New Insights into the Potential Inhibitory Effects of Native Plants from Cyprus on Pathogenic Bacteria and Diabetes-Related Enzymes

Abstract

Plants possess endless structural and chemical diversity, which is peerless with any synthetic library of small biomolecules, inspiring novel drug discovery. Plants are widely applied to encounter global health challenges such as antimicrobial resistance and diabetes. The objective of this work was to evaluate the antibacterial and antidiabetic potency of native plants grown in Cyprus. All plants were sequentially extracted with solvents of increasing polarity, namely hexane, acetone, methanol, and water. First, the phenolic and flavonoid contents of the extracts were assessed. Afterwards, the bacteriostatic and bactericidal potency of plant extracts were tested against a panel of six bacteria using the broth microdilution method, whereas the inhibitory effects on alpha-glucosidase and alpha-amylase enzymes were also determined with the employment of microplate assays. The results highlighted the superiority of Sarcopoterium spinosum as a potential enzyme inhibitor, while a knowledge base was also acquired for the inhibitory potential of all plants. Daucus carota, Ferula communis, and Tordylium.aegyptiacum displayed additionally outstanding bacteriostatic and bactericidal effects on Gram-positive bacteria at concentrations of 250 µg mL⁻¹ and 500 µg mL⁻¹. Overall, the present study describes the antibacterial and inhibitory activity against carbohydrate digestive enzymes of native plants grown in Cyprus delivering the first reports for many plant species.

1. Introduction

Natural products are considered an inexhaustible reservoir of successful drug leads, derived mainly from plant biodiversity [1]. Recently, Newman and Cragg reported that the majority of current drugs are natural products inspired by or derived from nature [2]. In addition, numerous patents have been registered for the potential health effects of diverse plants [3,4]. Noticeably, the use of herbal phytopharmaceuticals to treat diseases has also increased significantly over the past years. The annual growth rates reported exceed 15% and represent a significant share of the increase in the total world pharmaceutical market [5]. Significant research on the antimicrobial and antidiabetic properties of plants is constantly developing, as diabetes mellitus and antimicrobial resistance are essential elements of the top ten global public health threats [6,7].

Diabetes mellitus is the most common endocrine disorder resulting from a defect in insulin secretion, insulin resistance, or both. It is the third leading cause of morbidity and mortality, after heart attack and cancer [8]. A plethora of studies and clinical trials demonstrate that plants and their chemical constituents can treat diabetes and manage diabetic complications [9,10]. The inhibition of carbohydrate digestive enzymes such as alpha-glucosidase and alpha-amylase is a commonly applied strategy to manage diabetes mellitus. Both enzymes are responsible for the digestion of starch and glycogen, controlling the post-prandial glucose levels [11].

Antimicrobial resistance has been declared as one of the top ten global public health threats facing humanity by WHO in 2021 [12]. Although it is a natural phenomenon, resistance develops more rapidly through the misuse and overuse of antimicrobial agents [13]. Thus, the discovery of new antimicrobial agents is a mandatory need for healthcare workers and the pharmaceutical sector. Natural product research is poised to regain prominence in delivering new drugs to provide new solutions for the antibiotic crisis [14]. To date, numerous plant extracts and pure phytochemicals have been recommended as potential antimicrobial agents to treat diverse bacteria [15,16]. Furthermore, several herbal phytopharmaceuticals are commercially available to prevent bacterial growth for medicinal purposes, as well as to extend the shelf-life of food products [17,18]. In general, plant extracts and their constituents are strong inhibitors of Gram-positive bacteria, whereas the control of Gram-negative bacteria requires significantly higher concentrations of plant extracts or pure phytochemicals [19]. Foodborne bacteria cause a great number of diseases with significant effects on human health and the economy. Bacillus cereus, Cronobacter sakazakii, Esherichia coli, Listeria monocytogenes, Salmonella spp., and Staphylococccus aureus are common pathogenic bacteria that are responsible for foodborne infection or foodborne intoxication [20].

