pharmaceutics Article A Multifunctional Trypsin Protease Inhibitor from Yellow Bell Pepper Seeds: Uncovering Its Dual Antifungal and Hypoglycemic Properties Juliana Cotabarren 1, *,† , Brenda Ozón 1,† , Santiago Claver 1 , Florencia Geier 1 , Martina Rossotti 1 , Javier Garcia-Pardo 2, * and Walter David Obregón 1, * 1 Centro de Investigación de Proteínas Vegetales (CIPROVE), Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas, Universidad Nacional de la Plata, 47 y 115 s/N, La Plata B1900AVW, Buenos Aires, Argentina 2 Departament de Bioquimica i Biologia Molecular, Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain * Correspondence: cotabarren.juliana@biol.unlp.edu.ar (J.C.); javiergarciapardo@msn.com (J.G.-P.); davidobregon@biol.unlp.edu.ar (W.D.O.); Tel.: +54-221-423-5333 (ext. 57) (J.C. & W.D.O.); +34-93-586-8936 (J.G.-P.) † These authors contributed equally to this work. Abstract: Fungal infections are a growing public health concern worldwide and the emergence of antifungal resistance has limited the number of therapeutic options. Therefore, developing novel strategies for identifying and developing new antifungal compounds is an active area of research in the pharmaceutical industry. In this study, we purified and characterized a trypsin protease inhibitor obtained from Yellow Bell Pepper (Capsicum annuum L.) seeds. The inhibitor not only showed potent and specific activity against the pathogenic fungus Candida albicans, but was also found to be non-toxic against human cells. Furthermore, this inhibitor is unique in that it also inhibits α-1,4-glucosidase, positioning it as one of the first plant-derived protease inhibitors with dual biological activity. This exciting discovery opens new avenues for the development of this inhibitor Citation: Cotabarren, J.; Ozón, B.; as a promising antifungal agent and highlights the potential of plant-derived protease inhibitors as a Claver, S.; Geier, F.; Rossotti, M.; rich source for the discovery of novel multifunctional bioactive molecules. Garcia-Pardo, J.; Obregón, W.D. A Multifunctional Trypsin Protease Inhibitor from Yellow Bell Pepper Keywords: antifungal; bioactive compound; bioactive peptide; Capsicum annuum L.; Candida albicans; Seeds: Uncovering Its Dual protease; protease inhibitor; phytocystatin; trypsin inhibitor; natural product Antifungal and Hypoglycemic Properties. Pharmaceutics 2023, 15, 781. https://doi.org/10.3390/ pharmaceutics15030781 1. Introduction Academic Editor: Antonio Vassallo In the last decade, the increase in antifungal resistance among pathogenic fungi has become a serious concern worldwide. Recent studies have suggested that pathogenic fungi Received: 24 January 2023 are responsible of over 150 million severe fungal infections and about 1.7 million infectious- Revised: 17 February 2023 disease related deaths annually [1]. Fungi, which can be unicellular or multicellular Accepted: 21 February 2023 eukaryotic organisms, can survive in a wide range of environmental conditions. Due to Published: 27 February 2023 their eukaryotic nature, the number of currently available drugs to treat invasive fungal infections is limited to a reduced number of chemical entities [2]. These compounds perform their actions via three different main mechanisms: (I) inhibiting ergosterol biosynthesis Copyright: © 2023 by the authors. and/or its availability, (II) inhibiting the DNA/RNA biosynthesis, and (III) inhibiting Licensee MDPI, Basel, Switzerland. fungal cell wall biosynthesis and membrane sterols. The irresponsible use of antifungals This article is an open access article has led to the emergence of increasingly resistant pathogenic strains, leaving very few distributed under the terms and therapeutic options to treat aggressive fungal infections. Therefore, developing novel conditions of the Creative Commons strategies for identifying and developing novel antifungal compounds is an active area of Attribution (CC BY) license (https:// research in the pharmaceutical industry. creativecommons.org/licenses/by/ One promising strategy to develop novel antifungals is targeting their virulence 4.0/). factors [3]. Many pathogenic fungi produce molecules that facilitate the adhesion to Pharmaceutics 2023, 15, 781. https://doi.org/10.3390/pharmaceutics15030781 https://www.mdpi.com/journal/pharmaceutics Pharmaceutics 2023, 15, 781 2 of 13 the host tissues. Another common virulence strategy is the production and secretion of proteases, such as aspartic and serine proteases, which allows pathogenic fungi to survive and penetrate host tissues during infection. Given the relevant role of extracellular fungal proteases, it has been speculated that specific protease inhibitors (PIs) may be a promising strategy for the development of novel efficient antifungal drugs. Plants have traditionally been a crucial source for identifying and developing new molecules with antifungal properties. In recent years, medicinal plants and plant-derived extracts have been used as important sources for antifungal drug discovery [4,5]. Higher plants possess the ability to produce a wide variety of secondary metabolites with high chemical diversity, including proteins, peptides, sugars, and nucleosides. Many of these compounds, such as plant-derived proteins and peptides, are PIs with demonstrated antibacterial [6,7] and antifungal properties [8]. Plants are rich in small proteinaceous PIs as these molecules are involved in plant defense mechanisms against pathogens and predators [5,9,10]. In addition, plant proteins contain a relatively high content of disulfide bridges, which allows plant-derived PIs to effectively slow or inhibit the catalytic action of their target enzymes, even under extreme environmental conditions [5]. Compared to small chemical molecules, natural inhibitors are often less toxic, which leads to better tolerability. In addition, these proteins have showed a remarkable multifunctionality. Such properties can be exploited for antifungal drug development. In this study, we report the isolation and characterization of a thermostable trypsin protease inhibitor obtained from Yellow Bell Pepper (Capsicum annuum L.) seeds. We have evaluated the inhibitory capacity against a set of pathogenic bacteria and fungi. Ultimately, this inhibitor showed both potent and specific activity in vitro against pathogenic Candida albicans and hypoglycemic activity. Furthermore, our studies also suggest that this natu- rally occurring molecule is safe and has no toxicity against human cells. Taken together, the current results demonstrate that this novel inhibitor could effectively be used as an antifungal agent in pharmaceutical preparations to prevent invasive candida infections. 2. Materials and Methods 2.1. Materials The seeds of the Yellow Bell Pepper (Capsicum annuum) were hand-collected from local farmers around La Plata, Buenos Aires, Argentina. The seeds were processed as previously described elsewhere [11]. The reagents sodium chloride, Coomassie Blue G-250, N,N,N0 ,N0 -tetramethylethylenediamine (TEMED), tris (hydroxymethyl) aminomethane, sodium dodecyl sulphate (SDS), bovine serum albumin (BSA), β-mercaptoethanol (β ME), Nα-benzoyl-DL-arginine-p-nitroanilide (BApNA), and 4-nitrophenol-α-D-glucopyranoside (PNPG) were purchased from Sigma-Aldrich (San Luis, MO, USA). The proteases used in this study, such as Trypsin and α-Glucosidase, were obtained from Sigma-Aldrich. To perform PI purification, a Glyoxyl-agarose resin was obtained from FlukaTM. Fluconazole was purchased from Signa-Aldrich. All chemicals and reagents used in this study were of analytical grade, unless otherwise specified. 