Exploring the Functional Potential of the Xyrophytic Greek Carob (Ceratonia siliqua, L.) Cold Aqueous and Hydroethanolic Extracts

Featured Application

Cold infusions of Greek carob (Ceratonia siliqua L.) pods have a rich phenolic composition and can serve as a natural source of antimicrobial and antidiabetic agents. These extracts may be used in the development of functional beverages, dietary supplements, and plant-based therapeutics aimed at glycemic control and food preservation.

Abstract

The present study investigates the antimicrobial, antioxidant, and in vitro antidiabetic potential of cold infusions prepared from different parts of the Greek carob tree (Ceratonia siliqua L.), which is a xerophytic species. Carob samples, including green and ripe pods and leaves, were collected from an urban area of Attica, Greece, and extracted using food-grade solvents (water and a water–ethanol mixture, 90:10, v/v). The extracts were evaluated for antibacterial activity against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 6538 using automated turbidometry. In addition, total phenolic content and antioxidant and antiradical activities were determined via spectrophotometry; the phenolic profile was analyzed using liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-QToF-MS), and α-amylase inhibitory activity was assessed through an in vitro assay. All extracts exhibited statistically significant (p < 0.05) bacteriostatic effects, with green pods and leaves showing the highest activity. Ripe pods demonstrated the most potent α-amylase inhibition (up to 96.43%), especially when extracted with water–ethanol mixture (90:10, v/v). Liquid chromatography coupled with tandem quadrupole/time-of-flight mass spectrometry (LC-QToF-MS) analysis revealed a rich phenolic profile across all samples. While carob leaves showed no α-amylase inhibition, their phenolic profile suggests other potential health-related bioactivities. These findings support the development of carob-based functional food products and highlight the nutritional and pharmaceutical potential of this resilient Mediterranean crop.

1. Introduction

The carob tree (Ceratonia siliqua L.) belongs to the Fabaceae family and is native to the Mediterranean region, where it thrives in mild, dry climates and poor soils [1]. The flora of countries like Greece is characterized by rich biodiversity, and carob trees are part of the endemic plant species, particularly valued for their quality and contribution to the country’s agricultural heritage [2]. Furthermore, the carob tree is considered a xerophytic species that is well-adapted to arid and semi-arid climates due to its low water requirement; therefore, it can be used for sustainable land use in Mediterranean ecosystems [3,4]. In recent years, there has been a growing interest in exploring and utilizing xyrophytes, including Greek carobs, as they also offer significant potential for both nutrition and health. This heightened focus stems from a global shift toward natural and functional foods, as well as an increasing demand for sustainable and regionally unique crops. Greek carobs, in particular, have gained attention for their ability to combine rich cultural heritage with exceptional nutritional and bioactive properties, making them a valuable addition to modern diets and a subject of scientific research [5,6,7]. Their versatility and potential to contribute to health-promoting diets have positioned them as a promising candidate in the development of innovative food products and nutraceuticals. These fruits are rich in nutritional compounds, such as carbohydrates, minerals, essential oils, vitamins, and carotenoids. They also contain various bioactive compounds like polyphenols, which contribute to their unique aroma, flavor, and health-promoting properties [6,8].

Carob fruit, particularly Greek carobs, plays a significant role in various industries due to its versatile applications. In the food industry, carob is used for producing locust bean gum, which is used as a thickener (known as E410) and as a stabilizer in emulsions [9]. Additionally, it is widely used to create popular products such as drinks, syrups, and confections, including traditional carob syrup and seedless carob pods are often used as a substitute for chocolate or cacao, while carob by-products serve as livestock feed [10,11,12,13]. Furthermore, carob is highly valued in traditional medicine for preventing and managing various health issues [14] and has applications in the pharmaceutical, cosmetic, and textile industries.

