Introduction

The pharmaceutical and drug industries have relied heavily on plant products for many decades. Approximately 20% of medications used worldwide are derived from plants, as they are more widely available, cost-effective, and often exhibit more potent and safer effects than synthetic pharmaceuticals (Chaachouay & Zidane, 2024). According to Rahman et al. (2021), numerous natural substances are being utilized as essential therapeutics for various chronic and degenerative health conditions that are prevalent globally. These include cancer, Alzheimer’s disease, and diabetes mellitus. Context: There is a growing interest in the exploration of new natural compounds derived from plants (Akter et al., 2021).

Phenolic, tannin, and flavonoid compounds were among the many pharmacologically active substances extracted from the medicinal plants examined in this study (Tungmunnithum et al., 2021). These compounds are recognized for their pharmacological effects,

including hypoglycemic, anti-inflammatory, hepatoprotective, antifungal, antibacterial, and hypolipidemic activities (Bourais et al., 2023). Research conducted on four different plant species has demonstrated that various constituents of these plants exhibit anticancer properties against a wide range of tumors.

The pharmaceutical and drug industries have relied heavily on plant products for decades. Approximately 20% of medications used worldwide are derived from plants, as they are more widely accessible, cost-effective, and often exhibit more potent and safer effects than synthetic pharmaceuticals (Chaachouay & Zidane, 2024). The cinnamon plant, known as Cinnamomum verum, is particularly well-known for its medicinal and pharmacological properties (Singh et al., 2020). The Cinnamomum zeylanicum plant has been used as a medicinal condiment since ancient times. Both Sri Lanka and the southern states of India are its native habitats. It belongs to the Lauraceae family (Gulcin et al., 2019). It is a dry bark that has been stripped of its outer cork and the underlying parenchyma.

One of the most widely used medicinal herbs, C. verum can be utilized in various contexts (Chebabe et al., 2025). It is frequently used in the commercial sector as a component of candies, chewing gum, mouthwash, and toothpaste (Kumar et al., 2022). The plant contains many volatile oils, the most prominent of which are cinnamaldehyde, cinnamic acid, and cinnamate (Huang et al., 2021). Eugenol, the primary active component, is associated with several biological functions. This herb is found in nearly every pharmaceutical system worldwide, and each of these attributes plays an essential role in advancing human health (Nisar et al., 2021). The plant possesses several critical medical properties, including antimicrobial, wound healing, anti-inflammatory, anti-HIV, anti-anxiety, and anti-Parkinson’s disease activities (Singh & Yadav, 2024). Eugenol, cinnamaldehyde, cinnamyl acetate, copane, and camphor are the principal constituents of the C. verum plant (Pathak and Sharma, 2021).

Nerium oleander (NO), often called oleander, is a member of the Apocynaceae family and is native to the Mediterranean regions of several continents, including Africa and Europe (Sanna et al., 2019). It may grow as a small tree or an evergreen shrub with upright, coarse, dark green leaves. Despite its toxicity, it has been used as a medicinal agent for various conditions, including heart failure, malaria, dyspepsia, leprosy, and ringworm (Mouhcine et al., 2019). Both hot and cold extracts of N. oleander have been evaluated for their potential to inhibit viral growth. Similarly, N. oleander has been reported to exhibit hypolipidemic activity and has been used in folk medicine as a cardiotonic (Rauf et al., 2021).

Laurus nobilis (LN), which belongs to the family Lauraceae, is an evergreen tree cultivated in many warm regions worldwide, particularly in Mediterranean countries such as Morocco, Algeria, Spain, Portugal, Turkey, and Greece (Khodja et al., 2023). Historically, it has been used to treat ailments including diabetes, rheumatism, dermatitis, gastrointestinal disorders, snakebites, and migraines (Ansari et al., 2025).