2.2. Cell Lines and Pathogenic Microbial Strains Escherichia coli (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 29213), Klebsiella pneumoniae (ATCC 700603), Enterococcus faecalis (ATCC 29212), Candida albicans (CIPROVE), Candida tropicalis (CIPROVE), Candida glabrata (CIPROVE), Candida krusei (CIPROVE), Rhodotorula spp. (CIPROVE) and Saccharomyces cerevisiae (CIPROVE) were obtained from CIPROVE. 2.3. Crude Extract Preparation To prepare the extracts, Capsicum annuum seeds were washed with distilled water and stored at −20 ◦ C until PI extraction. Capsicum annuum dry seeds (about 30 g) were ground using a mechanic blender. The sample was grounded with 100 mL of 0.01 M phosphate buffer, 0.1 M NaCl, pH 7.4 placed in an ice bath to avoid possible protein denaturation. Pharmaceutics 2023, 15, 781 3 of 13 Afterwards, the mixture was incubated 3 h at 4 ◦ C and filtered using a gauze. The resultant homogenate was centrifuged at 7000× g for 30 min at 4 ◦ C, and the supernatant (from now on: YBPCE) was collected and frozen at −20 ◦ C until further processing. The total protein content present in the sample was as described in our previous publication [12] using the Bradford’s assay [13]. Bovine serum albumin (BSA) was used as standard (0.1 mg/mL). Next, the trypsin inhibitory activity present in the clarified homogenate was determined as described below. 2.4. Trypsin Inhibition Measurements Trypsin inhibitory activity of the different samples was determined using Nα-benzoyl- DL-arginine-p-nitroanilide (BApNA) as a substrate. To perform the inhibitory activity experiments, we adapted the method of Erlanger, Kokowsky and Cohen [14] to 96- well plate measurements. To perform the experiments, trypsin (0.25 mg/mL) was pre-incubated with increasing concentrations of the extract (concentrations ranging from 0 to 50 µg/mL) in 100 mM Tris-HCl buffer (pH 7.5) with 50 mM CaCl2 . The samples were pre-incubated for 10 min at 37 ◦ C, and then the BApNA substrate was added to each well at a final concentration of 1 mM. The hydrolysis of BApNA was monitored by recording increases in the absorbance at 410 nm using a Tecan Infinite M200 PRO spectrophotometer (Männedorf, Switzerland). The plates were incubated at 37 ◦ C for 10 min. One trypsin inhibitory unit (1 TIU) was defined as the decrease of 0.01 unit of absorbance at 410 nm per 10 min assay, at 37 ◦ C. All experiments were carried out in triplicate. 2.5. Heat Treatment and Affinity Chromatography Purification According to our previous studies, it is common that protease inhibitors present high physicochemical stability with minimal loss of inhibitory activity [5]. Accordingly, in the first purification step, the crude extract (named here as YBPCE) was subjected to 100 ◦ C for 5 min. After cooling at room temperature, thermally denatured proteins were removed by centrifugation for 30 min at 7000× g and 4 ◦ C. Afterwards, the total protein content and the inhibitory activity of the non-treated crude extract and heat-treated sample (named hereafter as YBPHT) were evaluated. For protein purification, a sample of YBPHT containing 40.4 ± 0.1 µg/mL of protein was loaded to a Trypsin-glyoxyl-agarose column (1.5 × 10 cm) previously equilibrated with 0.1 M Tris-HCl buffer (pH 8.0) containing 0.2 M NaCl. This protocol was optimized previously by our group [15]. In the first step of purification, the unbound proteins were eluted with equilibration buffer, and then affiliated proteins were eluted with HCl pH 2.0. The eluted fractions were adjusted to pH 7.0 with 0.1 M NaOH, and the fractions exhibit- ing trypsin inhibitory activity were pooled (see previous Section 2.3 Trypsin inhibition measurements). 2.6. Biochemical Characterization of the Inhibitor 2.6.1. Inhibitory Assays and Ki Determination The inhibition constant (Ki) of YBPTI was determined by performing a Dixon plot analysis (1/v vs. [I], where [I] is the inhibitor concentration). Previously, the inhibition of trypsin activity in the presence of different concentrations of the inhibitor and two different substrate concentrations was evaluated. The inhibition experiments were performed with increasing concentrations of the inhibitor YBPTI (0–3.2 µg/mL) and BApNA (1.0 and 2.0 mM). Bovine trypsin was assayed at a fixed concentration of 0.25 mg/mL, similarly to that described in [16]. The Ki was determined from a Dixon plot, where the reciprocal of the enzyme reaction rate was expressed as 1/v. The Ki value was derived from the intersection of the two lines plotted for two different BApNA concentrations. All the reactions were performed in triplicate. Pharmaceutics 2023, 15, 781 4 of 13 2.6.2. YBPTI Stability Studies The physico-chemical stability of the purified YBPTI was evaluated. First, the effect of the protease inhibitor under extreme temperatures was evaluated as previously de- scribed [12]. In brief, YBPTI aliquots were incubated at 100 ◦ C for different periods of time (i.e., 30, 60, 90, 120, 150 and 180 min). After incubation, the samples were cooled at room temperature and the residual trypsin inhibitory activity was determined as described in Section 2.4. Second, the effect of pH on YBPTI stability was studied. To perform the experiments, the residual activity after incubation at extreme pHs (pH 2 and pH 12) for 30 and 60 min at 25 ◦ C was determined. The residual trypsin inhibitory activity of YBPTI was measured as detailed above. 2.7. Biological Assays 2.7.1. Antimicrobial Activity against Pathogenic Bacterial and Fungal Strains The antifungal activity of the YBPCE, YBPHT, and purified YBPTI against a set of and pathogenic microbial strains (see Section 2.2) was evaluated using the agar diffusion assay. This method was based on the Kirby–Bauer test with slight modifications [17]. In essence, for the preparation of cell cultures of C. albicans, an inoculum from a fungal stock was transferred to a Petri dish containing Sabouraud agar and allowed to grow at 36 ◦ C for 2 days. The fungal suspension was adjusted with physiological solution to 0.3 McFarland scale (105 cells/mL). To perform the assays, 10 µL of each test sample was placed over the previously inoculated Sabouraud agar plates. Once the drop placed in the plate was dried, the plates were then incubated at 36 ◦ C for 24 h to allow fungal growth. After incubation, the diameters of the fungal growth inhibition were measured. 