It is well known that more than 90% of the world’s biodiversity has not been evaluated for any biological activity [21]. Cyprus is the third largest island of the Mediterranean basin and laid at the crossroad of three continents. Due to the peculiarity of its geographical position, the island is renowned for its richness of flora [22]. Although a wealth of herbal medicines is known, the inhibitory effects of native plants on diabetes-related enzymes and pathogenic bacteria are almost unexplored. Thus, the present comparative study aspires to evaluate the antibacterial and diabetes-related enzyme inhibitory potential of native plants from Cyprus. More specifically, the present work includes fourteen native plants with unknown antibacterial or carbohydrate digestive enzyme inhibition effects, two unexplored native plants (Teucrium micropodioides Rouy, Tordylium aegyptiacum L.), and two widely spread native plants (Helichrysum stoechas subsp. barrelieri (Ten.) Nyman, Sarcopoterium spinosum (L.) Spach), of which the above effects have been described in the literature.

2. Materials and Methods

2.1. Plant Materials

Eighteen native plants were harvested at the flowering stage in the Limassol District between March and June 2022. Voucher specimens were prepared for each plant and are housed in the department’s herbarium. Table 1 details the botanical names, common names, plant parts extracted, collection sites, and voucher specimen numbers of the native plants. The plant materials (1500–2000 g of fresh weight) were dried in an oven at 40 °C for 3 days. Dried samples were then ground to a fine powder using a Sage BCG820BSSUK (Breville Group Limited, Sydney, Australia) electric grinder.

2.2. Extraction of Plants

Five g of each plant material were mixed with 30 mL of hexane and placed in an ultrasonic bath (UCI-50, 35 KHz, Raypa-R. Espinar, S.L., Terrassa, Spain) for 60 min at 60 °C. Subsequently, the mixture was allowed to cool at room temperature and then centrifuged for 10 min at 2500 rpm. The supernatant was collected, and the remaining solid was extracted again with the next solvent following the order acetone, methanol, and water. The organic solvents were removed under nitrogen gas, and aqueous extracts were freeze-dried in order to obtain dry extracts. All extracts were stored at −20 °C until further analysis.

2.3. Assessment of Total Phenolic and Total Flavonoid Contents

The total phenolic content (TPC) of plant extracts was determined using a microplate Folin–Ciocalteu assay as described in a previous study [23]. For each determination, the extracts were dissolved in 20% (v/v) DMSO and filtered through a 0.45 μm membrane filter to remove any insoluble particles. TPCs of plant extracts were expressed as mg of gallic acid equivalents (GAE) per g of dry extract [23]. The total flavonoid content (TFC) of plant extracts was assessed with the employment of the aluminum chloride colorimetric assay. TFCs of plant extracts were expressed as mg of catechin equivalents (CE) per g of dry extract

2.4. Evaluation of the Antibacterial Potential of Plants

Three Gram-positive bacteria, namely Listeria monocytogenes ATCC 23074 (serotype 4b), Staphylococcus aureus ATCC 6538, and Bacillus cereus ATCC 6089, were grown in Listeria Agar (Merck^®, Darmstadt, Germany), Baird Parker Agar (Liofichem^®, Roseto degli Abruzzi, Italy), and Mannitol Egg Yolk Polymyxin (MYP) Agar (Merck^®, Germany), respectively. Furthermore, three Gram-negative bacteria, namely Salmonella enterica subsp. enterica serovar Enteritidis NCTC 5188, Escherichia coli ATCC 11775, and Cronobacter sakazakii ATCC 29544, were grown in Xylose Lysine Deoxycholate Agar (Merck^®, Germany), Tryptone Bile Glucuronic Agar (Merck^®, Germany), and Sakazakii Agar (Merck^®, Germany), respectively. A colony of each of the bacteria was inoculated into 10 mL of Brain–Heart Infusion Broth (BHI) (HIMEDIA^®, Mumbai, India) and incubated at 37 ℃. Antibacterial susceptibility testing was performed using the Broth microdilution method with slight modifications. More specifically, an aliquot of 50 μL of each plant extract was transferred, in triplicate, in a 96-well plate. An aliquot of 40 μL of BHI broth and 10 μL of microbial suspension were added to reach a final volume of 100 μL in each well. The final concentration of the plant extracts in the wells was 2000, 1000, 500, and 250 μg mL⁻¹. Microbial suspensions were adjusted so that the final concentration in the wells was 10⁶ cfu mL⁻¹. Screening for bacteriostatic/bactericidal activity of plant extracts was performed as described in our previous work by adding 10 μL of each well in BHI Agar plates, and the results were extracted after incubation for 24 h at 37 °C [22]. Controls of 10%, 5%, and 2.5% v/v DMSO and microbial cultures were also tested. Stock solutions of 10 mg mL⁻¹ for each plant extract were prepared using DMSO as a diluent. Aqueous and methanolic extracts were prepared in 50% v/v DMSO, while hexanic and acetonic extracts in pure DMSO. Stock solutions were further diluted using water to prepare working solutions. Bacteriostatic activity was expressed as minimum inhibitory concentration (MIC) and bactericidal activity as minimum bactericidal concentration (MBC). MIC and MBC values were calculated as μg dry extract mL⁻¹.