The carob pod consists of two main components: pulp (90%) and seeds (10%) [7]. The pulp is rich in carbohydrates (primarily sucrose), fibers, minerals [15], vitamins [16], and proteins [17], with low fat content [18]. It also contains secondary metabolites such as phenolic acids, flavonoids, and tannins, which exhibit numerous biological activities. These include antioxidant and anti-inflammatory properties [12], as well as antimicrobial, antiviral, anticarcinogenic, and anti-allergic activities [19]. Many recent studies have explored the biοactive compounds of carob extracts. Benyaich et al. (2025) compared Moroccan and Mediterranean varieties, emphasizing the diversity in phenolic content and associated biological activities [20]. Dahmani et al. (2025) and Serio et al. (2025) explored the antioxidant, vasorelaxant, and anti-inflammatory potential of carob leaves, while Dahmani et al. (2023) presented the ethnopharmacological profile of the plant, along with its traditional uses and bioactivity spectrum [21,22,23]. Ikram et al. (2023) presented the nutritional and clinical applications of carob, particularly its antidiabetic, hypocholesterolemic, and antimicrobial activities, whereas Khalil et al. (2025) focused on carob seeds as a source of flavonoid derivatives with anticancer potential [24,25]. Moreover, Mesías et al. (2025) and Mohamed Ahmed et al. (2025) investigated the antioxidant and antiglycation properties of carob flour and teas, respectively, providing evidence for their functional food applications [26,27]. Furthermore, the increasing global prevalence of diabetes, which is expected to impact approximately 600 million people by the year 2050, poses significant challenges for public health systems. This underscores the pressing need to develop natural anti-diabetic agents capable of effectively inhibiting glucose-inducing enzymes without triggering harmful side effects [28,29]. One of the clinical strategies for the treatment of type II diabetes is the inhibition of the hydrolytic enzyme α-amylase. Various studies have demonstrated that several natural compounds from edible plants can effectively inhibit glucose-inducing enzymes, such as α-glucosidase and α-amylase, offering potential alternatives for diabetes management with fewer side effects compared to synthetic drugs [30,31,32].

While the carob tree (Ceratonia siliqua L.) has been extensively studied for its nutritional and medicinal properties, most existing research has focused on methanolic, acetonic, or ethanolic extracts of carob pods or seeds [20,22,24]. Fewer studies have studied the bioactivity of cold aqueous and food-grade hydroethanolic extracts, particularly from different plant parts (green pods, ripe pods, and leaves) of Greek carob varieties. Moreover, although the antidiabetic and antimicrobial potential of carob has been reported, comparative studies evaluating α-amylase inhibition, antioxidant capacity, and LC-QToF-MS phenolic profiling across these plant parts using cold infusion techniques are scarce. Therefore, this study aims to bridge these gaps by (i) comparing the antimicrobial, antioxidant, and in vitro antidiabetic activities of cold aqueous and hydroethanolic extracts (water–ethanol, 90:10 v/v) from green pods, ripe pods, and leaves of the Greek carob tree and by (ii) characterizing their polyphenolic profiles using liquid chromatography coupled with tandem quadrupole/time-of-flight mass spectrometry (LC-QToF-MS). This food-grade approach provides novel insights into the valorization of a traditional Mediterranean crop through sustainable, low-impact processing. The findings are expected to provide a foundation for developing functional food supplements that capitalize on carob’s health-promoting properties.

2. Materials and Methods

2.1. Sampling and Extraction

Carob samples from different parts of the plant (green carob pods, mature carob pods, leaves) were collected from carob trees on the campus of the University of West Attica based in Egaleo, Athens, Greece (latitude of 37.996° N, longitude of 23.681° E) from April to November 2024 during three different harvest periods. Unripe green pods were collected in April and May 2024 and named “Green pod April” and “Green pod May” accordingly. Mature pods, named “Ripe pods”, and leaves were collected in November 2024. Upon arrival at the laboratory, carob pods and leaves were washed with tap water and cut into pieces, approximately 1–2 cm each. An amount of 20 g from each sample was weighed and placed in sterilized borosilicate glass laboratory bottles with 200 mL of either 100% water or water–ethanol (90:10, v/v) for 7 days at room temperature. Samples were then filtered with filter paper and subsequently micro-filtered with 20 nm syringe filters. The choice of food-grade solvents (water and water–ethanol mixture) for the extraction of carob parts was based on their possible use as infusions.