2.7.2. Antimicrobial Activity against Pathogenic Candida Albicans To further investigate the antifungal activity of YBPCE, YBPHT, and purified YBPTI on Candida albicans growth, the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) for all these samples was determined. To perform the experiments, C. albicans was seeded at a concentration of 105 cells/mL onto 96-well plates. The plates were then incubated at 36 ◦ C in 200 µL microplates in the absence and presence of different sample concentrations (i.e., YBPCE: 1653.1–2.3 µg/mL; YBPHT: 687.6–0.9 µg/mL; YBPTI: 51.1–0.07 µg/mL). Fluconazole (3.5 µg/mL) was also evaluated as reference drug with known antifungal properties. The absorbance of all the wells was measured (at 620 nm) at different time points of incubation (0, 2, 4, 16, 18 and 20 h) using a Tecan Infinite M200 PRO spectrophotometer (Männedorf, Switzerland) plate reader. All the growth inhibition experiments were performed in triplicate. The MIC was determined for each sample as the minimum sample concentration required to reduce yeast growth on 50%. The MFC was determined as the minimum sample concentration required to produce complete growth inhibition. After 20 h of incubation in the presence of these compounds, yeast cells were visualized using an optical microscope at 1000× magnification (Nikon Eclipse OPT-01514) by direct observation and with 30 min incubation with methylene blue (1:1 relation). The yeast cells grown in the absence of YBPTI were also determined as the control condition. 2.7.3. Plasma Membrane Permeabilization Experiments Plasma membrane permeabilization experiments were performed by investigating SYTOX Green (Molecular Probes Invitrogen, EUA) uptake, as described previously by [18] with some modifications. Briefly, a culture of C. albicans (105 cells/mL) was incubated in the absence or in the presence of YBPTI at the concentration of 5.7 µg/mL for 20 h. Aliquots of the suspension of yeast cells were incubated with 0.2 µM SYTOX Green (1:1 ratio) for 20 min at 25 ◦ C. Afterwards, the cells were observed in an optical microscope (IMLD Biosystems) equipped with a fluorescence filter set for fluorescein detection (excitation wavelengths: 450–490 nm; emission wavelength: 500 nm). Both negative and positive in the absence of peptide or with 3.5 µg/mL fluconazole, respectively, were performed as control conditions. Pharmaceutics 2023, 15, 781 5 of 13 2.7.4. Hypoglycemic Activity The α-Glucosidase inhibitory activity was evaluated by the [19] method, with slight modifications. The original protocol was adapted to a 96- well plate measurements, using the substrate 4-nitrophenol-α-D-glucopyranoside (PNPG). In brief, a fixed amount of α- glucosidase (0.5 U/mL) was preincubated with different concentrations of the inhibitor (ranging from 0 to 689 ng/mL) in 100 mM sodium phosphate buffer (pH 7.4). After 10 min pre-incubation at 37 ◦ C, the substrate was added to each reaction mixture at a final concentration of 0.2 mM. The hydrolysis of PNPG was recorded through the increase in the absorbance at 405 nm at 37 ◦ C every minute for 20 min. All the measurements were carried out in triplicate. 2.7.5. Cytotoxicity Assays The cytotoxicity of the purified trypsin inhibitor toward Hela (ATCC CCL-2) cells was evaluated using a MTT assay, similarly as described elsewhere [20,21]. To perform the experiments, the cells were seeded in 96-well plates at a concentration of 1.0 × 104 cells per well and incubated for 24 h. Afterwards, the cells were treated with the indicated inhibitor concentrations. After 24 h of incubation, aliquots of an MTT solution (0.5 mg/mL) were added to each well. The plates were then incubated for an additional 3 h at 37 ◦ C. After incubation, the supernatant was removed and 100 µL of DMSO were added to each well. The absorbance at 540 nm of each well was measured using a UV-vis microplate reader MultiSkan FC (Thermo fisher Scientific, Waltham, MA, USA). 3. Results and Discussion 3.1. Isolation and Purification of YBPTI The yellow bell pepper (Capsicum annuum L.) belongs to the genus Capsicum (vegetable pepper). Different species from this genus are commonly used as food products, either as fresh vegetables or as processed foods [22]. During the last few years, different reports have suggested that Capsicum annuum seeds are a rich source of protease inhibitors [11,23–26]. These reports demonstrated the presence of mainly serine proteinase inhibitors such as trypsin and chymotrypsin. Examples of such inhibitors are: PSI-1.1 and PSI-1.2, which have been isolated from paprika seeds [23], CaTI isolated from chilli pepper seeds [24,25], and CapA1 and CapA2 obtained from the leaves of Capsicum annum var. Phule Jyoti. More recently, PIJP was isolated from jalapeño pepper [26]. Since yellow bell pepper is a rich source of protease inhibitors, we prepared a crude extract of Capsicum annuum L. seeds (hereafter named as YBPCE) to evaluate its trypsin inhibitory capacity. The protein concentration of the sample was 802.4 ± 1.12 mg/mL (see Table 1), which is in agreement with previous studies [11]. We have next evaluated the trypsin inhibitory activity of the crude extract. The presence of the extract in the reaction caused a rapid decrease in the trypsin activity, showing the specific inhibitory activity of 0.55 TIU/mg. The effect of temperature (100 ◦ C) on the trypsin inhibitory activity of YBPCE is shown Table 1. After treatment, we observed a significant increase in the specific inhibitory activity of the sample between the heated to 100 ◦ C and unheated extract, suggesting that such a trypsin inhibitor is a thermostable molecule. In agreement, we observed a 2.1-fold of purification after heat treatment, suggesting that other non- thermostable proteins have been removed. Table 1. Trypsin inhibitory activity. Total Protein Total Inhibitory Specific Inhibitory Activity Purification Step Purity (Fold) b Yield (%) c Amount (mg) Activity (TIU) a (TIU/mg) Pharmaceutics 2023, 15, 781 6 of 13 Crude extract 80.24 ± 0.17 44 ± 0.05 0.55 ± 0.01 1 100 100 eat treatment 35.53 ± 1. Table 0.06 39.4 ±activity. Trypsin inhibitory 0.05 1.11 ± 0.01 2.1 89.5 Affinity chromatog- Total Protein Total Inhibitory Specific Inhibitory b c Purification Step 0.48 ± 0.02 37.1(TIU) ± 0.03a 77.29 ± Purity (Fold) 0.06 Yield (%) 140.5 84.3 Amount (mg) Activity Activity (TIU/mg) raphy Crude extract 80.24 ± 0.17 44 ± 0.05 0.55 ± 0.01 1 100 100 eat treatment aOne ± 35.53 trypsin 0.06 inhibitory 39.4unit (1 TIU) was defined ± 0.05 as the amount of 1.11 ± 0.01 2.1inhibitor that decreased 89.5 absorb- Affinity ance at 410 nm by 0.1 under the assay conditions; b The purification index (Purity) was calculated 0.48 ± 0.02 37.1 ± 0.03 77.29 ± 0.06 140.5 84.3 chromatography as the ratio between the specific inhibitory activity determined after each purification step as com- pared a Onetotrypsin the initial inhibitory inhibitory activity unit (1 TIU) present was defined inamount as the the crude extract. of inhibitor Yield of absorbance thatc decreased inhibitoryatactivity 410 nm after b The purification index (Purity) was calculated as the ratio between the specific each purification step compared to the crude extract (%). by 0.1 under the assay conditions; inhibitory activity determined after each purification step as compared to the initial inhibitory activity present in the crude extract. c Yield of inhibitory activity after each purification step compared to the crude extract (%). After the initial characterization, we decided to perform further purification of the trypsinAfter inhibitor found the initial in the yellow pepper characterization, we decidedseeds. to For this purpose, perform we selected further purification of the the heat- treated trypsinfraction, inhibitorand weinperformed found the yellow apepper high-speed centrifugation seeds. For this purpose,step to clarify