Scientia Pharmaceutica — Open Access Journal

Diabetes is a major metabolic disorder whose prevalence is increasing daily. Medicinal plants have played an important role in the prevention and treatment of type 2 diabetes via prophylactic and therapeutic management. In this study, Mangifera Indica leaf (MIL) extract was investigated for its promising anti-diabetic activity via an in vitro model. It was found that MIL extract possessed significant inhibition on alpha-amylase activity up to (51.4 ± 2.7)% at a concentration of 200 µg/mL. Moreover, glucose adsorption capacity of MIL was identified at (2.7 ± 0.19) mM glucose/g extract. Furthermore, the extract caused a significant increase in glucose uptake up to (143 ± 9.3)% in LO-2 liver cells. Notably, MIL extract was effective in scavenging (63.3 ± 2.1)% 1,1-diphenyl-2-picryl-hydrazyl (DPPH) and (71.6 ± 4.3)% 2,2-azinobis-3-ethyl benzothiazoline-6-sulfonic acid (ABTS)⁺ radicals and inhibiting (66 ± 4.9)% NO production from RAW264.7 cells without any cytotoxicity effects. Accordingly, M. indica leaves are suggested as a promising material for development of hypoglycemic products. Full article

According to our quantum and chemical calculations 4-methyl-2,2-dioxo-1H-2λ⁶,1-benzothiazine-3-carboxylic acid imidazolide is theoretically almost as reactive as its 2-carbonyl analog, and it forms the corresponding N-pyridyl-4-methyl-2,2-dioxo-1H-2λ⁶,1-benzothiazine-3-carboxamides with many aminopyridines. However, in practice, the sulfo group introduces significant changes at times and prevents the acylation of sterically hindered amines. One of these products was 2-amino-6-methylpyridine. Thus, it has been concluded that aminopyridines interact with imidazolide in aromatic form where the target for the initial electrophilic attack is the ring nitrogen. To confirm the structure of all substances synthesized, ¹H-NMR spectroscopy and X-ray diffraction analysis were used. From X-ray diffraction data it follows that in the crystalline phase the carbonyl and sulfo group may occupy different positions with respect to the plane of the benzothiazine bicycle: this position may be unilateral, typical for 4-methyl-2,2-dioxo-1H-2λ⁶,1-benzothiazine-3-carboxamides, versatile, and not yet encountered in compounds of this type. A comparison of these data with the results of the pharmacological screening conducted on the standard model of carrageenan inflammation showed that the N-pyridylamides of the first group demonstrated a direct dependence of their analgesic and anti-inflammatory activity on the mutual arrangement of the planes of the benzothiazine and pyridine fragments. The new molecular conformation of the benzothiazine nucleus provides a sufficiently high level of analgesic (but not anti-inflammatory) properties in all N-pyridylamides of the second group with an extremely weak dependence on the spatial arrangement of the pyridine cycle. All substances presented this article proved themselves in varying degrees as analgesics and antiphlogistics. Moreover, two of them—N-(5-methylpyridin-2-yl)- and N-(pyridin-3-yl)-4-methyl-2,2-dioxo-1H-2λ⁶,1-benzothiazine-3-carboxamides—exceeded the most effective drug of oxicam type Lornoxicam by these indicators. Full article

In the last decade, high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) combined with electrospray ionization (ESI) has been widely used for determining low concentrations of steroids, and derivatization has often been employed to enhance detection. In the present study, endogenous steroids were extracted using a Strata-XL polymeric reverse phase cartridge. The isolated steroids were reacted with 2-hydrazino-1-methylpyridine (HMP) at 50 °C for 30 min. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used in a positive mode with multiple reaction monitoring (MRM) for the quantification of testosterone (T) and its precursor, dehydroepiandrosterone (DHEA), in saliva samples collected from twenty young Saudi professional soccer players. The analytes were separated on an ACE Ultracore 2.5 Superphenylhexyl column (150 × 3.0 mm id). The extraction recovery during the pre-treatment was >89% and gave <±20% for inter- and intra-assay precision and accuracy. The limits of quantification (LOQ) were found to be 20 pg/mL for (T and DHEA) and 50 pg/mL for Epitestosterone (EPI). The results showed no significant variation in the concentration of T between pre and post training, whereas DHEA was significantly increased after short-term exercise. These results could indicate that there is no correlation between T and its precursor DHEA level following short term physical activity. EPI concentrations could not be detected with a LOQ of 50 pg/mL in the saliva samples. Full article