2.2. Evaluation of Antimicrobial Activity

Antimicrobial activity was assessed by measuring the growth rate of the tested microorganisms in the presence of various carob extracts. Bacterial growth curves were generated using automated turbidometry with the Bioscreen C instrument (Lab Systems, Helsinki, Finland) in 100-well honeycomb plates [33]. Antimicrobial activity was tested against 2 major pathogenic bacteria, the Gram-negative Escherichia coli ATCC 25922 and the Gram-positive Staphylococcus aureus ATCC 6538. The bacterial strains were incubated overnight at 37 °C in sterile Brain Heart Infusion (BHI, Condalab, Madrid, Spain) medium. Following vortexing of the culture tubes, 100 μL of the inoculum was diluted in 9 mL of sterile phosphate-buffered water (APHA) to obtain an initial optical density (OD) of approximately 0.1. Samples were prepared by adding 50 μL of the microorganism suspension (~10⁷ cfu/mL), 50 μL of the tested natural extract, and 200 μL of sterilized BHI broth in each well, resulting in a total volume of 300 μL. Two controls were prepared by using 50 μL of the bacterial suspension, 50 μL of either water or the hydroethanolic solution (water–ethanol 90:10, v/v), and 200 μL of sterilized BHI broth. All plate preparation steps were performed within a Class II Biosafety Cabinet. An OD measurement of each well was performed at 600 nm every 30 min for 48 h at 37 °C, with 10 s of shaking prior to each measurement. All experiments were conducted in triplicate. After incubation, the data were exported to Excel, and growth curves were generated by plotting OD against time for each sample. These curves represent the growth behavior of microorganisms under the specified conditions. Data analysis was conducted using the ComBase web resource (https://combase.errc.ars.usda.gov/, accessed on 10 June 2024) that fits primary growth curves to OD or CFU (Colony Forming Units) log data over time and estimates key microbial kinetic parameters, including the maximum specific growth rate (μmax) and lag time. Bacteriostatic activity was determined by observing a reduced growth rate (μ) during the logarithmic phase in the presence of the tested extract. This approach enables comparison between samples and controls while preserving the functional context of cold infusions.

2.3. Spectrophotometric Assays

2.3.1. Determination of Total Phenolic Content (TPC)

The total phenolic content (TPC) of the carob extracts was determined using a modified micromethod version of the Folin–Ciocâlteu colorimetric assay [34]. Ten microliters of each sample, standard solution, or blank [water or a water–ethanol (90:10, v/v) mixture] were added to 2500 μL of water and 200 μL of Folin–Ciocâlteu reagent. The mixture was thoroughly mixed and left to stand for 8 min. Then, 500 μL of saturated sodium carbonate solution was added, and it was mixed well. The cuvettes were left at 40 °C for 30 min. The absorbance of the cooled samples at room temperature was measured at 750 nm with a Vis spectrophotometer (Spectro 23, Digital Spectrophotometer, Labomed, Inc., Los Angeles, CA, USA). The TPC was expressed as milligrams (mg) gallic acid equivalents (GAE) per L of extract. A standard curve was used with 25–2600 mg/L of gallic acid (y = 0.0005x + 0.0786, R² = 0.9989).

2.3.2. Ferric Reducing/Antioxidant Power Assay (FRAP)

The ferric reducing antioxidant power (FRAP) assay was used to evaluate the antioxidant capacity of the extracts based on their ability to reduce Fe(III) to Fe(II). The assay, which involves the reduction of a ferric–TPTZ [2,4,6-tris(2-pyridyl)-s-triazine] complex to its ferrous form, was performed according to the method of Benzie and Strain (1996), with minor modifications [35,36]. The FRAP reagent was prepared by mixing 500 μL of 10 mM TPTZ in 40 mM of HCl, 500 μL of 20 mM FeCl₃·6H₂O, and 5.0 mL of 0.3 M acetate buffer (pH 3.6). In 96-well plates, 60 μL of extract or standard FeSO₄·7H₂O solution was combined with 52 μL of acetate buffer (300 mM, pH 3.6) and 88 μL of the freshly prepared FRAP reagent. The mixture was incubated at 37 °C for 20 min, and absorbance was measured at 593 nm using a Thermo Scientific Varioskan Flash Multimode Reader (Waltham, MA, USA). A standard calibration curve (y = 0.0003x + 0.0081, R² = 0.9969) was constructed using ten concentrations of FeSO₄·7H₂O (50–1800 μM), and results were expressed as mg FeSO₄·7H₂O per liter of extract.