we selected thethe sample heat- (see Materials treated andand fraction, Methods for details). we performed The resultant a high-speed sample centrifugation was step applied to clarify thetosample single-step (see Materials purification andon based Methods for details). The a glyoxyl-agarose resultant matrix sampleinwas prepared applied house, to single-step containing the target purification based on a glyoxyl-agarose matrix prepared in house, enzyme covalently immobilized on the resin. After purification by affinity containing the chromatog- target enzyme raphy, thecovalently immobilized trypsin inhibitory on the resin. activity of theAfter purification eluted fractionsbywasaffinity chromatography, evaluated. As shown in the trypsin inhibitory activity of the eluted fractions was evaluated. As shown in Figure 1A, Figure 1A, the purified inhibitor eluted in a single sharp peak with a specific inhibitory the purified inhibitor eluted in a single sharp peak with a specific inhibitory activity against activity trypsinagainst of 77.29trypsin TIU/mgof(Table 77.291). TIU/mg The high(Table 1). The specificity andhigh specificity efficiency of the and efficiency of purification themethod purification method allowed us to obtain a highly purified sample of allowed us to obtain a highly purified sample of the trypsin inhibitor from yellow the trypsin in- hibitor pepperfrom seedsyellow pepper suitable seedsinhibition for further suitable kinetics for further inhibition kinetics characterization. characterization. Figure 1. 1.Purification Figure Purificationand and kinetic characterizationofof kinetic characterization Yellow Yellow BellBell Pepper Pepper trypsin trypsin inhibitor inhibitor (YBPTI). (YBPTI). (A)(A) Affinity chromatography on immobilized trypsin. Fractions with trypsin inhibitory Affinity chromatography on immobilized trypsin. Fractions with trypsin inhibitory activity (grey activity (grey area) were pooled and named YBPTI, (B) Dixon plot (1/v vs. [I]) for identification of the Ki. Eachthe Ki. area) were pooled and named YBPTI, (B) Dixon plot (1/v vs. [I]) for identification of Each point point represents represents the mean the mean ofestimates. of three three estimates. 3.2.3.2. InhibitionKinetics Inhibition Kinetics and and Physicochemical Physicochemical Properties Propertiesof YBPTI of YBPTI Inhibitionkinetic Inhibition kinetic studies studiesofofYBPTI YBPTI against trypsin against activity trypsin was carried activity out following was carried a out following protocol developed in our laboratory [12]. Analysis of the data revealed that YBPTI has an a protocol developed in our laboratory [12]. Analysis of the data revealed that YBPTI has IC50 value of 3.9 µg/mL (2.05 × 10−7 −7 M) and a Ki value of 1.7 × 10−6 M−6(see Figure 1B). anThe IC50Kivalue of 3.9 μg/mL (2.05 × 10 M) and a Ki value of 1.7 × 10 M (see Figure 1B). value obtained for this inhibitor is in the range of other previously described The Ki value obtained for this trypsin inhibitors purified frominhibitor is in the natural sources, range such as theofprotease other previously described inhibitor isolated from tryp- sinB.inhibitors purified microplus larvae from (Ki = 1.20 natural − × 10 M)7 sources, such [27], the as the Kunitz protease inhibitor inhibitor isolated isolated from from Boophilus B. microplus microplus(Kilarvae = 1.08(Ki × 10 −7 M) = 1.20 × [28], 10−7 or M)the [27], the TcTI Kunitz trypsin inhibitor inhibitor isolated obtained from from Boophilus Torresea cearensis(Ki 1.4 ××10 (Ki == 1.08 − 6 microplus 10 M) −7 M) [29], [28],among or the others. TcTI trypsin inhibitor obtained from Torresea cea- rensis (Ki = 1.4 × 10−6 M) [29], among others. 5, x FOR PEER REVIEW 7 of 13 Pharmaceutics 2023, 15, 781 7 of 13 To evaluate the stability of YBPTI at different temperatures and pHs, the protein was incubated at 100 °C for various amounts of time and at pH 2 and 12 for 30 and 60 min at 25 °C. The residual trypsin inhibitory To evaluate activity the stability was then of YBPTI assessed. at different As shown temperatures in Figure and pHs, 2A, was the protein ◦ after incubating forincubated 30 min at 100 C at 100 °C,forthe residual various trypsin amounts of timeinhibitory and at pH activity of 30 2 and 12 for theand YBPTI 60 min at 25 ◦ C. The residual trypsin inhibitory activity was then assessed. As shown in Figure 2A, was 99.67 ± 5.55%; that is, almost all of its activity was maintained. Interestingly, after one after incubating for 30 min at 100 ◦ C, the residual trypsin inhibitory activity of the YBPTI hour of exposure towasthis extreme temperature, the YBPTI sample maintained 74.65 ± 4.03% 99.67 ± 5.55%; that is, almost all of its activity was maintained. Interestingly, after one of its trypsin inhibitory hour ofactivity, exposurewhich makestemperature, to this extreme this peptide theinhibitor a remarkable, YBPTI sample highly maintained 74.65 ± 4.03% stable molecule. Based on these of its trypsin findings, inhibitory wewhich activity, can conclude makes this that YBPTI peptide wasa remarkable, inhibitor found to be highly stableretaining stable at extreme pHs, molecule. approximately Based on these findings, 60% ofwe itscan conclude activity that1YBPTI after was found to be h of incubation stable at extreme pHs, retaining approximately 60% of its activity after 1 h of incubation at at both pH 2 and 12. both pH 2 and 12. Figure 2. Figure 2. Physicochemical Physicochemical stability of YBPTI. stability of YBPTI. (A) Effect (A) Effect of temperature of temperature and (B) pH andtreatment (B) pH treatment on theon the trypsinof trypsin inhibitory activity inhibitory YBPTI. activity In (A),of YBPTI. the In (A), asterisk (*)the asterisk (*) indicates indicates significantly significantly differentdifferent compared compared to time 0. In (B), the asteric (*) indicates significantly different compared to control conditions. In In to time 0. In (B), the asteric (*) indicates significantly different compared to control conditions. both cases, a one-way ANOVA (p < 0.05) and Tukeýs multiple comparison test was performed. both cases, a one-way ANOVA (p < 0.05) and Tukeýs multiple comparison test was performed. There are very few studies that have investigated the thermal stability of proteins at There are very100 few◦ C. studies Previously,that have investigated a reduced the high number of PIs with thermal stability stability of proteins at extreme temperaturesat and pHs have been studied. Most of these inhibitors showed 100 °C. Previously, a reduced number of PIs with high stability at extreme temperatures interesting biological activities, and pHs have beensuch as potential studied. Mostasofbiopesticides, with inhibitory these inhibitors activity on insect showed interesting intestinal biological proteases activi- or inhibition of larval growth: HSTI [30], C11PI [31], CFPI [32] and RsBBI1 [33]. Other ties, such as potential as biopesticides, previously with activities reported biological inhibitoryfor activity on insect these inhibitors intestinal include prote- antibacterial activity ases or inhibition ofagainst larvalS.growth: HSTI [30], C11PI [31], CFPI [32] and RsBBI1 [33]. aureus by LzaBBI [34] and anticoagulant activity in the extrinsic coagulation Other previously reported biological pathway activities