In order to detect new structural and biological patterns in a series of hetaryl-3-carboxylic acid derivatives, the optically pure (S)- and (R)-enantiomers of N-(1-arylethyl)-4-methyl- 2,2-dioxo-1H-2λ⁶,1-benzothiazine-3-carboxamides, their true racemates, and mechanical racemic mixtures have been synthesized in independent ways. The particular features of the ¹Н- and ¹³С-NMR spectra of all synthesized substances, liquid chromato-mass spectrometric behavior thereof under electrospray ionization conditions, and also the results of polarimetric and X-ray diffraction studies have been discussed. Pharmacological screening on a model of carrageenan inflammation has found a clear relationship between the spatial structure of the studied objects and biological activity thereof. Enantiomers with chiral centers having (S)-configuration showed weak inhibition of pain and inflammatory reactions, while their mirror (R)-isomers exhibited very powerful analgesic and antiphlogistic properties under the same conditions, with the level of specific activity exceeding that of Lornoxicam and Diclofenac. Taking obtained data into account, a noticeable decrease in the activity of mechanical racemic mixtures, consisting of one-half of the “wrong” (S)-enantiomers, is quite natural. The true racemate of N-(1-phenylethyl)-amide proved itself in a similar way, while 4-methoxy-substituted analog thereof stood out against this background with unexpectedly high analgesic and anti-inflammatory activities. A comparative analysis of X-ray diffraction data has found that crystalline and molecular structure of racemic N-[1-(4-methoxyphenyl)ethyl]-4-methyl-2,2-dioxo-1H-2λ⁶,1-benzothiazine-3-carboxamide is completely different from that of the original enantiomers and, moreover, very unusual for racemates. Obviously, it is the factor determining the unique character of the biological effects of the said substance. Full article

The aim of this study was to investigate the acute and subchronic toxicity of a film formulation that combines κ-Carrageenan and konjac glucomannan for soft capsule application. For the acute toxicity study, a dose of 2000 mg/kg body weight (bw) of the film suspension was administered orally to rats. The animals were observed for toxic symptoms and mortality daily for 14 days. In a subchronic toxicity study, the film suspension, at doses of 10, 30 and 75 mg/kg bw for 28 days, were orally administered to rats. After 28 days, the rats were sacrificed for hematological, biochemical and histological examination. In the acute toxicity study, neither signs of toxicity nor death among the rats were observed for up to 14 days of the experimental period. The results of the subchronic toxicity study show that there were no significant changes observed in the hematology and organ histology. Some alterations to the relative organ weight and blood biochemistry were observed, but they were considered to be temporary effects and not an indication of toxic effects. The overall findings of this study indicate that the film formulation of κ-Carrageenan and konjac glucomannan is non-toxic up to a dose of 75 mg/kg bw, which could be considered a safe dose for soft capsule application. Full article

Rhodiola rosea has been used in folk medicine as ethanolic macerates for a long time. This study aims to provide a quantitative and qualitative analysis and comparison of different ethanolic Rhodiola rosea rhizome macerates (35%, 70%, and 96% v/v) and accelerated solvent extraction (ASE) extracts prepared with 85% methanol, in order to shed light on the effectivity of different extraction methods. Extract samples were analyzed by UHPLC-DAD-ESI-MSⁿ on a ZORBAX SB-C18 column (100 × 2.1 mm, 1.8 μm) with a mobile phase consisting of water + 0.1% formic acid and acetonitrile. Qualitative analysis lead to the tentative identification of 18 compounds: Two cyanogenic glycosides (rhodiocyanoside A, lotaustralin), three phenylethanoids (salidroside, viridoside, 2-phenylethyl-vicianoside), two procyanidin and catechin derivatives (epigallocatechin-epigallocatechin gallate, epigallocatechin-3-O-gallate), five phenylpropanoids (cinnamyl alcohol, rosarin, rosavin, rosin, cinnamyl-(6’-O-β-d-xylopyranosyl)-O-β-glucopyranoside), two monoterpene alcohols (rhodioloside E, rosiridin) and four flavonols (rhodionidin, rhodiosin, rhodionin, kaempferol). Quantity was determined for salidroside, cinnamyl alcohol and its three major glycosides (rosarin, rosavin, rosin), as well as three flavonols (rhodionidin, rhodiosin, rhodionin). Methanolic ASE proved to be the superior extraction method for different substance groups. For macerates, high ethanol content increased yield and lowered hydrolysis of glycosides during extraction, but ethanolic macerates still showed low reproducibility and high fluctuations in quantity of marker compounds salidroside and rosavins, as well as flavonols. Rhodiola rosea rhizomes of wild origins seemed to underly great variability in chemical composition dependent on grow site. Full article