2.3.3. Scavenging Activity on 2,2′-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic Acid) Radical (ABTS•+)

The antiradical activity of carob extracts was evaluated using a slightly modified version of the method described by Re et al. (1999) [37]. This assay measures the ability of compounds to scavenge the stable ABTS radical cation (ABTS•+), with Trolox—a water-soluble analog of vitamin E—used as the reference antioxidant. The ABTS•+ stock solution was generated by reacting 7 mM ABTS•+ with 2.45 mM sodium persulfate (Na₂S₂O₈) in a 1:1 volume ratio, followed by incubation in the dark at room temperature for 16 h. Prior to use, the solution was diluted with ethanol to achieve an absorbance of 0.7–0.8 at 734 nm. For the assay, 15 μL of carob extract (20–25 mg mL⁻¹) or Trolox standard solution was added to 1500 μL of the diluted ABTS•+ solution. After 1 min of stirring in the dark, the reduction in absorbance was recorded using a UV-Vis spectrophotometer (Novaspek III, Amersham Biosciences, Piscataway, NJ, USA). A 6.0 mM stock solution of Trolox was prepared in ethanol. Appropriate blank solutions were included to account for potential errors due to ABTS•+ dilution. More specifically, the corrected absorbance is given by the equation Af = Ai + (Aa − Ab), where Af is the measured absorbance of the sample (1500 mL ABTS•+ with 15 mL extract), Aa is the absorbance of ABTS•+ solution, and Ab is the absorbance of a solution of 1500 mL ABTS•+ with 15 mL ethanol. The antiradical activity of the extracts was expressed as mg Trolox equivalents (TE) per L of extract, using a standard curve with 0.20–1.50 mM Trolox (y = −0.2876x − 0.002, R² = 0.9995).

2.4. Phenolic Profile by LC-QToF-MS Analysis

2.4.1. Reagents and Materials

LC-MS grade acetonitrile and acetic acid were purchased from Merck (Darmstadt, Germany), while ultrapure water was obtained using a Genie Water System (RephiLe Bioscience Ltd., Shanghai, China). Analytical standards of gallic acid, protocatechuic acid, 4-hydroxybenzoic acid, p-coumaric acid, gentisic acid, vanillic acid, ferulic acid, syringic acid, myricitrin, phloroglucinol, abscisic acid, epicatechin, trans-cinnamic acid, and (–)-catechin were provided by Extrasynthese (Genay, France).

2.4.2. Mass Spectra Analysis

Phenolic compounds were identified using an Agilent 6530 quadrupole time-of-flight (QToF) mass spectrometer equipped with an electrospray ionization (ESI) source, coupled to a 1290 Infinity UHPLC system (Agilent Technologies, Santa Clara, CA, USA). The system operated in negative ionization mode with the following optimized settings: capillary voltage at 4000 V, nebulizer pressure at 45 psi, drying gas flow at 10 L/min (300 °C), fragmentor voltage at 150 V, and skimmer voltage at 65 V. Auto MS/MS acquisition was performed using a collision energy slope of 5 V and an offset of 2.5 V. Internal reference masses (m/z 112.9856 and 1033.9881) were used for continuous calibration. Chromatographic separation was carried out on a Nucleoshell EC C18 column (100 × 4.6 mm, 2.7 μm; Macherey-Nagel, Düren, Germany) with a binary mobile phase consisting of 0.1% acetic acid in water (solvent A) and acetonitrile (solvent B), at a flow rate of 1.0 mL/min. The elution gradient was as follows: 0 min, 10% B; 8 min, 30% B; 12 min, 40% B; 16 min, 50% B; and 18–33 min, 10% B. The injection volume was 10 μL, and the column temperature was maintained at 30 °C.

Data processing was performed using MassHunter software (versions B.06.00 and B.07.00). Compound identification was based on comparison of retention times, accurate mass measurements, and MS/MS fragmentation patterns with those of authentic standards. In the case of quercetin-3-glucoside, annotation was further supported by reference to the Human Metabolome Database (https://www.hmdb.ca, accessed on 15 January 2025).