Eucalyptus camaldulensis (EC) belongs to the Myrtaceae family and is commonly known as the river red gum. It is endemic to the continents of Africa and Australia. This tree has smooth, cream-colored or white bark; mature leaves that are lance-shaped or curved; flower buds arranged in clusters of seven or nine; white flowers; and hemispherical fruit with valves that extend above the rim (Kurek et al., 2020). It is considered a source of compounds with physiologically active qualities (Elshafie, 2023). The leaf extract of E. camaldulensis has demonstrated several beneficial properties, including antioxidant and antimicrobial effects (Mahmoud Dogara et al., 2024).

Previous studies have demonstrated that C. verum, N. oleander, L. nobilis, and E. camaldulensis possess high nutritional value and considerable biological benefits. These effects include antioxidant, antiproliferative, and enzyme-inhibitory activities. However, to the best of our knowledge, no reports exist regarding the enzyme-inhibiting impact of the studied herbaceous plant species. Consequently, this study aimed to evaluate the enzyme inhibitory effects (anti-amylase and anti-glucosidase) of various extracts derived from different biological sources. Based on the results obtained, there may be potential interest in developing novel natural products for the pharmaceutical and food industries.

Materials and Methods

Collection of plant materials and preparation of extracts

The leaves of the herbaceous plants C. verum (CV) and L. nobilis (LN) were collected from spice markets in Riyadh; N. oleander (NO) was obtained from Riyadh Gardens, and E. camaldulensis (EC) from Al-Qassim. A taxonomist from the University of King Saud’s Department of Botany verified the botanical identity of the plants after placing the voucher specimens in the herbarium. The leaves of the plant materials (CV, NO, LN, and EC) were dried at room temperature. A laboratory mill was used to grind the dried samples into a fine powder. Fifteen grams of each powdered component were extracted separately using methanol.

Preparation of the extract

Forty grams of the powdered components were extracted separately using 70% methanol, with a solid-to-liquid ratio of 1:5. The mixtures were then kept on a shaker for 3 days at +4 °C. The resulting mixtures were filtered through Whatman filter paper. The filtrates were concentrated and dried using a rotary evaporator at 50 ºC (Yamato RE300, Japan) until a thick, dry substance was formed, following Yang’s method (Yang et al., 2014). Distilled water was used to dissolve the powder for laboratory experiments.

α-Glycosidase inhibition studies

The leaf extract of E. camaldulensis has been shown to contain several advantageous properties, including antioxidant and antimicrobial effects (Mahmoud Dogara et al., 2024). To achieve this objective, a range of concentrations of WEC and EEC were introduced into phosphate buffer, which had a volume of 75 microliters and a pH of 7.4. Next, twenty microliters of α-glucosidase solution were added to the specified buffer, and the mixture was incubated for ten minutes. A 50 μL aliquot of p-nitrophenyl-D-glycopyranoside (p-NPG) was then added to the final mixture. The mixture was re-incubated at the physiological temperature of 37 °C, and absorbance was determined by spectrophotometric analysis at a wavelength of 405 nm.

α-Amylase activity

According to Visvanathan et al. (2020), the inhibitory effects of E. camaldulensis extract (EEC) on the α-amylase enzyme were tested at a specific concentration. Briefly, 1 g of starch was dissolved in 40 mL of a 0.4 M alkaline solution, and the mixture was heated at 80 °C for 30 minutes. After the solution was completely cooled and the pH adjusted to 6.9, deionized water was added to bring the total volume to 100 milliliters. Then, mixtures containing varying concentrations of EEC and an equal volume (35 μL) of starch and phosphate buffer (pH 6.9) were combined. Next, 20 μL of α-amylase solution was added to the final mixture, which was incubated at 30 °C for one hour. Finally, the reaction was quenched by adding 50 μL of hydrochloric acid (0.1 M), and the absorbance was measured at 580 nm.