2.5. Evaluation of the Inhibitory Potential of Plants on Diabetes-Related Enzymes

The determination of alpha-glucosidase inhibitory effect of plant extracts was assessed by mixing the extract solution (100 μL, 0.5 mg mL⁻¹) with 50 μL of 0.1 mM phosphate buffer (pH = 6.8) containing alpha-glucosidase (1.0 U mL⁻¹). The mixture was incubated at 37 ℃ for 10 min. Subsequently, 50 μL of p-nitrophenyl-α-D-glucopyranoside (PNG) (5 mM in 0.1 mM phosphate buffer, pH = 6.8) was added, and the reaction mixture was allowed to stand for 5 min. Finally, the absorbance was measured at 405 nm against a blank solution, where PNG was replaced with buffer. Control which represents 100% enzyme activity was prepared by replacing the extract solution with 20% (v/v) DMSO.

For the assessment of alpha-amylase inhibitory potential, 100 μL of the extract solution (10 mg mL⁻¹) and 100 μL of alpha-amylase solution (2 U mL⁻¹ in 20 mM of sodium phosphate containing 6.7 mM of NaCl, pH 6.9) were incubated at 35 °C for 10 min. Then, 200 μL of soluble starch (1% w/v in the same buffer) was added, and the mixture was incubated again at 35 °C for 20 min. The reaction was terminated by adding 200 µL of 3,5-dinitrosalicylic acid (DNS) reagent. Afterwards, the mixture was boiled for 10 min, cooled down, and diluted with deionized water (1:10, v/v) before the measurement of absorbance at 540 nm. The absorbance of the reaction mixture was measured against a blank sample containing the extract solution, starch solution, and DNS (without enzyme). The results for both assays were expressed as the % inhibitory activity of the extracts compared to the control [22].

2.6. Statistical Analysis

All measurements were performed in triplicate, and the results obtained were expressed as mean values ± standard deviation (SD). The means were compared, and statistically significant differences were determined through one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (at a 95% confidence level). The differences between individual means were considered significant at p < 0.05. All statistical analyses were performed using RStudio statistical software (version 1.3.1073).