2.5. In Vitro Antidiabetic Activity

In vitro antidiabetic activity of carob extracts was assessed with the α-amylase inhibitory assay. An amount of 1 g of starch azure (Sigma-Aldrich, St. Louis, MO, USA) was suspended in 100 mL of 100 mM phosphate buffer (pH 6.8) to prepare the substrate solution. The tubes containing 200 μL substrate solution were preincubated at 37 °C for 10 min. Subsequently, 500 μL of phosphate buffer (pH 6.8) was added to all tubes. Then, 0.2 mL of each sample solution was introduced into the tubes containing the substrate solution, followed by the addition of 0.1 mL of Aspergillus oryzae α-amylase (Sigma-Aldrich, St. Louis, MO, USA), prepared in phosphate buffer (pH 6.8) at a concentration of 5 mg/mL. The mixture was incubated at 37 °C for 10 min, after which 350 μL of DNS solution (43 mM DNS in 0.0571 M tartarate acid, 0.4 M NaOH) was supplemented to stop the reaction. The absorbance was measured at 540 nm with a spectrophotometer (Ultrospec 2100 pro, Amersham Biosciences, Piscataway, NJ, USA).

The α-amylase inhibitory activity (AIA) was calculated using the following formula:

$$\mathrm{AIA} \% = {\displaystyle \frac{\left[\right(Ac+ - Ac-) - (As - Ab\left)\right]}{(Ac+ - Ac-) } \times 100}$$

where Ac+ is the absorbance representing 100% enzyme activity (solvent with enzyme), Ac− is the absorbance for 0% enzyme activity (solvent without enzyme), As is the absorbance of the test sample with enzyme, and Ab is the absorbance of the blank (test sample without enzyme).

2.6. Statistical Analysis of Results

Three measurements were conducted in all analyses in order to calculate the average values and standard deviation. Statistical analysis was performed by IBM SPSS 29.0 (IBM Corp., Armonk, NY, USA).

3. Results

3.1. Antimicrobial Activity

The growth curves of the microorganisms as produced by Bioscreen C indicated that all carob extracts exerted bacteriostatic activity against the tested bacteria, both the Gram-negative Escherichia coli ATCC 25922 and the Gram-positive Staphylococcus aureus 6538. The growth rates of the bacteria, which were incubated in the presence of both aqueous and ethanolic carob extracts (Figure 1 and Figure 2), were found to be significantly (p < 0.05) lower than the growth rate of the controls (bacteria grown in nutrient broth). The highest bacteriostatic activity was observed by the green pods and the leaves as compared to the ripe pods. It was also observed that the hydroethanolic solution exerted bacteriostatic activity in both bacteria when it was used as a control.

3.2. Total Phenolic Content (TPC), Antioxidant and Antiradical Activity

The total phenolic content (TPC) and antioxidant and antiradical activity of the carob extracts are presented in Figure 3. Total phenolic content expressed as mg GAE/L ranged from 369.5 to 789.5 mg GAE/L in 100% water extracts and from 492.4 to 1124.1 mg GAE/L in water–ethanol, 90:10, v/v extracts. Total phenolic content was higher in all hydroethanolic carob extracts, indicating that the addition of 10% ethanol in the solvent mixture results in more efficient extraction of total phenolic compounds. The highest TPC was observed in green pod hydroethanolic extracts collected in April and May, as well as in hydroethanolic leaf extracts; however, there was no statistical difference between the leaves and the green pods. Similar trends were observed in antioxidant and antiradical activities of extracts. More specifically, in all hydroethanolic extracts, statistically significantly higher antioxidant and antiradical activity was detected than in aqueous extracts, except in the antioxidant activity of ripe pods.

3.3. Phenolic Profile by LC-QToF-MS Analysis

All carob extracts, either extracted with 100% water or with water–ethanol 90:10 v/v, have shown a rich phenolic profile in which 15 phenolic compounds were identified, suggesting strong bioactive potential (Figure 4). The corresponding MS spectra for each compound are provided in the Supplementary Information. Regarding the distribution of phenolic compounds across samples, ripe pods showed the richest phenolic profile in which all compounds were present, along with the profiles of aqueous extracts of green pods and leaves. The phenolic compounds missing from the profiles were protocatechuic acid from ethanolic extracts of both green pods, vanillic acid from the ethanolic extract of green pod May, and carob leaves and trans-cinnamic acid from the aqueous extract of ripe pods. Carob leaves (especially with 90% H₂O–10% EtOH) also showed a very broad spectrum of phenolics, despite not showing α-amylase inhibition. This may suggest different bioactivity pathways (e.g., antioxidant, anti-inflammatory) not related to α-amylase inhibition.