by MpBBI for these [35]. Despite inhibitors the great includepotential pharmaceutical antibacterial of theseactivity highly stable plant-derived inhibitors, the biological activities of these PIs against S. aureus by LzaBBI [34] and anticoagulant activity in the extrinsic coagulation have been little explored. Overall, our results show that YBPTI is a highly stable trypsin inhibitor, with few pathway by MpBBI [35]. Despite the great pharmaceutical potential of these highly stable reports of other proteins with similar stability at extreme temperatures and pHs. This plant-derived inhibitors, stability,the biological combined with activities of these PIs its trypsin inhibitory havemakes activity, beenYBPTIlittle aexplored. potentially useful Overall, our results protein for show that YBPTI pharmaceutical is a highly stable trypsin inhibitor, with few applications. reports of other proteins with similar stability at extreme temperatures and pHs. This sta- 3.3. Antifungal Activity of YBPTI bility, combined with its trypsin inhibitory activity, makes YBPTI a potentially useful pro- The antifungal activity of YBPTI was evaluated against a set of fungal strains. As tein for pharmaceutical shownapplications. in Figure 3, a clear area of growth inhibition was observed for C. albicans and S. cerevisiae, tested with 5 µg of YBPTI, and an inhibition halo of 21 mm was observed 3.3. Antifungal Activity for C.ofalbicans, YBPTIwhen 66 µg of YBPTI were added to the plate. Interestingly, no inhibitory effect of the Capsicum annuum L. inhibitor was observed for other fungal or bacterial strains The antifungal activity of YBPTI was evaluated against a set of fungal strains. As (Figure 3 and Table S1). shown in Figure 3, a clear area of growth inhibition was observed for C. albicans and S. cerevisiae, tested with 5 μg of YBPTI, and an inhibition halo of 21 mm was observed for C. albicans, when 66 μg of YBPTI were added to the plate. Interestingly, no inhibitory effect of the Capsicum annuum L. inhibitor was observed for other fungal or bacterial strains (Figure 3 and Table S1). , x FOR PEER REVIEW Pharmaceutics 2023, 15, 781 8 of 13 8 of 13 Figure 3. Growth inhibition of fungal strains by YBPTI. The growth inhibition of seven fungal Figure 3. Growth inhibition of fungal strains by YBPTI. The growth inhibition of seven fungal strains strains was evaluated by agar diffusion: (A) C. albicans, (B) C. krusei, (C) Rodothorula, (D) C. tropicalis, was evaluated by agar(E)diffusion: (A) C. albicans, (B) C. krusei, (C) Rodothorula, (D) C. tropicalis, (E) C. C. glabrata, (F) S. cerevisiae. In (A–F) 5 µg of YBPTI were tested. In (G), C. albicans was assayed glabrata, (F) S. cerevisiae. withIn66(A–F) 5 μgThe µg YBPTI. of treatments YBPTI were are: tested. In (G), (i) Nystatin, C. albicans(iii) (ii) Fluconazole, was assayed distilled with water, (iv)66 100 mM μg YBPTI. The treatments are: (i) Nystatin, (ii) phosphate buffer pH 7.4, (v) YBPTI. Fluconazole, (iii) distilled water, (iv) 100 mM phos- phate buffer pH 7.4, (v) YBPTI. Given that C. albicans is a pathogen of great importance in clinics, we decided to further investigate the inhibitory activity of YBPTI against this microorganism. The effect Given that C. albicans is a pathogen of great importance in clinics, we decided to fur- of the purified YBPTI was evaluated using serial dilutions in SB broth by adding increasing ther investigate the sample inhibitory activity ofThe concentrations. YBPTI minimumagainst this microorganism. inhibitory concentration (MIC) Thewas effect of determined the purified YBPTI towas be evaluated 5.7 µg/mL, usingshowing serial dilutions a fungicidal in SB effect broth by adding of MFC/MIC increasing < 4 against this pathogen. sample concentrations. The minimum inhibitory concentration (MIC) was determined which Under similar experimental conditions, fluconazole showed a MIC of 3.5 µg/mL, to is be 5.7 μg/mL, showingcertainly close to theeffect a fungicidal MIC observed of MFC/MICfor YBPTI < obtained 4 against from thisyellow pepper Under pathogen. seeds. Next, we performed a morphological analysis by light microscopy to confirm the toxic similar experimental conditions, fluconazole showed a MIC of 3.5 μg/mL, which is cer- effect of the YBPTI on C. albicans (Figure 4). Through direct observation it was possible tainly close to the MIC observed the to demonstrate forpresence YBPTI obtained from and of agglomerated yellow pepper darkened cellsseeds. (Figure 4B), unlike the Next, we performed a morphological control cells that showed normal analysis by light fungal growth and microscopy morphology (Figure to confirm the 4A). Then, SYTOX toxic effect of the YBPTI on C. albicans (Figure 4). Through direct observation it was pos- yeast Green was used to confirm that YBPTI is able to disrupt the plasma membrane of cause cell death. Overall, these results suggest that YBPTI from Capsicum annuum sible to demonstratecells theandpresence of agglomerated and darkened cells (Figure 4B), unlike L. has the potential to be developed as a natural drug for treating infections caused by the control cells that C. showed albicans. normal fungal growth and morphology (Figure 4A). Then, SYTOX Green was usedAs tomentioned confirm that above, YBPTI manyispathogenic able to disrupt the plasma fungi produce membrane extracellular of that proteases yeast cells and causecancell playdeath. an active role in the Overall, development these of diseases. results suggest thatVirulence YBPTI from and optimal growth of Capsicum annuum L. has the fungi depend potential toonbeseveral secreted as developed extracellular a naturalproteases, drug for among whichinfections treating serine proteases are of particular interest. This proteolytic system allows fungi to survive and penetrate caused by C. albicans. tissues. One of the first examples of this phenomenon was studied for the first time in As mentioned tomatoes above, many infectedpathogenic with Phytophthorafungi produce infestans [36], extracellular proteases in which the increase thatlevels in trypsin can play an active role in the development and trypsin of diseases. inhibitors were correlated withVirulence and optimal plant resistance growthInofrecent to the pathogen. years, an increasing number of antimicrobial peptides fungi depend on several secreted extracellular proteases, among which serine proteases rich in cysteine residues have been isolated from plants, particularly from seeds, such as Abelmoschus moschatus [37]. Indeed, are of particular interest. This proteolytic system allows fungi to survive and penetrate previous studies on other varieties of Capsicum annuum L. have also shown that they have tissues. One of the peptide first examples of this inhibitors with phenomenon antifungal activity duewas studied to their plasmaformembrane the firstpermeabilizing time in tomatoes infected with Phytophthora capacity infestans [24,25]. Similarly, other[36], serineinand which the increase metalloprotease in trypsin inhibitors levels have been studied and have been reported to possess antimicrobial activities and trypsin inhibitors were correlated with plant resistance to the pathogen. In recent [10]. years, an increasing number of antimicrobial peptides rich in cysteine residues have been isolated from plants, particularly from seeds, such as Abelmoschus moschatus [37]. Indeed, previous studies on other varieties of Capsicum annuum L. have also shown that they have peptide inhibitors with antifungal activity due to their plasma membrane permeabilizing capacity [24,25]. Similarly, other serine and metalloprotease inhibitors have been studied and have been reported to possess antimicrobial activities [10]. Pharmaceutics 2023, 15, x FOR PEER REVIEW 9 of 13 Pharmaceutics 2023, 15, 781 9 of 13 Effectof Figure4.4.Effect Figure ofYBPTI YBPTI on C. albicans on C. albicans cell cellmorphology. morphology.