3. Results and Discussion

3.1. Total Phenolic Content (TPC) and Total Flavonoid Content (TFC) of Native Plants

Several biological and health effects of plants have been associated with the presence of phenolic compounds [24,25]. Previous studies also support the antibacterial and carbohydrate digestive enzyme inhibitory potential of many phenolic compounds including phenolic acids, flavonoids, etc. [26,27,28]. Thus, the TPCs and TFCs of the native plants were first estimated. The serial exhaustive extraction method using a solvent of increasing polarity, from non-polar (hexane) to polar (water), was used to extract the plant materials. This strategy allows us to study thoroughly the phenolic contents as well as the bioactivity of plants. The results showed a great diversity of phenolic contents in 18 native plants studied. A strong impact of extraction solvent was also found. As expected, the methanol and water are the most appropriate solvents to recover phenolic compounds from plant material. This solubility preference of phenolic compounds to polar solvents is explained by the presence of multiple hydroxyl groups and glucosides in their moiety [29]. Figure 1 demonstrates that TPC values were ranged from 27.3 ± 0.7 mg gallic acid equivalents (GAE) g⁻¹ to 2159.0 ± 2.9 mg GAE g⁻¹ for methanolic extracts and from 43.9 ± 1.4 mg GAE g⁻¹ to 524.1 ± 12.4 mg GAE g⁻¹ for aqueous extracts of native plants. On the other hand, the lowest TPCs were found for hexanic extracts of plants; all values were lower than 18.5 ± 0.6 mg GAE g⁻¹. Finally, the efficacy of acetone was between hexane and polar solvents. The results also showed that TPC values are also strongly influenced by genetic factors. Sarcopoterium spinosum is the richest plant in phenolic compounds among native plants studied, followed by Lithodora hispidula and Micromeria nervosa. Daucus carota, Echium angustifolium, Prasium majus, and Smyrnium olusatrum also contain significant amounts of phenolic compounds.

In the next step, the TFCs of native plants were assessed as flavonoids are a sub-group of phenolics with potent antibacterial and antidiabetic properties [28,30]. TFC values of plant extracts were strongly affected by extraction solvent and genetic factors; their contents fluctuated between 0.1 ± 0.0 mg catechin equivalents (CE) g⁻¹ and 314.5 ± 23.0 mg CE g⁻¹ (Figure 2). Regarding extraction solvents, the use of water as an extractor matrix for flavonoids is recommended for 10 plants. The latter is rather attributed to the presence of glycosylated flavonoids in these plants. In addition, the superiority of acetone was revealed for one-third of the plants studied. Pure acetone solubilizes less polar flavonoids, such as aglycones of isoflavones, flavanones, methylated flavones, and flavonols [31]. It is obvious that each plant requires a different solvent for the efficient recovery of flavonoids due to the structural diversity of flavonoids present in native plants. The results also highlighted that Lithodora hispidula subsp. versicolor (314.5 ± 23.0 mg CE g⁻¹), Solanum villosum (276.3 ± 19.4 mg CE g⁻¹), M. nervosa (252.9 ± 13.5 mg CE g⁻¹), and Tordylium aegyptiacum (244.3 ± 6.6 mg CE g⁻¹) had the highest TFCs among plants studied. The concentration of flavonoids in P. majus (193.3 ± 21.4 mg CE g⁻¹), L. hispidula (175.7 ± 9.1 mg CE g⁻¹), and Thymelaea tartonraira (158.8 ± 0.4 mg CE g⁻¹) was also remarkable, whereas the rest of the native plants contain significantly lower amounts of flavonoids (<120 mg CE g⁻¹).

3.2. Inhibitory Effects of Native Plants on Gram-Positive and -Negative Bacteria

The bacteriostatic and bactericidal activity of native plants grown in Cyprus was additionally determined. Table 2 summarizes the antibacterial effects of all extracts against the bacteria tested. The results demonstrated that Gram-positive bacteria were more susceptible to plant extracts than Gram-negative bacteria. Differences in bacterial cell wall and mode of action between bacteria are responsible for this dissimilar response to plant extracts [19]. Regarding Gram-positive bacteria, the native plants tested were efficient against S. aureus and B. cereus. Furthermore, only F. communis and T. micropodioides had antibacterial activity against L. monocytogenes at decent concentrations. This is the first report on the anti-Listeria potential of these native plants. A strong inhibitory effect of six and three native plants was found for S. aureus and B. cereus, respectively. D. carota, F. communis, and T. aegyptiacum extracts exerted bacteriostatic and bactericidal activity for both bacteria; MIC and MBC values were estimated at 250 µg mL⁻¹ and 500 µg mL⁻¹, respectively. Similar inhibitory effects on S. aureus were observed for H. stoechas, S. spinosum, and T. micropodioides. Furthermore, seven native plants presented significant growth inhibition of S. aureus and B. cereus. Five native plants had weak bacteriostatic activity for the same bacteria since their MICs were higher than 2000 µg mL⁻¹. As mentioned above, the majority of native plants cannot be considered as significant antibacterial agents for Gram-negative bacteria. However, the results revealed bacteriostatic activity on E. coli for A. neapolitanum, D. carota, F. communis, G. italicus, M. officinarum, T. micropodioides, and T. aegyptiacum at a concentration of 1000 µg mL⁻¹. In addition, hexanic extract of T. micropodioides inhibited the growth of Salmonella enterica and Cronobacter sakazakii. This antibacterial potential of T. micropoioides against Gram-negative bacteria was demonstrated for the first time, whilst there is only available information for other species of Teucrium. More specifically, Teucrium species inhibited the growth of E. coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Proteus mirabilis using concentrations between 1024 μg mL⁻¹ and 8192 μg mL⁻¹ [32]. Regarding their active phytoconstituents, the amount of literature is really poor, but it is known that they contain diterpenes and essential oils such as alpha- and beta-pinene, which demonstrate substantial antimicrobial activity [33,34].