3.4. In Vitro Inhibition of α-Amylase Activity

The % of in vitro inhibition of α-amylase activity (AIA%) for different carob extracts is presented in Figure 5. The extracts of ripe carob pods and green carobs have shown α-amylase inhibitory activity ranging from 58.82 to 96.43%, but not the extract of leaves. Although water alone as a solvent was efficient in extracting phytochemicals that exerted α-amylase inhibitory activity, it was also shown that across all pod samples, the ethanolic solvent (90% H₂O–10% EtOH) consistently increased AIA% compared to water alone. This suggests that the ethanol–water mixture is more effective in extracting bioactive compounds responsible for α-amylase inhibition. Ripe pods showed the highest inhibition, especially when extracted with the ethanol mixture (up to 96.43%). Green pods showed moderate inhibition, with April samples having higher AIA% than May in the water extracts, possibly because the bioactive content of pods may vary with pod development stage and harvest time. The carob leaves showed zero inhibition (0%) in both solvent systems (not displayed in the graph), indicating the absence of α-amylase inhibitory compounds in the leaves under these extraction conditions.

4. Discussion

4.1. Antimicrobial Activity

In this study, the bacteriostatic activity of cold infusions using food-grade solvents (water and a 90% water–10% ethanol mixture) from carob pods and leaves was observed against the pathogenic bacteria Escherichia coli ATCC 25922 (Gram-negative) and Staphylococcus aureus ATCC 6538 (Gram-positive). Scientific studies have investigated the antimicrobial properties of carob (Ceratonia siliqua) extracts, yielding varied results. A study published in 1997 evaluated the antimicrobial activity of carob extract against several food-related bacteria, including Staphylococcus aureus, Pseudomonas fragi, Listeria monocytogenes, Salmonella enteritidis, and Shewanella putrefaciens. The results indicated a weak inhibitory effect against these bacteria, suggesting limited antimicrobial potential in this context [38]. In another earlier study, the antimicrobial activities of various organic solvents and water extracts of Ceratonia siliqua L. leaves were evaluated by the disc diffusion method against pathogenic bacteria, and the ethanolic and water extracts had a mild antimicrobial activity against two strains of Escherichia coli and two strains of Staphylococcus aureus, among others [39]. Research conducted in 2012 assessed the methanolic extract of carob leaves for its ability to inhibit Listeria monocytogenes. The extract demonstrated inhibitory activity, with a minimum inhibitory concentration (MIC) of 28.12 μg/mL. Chemical analysis identified compounds such as gallic acid, (-)-epigallocatechin-3-gallate, and myricitrin, which may contribute to this antibacterial effect [40]. A 2014 study evaluated acetone and ethanol extracts of carob leaves and pods against Pectobacterium atrosepticum, a pathogen responsible for potato soft rot. The acetone extract of carob leaves exhibited the strongest inhibitory effect, with an IC50 value of 1.5 mg/mL, indicating potential use in controlling this phytopathogen [41]. Research published in 2019 investigated the antibacterial activity of carob syrup against various pathogenic bacteria, including Escherichia coli, Listeria monocytogenes, Staphylococcus aureus and Salmonella enterica. The study found that carob syrup exhibited inhibitory effects on these bacteria, with the most pronounced activity observed against L. monocytogenes [42]. In another study, tea infusion prepared with carob leaves was found to have a significant antibacterial and fungal effect [43]. These studies suggest that carob extracts possess antimicrobial properties, with effectiveness varying depending on the type of extract, bacterial strain, and experimental conditions. Various bioactive compounds such as phenolic acids and flavonoids in carob extracts may contribute to these antimicrobial effects, since plant polyphenols may exert antibacterial action through multiple mechanisms, including disrupting bacterial cell membranes and walls, inhibiting DNA replication and metabolism, interfering with biofilm formation, and affecting bacterial enzymes and protein functions [44]. In a study by Rivas-Gastelum et al. (2025), in silico analyses demonstrated that phenolic compounds and flavonoids such as quercetin have the capacity to inhibit the activity of DNA gyrase B, an enzyme playing a crucial role in bacterial DNA replication and thus exhibiting antibacterial activity [45].

However, the overall antimicrobial efficacy appears to be weak to moderate, and further research is needed to fully elucidate the potential applications of carob extracts in antimicrobial treatments [45].

