Antilipase activities of cultivated peppermint and rosemary essential oils: in vitro and in silico studies

Abstract

Background/aim

The growing interest in essential oils clearly indicates the power of nature and aligns with our increasing need to find therapeutic solutions in the natural world. This study aimed to investigate the inhibitory effects of the essential oils of Mentha × piperita and Salvia rosmarinus, harvested from the Laghouat region of Algeria, against Candida rugosa lipase (CRL) and pancreatic lipase through both in vitro and in silico studies.

Materials and methods

Essential oils were extracted via hydrodistillation and analyzed using gas chromatography–mass spectrometry and spectrophotometry. Their antilipase activities were assessed using an inhibition assay, and molecular docking was performed with AutoDock Vina to explore interactions between essential oil compounds and lipase enzymes.

Results

Spectrophotometric analysis demonstrated significant inhibitory activity for each essential oil against CRL lipase, with IC₅₀ values of 0.56 ± 0.005 and 0.69 ± 0.008 mg/mL for peppermint and rosemary oils, respectively. These results were satisfactory in comparison to those achieved with orlistat. Molecular docking studies revealed the mechanisms of major compounds in each essential oil, demonstrating that these compounds inhibited CRL (PDB ID: 1CRL) and pancreatic lipase (PDB ID: 1LPB) with repeated hydrophobic interactions. The interactions were observed to be consistent with His449, Gly123, Gly124, Phe344, and Ser152 for many molecules.

Conclusion

This study highlights opportunities for essential oils and their bioactive components to be utilized as adjuvants in the management of obesity and other lipase-related disorders.

1. Introduction

Natural products have gained considerable attention as a rich source of biologically active compounds. There is an increasing need to incorporate these natural substances into pharmaceutical research to develop effective therapies with fewer side effects (Clark, 1996; Amaral-Machado et al., 2020; Sauter, 2020; Zhang et al., 2020; Najmi et al., 2022). Among these compounds, many have shown potential in treating various medical conditions, including obesity. Obesity constitutes a global health threat (Blüher, 2019; Kopelman, 2000; Safaei et al., 2021). According to the World Health Organization, in 2022, 2.5 billion adults aged 18 and older were classified as overweight, with 890 million of them considered obese.1 This alarming figure continues to rise, and if left unchecked, the prevalence of obesity will keep increasing in the coming years. Obesity is not only a major health concern but also a contributor to several serious conditions, such as type 2 diabetes (Kopelman, 2000; Safaei et al., 2021), liver disease, and cardiovascular complications (Kopelman, 2000; Lopez-Jimenez et al., 2022). It also imposes daily challenges on affected individuals, leading to increased sedentary behavior, which negatively impacts the global economy (Hammond and Levine, 2010; Okunogbe et al., 2021). One of the key contributors to obesity is the excessive activity of the enzyme lipase (Lunagariya et al., 2014; Rocha et al., 2023), which breaks down triglycerides into glycerol and fatty acids that are stored as triglycerides in adipocytes (Miyake, 2001; Birari and Bhutani, 2007; Kumar and Chauhan, 2021; Rocha et al., 2023). The prolonged accumulation of adipose tissue leads to weight gain and, ultimately, obesity.

Peppermint (Mentha × piperita) is a member of the family Lamiaceae renowned for its distinctive flavor and medicinal attributes, which are primarily attributed to its chemical constituents. Since antiquity, the essential oil of peppermint has been utilized in medicine and disinfection and has served various other functions, including use as a deodorant. Peppermint oil is a well-known antioxidant that protects cells from damage caused by free radicals and has antiinflammatory, antibacterial, and antifungal properties (Burbott et al., 1983; McKay and Blumberg, 2006; Khalvandi et al., 2019).

Rosemary (Salvia rosmarinus) is a shrub species of the family Lamiaceae. The essential oil of rosemary is extensively utilized in traditional medicine and aromatherapy for its numerous health advantages. It contains compounds that improve memory and concentration; its inhalation is believed to augment cognitive performance, mitigate pain and muscular inflammation, and reduce respiratory issues such as congestion, asthma, sore throats, and colds. It possesses a calming antibacterial effect and enhances intestinal health (Soliman et al., 1994; Boutabia et al., 2016; Hannour et al., 2018; Lešnik et al., 2021; Annemer et al., 2022).