The article presents the synthesis of 2-arylimino-4-methyl-2,3-dihydro-1,3-thiazoles via Hantzsch reaction of thioureas and 3-chloropentane-2,4-dione or ethyl 2-chloro-3-oxobutanoate. The structure of synthesized compounds was confirmed by LCMS, ¹H, and ¹³C NMR spectra. Cardioprotective activity of synthesized thiazole derivatives were studied in vitro on the isolated rings of the thoracic aorta of laboratory rats. Based on pharmacological studies, the tested compounds possessed a moderate to high cardioprotective effect. A prospective 1-[2-(4-methoxyphenylimino)-4-methyl-3-(4-methylpiperazine-1-yl)-2,3-dihydro-1,3-thiazole-5-yl] ethan-1-one hydrochloride 4c was identified. The mentioned compound has delayed the development of constrictor responses of isolated rings of the thoracic rat aorta and exceeds the activity of L-carnitine by 18.2% and meldonium by 12.9%. The compound 4c may be proposed as a potential cardioprotective agent for in-depth pharmacological studies. Full article

Treatment of certain central nervous system disorders, including different types of cerebral malignancies, is limited by traditional oral or systemic administrations of therapeutic drugs due to possible serious side effects and/or lack of the brain penetration and, therefore, the efficacy of the drugs is diminished. During the last decade, several new technologies were developed to overcome barrier properties of cerebral capillaries. This review gives a short overview of the structural elements and anatomical features of the blood–brain barrier. The various in vitro (static and dynamic), in vivo (microdialysis), and in situ (brain perfusion) blood–brain barrier models are also presented. The drug formulations and administration options to deliver molecules effectively to the central nervous system (CNS) are presented. Nanocarriers, nanoparticles (lipid, polymeric, magnetic, gold, and carbon based nanoparticles, dendrimers, etc.), viral and peptid vectors and shuttles, sonoporation and microbubbles are briefly shown. The modulation of receptors and efflux transporters in the cell membrane can also be an effective approach to enhance brain exposure to therapeutic compounds. Intranasal administration is a noninvasive delivery route to bypass the blood–brain barrier, while direct brain administration is an invasive mode to target the brain region with therapeutic drug concentrations locally. Nowadays, both technological and mechanistic tools are available to assist in overcoming the blood–brain barrier. With these techniques more effective and even safer drugs can be developed for the treatment of devastating brain disorders. Full article

Proteins play crucial roles in the transportation and distribution of therapeutic substances, including metal ions in living systems. Some metal ions can strongly associate, while others show low affinity towards proteins. Consequently, in the present work, the binding behaviors of Ca²⁺, Ba²⁺, Ag⁺, Ru³⁺, Cu²⁺ and Co²⁺ with bovine serum albumin (BSA) were screened. BSA and the metal ions were allowed to interact at physiological pH and their binding interactions were screened by using FT-IR spectroscopy. Spectra were collected by using hydrated films over a range of 4000–400 cm⁻¹. The interaction was demonstrated by a significant reduction in the spectral intensities of the amide I (C=O stretching) and amide II bands (C–N stretching coupled to NH bending) of the protein after complexation with metal ions. The binding interaction was further revealed by spectral shifting of the amide I band from 1651 cm⁻¹ (free BSA) to 1653, 1654, 1649, 1655, 1655, and 1654 cm⁻¹ for BSA–Ca²⁺, BSA–Ba²⁺, BSA–Ag⁺, BSA–Ru³⁺, BSA–Cu²⁺ and BSA–Co²⁺ complexes, respectively. The shifting of the amide I band was due to the interactions of metal ions with the O and N atoms of the ligand protein. Estimation of the secondary protein structure showed alteration in the protein conformation, characterized by a marked decrease (12.9–40.3%) in the α-helix accompanied by increased β-sheet and β-turn after interaction with the metal ions. The interaction results of this study were comparable with those reported in our previous investigation of metal ion–BSA interactions using affinity capillary electrophoresis (ACE), which has proven the accuracy of the FT-IR technique in the measurement of interactions between proteins and metal ions. Full article