4.2. Polyphenolic Profile and Antioxidant Activity

The analysis of total phenolic content (TPC), antioxidant capacity (FRAP), and antiradical activity (ABTS•+) revealed significant variations among the different carob extracts. These variations depended on both the plant part and the extraction solvent used. Notably, the hydroethanolic extracts (water–ethanol 90:10 v/v) consistently exhibited higher TPC values compared to aqueous extracts, suggesting that the presence of ethanol enhanced the solubility and extraction of polyphenolic compounds. It is well established that solvent polarity and temperature play a crucial role in extracting different classes of plant polyphenols [8,45]. In our study, we used a cold infusion process with food-grade solvents such as water and a water–ethanol 90:10 v/v mixture. The hydroethanolic solvent was shown to enhance total phenolic content, antioxidant activity, and antiradical activity compared to water alone. This aligns with previous findings indicating that binary solvent mixtures are preferred over their neat counterparts [46] and hydroethanolic solvents often increase the extraction efficiency of polyphenols due to their moderate polarity [9]. Furthermore, plant metabolite composition is influenced by environmental factors, such as soil type, temperature, sunlight exposure, and precipitation, all of which are related to the geographical origin of the plant material [7,16]. Studies comparing carob samples from different Mediterranean regions have demonstrated that there is substantial variability in phenolic content and biological activity [2,20].

The polyphenolic profile of the carob extracts in cold infusions using food-grade solvents (water and a 90% water–10% ethanol mixture), as identified by LC-QToF-MS analysis, revealed 15 bioactive compounds, each contributing distinct functional properties. The presence of gallic acid, a well-documented antioxidant, antimicrobial, and anti-inflammatory agent, underscores the therapeutic potential of the extracts [47,48]. Protocatechuic acid and gentisic acid are both known for their antioxidant and cytoprotective activities [49,50], while catechin and epicatechin, two flavan-3-ols, are associated with strong free radical scavenging and cardioprotective effects [51]. Vanillic acid and syringic acid exhibit antimicrobial, antioxidant, and potential neuroprotective actions [52,53]. The identification of p-coumaric acid and ferulic acid, both hydroxycinnamic acids, suggests anti-inflammatory, antimicrobial, and UV-protective roles [54,55]. Myricitrin, a flavonoid glycoside, has demonstrated antidiabetic, anti-inflammatory, and hepatoprotective effects [56]. Abscisic acid, though primarily known as a plant hormone, has been linked to glucose homeostasis and anti-diabetic properties in mammals [57]. Trans-cinnamic acid exhibits antimicrobial, antioxidant, and antidiabetic effects [58]. The high concentration and diversity of these polyphenols, in ripe and green pods and carob leaves, highlight the significant health-promoting potential of Greek carob and justify its use in functional food and nutraceutical formulations.

In a study by Christou (2021), ultrasound-assisted extraction of ripe carob pulp revealed that gallic acid was the predominant phenolic compound, accounting for approximately 65% of the total phenolic content. Other identified compounds included catechin, naringenin, cinnamic acid, quercetin, catechol, ferulic acid, gentisic acid, and additional gallic acid derivatives [19]. Similarly, Goulas (2020) analyzed carob fruits using acidic acetone and acetone–water extraction and detected compounds such as myricetin, catechin, epicatechin, quercetin, rutin, and syringic acid, with their presence depending on the extraction solvent used [8]. In another study, aqueous extracts of Egyptian carob pods showed high concentrations of gallic acid, followed by catechin, protocatechuic acid, and cinnamic acid. Lower levels of p-coumaric acid, rutin, gentisic acid, p-hydroxybenzoic acid, vanillic acid, and ferulic acid were also reported [59]. Finally, Roseiro (2013) found that aqueous decoctions of carob kibbles contained a range of antioxidant compounds, including gallic acid, catechin, epigallocatechin gallate, gallocatechin gallate, and epicatechin [60].

