Introduction

In contemporary times, medicinal plants represent an essential therapy for some people especially in Western countries due to the undesirable effects of synthetic chemical drugs and the substantial financial benefits1,2. However, despite the difficulty of estimating the exact commercial value of plants and plant extracts, it is clear that folk medicine-related industries grow by more than 4% annually3,4. The standing of medicinal plants and non-conventional plants is attributed to the high multiplicity of bioactive components, like flavonoids, alkaloids, lignans, triterpenoids, essential oils, carotenoids, tocopherols, and vitamins5,6. Some of these bioactive molecules demonstrate remarkable antioxidant properties which are essentially searched for their valuable impact on various chronic diseases, like Alzheimer’s disease (AD), skin wrinkles, pigmentation, obesity, diabetes mellitus, and cancer7,8,9.

Natural products including essential oils have been recognized via scientific reported literature for their activities against AD. Tumeric (Curcuma longa)10, guava (Psidium guajava)11, and bitter orange (Citrus aurantium)12 showed potential against acetylcholinesterase and butyrylcholinesterase. Tyrosinase is a key enzyme in melanogenesis. The antityrosinase activity of many plants has also been studied for the development of functional cosmetics because the inhibition of this enzyme can attenuate melanin synthesis to induce a whitening effect. Several medicinal plants have been reported for tyrosinase inhibitory effects such as Milk thistle (Silybum marianum), lemongrass (Cymbopogon citrates), and licorice (Glycyrrhiza glabra)13.

Recently, there has been renewed interest in functional food products and plant-based therapies that modify the physiological properties for the management of diabetes mellitus and aging symptoms14,15. One of the targeted antidiabetic approaches is to inhibit carbohydrate-metabolizing enzymes like α-glucosidase and α-amylase3. Thus, hyperglycemia can be managed in the type-II diabetic patients and borderline patients3,16. Arrays of previous in vitro studies have shown the potential role of several plants as α-amylase and α-glucosidase inhibitors such as cranberry (Vaccinium macrocarpon)17, black plum (Syzygium cumini)18, turmeric (Curcuma longa)19, Black pepper (Piper nigrum)20, soybean (Glycine max)21 Fenugreek (Trigonella foenum-graecum ) and turmeric (Curcuma longa)22. Consequently, the plant-derived α-glucosidase and α-amylase inhibitors provide an attractive strategy to restrain postprandial hyperglycemia.

The Pimenta genus is one of the evergreen flowering trees that incorporates about fifteen species and belongs to the family Myrtaceae23,24. Pimenta plants are distributed in West Indies, Central America and Jamaica25. In traditional medicine, different Pimenta species were employed for treatment of common cold, viral infections, bronchitis, dental pain, muscle aches, rheumatic pains, and arthritis26. Moreover, Pimenta essential oils exert antimicrobial, antiseptic, anesthetic, and analgesic effects25. From the phytochemical aspect, the isolated essential oils of Pimenta plants are characterized by an abundance of eugenol, methyl eugenol, β-caryophyllene, and myrcene27. Whereas limonene, 1,8-cineole, terpinolene, β-selinene, and methyl eugenol were reported to be the predominant components in some studies28. P. racemosa (Mill.) J. W. Moore is one of the most characteristic aromatic plants of this genus, due to its high essential oil content and diverse medicinal targets29.P. racemosa is native to Venezuela, Puerto Rico, and Jamaica in the Caribbean30. It is characterized by the richness of essential oil which is commonly named “bay oil” or “Myrcia oil”30. Traditionally, P. racemosa was utilized for different diseases in the Dominican Republic of the Caribbean basin; the leaf oil is used for rheumatism and toothache due to its anti-inflammatory and analgesic effects31. The infusion of the bark is used against hypertension30. The leaf essential oil was reported to possess antimicrobial effects against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Aspergillus niger, Penicillium verrucosum and Cladosporium cladosporioides32. Several biological studies have demonstrated its anti-cancer33, anti-inflammatory34, nematicidal activity35, and insecticidal activities36.

In vitro antidiabetic assays are important to determine the activity of plant extract or compounds hence saving time, cost, and effort37. Interestingly, no previous data have been reported on the antioxidant or enzyme inhibition data of P. racemosa leaf oil. Thus, the current study was designated to explore the chemical composition of essential oil isolated from P. racemosa leaves growing in Egypt via GC-MS chemical analysis, alongside investigation of its antioxidant property and enzyme inhibitory activity against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), tyrosinase, α-glucosidase, and α-amylase.