The present work provides unprecedented bacteriostatic and bactericidal data for native plants grown in Cyprus, an isolated ecosystem at the crossroads of three continents. The usefulness of new insights into the antimicrobial properties of all native plants is non-negotiable. This study also highlights that the antibacterial potency of D. carota, F. communis, and T. aegyptiacum is promising and needs further investigation. Although D. carota tubers and seeds have been investigated, the antimicrobial properties of its aerial parts are poorly examined. A previous study also evaluated the antimicrobial properties of essential oils of D. carota leaves. Their potential is mostly linked with high amounts of monoterpenes and sesquiterpenes, but these compounds may not be present in their methanolic and acetonic extracts [35]. F. communis is the second Apiaceae plant in the shortlist of most active native plants. The inhibitory effect of alcoholic and organic extracts derived from F. communis has been determined, but MIC values tested were significantly higher (2000–12,000 µg mL⁻¹) for other strains of B. cereus, E. coli, and S. aureus [36]. T. aegyptiacum also belongs to the Apiaceae family and is distributed to the Cyprus and Middle-East regions. This is the first report on the potent antibacterial activity of T. aegyptiacum extracts. The strong inhibitory effects of other Tordylium species on Gram-negative and Gram-positive bacteria have been documented [37,38]. The phytochemical composition of T. aegyptiacum is not available in the literature, but it is documented that Tordylium plants contain a mixture of known antimicrobial agents such as quercetin and kaempferol derivatives, as well as coumarins [39].

3.3. Inhibitory Effects of Native Plants on Diabetes-Related Enzymes

The high blood sugar levels, insulin resistance, and relative lack of insulin are the main characteristics of type 2 diabetes, which represents over 90% of diabetes cases worldwide [40]. The reduction in or inhibition of carbohydrate absorption by inhibiting digestive enzymes such as alpha-glucosidase and alpha-amylase is one of the most widely used strategies to reduce postprandial hyperglycemia [27]. At first, the efficiency of all extracts derived from Cypriot native plants against alpha-glucosidase was determined (Figure 3). Considering that an effective enzyme inhibition is higher than 50%, the most active extracts are produced by the use of hexane as the extractor solvent, followed by acetone. Additionally, polar solvents such as water and methanol cannot be considered suitable extractors of alpha-glucosidase inhibitors. The polar extracts of S. spinosum were an exception as they effectively inhibited α-glucosidase. Regarding the plant materials, the superiority of S. spinosum was indisputable as its extracts present enzyme inhibition between 86% and 99%. Our results demonstrated clearly that the methanolic and acetonic extracts of S. spinosum were potent inhibitors of alpha-glucosidase. The prominent inhibitory effect on alpha-glucosidase activity of S. spinosum leaves and fruits was also reported in previous studies [41,42]. However, there are no available data for their active ingredients since research has mainly focused on S. spinosum roots. The hexanic extract of D. carota aerial parts is also the next promising extract, as its enzyme inhibition is over 84%. This is the first evidence of the inhibitory potential of D. carota aerial parts. Its potential is rather not linked with polyphenols since both TPCs and TFCs were found at low concentrations. The alpha-glucosidase inhibition of M. nervosa is also demonstrated for the first time; the acetonic extract had the highest potential. Hexanic extracts of L. hispidula (66.4%), T. aegyptiacum (65.1%), M. officinarum (58.8%), P. majus (57.4%), H. stoechas (51.7%), and acetonic extract of T. aegyptiacum (60.7%) present remarkable inhibitory effects on alpha-glucosidase; their potentials were unknown in the literature.