Calculating the phenolic contents

The Folin–Ciocalteu technique (Nikolaeva et al., 2022) was used to determine the total phenolic content of each extract. Briefly, 2.5 mL of Folin–Ciocalteu reagent (10% w/v) was mixed with 1 mL of extract solution at concentrations ranging from 100 to 500 µg/mL. After 5 minutes, 2.0 mL of 75% Na₂CO₃ was added, and the mixture was incubated for 10 minutes at 50 °C with intermittent stirring. Subsequently, the sample was cooled before measuring its absorbance at 765 nm using a UV spectrophotometer (Shimadzu, UV-1800), compared to a blank sample without any extract. The results were expressed as milligrams of gallic acid equivalents per gram (mg GAE/g) of dry extract.

Calculating the tannin contents

The method described by Sharma et al. (2021) was used to determine the total tannin content (TTC). The procedure combined 1.5 mL of purified Milli-Q water, 0.1 mL of Folin–Ciocalteu phenol reagent, and 0.1 mL of the extracted samples. The mixture was allowed to stand for 8 minutes. Afterwards, the solution was neutralized by adding 0.3 mL of 30% Na₂CO₃. Subsequently, the components were mixed thoroughly and incubated at ambient temperature for 20 minutes in the dark. Absorbance was measured at 700 nm. The tannin content was calculated and expressed as milligrams per gram of dry weight (DW).

Calculating the flavonoid contents

The Dowd technique was used to measure the flavonoid concentration in each extract (Nikolaeva et al., 2022). A solution containing 0.2 mL of AlCl₃ in methanol (10% w/v), 0.2 mL of 1 M potassium acetate, and 5.6 mL of distilled water was mixed with either 1 mL of extract solution or 1 mL of quercetin, both with concentrations ranging from 25 to 200 µg/mL. After allowing the mixture to stand at room temperature for 30 minutes, absorbance was measured at 415 nm relative to the blank. The results were expressed as milligrams of quercetin equivalents per gram (mg QE/g) of dry extract.

DPPH radical scavenging assay

The free radical scavenging activity of the extracts was evaluated using the DPPH radical scavenging assay, following methodologies established by Murshed et al. (2024). This assay measures the ability of plant extracts to donate hydrogen atoms by decolorizing a methanol solution of DPPH (2,2-diphenyl-1-picrylhydrazyl). The violet/purple color of DPPH fades to yellow upon interaction with antioxidants. A 0.1 mM solution of DPPH in methanol was prepared and mixed with varying concentrations (12.5–150 μg/mL) of extract in methanol (2.4 mL DPPH solution combined with 1.6 mL extract). The reaction mixture was vortexed thoroughly and incubated in darkness at room temperature for 30 minutes before measuring absorbance at 517 nm using a spectrophotometer. Butylated hydroxytoluene (BHT) served as the reference compound.

The percentage DPPH radical scavenging activity was calculated using:

where A₀ represents the absorbance of the control, and A₁ is that of the extract or standard. The percentage inhibition versus concentration graph was used to calculate the IC₅₀ value; experiments were performed in triplicate at each concentration.

ABTS radical cation decolorization assay

In this assay, color loss is measured when an antioxidant interacts with ABTS⁺ radical cations, converting them into ABTS and causing decolorization. The method described by Kut et al. (Hirsch et al., 2022) outlines the assessment of antioxidant activity through this process. ABTS radical cations are generated by combining potassium persulfate (2.45 mM) with an aqueous stock solution of ABTS (7 mM). The working solution is prepared by mixing equal volumes of both stock solutions and incubating them for 16 hours at 25 °C in darkness before dilution with methanol to achieve an absorbance of 0.70 ± 0.2 at 734 nm, measured by spectrophotometry. Fresh solvent was used for each experiment, with Trolox as the antioxidant standard. The calibration curve included concentrations ranging from 0 to 500 µM. In test tubes, diluted samples (1 mL) were mixed with an equal volume of ABTS⁺ radical cation solution, and absorbance was measured after seven minutes at 734 nm to calculate Trolox equivalent antioxidant capacity (TEAC), expressed as Trolox equivalents (µM).

Evaluation of cytotoxicity (MTT Assay)

Cell culture

The Hep-G2/2.2.15 human hepatoblastoma cell line was obtained from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere containing 5% CO₂ at 37 °C.