(A) (A)Representative Representative optical microscopy optical microscopy images im- of control ages C. albicans, of control and (B) C. albicans, andthe (B)same cells treated the same with YBPTI cells treated (5.7 µg/mL). with YBPTI (C) C. albicans (5.7 μg/mL). stained (C) C. albicans with methylene stained blue (C) with methylene in the blue (C) absence or (D) or in the absence presence of YBPTI (D) presence (5.7 µg/mL). of YBPTI (E–H) C. (5.7 μg/mL). albicans (E–H) C. permeabilization albicans assay using permeabilization assay SYTOX green. green. using SYTOX Representative optical Representative microscopy optical images microscopy of con- images of control trol C. C. albicans albicans stainedwith stained withSYTOX SYTOXgreen greeninin(E) (E)presence presence oror (F) (F) absence of YBPTI YBPTI (5.7 (5.7μg/mL). µg/mL). (G,H) (G,H)shows showsfluorescence fluorescencemicroscopy microscopyimages imagesofof(E,F), (E,F),respectively. respectively.InIn(E,F), (E,F),SYTOX SYTOXfluorescence fluorescence emission is shown in green. emission is shown in green. 3.4.Hypoglycemic 3.4. HypoglycemicActivityActivityofofYBPTI YBPTI Amongthe Among therisk riskgroups groupsthat thatare aremore more susceptible susceptible to to infection infection by Candida spp. by Candida spp. are are patientswith patients withdiabetes diabetes mellitus mellitus (DM) (DM) [38], [38], whose whose infections infections are aggravated are aggravated in cases in cases of un-of uncontrolled controlled hyperglycemia hyperglycemia [39].[39]. For reason, For this this reason, it hasitbeen has been previously previously suggested suggested that that reg- regulating ulating bloodblood glucose glucose levels levels may may be an be an important important factor factor forfor thethe preventionand prevention andtreatment treatment ofofcomorbid comorbidinvasive invasivecandida candidainfections. infections. In this study, we have further investigated In this study, we have further investigatedthe theinhibitory inhibitoryactivity activityYBPTI YBPTI against againstα-1,4- α- glucosidase. As shown in Figure 5, YBPTI was able to inhibit the 1,4-glucosidase. As shown in Figure 5, YBPTI was able to inhibit the enzyme α-1,4-gluco- enzyme α-1,4-glucosidase with an sidase IC50 with anvalue IC50 of 75.33of±75.33 value 1.17 ng/mL. It is expected ± 1.17 ng/mL. that inhibiting It is expected the activity that inhibiting of α-1,4- the activity ofglucosidase leads toleads α-1,4-glucosidase a decrease in the release to a decrease of free glucose in the release from complex of free glucose carbohydrates, from complex carbo- thereby lowering local blood glucose levels [40]. Indeed, hypoglycemic-lowering hydrates, thereby lowering local blood glucose levels [40]. Indeed, hypoglycemic-lower- activity tests ing have been activity tests rarely described have been rarelyfor PIs, being described formostly reported PIs, being for reported mostly peptides for derived from peptides derived from protein hydrolysates [41,42]. In this regard, peptides from whey protein have protein hydrolysates [41,42]. In this regard, peptides from whey protein hydrolysates hy- been shown drolysates haveto have beenstrong shownα-1,4-glucosidase inhibitory activity,inhibitory to have strong α-1,4-glucosidase with an IC50 of 3.5with activity, mg/mL, an according IC50 to research of 3.5 mg/mL, by Konrad according et al. [41]. to research bySimilar Konradlevelset al. of inhibition [41]. Similar of α-1,4-glucosidase levels of inhibition were reported by Matsui et al. [43] and Yu et al. [44] for different peptides. Pharmaceutics 2023, 15, x FOR PEER REVIEW 1 of α-1,4-glucosidase were reported by Matsui et al. [43] and Yu et al. [44] for dif Pharmaceutics 2023, 15, 781 10 of 13 peptides. Figure 5. Effect of YBPTI on the α-glucosidase activity. YBPTI showed a conce inhibition of α-glucosidase activity. Note that the derived IC50 value is 75.33 ± 3.5. Cytotoxicity of YBPTI against HeLa Cells To Figure evaluate 5. Effect of YBPTIthe on thetoxicity of activity. α-glucosidase YBPTI against YBPTI showed ahuman cells, in vitro c concentration-dependent Figure with 5. Effect HeLa inhibition of YBPTI cells of α-glucosidase on theNote wasactivity. carried α-glucosidase out, activity. similarly that the derived IC50to YBPTI those value showed described is 75.33 a concentration-dep previously ± 1.17 ng/mL. inhibition of α-glucosidase activity. Note that the derived IC50 value is 75.33 ± 1.17 ng/mL. molecules [45,46]. 3.5. Cytotoxicity of YBPTIThese assays against HeLa Cells were performed using the range of con YBPTI inhibited To evaluate 3.5. Cytotoxicity the growth the toxicity of YBPTI of Candida of YBPTI against against HeLa albicans human cells, Cells in vitroin the range cytotoxicity assays0.15 with μg/mL HeLa cells was carried out, similarly to those described previously for other bioactive shown molecules in[45,46]. Figure To evaluate the6,toxicity These a value of of assays were around YBPTI performed 5 μg/mL against human using the would range ofcells, in maintain almosta vitro cytotoxicity concentrations where while YBPTI with ainhibited HeLa value ofgrowth cells the was 40 μg/mL carried out,would of Candida albicansmaintain similarly in tothe range those close topreviously 0.15 µg/mL described 80% to 40 cell µg/mL. viability. for other bioT As shown mated in Figure IC50 6, a value is certainly of around higher5 µg/mL would maintain almost 90% cell viability molecules [45,46]. These assays werethan 50 μg/mL, performed using thea value range almost 10-fold o of concentrations while a value of 40 µg/mL would maintain close to 80% cell viability. Thus, the approxi- YBPTI MIC mated inhibited determined the against IC50 is certainly growth of C.Candida higher than albicans. 50 µg/mL, albicans a value in the range almost 10-fold 0.15 μg/mL over the to 40 μg/m observed shown in Figureagainst MIC determined 6, a value of around 5 μg/mL would maintain almost 90% cell vi C. albicans. while a value of 40 μg/mL would maintain close to 80% cell viability. Thus, the ap mated125 IC50 is certainly higher than 50 μg/mL, a value almost 10-fold over the obs MIC determined against C. albicans. 100 viability (%) 125 75 100 Cell(%) 50 75 Cell viability 25 50 25 0 10 20 40 0 15 3 6 2 5 5 0. 0. 1. 2. 0. 0 YBPTI concentration (μg/ml) 10 20 40 0 15 3 6 2 5 5 0. 0. 1. 2. 