This study aimed to explore safe and effective solutions to obesity by inhibiting lipase activity using natural inhibitors derived from the essential oils of M. × piperita and S. rosmarinus sourced from the Laghouat region of Algeria. The chemical composition of peppermint and rosemary essential oils was determined using gas chromatography–mass spectrometry (CG-MS) and then the efficacy of these essential oils in inhibiting Candida rugosa lipase (CRL) was evaluated. Subsequently, binding affinities and interactions between the constituents of the oils and CRL and human lipase were investigated with molecular docking studies.

2. Materials and methods

2.1. Plant material

This study was carried out at the ETS Aquacol Laghouat aquacultural farm in Laghouat, Algeria (33°47′N, 2°50′E; elevation of 750 m). The province of Laghouat is bordered by Tiaret to the north, El Bayadh to the west, Ghardaia to the south, and Djelfa to the east. It is located in the heart of the Saharan Atlas range and extends across the steppe. The climate2 is continental, with extremely hot summers and freezing winters. Rainfall in the region is uneven, averaging 180 mm per year, with significant droughts in some years (Boumeddiene et al., 2023).3 The site has arid sandy soils with high water permeability and pH of 6.9.

We planted M. × piperita using seedlings received from Haffaci Ahmed, a member of the Professional Council of Aromatic and Medicinal Plants of Laghouat. We planted the seedlings in the field in the spring of 2022 in a location that receives direct sunlight for about 6–8 h each day, as peppermint thrives in sunny conditions, and we watered the plants three times a week. This watering schedule helped keep the soil consistently moist without becoming waterlogged. To preserve the plants from overharvesting and prevent desertification, we planted S. rosmarinus from cuttings collected in the El-Ghicha region of Laghouat (33°56′14″N, 2°08′54″E; elevation of 1207 m). Rosemary seedlings were planted in the spring of 2021 and watered once a week after making sure the soil was dry because rosemary plants do not tolerate wet soil. They were planted in an isolated region to prevent cross-hybridization. We used a drip irrigation system with water from aquaculture ponds. This water was rich in organic and mineral matter, which included essential nutrients such as nitrogen, phosphorus, and potassium and other micronutrients. These nutrients significantly enhance soil fertility, ensuring plant health and promoting vigorous growth (Zajdband, 2011; Hasimuna et al., 2023; Ignowski et al., 2023).4

2.2. Essential oil extraction procedure

Leaves of M. × piperita and S. rosmarinus were collected in July 2022 and dried in a dark, well-ventilated chamber. Once adequately dried, the leaves underwent essential oil distillation in October 2022. Using steam distillation, the essential oil was extracted from the leaves of both M. × piperita and S. rosmarinus. Briefly, the dried plant parts were placed in an alembic distillation unit (BF50 50L, Biofleur, Algeria). Steam was passed through the material for 3 h and the distillate was collected in a graduated cylinder. The oily layer was separated from the aqueous layer using a separating funnel, dried over anhydrous sodium sulfate, and stored at −4 °C until further analysis. The essential oil yield was calculated as follows:

$$T\%=\left(\frac{m}{m_0}\right)\times 100$$

Here, T% is the content expressed in %, m is the mass in grams of essential oil recovered, and m₀ is the initial mass of plant material in grams.

2.3. Determination of essential oil composition using GC-MS

We tested the samples via the Technical Platform of Physico-Chemical Analysis (PTAPC-CRAPC) of Laghouat in Algeria employing a GCMSQP2020 instrument (Shimadzu, Kyoto, Japan). This instrument was equipped with a fused Rxi-5ms capillary column (Phase: Crossbond, 5% diphenyl/95% dimethyl polysiloxane) with dimensions of 30 m × 0.25 mm and film thickness of 0.25 μm (Restek, Bellefonte, PA, USA). This column has a phase identical to HP-1ms, HP-1msUI, DB-1ms, DB-5ms, DB-1msUI, Ultra-1, VF-1ms, ZB-1, and ZB-1ms and is also considered equivalent to the USP G1, G2 and G38 phases. Diluted material (0.5 μL) in n-hexane was injected using a split ratio of 1:80. The injector and detector temperatures were set at 250 °C and 310 °C, respectively. The column temperature was initially fixed at 50 °C for 2 min and then raised to 310 °C at a rate of 3 °C/min, and then it was held at 310 °C for 2 min. The carrier gas employed was helium (99.995% purity) at a flow rate of 1 mL/min. The parameters for the mass spectrometer were as follows: ionization voltage of 70 eV, ion source temperature of 200 °C, and electron ionization mass spectra generated within the mass range of 45–600 m/z. The retention indices were determined using the following equation:

$$LRI=100\times \left(\frac{t_x-t_n}{t_{n+1}-t_n}\right)+100\times n$$

Here, LRI is the linear retention index, t_x is the retention time of the targeted component, t_n is the retention time of the C_n alkane (lower bound n-alkane), and t_n+1 is the retention time of the C_n+1 alkane (higher bound n-alkane). The n-alkane series in these analyses was (n–C7–C33).

2.4. Chemicals and reagents

CRL and p-nitrophenyl laurate (p-NPL) were acquired from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals and solvents utilized were of analytical quality and were also procured from Sigma-Aldrich.

2.5. Evaluation of in vitro antioxidant activity

We performed free radical scavenging and ABTS inhibition tests to evaluate the in vitro antioxidant activity of the M. × piperita and S. rosmarinus essential oils.

2.5.1. DPPH assay

Our spectrophotometric method, based on the free radical DPPH^•, was performed with a Multiskan GO microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at 517 nm. Extracted samples were dissolved in ethanol at concentrations of 0.1 to 1 mg/L, 120 μL of the sample solution was mixed with 120 μL of DPPH solution, and the absorbance of the mixture was measured after 30 min of incubation at ambient temperature in darkness (Gulcin and Alwasel, 2023). The radical scavenging activity (I%) was calculated using the following formula:

$$I\%=\left(\frac{A_0-A_1}{A_0}\right)\times 100$$

Here, I% is the percentage of inhibition, A₀ is the absorption of the DPPH solution without the essential oil, and A₁ is the absorption of the solution with the essential oil.

Percentage data were utilized to construct a graph illustrating the variation in absorbance in relation to the concentration of the essential oil samples. This analysis was used to determine the IC₅₀ values, representing the concentration of essential oil (mg/mL) required to inhibit 50% of the free radicals (DPPH). Trolox and quercetin were employed as reference antioxidant standards for comparisons.

2.5.2. ABTS assay

An ABTS radical cation solution (100 μL of ABTS, 15 μL of hydrogen peroxide, and 100 μL of peroxidase enzyme, with the volume completed to 100 mL with water) was prepared. For the measurement of the radical scavenging activity, 200 μL of diluted ABTS radical cation solution was mixed with 40 μL of a sample solution of essential oils at different dilutions (0.1 to 1 mg/mL), and the absorption was measured with the Multiskan GO microplate reader after 6 min at 417 nm. A calibration curve with Trolox and quercetin standards was measured in the range of 0.1–1 mg/mL (Ilyasov et al., 2020). Values of I% were calculated as described for the DPPH assay.

2.6. C. rugosa lipase assay

The p-NPL substrate was initially dissolved in isopropanol at a concentration of 1 mM. Subsequently, it was diluted at a ratio of 1:10 (v/v) in a buffer solution containing gum arabic with the pH adjusted to 7. To assess the lipase inhibitory activity, volumes of 20 μL of successive essential oil dilutions (0.1 to 1 mg/mL) were preincubated with 20 μL of enzyme solution (1 mg/mL) for 15 min at 37 °C. Subsequently, 180 μL of the p-NPL substrate was introduced. The reaction mixture was incubated for an additional 15 min at 37 °C. The absorbance of p-nitrophenol, indicated by the yellow color arising from the reaction, was promptly measured using the Multiskan GO microplate reader at 405 nm. For the negative control, the extract was replaced by an equivalent volume of methanol. Positive controls were employed to evaluate lipase activity in the presence and absence of the inhibitor. The percentage of lipase inhibition (I%) was calculated, followed by the determination of IC₅₀ values. These results were then compared to results for orlistat and quercetin, which were used as reference compounds. The percentage inhibition of lipase was determined as follows:

$$Inhibition\%=\left(1-\frac{A-A_i}{A_0}\right)\times 100$$

Here, A is the absorbance of the sample containing essential oil, A₁ is the absorbance of the sample devoid of lipase (i.e., the blank), and A₀ is the absorbance of the control without essential oil. The 50% inhibitory concentration (IC₅₀) was determined from the curve generated by graphing the percentage of inhibition against the final concentrations of essential oil (Lante et al., 2004).