An MTT assay (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, cat#475989-1GM, Sigma-Aldrich, Germany) was used to assess the cytotoxicity of the plant extract V. vinifera. Briefly, cells were plated at a density of 5 × 10⁴ cells/mL in a 96-well culture plate and incubated for 24 h. The cells were then treated with extract concentrations of 0, 2.5, 5, 10, 25, 50, and 100 μg/mL. Doxorubicin was used as a positive control. After 48 h of incubation, 10 μL of MTT solution (5 mg/mL in phosphate-buffered saline, PBS) was added to each well. Subsequently, 100 μL of acidified isopropanol was added to dissolve the formazan crystals. The plate was shaken for 10 minutes, and absorbance was measured at 570 nm using a microplate reader (BioTek, USA). Cell viability (%) was calculated as follows:

The IC₅₀ values (concentration of extract causing 50% inhibition) were determined from the dose-response curve of cell viability percentage using OriginPro software.

Statistical analysis

The experiment outcomes represent the average of three duplicate analyses. The mean ± standard deviation of the experimental data was calculated and analyzed. One-way analysis of variance (ANOVA) was performed. Duncan’s multiple-range test was used to identify significant differences among means. Significance was defined as P < 0.05, and highly significant as P < 0.01.

Results and Discussion

Regulating blood glucose levels is a considerable challenge for individuals with diabetes. Inhibition of carbohydrate-digesting enzymes, such as α-amylase and α-glucosidase, is an effective strategy. α-Amylase catalyzes the hydrolysis of (1,4) glycosidic linkages in complex carbohydrates, such as starch or glycogen (Kaur et al., 2025), whereas α-glucosidase catalyzes the final step in carbohydrate hydrolysis (Lu et al., 2023). Consequently, the inhibitor acarbose was developed to reduce blood glucose levels in patients with diabetes mellitus. Novel and safe inhibitors derived from natural sources could serve as effective therapies for Alzheimer’s disease (AD), skin disorders (SD), and diabetes mellitus (DM) (Mohd Zaid, 2023).

The inhibitory activities of C. verum, N. oleander, L. nobilis, and E. camaldulensis extracts against α-amylase and α-glucosidase were tested using a microplate reader (Table 1). The extracts exhibited inhibitory effects on the enzymes tested. Generally, methanolic extracts showed vigorous activity. Batiha et al., (2020) and Chandorkar et al., (2021) reported similar results for C. verum, N. oleander, L. nobilis, and E. camaldulensis extracts and other plant extracts. These findings suggest that organic solvents may be suitable for enzyme inhibitory assays. Additionally, the enzyme-inhibitory activities of the extracts varied significantly according to the plant species used.

Table 2 presents the findings. All extracts showed inhibitory potential against the tested enzymes. The methanolic extracts of L. nobilis and N. oleander exhibited stronger inhibition of α-amylase activity than the other extracts, with values of 0.586 ± 0.015 and 0.564 ± 0.0001 mmol acarbose/g extract, respectively. C. verum and E. camaldulensis showed lower inhibition values of 0.230707 ± 0.017 and 0.369 ± 0.006 mmol acarbose/g extract, respectively (Figure 1).

Additionally, methanolic extracts of N. oleander and L. nobilis demonstrated stronger inhibition of α-glucosidase activity than the other extracts, with values of 8.242 ± 0.113 and 7.570 ± 0.107 mmol acarbose/g extract, respectively. In comparison, C. verum and E. camaldulensis showed lower inhibition values of 2.0476 ± 0.0468 and 1.4668 ± 0.08 mmol acarbose/g extract, respectively (Figure 2). Christoforidi et al., (2022) and Mutlu-Ingok et al., (2022) reported similar findings for some of these and other plant extracts. These results suggest that organic solvents may be appropriate for enzyme inhibitory assays.

The lowest α-amylase and α-glucosidase inhibitory potentials were observed in C. verum and E. camaldulensis, with values of 0.231 ± 0.02 and 1.466 ± 0.08, and 0.369₄ ± 0.006 and 2.048 ± 0.05 mmol acarbose/g extract, respectively.