Figure 4 summarizes the inhibitory potentials of native plants grown in Cyprus on alpha-amylase. The results clearly demonstrated that the inhibitory effect of extracts was less efficient against alpha-amylase than alpha-glucosidase, albeit a higher concentration of extracts was used. Researchers attributed the above trend to the fact that phytoconstituents are partially absorbed by starch during gelatinization, and different interactions between the enzyme and active constituents may occur [43]. In contrast with alpha-glucosidase’s results, hexanic extracts had the weakest effects among the tested extracts. All hexanic extracts exerted alpha-amylase inhibition below 11%. On the other hand, polar solvents seem to be a better choice to extract alpha-amylase inhibitors from plant materials. The results highlighted the inhibitory potential of S. spinosum extracts on alpha-amylase enzyme as it is also mentioned for alpha-glucosidase. Although the inhibitory effect of aerial parts of S. spinosum on alpha-amylase has been previously described, the active phytoconstituents must be investigated [41,42]. Unfortunately, the other native plants tested had weak-to-moderate activity against alpha-amylase. The utilization of different extraction methods and solvents for the recovery of alpha-amylase inhibitors from A. aestivus, P. majus, S. vilosum, and T. tartonraira will perhaps produce more promising findings. The inhibitory effects of these plants on the alpha-amylase enzyme are described for the first time, and their active phytoconstituents are unknown. Although their inhibitory potential is not high, these plants may contain novel strong inhibitors at low concentrations.

In summary, the present work demonstrates for the first time the inhibitory potential of twelve native plants grown in Cyprus on carbohydrate digestive enzymes. Although the findings were not encouraging for the majority of native plants, the study delivers new knowledge for researchers. The results additionally highlight the great potential of S. spinosum to serve as an inhibitor for both enzymes. Previous studies mainly investigated the inhibitory properties of S. spinosum roots, while the results presented here demonstrate that aerial parts are also active. Recently, a phytochemical study reported that aerial parts of S. spinosum mainly contain ellagitannins such as casuarictin, corilagin, pedunculagin, and castalagin, as well as amounts of glycosylated derivatives of quercetin and triterpenoids, namely tormentic acid and its derivatives [44]. It is known that ellagitannins, quercetin glucosides, and tormentic acid act as remarkable inhibitors of diabetes-related enzymes [45,46,47,48]. Thus, further purification and testing of S. spinosum are strongly recommended to identify the active components that are able to act in an additive and/or synergistic manner. Finally, the alpha-glucosidase inhibitory effects of a panel of unexplored native plants were assessed, delivering new knowledge for their potential use as inhibitors of carbohydrate digestive enzymes.

4. Conclusions

The present study provides insight into the antibacterial and in vitro antidiabetic activity of a wide variety of native plants grown in Cyprus. This work presents for the first time the antibacterial and diabetes-related enzyme inhibitory potential of many native plants. The results contribute to the ongoing research attempts to discover novel antibacterial and antidiabetic agents for the food and pharmaceutical industry. The results highlight the strong bactericidal potency on Gram-positive bacteria of three Apiaceae plants, namely D. carota, F. communis, and T. aegyptiacum. These plants also exert remarkable bacteriostatic activity against E. coli, a Gram-negative bacterium. Our findings also support that twelve native plants can be considered as possible antibacterial factors. In addition, the present work reports for the first time the inhibitory potential of twelve native plants grown in Cyprus on carbohyd rate digestive enzymes. More specifically, a panel of six native plants significantly inhibited the activity of the alpha-glucosidase enzyme. Among the plants studied, S. spinosum is the most promising plant for the discovery of new inhibitors for both digestive enzymes. The implementation of this study strongly recommends the further investigation of native plants to identify the antibacterial and antidiabetic constituents of plant materials as well as to decode their mode of action, opening new horizons for their utilization in drug discovery.