were assigned as 1 (pH 6.8) and 2 (pH 2.5) and characterized by spectroscopic, thermal, and microscopic methods. Spectroscopic investigation confirmed the presence of TPPS(4) and chitosan in both products and that the porphyrin is highly self-associated. H-type (stacked) of TPPS(4) aggregation was proposed for 1 and J-type (tilted) for 2. Thermal analysis revealed different pyrolysis routes of the complexes depending on their structural diversity. Light microscopic analysis indicated fibrous and lamellar microstructures, respectively, for 1 and 2. SEM and AFM analysis showed that both complexes consist of compact nanostructures; their size and interconnection is different for 1 and 2. Based on structural inferences, self-assembling hierarchy models were proposed for both of the TPPS(4)-chitosan supramolecular complexes.

Antifungal Polyamides of Hydroxycinnamic Acids from Sunflower Bee Pollen.

The aim of the bioassay-guided fractionation was the selection of the most potent group of compounds responsible for the protection of sunflower bee pollen grains. Synthesis of prospective antifungal polyamides of hydroxycinnamic acids was based on previous structural elucidation of ethanol soluble fraction by 1H,1H-PFG-COSY, 1H,13C-HSQC, FT-IR, FT-Raman, and LC-MS experiments. The main compounds found were tri- p-coumaroylspermidines accompanied by other HCAA of spermidine and putrescine. Several model HCAA derivatives were prepared to test their antifungal activity against widespread spoilage fungi ( A. niger 42 CCM 8189, F. culmorum DMF 0103, and P. verrucosum DMF 0023). A. niger CCM 8189 and F. culmorum DMF 0103 exhibited higher resistance to the antifungal effects of hydroxycinnamic acid amides, whereas P. verrucosum DMF 0023 was the most sensitive strain. It has been discovered the effect of HCAA polarity on the role of secondary metabolites in the microbial protection of pollen grains. The combination of bioassay-guided fractionation, structural elucidation, selection of prospective compounds, and their synthesis to determine their antifungal properties could be considered as an original approach.