The levels of total phenolics varied among the extracts. The highest phenolic content was observed in the methanolic extract of E. camaldulensis at 63.56 mg GAE/g of extract, followed by L. nobilis and C. verum with 57.397 and 54.324 mg GAE/g of extract, respectively. Conversely, the lowest phenolic content was recorded for N. oleander at 26.44 mg GAE/g of extract (Figure 3).

The differences found in the phenolic composition align with those reported in other comparable investigations (Limam et al., 2020). This outcome is likely due to the higher polarity of methanol, which enables a more efficient extraction of the highly polar antioxidant and phenolic compounds present in eucalyptus leaves (Johnson et al., 2020).

The phytochemical screening results in Figure 4 illustrate the presence of various components in the methanolic extracts. C. verum, N. oleander, L. nobilis, and E. camaldulensis extracts exhibited a weak to strong presence of essential phytochemicals. The levels of total tannins varied among the extracts. The highest tannin content was recorded at 34.80 mg GAE/g of extract for methanolic E. camaldulensis, followed by L. nobilis and C. verum with 30.84 and 29.37 mg GAE/g of extract, respectively. In contrast, the lowest tannin content was observed in N. oleander at 12.98 mg GAE/g of extract (Figure 4).

Tannins are associated with medicinal properties, including anti-inflammatory, antidiabetic, analgesic, and central nervous system effects (Omar et al., 2022). The identification of proteins, fatty acids, and carbohydrates highlights the nutritional value of cinnamon spice (Cinnamomum verum) (Al Dhaheri et al., 2023). Phytochemical screening of oleander (Nerium oleander) leaves confirms the presence of tannins. The phenolic and flavonoid content of oleander leaf extracts varied based on the polarity of the solvent (Redha, 2020). The tannin content recorded—12.98 mg GAE/g—is higher than the 10.89 mg/g DW found in the ethanolic extract of Laurus nobilis leaves. Phytochemical screening of Eucalyptus camaldulensis also reveals a high tannin concentration in its leaves (Hussain et al, 2025).

The total flavonoid content varied across the extracts, as shown in Table 3. The results indicated that the methanolic extract of Eucalyptus camaldulensis had the highest flavonoid concentration, measuring 37.56 mg QE/g of extract. This was followed by Cinnamomum verum, with a flavonoid content of 21.51 mg QE/g of extract. Conversely, Nerium oleander and Laurus nobilis extracts exhibited the lowest flavonoid concentrations, measuring 8.13 and 6.09 mg QE/g, respectively (Figure 5). These findings are supported by several other studies (Ahmed et al., 2020; Ayouaz et al., 2020).

C. verum, N. oleander, L. nobilis, and E. camaldulensis extracts exhibit antioxidant activity that can help protect against various diseases. In the current study, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay was employed to evaluate the antioxidant potential of the extracts. Results showed that increasing the concentration of extracts enhanced their DPPH radical scavenging ability. At 500 μg/mL, the DPPH scavenging percentages were 89.10 ± 0.36, 90.64 ± 0.61, 82.10 ± 0.53, and 80.39 ± 0.24, respectively, with corresponding IC₅₀ values of 13.95 ± 0.35, 41.39 ± 0.29, 12.60 ± 0.51, and 8.84 ± 0.04 μg/mL. For the ABTS assay at 250 μg/mL, scavenging percentages were 90.88 ± 0.35, 94.92 ± 1.25, 93.61 ± 0.38, and 88.88 ± 0.44, with IC₅₀ values of 16.25 ± 0.25, 111.54 ± 6.02, 30.32 ± 3.22, and 38.65 ± 1.57 μg/mL, respectively (Tables 1 and 2). The radical-scavenging activity of the phenolic compounds is primarily responsible for the antioxidant effects observed in these plant extracts.