Nigella sativa oil has been known to have potent anti-inflammatory activity. This research aimed to determine the anti-inflammation activity of Nigella sativa oil in a simple balm stick by topical application. The activity was checked using two methods: carrageenan-induced paw oedema and granuloma pouch on rats. The results showed that balm sticks which contained 10% Nigella sativa could overcome both acute and sub-acute inflammation showing by high oedema inhibition (60.64%), low leucocytes count (43.55% lower than control) as well as a notable TNF-α concentration (50% lower than control) on the inflamed area. In conclusion, topical application of a Nigella sativa balm stick was effective for both acute and sub-acute forms of inflammation. Full article

Berberis goudotii is an endemic Colombian plant found in the paramo ecosystem. It has been used in food preparation and as a medicinal plant for diverse treatments. Additionally, it is used as a mouthwash to strengthen the gums and combat throat irritations and periodontitis. The present research evaluated Berberis goudotii aerial parts extract and fractions antimicrobial activities. Ultrasonic-assisted extraction was used to attain total ethanol-water extract. Solid-liquid fractionation was used to obtain hexane fraction. The residue was dispersed in water and liquid-liquid fractionation was carried-out to acquire dichloromethane, butanol and water fractions. Preliminary phytochemical analysis was performed on total extract and phenol, polyphenol, flavonoid, and proanthocyanidin, while tannin content was quantified. Antimicrobial activity assessment was performed by agar diffusion method using disks and wells employing Ceftazidime as a positive control against Streptococcus mutans, Streptococcus sobrinus, Lactobacillus acidophilus, Lactobacillus casei, Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum. Antimicrobial activity was determined as relative percentage inhibition (RPI), minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Phenols (92.5 ± 7.7 mg GA/10 g), polyphenols (87.7 ± 8.1 mg PG/10 g) and tannins (44.1 ± 4.3 mg PG/10 g) were among the highest secondary metabolites observed. Total extract presented an MBC of 1.0 µg/µL against cariogenic bacteria (Streptococcus mutans and Streptococcus sobrinus) and 0.12 µg/µL against bacteria associated with periodontal disease (Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum). Butanol and hexane fractions showed antiperiodontal activity with MBC of 0.12 and 1.0 µg/µL, respectively. In conclusion, Berberis goudotii total extract demonstrated antimicrobial activity against cariogenic and periodontal microorganisms, on the other hand, hexane and butanol fractions displayed antiperiodontal activity. Full article

Usnic acid is known for its remarkable antimicrobial activity. The aim of this research was to formulate hydrogel of usnic acid and evaluate the antibacterial activity against Propionibacterium acne. Due to low solubility of usnic acid, solid dispersion was prepared using PVP K-30. In this study, intact usnic acid (UA) and usnic acid-solid dispersion (UA-SD) was formulated in hydrogel using several gelling agents: Aqupec HV-505, sodium alginate and HPMC K 100M. Concentration of each gelling agent was optimized for hydrogel base. All of hydrogel base showed homogenous gel, pH at range 5.37–6.33 and viscosity in range 259.07–10,759.00 cps. Hydrogel was prepared by dispersing 1% intact UA and 3% UA-SD in three different gelling agents. The hydrogel was evaluated for pH, viscosity, stability test for two months and microbiology test. The amount of usnic acid in hydrogel was determined by spectrophotometry UV-Vis. Hydrogel UA showed non-homogenous gel, while hydrogel usnic UA-SD was homogenous. The pH of all hydrogel was in range 5.5–6.4 and viscosity was 2,017.03–3,866.52 cps. All the hydrogel was stable and diameter inhibition of hydrogel was in a range 20–32 mm. The amount of usnic acid in hydrogel was in range 96.9–99.23%. In conclusion, hydrogel UA-SD is promising preparation in handling acne. Full article

(1) Background: Clitoria ternatea (butterfly pea), a plant species belonging to the Leguminosae (Fabaceae) family, is useful for medical treatments and has been used in folk medicines and to cure different diseases. The antioxidation ability of the total phenolic compounds of butterfly pea is useful for preserving flavor, and colour and for preventing vitamin destruction in processed foods. In this study, a butterfly pea flower fermentation solution was added to cosmetics as a whiting ingredient. (2) Methods: After the phenolics, flavonoids and ascorbic acid content of the butterfly pea flower extraction had been determined, lactic acid bacteria fermented the extraction. The whitening and moisturizing effect was assayed by SSC3 and NF333 analyzers. (3) Results: This study demonstrated that the butterfly pea flower fermentation solution has free radical scavenging ability, a reducing power in high concentrations, a moisturizing effect, and a whiting effect. (4) Conclusions: The results showed that the butterfly pea flower fermentation solution not only inhibits redness, itching, allergies, and irritation to the skin, but also has antioxidation properties and promotes moisture retention and whitening effects, and the results increase as the concentration increases. Therefore, butterfly bean flowers may be suitable as a raw material for natural beauty care products. Full article