Materials and methods

Plant material and oil isolation

P. racemosa (Myrtaceae) were collected as fresh leaves in March 2023 from El-Zohrya Garden (30°02′45″N 31°13′28″E), Zamalek, Cairo Governorate, Egypt. The collection of plant material was permitted and established in compliance with the national guidelines. A voucher specimen under code PHG-P-PR-473 was put at the Herbarium of the Department of Pharmacognosy, at the Faculty of Pharmacy, Ain Shams University, Cairo, Egypt. The leaves were accurately authenticated by Mrs. Treize Labib, a taxonomy expert at El-Orman Botanical Garden, Giza-Egypt. About 1 kg of the fresh leaves were finely cut, and hydrodistilled using a Clevenger apparatus for a period of 4 h. Afterwards, the essential oil isolation was accomplished and stored in a sealed glass tube at -5 °C waiting for GC-MS analysis and further biological exploration.

Chemicals

The chemicals were purchased from Sigma-Aldrich (Darmstadt, Germany). They were: 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS), 1,1-diphenyl-2-picrylhydrazyl (DPPH), electric eel acetylcholinesterase (AChE) (type-VI-S, EC 3.1.1.7), horse serum butyrylcholinesterase (BChE) (EC 3.1.1.8), galantamine, acetylthiocholine iodide (ATChI), butyrylthiocholine chloride (BTChI) 5,5-dithio-bis(2-nitrobenzoic) acid (DTNB), tyrosinase (EC1.14.18.1, mushroom), glucosidase (EC. 3.2.1.20, from Saccharomyces cerevisiae), amylase (EC. 3.2.1.1, from the porcine pancreas), sodium molybdate, hydrochloric acid, sodium hydroxide, trolox, ethylenediaminetetraacetate (EDTA), neocuproine, cupric chloride, ammonium acetate, ferric chloride, 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ), ammonium molybdate, ferrozine, ferrous sulphate hexahydrate, kojic acid, and acarbose. All chemicals were of analytical grade.

Gas chromatography/mass spectrometry (GC-MS) analysis

At the pharmacognosy department of Ain Shams University, Cairo, Egypt, the isolated oil was analyzed with a Shimadzu GCMS-QP 2010 (Kyoto, Japan) and a TRACE GC Ultra Gas Chromatograph (THERMO Scientific Corp., USA), coupled to a thermo-mass detector as previously reported38,39,40. The column specifications (Restek, USA) include a TG-5MS capillary with an internal diameter of 30 m x 0.25 mm i.d. and film thickness of 0.25 μm, directly attached to a quadrupole mass spectrometer (SSQ7000; Thermo-Finnigan). The carrier gas is helium with a constant flow rate of 1.0 mL/min. The sample is diluted with a ratio 1% v/v, the injected volume = 1 µL, and a split ratio of 1:15. Initially, the oven temperature of 80 °C was maintained for 2 min (isothermal), and then 300 °C was reached by raising 5.0 °C/min and retaining it for 5 min (programmed). The temperatures of the injector and detector were set at 280 °C. Using an electron ionization (EI) mode of 70 eV, a scanned spectrum at m/z 35–500 was used to obtain mass spectra of the ions: interface temperature = 280 °C, ion source temperature = 200 °C. A peak area normalization technique was used to determine the relative proportions of the hexane extract constituents.

Percentage peak area method

The percentage peak area method uses the area of the target component peak as a proportion of the total area of all detected peaks to analyze quantity. This method is used to determine an approximate concentration of a sample mixture or changes in the concentration of a known sample mixture.

GC-MS identification of chemical components of the essential oil

To tentatively identify oil components, the GC-FID and GC-MS spectra were used, and fragmentation patterns, mass numbers, and Kovats retention indices were compared with those published by Wily, NIST, and literature sources18,41,42,43,44,45,46. Under the same conditions, homologous series of n-alkanes (C8-C28) were injected, and retention indices were calculated. In this method, peak areas were calculated as a ratio of the peak areas of each compound to the area of the entire FID chromatogram (100%).