0. Figure 6. Cytotoxicity of YBPTI against HeLa cells. Effect of YBPTY on the viability of HeLa cells YBPTI concentration (μg/ml) Figure 6.incubation. after 24 h Cytotoxicity of YBPTI against HeLa cells. Effect of YBPTY on the via after Figure24 6. h On incubation. Cytotoxicity the of findings, basis of these YBPTI against we can HeLa cells. state that ourEffect of did inhibitor YBPTY on the not show viability of HeL a reduction in cell after 24 viability at the assayed inhibitor concentrations in the range 0.15 to 40 µg/mL, which h incubation. demonstrates On thethe safetyof basis of these this natural compound findings, wefor can the potential treatment state that C. albicans ourofinhibitor did n infections, as well as other pharmaceutical applications. tion On the basis in cell of these viability findings, at the we can assayed state that inhibitor our inhibitor did concentrations innot theshow rangear tion in cell viability at the assayed inhibitor concentrations in the range 0.15 to 40 μ which demonstrates the safety of this natural compound for the potent which demonstrates the safety of this natural compound for the potential treatmen albicans infections, as well as other pharmaceutical applications. albicans infections, as well as other pharmaceutical applications. 4. Conclusions 4. Conclusions Pharmaceutics 2023, 15, 781 11 of 13 4. Conclusions The increasing antifungal resistance among pathogenic fungi is a serious global con- cern. The limited number of currently available drugs to treat invasive fungal infections has led to the emergence of increasingly resistant strains, leaving few therapeutic options. Targeting virulence factors, such as extracellular proteases, is a promising strategy for the development of novel antifungal drugs. Herein, we report the purification and characterization of a thermostable trypsin inhibitor from Capsicum annuum L. seeds with dual antifungal and hypoglycemic proper- ties. This natural inhibitor was obtained from Yellow Bell Pepper (Capsicum annuum L.) seeds by heat-treatment and affinity purification with immobilized trypsin. The purified inhibitor showed potent and specific activity in vitro against pathogenic Candida albicans, and was found to be safe and non-toxic against human cells. Additionally, the inhibitor also exhibited α-1,4-glucosidase inhibition activity, positioning this inhibitor as one of the first plant-derived molecules with such a particular dual combination of biological activities. Our results demonstrate that this novel inhibitor could potentially be used as an antifungal agent in pharmaceutical preparations to prevent invasive candida infections. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/pharmaceutics15030781/s1, Table S1: Evaluation of the antibacterial activity of Capsicum annuum L. crude extract (YBPCE) and heat-treated sample (YBPHT). Author Contributions: J.C., B.O., S.C., F.G., M.R. and W.D.O. performed experiments, analyzed the data, and wrote the initial draft of the manuscript. J.C., J.G.-P. and W.D.O. assisted with the design of experiments and with the writing and revision of the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: J.C. and W.D.O. are members of the Researcher Career Program of the CONICET. B.O. is a doctoral fellow from National Agency of Scientific and Technological Promotion (ANPCyT, Argentina). The authors acknowledge the support from Universidad Nacional de La Plata (Argentina) under grants PPID X/014 and PPID X/038 and the ANPCyT under Project PICT-2016-4365. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available in this article or in the associated Supporting Information file. Conflicts of Interest: The authors declare no conflict of interest. References 1. Kainz, K.; Bauer, M.A.; Madeo, F.; Carmona-Gutierrez, D. Fungal Infections in Humans: The Silent Crisis. Microb. Cell 2020, 7, 143–145. [CrossRef] [PubMed] 2. Lee, Y.; Puumala, E.; Robbins, N.; Cowen, L.E. Antifungal Drug Resistance: Molecular Mechanisms in Candida Albicans and Beyond. Chem. Rev. 2021, 121, 3390–3411. [CrossRef] 3. Burchacka, E.; Pi˛eta, P.; Łupicka-Słowik, A. Recent Advances in Fungal Serine Protease Inhibitors. Biomed. Pharmacother. 2022, 146, 112523. [CrossRef] [PubMed] 4. Otvos, R.A.; Still, K.B.M.; Somsen, G.W.; Smit, A.B.; Kool, J. Drug Discovery on Natural Products: From Ion Channels to NAChRs, from Nature to Libraries, from Analytics to Assays. SLAS DISCOVERY Adv. Life Sci. RD 2019, 24, 362–385. [CrossRef] 5. Cotabarren, J.; Lufrano, D.; Parisi, M.G.; Obregón, W.D. Biotechnological, Biomedical, and Agronomical Applications of Plant Protease Inhibitors with High Stability: A Systematic Review. Plant Sci. 2020, 288, 110398. [CrossRef] 6. Cisneros, J.S.; Cotabarren, J.; Parisi, M.G.; Vasconcelos, M.W.; Obregón, W.D. Purification and Characterization of a Novel Trypsin Inhibitor from Solanum Tuberosum Subsp. Andigenum Var. Overa: Study of the Expression Levels and Preliminary Evaluation of Its Antimicrobial Activity. Int. J. Biol. Macromol. 2020, 158, 1279–1287. [CrossRef] [PubMed] 7. Cotabarren, J.; Claver, S.; Payrol, J.A.; Garcia-pardo, J.; Obregón, W.D. Purification and Characterization of a Novel Thermostable Pa- Pain Inhibitor from Moringa Oleifera with Antimicrobial and anticoagulant Properties. Pharmaceutics 2021, 13, 512. [CrossRef] [PubMed] 8. Gutierrez-Gongora, D.; Geddes-Mcalister, J. From Naturally-Sourced Protease Inhibitors to New Treatments for Fungal Infections. J. Fungi 2021, 7, 1016. [CrossRef] Pharmaceutics 2023, 15, 781 12 of 13 9. Shamsi, T.N.; Parveen, R.; Fatima, S. Characterization, Biomedical and Agricultural Applications of Protease Inhibitors: A Review. Int. J. Biol. Macromol. 2016, 91, 1120–1133. [CrossRef] 10. Kim, J.Y.; Park, S.C.; Hwang, I.; Cheong, H.; Nah, J.W.; Hahm, K.S.; Park, Y. Protease Inhibitors from Plants with Antimicrobial Activity. Int. J. Mol. Sci. 2009, 10, 2860–2872. [CrossRef] 11. Cotabarren, J.; Tellechea, M.E.; Avilés, F.X.; Lorenzo Rivera, J.; Obregón, W.D. Biochemical Characterization of the YBPCI Miniprotein, the First Carboxypeptidase Inhibitor Isolated from Yellow Bell Pepper (Capsicum Annuum L). A Novel Contribution to the Knowledge of Miniproteins Stability. Protein Expr. Purif. 2018, 144, 55–61. [CrossRef] 12. Cotabarren, J.; Broitman, D.J.; Quiroga, E.; Obregón, W.D. GdTI, the First Thermostable Trypsin Inhibitor from Geoffroea Decorticans Seeds. A Novel Natural Drug with Potential Application in Biomedicine. Int. J. Biol. Macromol. 2020, 148, 869–879. [CrossRef] [PubMed] 13. Bradford, M.M. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal. Biochem. 1976, 72, 248–254. [CrossRef] 14. Erlanger, B.F.; Kokowsky, N.; Cohen, W. The Preparation and Properties of Two New Chromogenic Substrates of Trypsin. Arch. Biochem. Biophys. 1961, 95, 271–278. [CrossRef] 15. Obregón, W.D.; Ghiano, N.; Tellechea, M.; Cisneros, J.S.; Lazza, C.M.; López, L.M.I.; Avilés, F.X. Detection and Characterisation of a New Metallocarboxypeptidase Inhibitor from Solanum Tuberosum Cv. Desirèe Using Proteomic Techniques. Food Chem. 2012, 133, 1163–1168. [CrossRef] 16. Tellechea, M.; Garcia-Pardo, J.; Cotabarren, J.; Lufrano, D.; Bakas, L.; Avil?s, F.; Obregon, W.; Lorenzo, J.; Tanco, S. Microplate Assay to Study Carboxypeptidase A Inhibition in Andean Potatoes. Bio-Protocol 2016, 6, e2032. [CrossRef] 17. Bauer, A.W.; Kirby, W.M.; Sherris, J.C.; Turck, M. Antibiotic Susceptibility Testing by a Standardized Single Disk Method. Am. J. Clin. Pathol. 1966, 45, 493–496. [CrossRef] 18. Thevissen, K.; Terras, F.R.G.; Broekaert, W.F. Permeabilization of Fungal Membranes by Plant Defensins Inhibits Fungal Growth. Appl. Environ. Microbiol. 