The essential oil obtained from the leaves of Lippia alba (Mill.) N.E. Brown (Verbenaceae) has shown great pharmacological potential as an analgesic, antispasmodic, and antimicrobial agent. The aim of this study was to evaluate the modulatory effect of Lippia alba essential oil (LaEO I) on the activity of clinically used antimicrobial agents on Salmonella enterica serovar Typhi (Salmonella typhi) and Shigella dysenteriae biofilms. The Minimum Inhibitory Concentration of LaEO I (MIC_{LaEO I}) was determined by the microdilution method, and the effect of LaEO I on the activity of clinically used antimicrobials was assessed by the Checkboard method. The values obtained from MIC_{LaEO I} and ciprofloxacin were used to evaluate the effect of time of exposure on cell viability. LaEO I main components were geranial (34.2%), neral (25.9%), and myrcene (12.5%). The MIC_{LaEO I} was 1 mg/mL for both strains. LaEO I positively modulated the action of ciprofloxacin, cefepime, and ceftriaxone. After the first hour of treatment with MIC_{LaEO I}, the cell viability of the strains showed a 5 log10 CFU/mL reduction, and the LaEO I-CIP association was able to inhibit growth during the first 6 h of the test. Regarding the anti-biofilm activity, MIC_{LaEO I} was able to reduce the biofilm mass of Salmonella typhi by 61.2% and of Shigella dysenteriae by 38.9%. MIC_{LaEO I} was not able to eradicate the preformed biofilm; however, there was a reduction in the biofilm microbial viability. LaEO I has the potential to be used as an antimicrobial agent and interferes with biofilm formation; also, it is able to reduce cell viability in preformed biofilm and synergistically modulate the activity of ciprofloxacin. Full article

The aim of this study was to investigate the effect of adenosine in non-occluded or occluded femoral arteries (FA) that were isolated from healthy or diabetic Wistar rats. Determining the role of endothelium, and a transmembrane flow of potassium ions in adenosine actions were also of interest. Diabetes was experimentally induced by alloxan, while the vascular occlusion was performed for 45 min on randomly selected FA. Vascular tone changes were continuously recorded. Selected markers of endothelial dysfunction were measured in animal serum. Thus, adenosine produced a concentration-dependent relaxation of rat FA, which was endothelium-dependent, too, except in a group of diabetic animals. Moreover, serum asymmetric dimethylarginine (ADMA) levels were higher in diabetic animals, thus reflecting endothelial dysfunction (ED). Still, an occlusion of FA enhanced the relaxation effect of adenosine in endothelium-intact rings from diabetic animals. Oppositely, in the presence of high potassium concentration in the buffer, adenosine-induced relaxation was significantly reduced in all of the investigated groups/subgroups. These results suggest that in diabetic animals, an occlusion of FA most probably reversed adenosine-induced relaxation from endothelium-independent into an endothelium-dependent relaxation, thus indicating the possible protective mechanism against ischemic episodes of FA in the presence of diabetes. Full article