Laurus nobilis extracts have been reported to be rich in polyphenols. Our findings are consistent with those of Dobroslavic et al. (2022), who observed that L. nobilis extract exhibited significant antioxidant capacity, attributed to the high degree of hydroxylation in its phenolic compounds. This structural feature enhances the extract’s ability to donate protons and stabilize free radicals such as DPPH. Notably, L. nobilis demonstrated strong antioxidant activity against DPPH radicals, as also reported by El Faqer et al. (2024). According to Stagos (2019), the antioxidant activity of these extracts is primarily driven by polyphenols acting as effective reducing agents.

The distinct chemical compositions—particularly in phenolics, tannins, and flavonoids—among C. verum, N. oleander, L. nobilis, and E. camaldulensis explain the observed variations in their enzyme inhibitory and antioxidant activities. While L. nobilis and N. oleander exhibited superior enzyme inhibition, E. camaldulensis consistently demonstrated higher total phenolic, tannin, and flavonoid contents, which contributed to its strong antioxidant capacity. These findings highlight the critical role of plant species in determining the therapeutic potential of natural extracts.

The superior antioxidant activity of E. camaldulensis and L. nobilis, demonstrated by their lower DPPH IC₅₀ values (8.83 and 12.50 µg/mL, respectively) and ABTS IC₅₀ values (38.65 and 30.32 µg/mL), compared to N. oleander and C. verum (DPPH IC₅₀: 32.10 and 13.95 µg/mL; ABTS IC₅₀: 111.53 and 16.25 µg/mL), strongly correlates with their higher phenolic content. This finding aligns with established literature that links phenolic-rich extracts to enhanced radical scavenging capacity (Mohammed et al., 2021; Ouattara et al., 2024).

The Hep-G2/2.2.15 human hepatoblastoma cell line was treated with serial concentrations (0, 2.5, 5, 10, 25, 50, and 100 μg/mL) of methanolic extracts from C. verum, N. oleander, L. nobilis, and E. camaldulensis for 48 hours. The IC₅₀ values for the extracts on the Hep-G2/2.2.15 cells were determined as 54.48 ± 21.61, 44.20 ± 0.0075, 137.48 ± 12.30, and 54.48 ± 21.61 μg/mL, respectively.

The superior α-amylase and α-glucosidase inhibitory activities observed in the methanolic extracts of L. nobilis and N. oleander—and to a lesser extent, C. verum and E. camaldulensis—are likely due to a complex interplay of their unique phytochemical compositions. While polyphenols such as flavonoids, tannins, and phenolic acids are consistently implicated in enzyme inhibition across many plants, other specialized metabolites also play significant roles. For instance, essential oil components like terpenoids and specific compounds such as cardiac glycosides in N. oleander contribute to the distinct inhibitory profiles and potency variations among these species. Further research focusing on the isolation and characterization of individual bioactive compounds from these potent extracts is essential to elucidate the precise mechanisms underlying their enzyme inhibitory effects.

Conclusions

This study underscores the significant therapeutic potential of Cinnamomum verum, Nerium oleander, Laurus nobilis, and Eucalyptus camaldulensis as rich sources of bioactive compounds. Methanolic extracts from these plants effectively inhibit α-amylase and α-glucosidase enzymes, with L. nobilis and N. oleander exhibiting particularly strong inhibitory activity, suggesting their promise as natural alternatives to synthetic inhibitors such as acarbose. Additionally, their potent antioxidant capacities, closely correlated with their phenolic content—especially notable in E. camaldulensis and L. nobilis—highlight their potential in mitigating oxidative stress-linked diseases. These findings open promising avenues for both industrial and clinical applications, positioning these plants as valuable natural antidiabetic agents that provide safer and more affordable options, particularly in developing regions. Furthermore, their antioxidant properties support the development of functional foods and dietary supplements aimed at preventing or alleviating chronic conditions such as cardiovascular diseases. The sustainability and widespread availability of these plant resources further enhance their appeal for pharmaceutical and food industry use, contributing to the advancement of a bioeconomy focused on natural product innovation.