Antioxidant and enzyme inhibitory assays

The essential oil was subjected to six spectrophotometric tests to determine its antioxidant potential. The essential oil solution was carefully formulated in ethanol at a concentration of 2 mg/mL. All chemicals were freshly prepared, with special attention to keeping the enzymes and their corresponding substrates in an ice bath for upcoming. We prepared an ethanolic solution of the essential oil, and then transferred it to the reaction medium. To assess the antioxidants’ aptitude to neutralize free radicals, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays were employed47. Also, the reduction capabilities of isolated essential oil were evaluated through ferric ion-reducing antioxidant power (FRAP) and cupric-reducing antioxidant capacity (CUPRAC) tests47. Furthermore, phosphomolybdenum (PBD) and ferrozine assays were used to measure the antioxidant activity and metal-chelating capability of the essential oil, respectively47,48. Apart from metal chelating ability (MCA), each of these assays was valued using a Trolox standard (mg TE/g). As for MCA, its comparison was estimated according to the EDTA equivalent per gram of essential oil (mg EDTAE/g). All procedures are detailed in our previous works47,48. To explore the inhibitory effects of the tested oil on various enzymes, we applied acetylcholinesterase (AChE), butyrylcholinesterase (BChE), tyrosinase, and amylase, and glucosidase and the experimental procedures were described previously in our earlier work48,49. As a measure of AChE and BChE inhibition, we measured the inhibition using milligram galantamine equivalents per gm of essential oil (mg GALAE/g), the inhibition of tyrosinase expressed as mg kojic acid equivalents per gm essential oil (mg KAE/g), and the inhibition of amylase expressed in milligrams of acarbose equivalents per gm essential oil (mg ACAE/g). The measurements of these antioxidant and enzyme assays were carried out using a microplate reader ((Thermo Multiskan GO, Thermo, United States). Each assay was carried out with three technical replicates. The details of each essay were shown in the supplementary file. Further, the calibration curves of all used standards are illustrated in Table S1.

Results and discussion

GC-MS analysis of leaf essential oil from P. racemosa

The GC-MS analysis was employed to characterize the volatile components of essential oil isolated from P. racemosa leaves. It revealed a depiction of seventeen compounds constituting about 99.76% of the total oil components as illustrated in Table 1 and Fig 1. The Phenylpropanoids are the chief class of oil components representing 72.35%, among them eugenol (70.87%) and chavicol (1.48%). Hydrocarbon monoterpenes represent the second major class of oil components with a total percentage of 23.41% predominated by β-myrcene (12.88%), D-limonene (8.35%), and p-cymene (1.12%). The oxygenated monoterpenes accounted for about 2.33% of total oil including β-linalool (2.14%) and terpinene-4-ol (0.19%). Hydrocarbon sesquiterpene showed a minor composition percentage of oil (0.32%) represented by (E)-β-caryophyllene oxide (0.16%) and τ-cadinol (0.16%). The chemical structures of the identified major oil components are illustrated in Fig. 2. Certain Egyptian studies reported a predominance of monoterpene hydrocarbons (64.40%) predominated by β-myrcene (39.60%) and eugenol (31.00%) in P. racemosa leaf oils25. While the Cuban type of P. racemosa leaf oil demonstrated a high content of 1,8-cineole (14.69%)50. In Benin, the leaf essential oil was quantified with eugenol (45.20%), myrcene (25.10%), chavicol (7.10%), and limonene (3.0%) as major compounds30. Unlike that, another study in Santo Domingo demonstrated the presence of α-terpineol acetate (27%), α-terpineol (20%), and methoxy eugenol (12.6%)31. It is assumed that these differences in the chemical composition of the above-mentioned essential oils can be ascribed to differences in the cultivation environment, geographical resources, climate, genotype, and collection time51. Few literatures about the chemical nature of the essential oil of P. racemosa var. racemose were reported which matched our result. Contreras-Moreno et al., and Tucker et al., revealed the presence of eugenol as the major compound in the same chemotype (60.4–82.9%; 44.41–68.93% respectively)52,53. The main oil components previously identified in P. racemosa, their percentage, and the location are illustrated in Table 2.

Fig. 1
figure 1

GC-MS chromatogram of the leaf essential oil isolated from P. racemosa grown in Egypt.