1999, 65, 5451–5458. [CrossRef] 19. Kim, Y.M.; Wang, M.H.; Rhee, H.I. A Novel α-Glucosidase Inhibitor from Pine Bark. Carbohydr. Res. 2004, 339, 715–717. [CrossRef] 20. Mosmann, T. Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays. J. Immunol. Methods 1983, 65, 55–63. [CrossRef] 21. Twentyman, P.R.; Luscombe, M. A Study of Some Variables in a Tetrazolium Dye (MTT) Based Assay for Cell Growth and Chemosensitivity. Br. J. Cancer 1987, 56, 279–285. [CrossRef] [PubMed] 22. Anaya-Esparza, L.M.; de la Mora, Z.V.; Vázquez-Paulino, O.; Ascencio, F.; Villarruel-López, A. Bell Peppers (Capsicum Annum L.) Losses and Wastes: Source for Food and Pharmaceutical Applications. Molecules 2021, 26, 5341. [CrossRef] 23. Antcheva, N.; Pintar, A.; Patthy, A.; Simoncsits, A.; Barta, E.; Tchorbanov, B.; Pongor, S. Proteins of Circularly Permuted Sequence Present within the Same Organism: The Major Serine Proteinase Inhibitor from Capsicum Annuum Seeds. Protein Sci. 2001, 10, 2280–2290. [CrossRef] [PubMed] 24. Ribeiro, S.F.F.; Silva, M.S.; Da Cunha, M.; Carvalho, A.O.; Dias, G.B.; Rabelo, G.; Mello, É.O.; Santa-Catarina, C.; Rodrigues, R.; Gomes, V.M. Capsicum Annuum L. trypsin Inhibitor as a Template Scaffold for New Drug Development against Pathogenic Yeast. Antonie van Leeuwenhoek. Int. J. Gen. Mol. Microbiol. 2012, 101, 657–670. [CrossRef] 25. Ribeiro, S.F.F.; Fernandes, K.V.S.; Santos, I.S.; Taveira, G.B.; Carvalho, A.O.; Lopes, J.L.S.; Beltramini, L.M.; Rodrigues, R.; Vasconcelos, I.M.; Da Cunha, M.; et al. New Small Proteinase Inhibitors from Capsicum Annuum Seeds: Characterization, Stability, Spectroscopic Analysis and a CDNA Cloning. Biopolymers 2013, 100, 132–140. [CrossRef] 26. Tamhane, V.A.; Chougule, N.P.; Giri, A.P.; Dixit, A.R.; Sainani, M.N.; Gupta, V.S. In Vivo and in Vitro Effect of Capsicum Annum Proteinase Inhibitors on Helicoverpa Armigera Gut Proteinases. Biochim. Biophys. Acta–Gen. Subj. 2005, 1722, 156–167. [CrossRef] 27. Tanaka, A.S.; Andreotti, R.; Gomes, A.; Torquato, R.J.S.; Sampaio, M.U.; Sampaio, C.A.M. A Double Headed Serine Proteinase Inhibitor—Human Plasma Kallikrein and Elastase Inhibitor—from Boophilus Microplus Larvae. Immunopharmacology 1999, 45, 171–177. [CrossRef] 28. Sasaki, S.D.; de Lima, C.A.; Lovato, D.V.; Juliano, M.A.; Torquato, R.J.S.; Tanaka, A.S. BmSI-7, a Novel Subtilisin Inhibitor from Boophilus Microplus, with Activity toward Pr1 Proteases from the Fungus Metarhizium Anisopliae. Exp. Parasitol. 2008, 118, 214–220. [CrossRef] 29. Sampaio, C.A.; Oliva, M.L.; Tanaka, A.S.; Sampaio, M.U. Proteinase Inhibitors in Brazilian Leguminosae. Memórias Do Inst. Oswaldo Cruz 1991, 86, 207–209. [CrossRef] 30. Aguirre, C.; Valdés-Rodríguez, S.; Mendoza-Hernández, G.; Rojo-Domínguez, A.; Blanco-Labra, A. A Novel 8.7 KDa Protease Inhibitor from Chan Seeds (Hyptis Suaveolens L.) Inhibits Proteases from the Larger Grain Borer Prostephanus Truncatus (Coleoptera: Bostrichidae). Comp. Biochem. Physiol. B Biochem. Mol. Biol. 2004, 138, 81–89. [CrossRef] 31. Swathi, M.; Lokya, V.; Swaroop, V.; Mallikarjuna, N.; Kannan, M.; Dutta-Gupta, A.; Padmasree, K. Structural and Functional Characterization of Proteinase Inhibitors from Seeds of Cajanus Cajan (Cv. ICP 7118). Plant Physiol. Biochem. 2014, 83, 77–87. [CrossRef] [PubMed] 32. Dantzger, M.; Vasconcelos, I.M.; Scorsato, V.; Aparicio, R.; Marangoni, S.; Macedo, M.L.R. Bowman-Birk Proteinase Inhibitor from Clitoria Fairchildiana Seeds: Isolation, Biochemical Properties and Insecticidal Potential. Phytochemistry 2015, 118, 224–235. [CrossRef] [PubMed] Pharmaceutics 2023, 15, 781 13 of 13 33. Mohanraj, S.S.; Tetali, S.D.; Mallikarjuna, N.; Dutta-Gupta, A.; Padmasree, K. Biochemical Properties of a Bacterially-Expressed Bowman-Birk Inhibitor from Rhynchosia Sublobata (Schumach.) Meikle Seeds and Its Activity against Gut Proteases of Achaea Janata. Phytochemistry 2018, 151, 78–90. [CrossRef] [PubMed] 34. Martins, T.F.; Vasconcelos, I.M.; Silva, R.G.G.; Silva, F.D.A.; Souza, P.F.N.; Varela, A.L.N.; Albuquerque, L.M.; Oliveira, J.T.A. A Bowman-Birk Inhibitor from the Seeds of Luetzelburgia Auriculata Inhibits Staphylococcus Aureus Growth by Promoting Severe Cell Membrane Damage. J. Nat. Prod. 2018, 81, 1497–1507. [CrossRef] [PubMed] 35. Lazza, C.; Trejo, S.; Obregon, W.; Pistaccio, L.; Caffini, N.; Lopez, L. A Novel Trypsin and α-Chymotrypsin Inhibitor from Maclura Pomifera Seeds. Lett. Drug Des. Discov. 2010, 7, 244–249. [CrossRef] 36. Woloshuk, C.P.; Meulenhoff, J.S.; Sela-Buurlage, M.; Van Den Elzen, P.J.M.; Cornelissen, B.J.C. Pathogen-Induced Proteins with Inhibitory Activity toward Phytophthora Infestans. Plant Cell 1991, 3, 619–628. [CrossRef] [PubMed] 37. Dokka, M.K.; Davuluri, S.P. Antimicrobial Activity of a Trypsin Inhibitor from the Seeds of Abelmoschus Moschatus L. Int. J. Curr. Microbiol. Appl. Sci. 2014, 3, 184–199. 38. Mohammed, L.; Jha, G.; Malasevskaia, I.; Goud, H.K.; Hassan, A. The Interplay Between Sugar and Yeast Infections: Do Diabetics Have a Greater Predisposition to Develop Oral and Vulvovaginal Candidiasis? Cureus 2021, 13. [CrossRef] [PubMed] 39. Rodrigues, C.F.; Rodrigues, M.E.; Henriques, M. Candida Sp. Infections in Patients with Diabetes Mellitus. J. Clin. Med. 2019, 8, 76. [CrossRef] 40. Jaiswal, N.; Bhatia, V.; Srivastava, S.P.; Srivastava, A.K.; Tamrakar, A.K. Antidiabetic Effect of Eclipta Alba Associated with the Inhibition of Alpha-Glucosidase and Aldose Reductase. Nat. Prod. Res. 2012, 26, 2363–2367. [CrossRef] 41. Konrad, B.; Anna, D.; Marek, S.; Marta, P.; Aleksandra, Z.; Józefa, C. The Evaluation of Dipeptidyl Peptidase (DPP)-IV, α- Glucosidase and Angiotensin Converting Enzyme (ACE) Inhibitory Activities of Whey Proteins Hydrolyzed with Serine Protease Isolated from Asian Pumpkin (Cucurbita Ficifolia). Int. J. Pept. Res. Ther. 2014, 20, 483–491. [CrossRef] [PubMed] 42. Wang, R.; Zhao, H.; Pan, X.; Orfila, C.; Lu, W.; Ma, Y. Preparation of Bioactive Peptides with Antidiabetic, Antihypertensive, and Antioxidant Activities and Identification of α-Glucosidase Inhibitory Peptides from Soy Protein. Food Sci. Nutr. 2019, 7, 1848–1856. [CrossRef] [PubMed] 43. Matsui, T.; Oki, T.; Osajima, Y. Isolation and Identification of Peptidic α-Glucosidase Inhibitors Derived from Sardine Muscle Hydrolyzate. Z. Für Naturforsch. Sect. C J. Biosci. 1999, 54, 259–263. [CrossRef] 44. Yu, Z.; Yin, Y.; Zhao, W.; Yu, Y.; Liu, B.; Liu, J.; Chen, F. Novel Peptides Derived from Egg White Protein Inhibiting Alpha- Glucosidase. Food Chem. 2011, 129, 1376–1382. [CrossRef] 45. Covaleda-Cortés, G.; Hernández, M.; Trejo, S.A.; Mansur, M.; Rodríguez-Calado, S.; García-Pardo, J.; Lorenzo, J.; Vendrell, J.; Chávez, M.A.; Alonso-del-Rivero, M.; et al. Characterization, recombinant production and structure-function analysis of NvCI, A picomolar metallocarboxypeptidase inhibitor from the marine snail nerita versicolor. Mar. Drugs 2019, 17, 511. [CrossRef] [PubMed] 46. Maculins, T.; Garcia-Pardo, J.; Skenderovic, A.; Gebel, J.; Putyrski, M.; Vorobyov, A.; Busse, P.; Varga, G.; Kuzikov, M.; Zaliani, A.; et al. Discovery of protein-protein interaction inhibitors by integrating protein engineering and chemical screening platforms. Cell Chem. Biol. 2020, 27, 1441–1451. [CrossRef] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.