There are multiple mechanisms by which polyphenols exert their antioxidant activity. For example, gallic acid, which was found in all carob extracts, exhibits potent antioxidant activity, including radical scavenging, inhibition of lipid peroxidation, metal ion chelation, and maintenance of endogenous antioxidant defense systems. Its specific mechanisms in different environments involve hydrogen atom transfer (HAT) in vacuum and sequential proton loss electron transfer (SPLET) in polar media like water [51]. Moreover, catechins, which were also detected in all carob extracts, exert their antioxidant activity through both direct and indirect mechanisms to combat oxidative stress. Directly, they scavenge reactive oxygen species (ROS) and chelate metal ions, while indirectly, they modulate the activity of endogenous antioxidant enzymes and detoxification enzymes [61].

4.3. Inhibition of α-Amylase/Antidiabetic Activity

This study has shown that carob pods, both ripe and unripe, extracted in cold infusions using food-grade solvents (water and a 90% water–10% ethanol mixture), have the potential to inhibit in vitro the enzyme α-amylase. This is notable considering that the extraction method used (cold infusion, food-grade solvents) is milder than those in many prior studies, such as those using methanol or hot extraction. Moreover, several scientific studies have investigated the antidiabetic properties of carob (Ceratonia siliqua L.) extracts, demonstrating promising results in the inhibition of intestinal glucose absorption and improved glucose tolerance, as well as enzyme inhibition and antihyperglycemic effects. Rtibi et al. (2017) [62] examined the effects of immature carob pod aqueous extract (ICPAE) on glucose metabolism. Their findings revealed that ICPAE significantly inhibited sodium-dependent glucose transport in isolated mice jejunum and enhanced glucose tolerance in vivo, suggesting that these compounds may be used as a food supplement in hyperglycemia and diabetes treatments. Qasem et al. (2018) [63] evaluated the methanolic extract of carob pods for its inhibitory effects on α-amylase and α-glucosidase enzymes, which are crucial in carbohydrate digestion. The extract exhibited significant inhibition of both enzymes in vitro. In streptozotocin–nicotinamide-induced diabetic rats, carob exerted an in vitro inhibitory activity against α-amylase and α-glucosidase with IC₅₀ of 92.99 ± 0.22 and 97.13 ± 4.11 µg mL⁻¹, respectively. In a systematic review and meta-analysis on the effects of carob extract on glucose metabolism and the intestinal microbiome, the authors have concluded that carob extracts have beneficial effects on glucose metabolism, supporting their potential use in managing hyperglycemia and diabetes [64]. These studies collectively suggest that carob extracts possess antidiabetic properties, including the inhibition of glucose absorption, enhancement of glucose tolerance, and protective effects on pancreatic cells. Regarding the mechanism of α-amylase inhibition, several studies have shown that some phenolic compounds, such as flavonoids, have the capacity to bind to the active site or near the active site of the enzyme, inducing conformational changes in the three-dimensional structure of the enzyme, thus decreasing its activity [65]. These studies include techniques such as molecular docking and enzyme kinetic analysis. However, further research, particularly clinical trials, is necessary to fully elucidate the mechanisms and confirm the efficacy of carob extracts in diabetes management.

5. Conclusions

In the present study, we employed cold infusion techniques to extract bioactive components from carob to reflect traditional, food-grade sustainable methods that are relevant for consumer use. The study highlights the rich polyphenolic composition and promising bioactivity of cold aqueous and hydroethanolic extracts from Greek carob pods and leaves. At the tested extract concentration (20 g plant material in 200 mL solvent; equivalent to 100 mg/mL), all samples demonstrated antimicrobial activity, with green pods and leaves showing the greatest bacteriostatic effects against Escherichia coli and Staphylococcus aureus. The most potent α-amylase inhibition was observed in ripe pod extracts, reaching up to 96.43% inhibition, particularly with the 90:10 water–ethanol mixture. Total phenolic content ranged from 369.5 to 789.5 mg GAE/L in aqueous extracts and from 492.4 to 1124.1 mg GAE/L in hydroethanolic extracts, supporting the strong antioxidant and antiradical activity measured by FRAP and ABTS assays.

These findings support the development of carob-based infusions as natural health-promoting products and sustainable food applications. Among the limitations of this study are the lack of in vivo assays and postprandial studies that would further support the in vitro data. Future research could focus on establishing dose–response relationships for antimicrobial activity, determining IC₅₀ values for more accurate classification of bioactivity. The exploration of synergistic effects of phenolic compounds in food matrices would also yield valuable information. In addition, comparisons with conventional therapeutics and studies on additional pathogens could further validate the applications of these extracts in food preservation and glycemic control strategies.