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Table 1 GC-MS chemical composition (%) of the essential oil from P. racemosa grown in egypt.
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Fig. 2
figure 2

The structures of the volatile components detected in the essential oil of P. racemosa leaves grown in Egypt [1: α-Pinene, 2: β-Myrcene, 3: α-Phellandrene, 4: p-Cymene, 5: D-Limonene, 6: (E)-β-Ocimene, 7: β-Linalool, 8: Terpinen-4-ol, 9: Chavicol, 10: Eugenol, 11: α-Copaene, 12: (E)-β-Caryophyllene, 13: α-Murolene, 14: 3-Cyclohexene-1-carboxaldehyde, 15: 4-(4-methyl-3-pentenyl)-, 16: δ-Cadinene, (E)-β-Caryophyllene oxide, 17: τ-Cadinol].

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Table 2 The main oil components previously identified in P. racemosa, their percentage, and the location.
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Assessment of antioxidant activity of the essential oil isolated from P. racemosa

Reactive oxygen species are linked to various human diseases and aging. The elevated levels of free radicals, like reactive oxygen and nitrogen species, are central to oxidative damage in biomolecules (e.g., DNA, proteins, and lipids) and play a role in food and material degradation60,61,62. These factors are also associated with chronic diseases and aging63. Various assays are available to assess the antioxidant capacity of samples, and this study employs six different types including 2,2-diphenyl-1-picrylhydrazyl (DPPH), [2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] (ABTS), cupric reducing antioxidant capacity (CUPRAC), ferric reducing ability of plasma (FRAP), and phosphomolybdenum (PBD) assay as illustrated in Table 3. The essential oil isolated from P. racemosa leaves demonstrated remarkable antioxidant capabilities in both anti-radical and radical scavenging assays. Specifically, it exhibited the highest FRAP activity with a reducing power of 1506.62 mg Trolox equivalent (TE)/g, followed by the ABTS scavenging test at 1346.85 mg TE/g. Additionally, the essential oil displayed 1032.83 mg TE/g DPPH scavenging activity and a reducing power of 1001.03 mg TE/g in the CUPRAC assay. These antioxidant properties can be attributed to its major phenolic component, eugenol, as well as other hydrocarbon monoterpenes constituents. Furthermore, the essential oil showed a metal chelation activity (MCA) of 25.63 mg EDTAE/, and (PBD) 209.59 mmol TE/g. These results support the use of P. racemosa leaf essential oil as a natural antioxidant, addressing concerns about the safety of synthetic antioxidants.

Our findings closely align with the results reported by Al-Gendy et al.58, indicating that the P. racemosa flower essential oil exhibits a remarkably high scavenging capacity of 85.96%. These findings align with prior research confirming the antioxidant properties of P. racemosa essential oils64. Phenolic compounds exert antioxidant activity via different mechanisms; transference of hydrogen atom or single electron, transition metal chelation, or sequential proton loss electron transfer65. The robust antioxidant activity observed in the essential oil of P. racemosa can be attributed to its significant eugenol content, a well-established phenolic compound renowned for its antioxidative properties, as substantiated by previous studies66,67,68. Eugenol’s effectiveness as an antioxidant is, in part, attributed to the presence of a methoxy group in the ortho position of the phenolic hydroxyl, facilitating the homolytic dissociation of the O–H bond69. Additionally, chavicol, another phenolic compound identified in P. racemosa essential oil in this study, is recognized for its antioxidant activity70. Notably, this essential oil also contains a substantial concentration of β-myrcene, which serves as a major oxidation product71. Moreover, it is important to mention that the synergy among various compounds, even at lower concentrations, may contribute to the overall antioxidant effect.

Table 3 Total antioxidant effects of the tested essential oil in different in vitro assays.
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Assessment of enzyme inhibitory activities of the essential oil isolated from P. racemosa

The study of enzyme inhibition is extensively researched due to its pivotal role in comprehending enzyme mechanisms72 and its significance in both human and animal pharmaceuticals within the realm of pharmacological research73,74. For example, in the case of AD, which affects over 35 million individuals globally, inhibiting cholinesterase enzymes is a key therapeutic approach75. Enzyme’s acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) decrease acetylcholine levels, alleviating symptoms as the disease advances. Researchers are particularly interested in selective AChE and BChE inhibitors76. Plant bioactive compounds hold promise in effectively targeting acetylcholine-regulating enzymes for therapeutic purposes in diseases like AD77. Tyrosinase, another enzyme plays a central role in melanin synthesis and enzymatic browning in various contexts, including mammalian melanogenesis and fruit/fungi processes78. Inhibiting tyrosinase activity has broad applications in medicine and cosmetics, addressing hyperpigmentation and serving as a focal point in this study’s investigation79.