The analysis of our previous studies on the search for synthetic analgesics among N-R-amides of bicyclic hetaryl-3-carboxylic acids has been performed; on its basis N-hetaryl(aryl)-alkyl-4-methyl-2,2-dioxo-1H-2λ⁶,1-benzothiazine-3-carboxamides have been selected as new study objects. The “one pot synthesis” of these compounds, which is simple to perform and at the same time highly effective, has been offered. The method consists in the initial reaction of 4-methyl-2,2-dioxo-1H-2λ⁶,1-benzothiazine-3-carboxylic acid and N,N′-carbonyldiimidazole in anhydrous N,N-dimethylformamide with the subsequent amidation of imidazolide formed with hetarylalkyl- or benzylamines in the same solvent. The peculiarities of ¹H- and ¹³C-NMR spectra of the substances obtained, as well as their electrospray ionization liquid chromato-mass spectra are discussed. According to the results of the pharmacological tests carried out on the model of carrageenan inflammation it has been found that all without exception N-hetaryl(aryl)alkyl-4-methyl-2,2-dioxo-1H-2λ⁶,1-benzothiazine-3-carboxamides demonstrate the statistically significant analgesic and anti-inflammatory properties. Among the substances presented in this article analgesics and antiphlogistics, which increase the pain threshold and suppress the inflammatory response more effectively than Lornoxicam and Diclofenac in the same doses, have been identified. The molecular and crystal structures of a large group of the substances synthesized have been studied by X-ray diffraction analysis. Comparison of these data with the results of biological tests has revealed the fact of excellent correlation between the molecular conformations of N-hetaryl(aryl)alkyl-4-methyl-2,2-dioxo-1H-2λ⁶,1-benzothiazine-3-carboxamides recorded in the crystal and the potency of their analgesic effect. N-Thiophen-2-ylmethyl- and N-4-methoxybenzyl-amides of 4-methyl-2,2-dioxo-1H-2λ⁶,1-benzothiazine-3-carboxylic acid has shown a high analgesic and anti-inflammatory effect, therefore, they deserve more careful research. Full article

As the demand for healthy products targeted to prevent or ameliorate bowel disease and digestive disorders of the intestinal tract is increasing, this review describes non-starch polysaccharides, such as β-glucan, arabinoxylan, galactomannan, fructan, and heteropolysaccarides from mucilages, as useful sources for adequate and tailor-made products aimed for regulation of the colon and wellbeing effects on the gut microbiota. Their monosaccharide composition, structure, molecular dimensions, physicochemical characteristics and growth stimulation of lactobacilli and bifidobacteria in the gut microbiota is reported. Arabinoxylan from wheat and rye grains is discussed as an ingredient for gluten and lectin-free bread and baked goods. Galactomannans from legumes and their partially hydrolysed products are presented as sources for specific healthy products against bowel disease and digestive discomfort. Commercial fructan products obtained from inulin, fructan of agave, and fructooligosaccharides are discussed in detail as a selective substrate for fermentation by health-promoting bacteria in the colon, such as lactobacilli and bifidobacteria. Structurally different heteropolysaccharides from mucilages of traditional medicinal plants, such as seeds from psyllium, flax, chan, chia, and basil or cladodes from Opuntia spp., are discussed as useful sources of dietary fibre, with prebiotic characteristics and digestive regulation in the intestinal tract as well. Full article

The aim of this study was to increase the antibacterial spectrum of modified hen egg white lysozyme (HEWL) with thermal and chemical treatments against Gram-negative bacteria. The antibacterial activity of heat-denatured HEWL and chemical denatured HEWL against Gram-negative and Gram-positive bacteria was evaluated in 15 h of incubation tests. HEWL was denatured by heating at pH 6.0 and pH 7.0 and chemical denaturing was carried out for 1.0, 1.5, 2.0, and 4.0 h with DL-Dithiothreitol (DTT). HEWL modified by thermal and chemical treatments was characterized using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis method. Heat-denatured HEWL lytic activity against Micrococcuslysodeikticus lessened with increasing temperature and time of incubation with the chemical agent (DTT). The loss of lytic activity in modified HEWL suggests that the mechanism of action of the antibacterial activity is not dependent on the lytic activity. Thermal and chemical treatments of HEWL enabled the production of oligoforms and increased antibacterial activity over a wider spectrum. Heat-denatured HEWL at pH 6.0 and chemically-denatured HEWL increased the HEWL antibacterial spectrum against Gram-negative bacteria (Escherichia coli ATCC 25922). HEWL at 120 °C and pH 6.0 (1.0 mg/mL) inhibited 78.20% of the growth of E. coli. HEWL/DTT treatment for 4.0 h (1.0 mg/mL) inhibited 68.75% of the growth E. coli. Heat-denatured HEWL at pH 6.0 and pH 7.0 and chemically-denatured HEWL (1.0, 1.5, 2.0, and 4.0 h with DTT) were active against Gram-positive bacteria (Staphylococcus carnosus CECT 4491T). Heat-denatured and chemical-denatured HEWL caused the death of the bacteria with the destruction of the cell wall. LIVE/DEAD assays of fluorescent dye stain of the membrane cell showed membrane perturbation of bacteria after incubation with modified HEWL. The cell wall destruction was viewed using electron microscopy. The results obtained in this study suggest that heat-denatured HEWL at pH 6.0 and chemical-denatured HEWL treatments increase the HEWL antibacterial activity against Gram-negative bacteria. Full article