In the current study, in terms of enzyme inhibitory activity, the essential oil of P. racemosa displayed significant AChE and BChE inhibitory activities, with values of 1.96 mg GALAE/g and 1.42 mg GALAE/g, respectively (Table 4). Additionally, the essential oil exhibited a moderate level of tyrosinase inhibitory activity, measuring 38.83 mg KAE/g (Table 4). These findings underscore the potential of this essential oil in enzyme-related applications, with notable effects on acetylcholinesterase AChE and BChE inhibition, as well as a modest influence on tyrosinase inhibition.

Diabetes mellitus is a widespread epidemic, characterized by the inhibition of enzymes like α-amylase and α-glucosidase that convert dietary starch into glucose80. Plant extracts have shown promise in inhibiting these enzyme activities, offering potential health benefits, and serving as a focus in this study81. In the present study, for the 1st time, the essential oil extracted from the P. racemosa demonstrated a lower level of α-amylase inhibition activity, measuring at 0.08 mmol ACAE/g, while it exhibited a significantly higher α-glucosidase inhibitory activity, with a value of 2.38 mmol ACAE/g (Table 4).

In the current investigation, the plant under study exhibited a moderate inhibitory effect on the specified enzymes, as opposed to its antioxidant properties. No research has been conducted to date that demonstrates the inhibitory effects of P. racemosa extract or essential oil on AChE, BChE, and tyrosinase activities. Nevertheless, previous research by Adefegha et al., and Bilgicli et al., indicated that eugenol exhibited a more pronounced inhibitory effect on both AChE and BChE activities82]– [83. This compound possesses a diverse array of pharmacological properties, including neuromodulatory and antioxidant characteristics. Moreover, research conducted by Varela et al. and Marongiu et al. aligns with our findings, supporting the notion that eugenol exhibits a relatively less potent tyrosinase inhibition84]– [85. In our present study, we observed that P. racemosa essential oil displays a heightened level of α-glucosidase inhibitory activity when compared to its α-amylase inhibitory potential. In contrast to our current investigation, a previous study has validated that P. racemosa exhibits a greater inhibitory effect on α-glucosidase in comparison to α-amylase. To illustrate, the application of 2.00 mg of P. racemosa leaf essential oil led to a notable 63.90% increase in α-amylase inhibitory activity and a 53.00% increase in α-glucosidase inhibitory activity86. Similarly, several studies confirmed the pronounced α-glucosidase inhibitory properties of eugenol83,87]– [88. β-Myrcene is a monoterpene hydrocarbon that exerts antioxidant activity89 as well as neuroprotective activity where it could inhibit the acetylcholinesterase activity in different brain regions of AlCl3 and D - galactose-induced Alzheimer disease mice90. The essential oils of citrus have been reported by Matsuura et al. (2006) to possess strong antityrosinase activity due to the presence of β-myrcene91. Limonene, the 3rd predominant component of the Pimenta oil, is the most abundant compound in the peel oil of orange. The latter showed strong activity against α-amylase and α-glucosidase92, and anti-tyrosinase inhibitory activity. The presence of the major compounds as well as the synergistic effect with other minor compounds are responsible for the activity exhibited by the oil.

Table 4 The enzyme inhibitory effects of the tested essential oil.
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Conclusion

Our findings proved that the essential oil of P. racemosa possesses potent antioxidant properties as confirmed in six in vitro assays, suggesting that it represents a highly promising resource for the development of therapeutic molecules and health-promoting agents. Our study, for the first time, indicates that the essential oil derived from P. racemosa substantially showed inhibitory activities against acetylcholinesterase, butyrylcholinesterase, tyrosinase, α-glucosidase, and α-amylase enzymes. This emphasizes the potential for exploiting P. racemosa-based preparations or adjuvant therapies for the management of chronic diseases such as Alzheimer disease, diabetes mellitus, and skin-related applications. However, further in vivo studies are recommended as an important phase in screening the plant oil for physiological, pharmacological, and toxicological properties.