2.7. Computational analysis

2.7.1. Biological activity prediction (PASS)

PASS (“Prediction of Activity Spectra for Substances”) is structure–activity relationship software that provides ligand-based virtual screening to find ligands that bind to different receptors and/or molecules with certain biological activities (Abdou et al., 2017; Stasevych et al., 2017; Yildirim et al., 2017; James and Ramanathan, 2018; Pogodin et al., 2018). We used the PASS web server5 to identify the key compounds within M. × piperita and S. rosmarinus essential oils that could serve as potential inhibitors of CRL. The predictive analysis used the structural molecular formulae (SMILES) obtained from the PubChem database.6 The outcome of the PASS prediction was a ranked list of potential biological activities, organized in decreasing order of Pa – Pi values, where Pa represents the likelihood of classification as “active” and Pi denotes the chance of classification as “inactive.” We defaulted to Pa > Pi as the threshold for differentiating between “active” and “inactive” molecules. PASS-based virtual screening for a chemical library thus yields a compilation of molecules identified as “active,” or suitable for biological evaluation (Filimonov et al., 2014; Pogodin et al., 2018).

2.7.2. ADMET evaluation

ADMET, which stands for “absorption, distribution, metabolism, excretion, and toxicity,” is a critical framework in drug research. Many drug candidates have failed during clinical trials or advanced stages of drug development due to inadequate efficacy or the presence of adverse side effects, underscoring the importance of evaluating these pharmacokinetic and toxicological properties early in the drug discovery process (Lucas et al., 2019). We evaluated the ADMET properties of the principal constituents of M. × piperita and S. rosmarinus essential oils using the SwissADME,7 ADMETlab 2.0 (Xiong et al., 2021),8 and admetSAR 3.09 online servers. Quantitative predictions were performed using a set of rules that defined the molecular chemical structure’s ADMET characteristics based on available drug data (Alsawalha et al., 2019).

2.7.3. Molecular docking

The studied compounds, including menthol, camphene, and orlistat, were obtained from the PubChem database.10 For assembly, we utilized Discovery Studio Visualizer Version 2024.11 PDB files for CRL (PDB ID: 1CRL), with resolution of 2.06 Å, and human pancreatic lipase (PDB ID: 1LPB), with resolution of 2.46 Å, were downloaded from the Protein Data Bank.12 We used AutoDock Tools Version 1.5.7 to prepare the enzymes for docking investigations, eliminating all water molecules, heteroatoms, cocrystallized solvents, and ligands. The polar hydrogens and partial charges were included in the structures (Morris et al., 2008; Valdés-Tresanco et al., 2020). Docking simulations were conducted using AutoDock Vina software (Trott and Olson, 2009; Di Muzio et al., 2017). The box’s center was established and the docking box was shown and delineated with AutoDock Tools. The docking was conducted blindly using a grid box of 72 × 68 × 68, with grid points spaced apart at 1 Å, concentrated at the protein locations (x = 72.33; y = 55.391; z = −20.425). All default parameters were employed. Fifty runs were conducted. The seed being searched was arbitrary. The favored conformations were those with the lowest binding energy inside the active site. The ratios of repetitions of the optimal solutions were established. The obtained docking findings were directly imported into Discovery Studio Visualizer Version 2024 (Serseg and Benarous, 2018; Serseg et al., 2020; Eberhardt et al., 2021).

2.8. Statistical analysis

Inhibitory activity was evaluated in all experiments, all of which were performed at least in triplicate, and results were presented as mean values with standard deviations. Data were analyzed at a significance level of p < 0.05. All results were generated using Microsoft Office Excel 2024.

3. Results and discussion

3.1. Extraction yield and composition of essential oils

The yield of peppermint was 1.81% in this study, Abbas et al. (2024) reported a yield of 0.2% for peppermint essential oil in Pakistan (District Khushab, Punjab), while in Ordu, Türkiye, the obtained yield of peppermint essential oil ranged from 0.72% to 1.82% (Saka et al., 2024).

The yield of the rosemary samples was 1.6% in this study, a value comparable to that obtained in the municipality of Bibans in the Algerian province of Bordj Bouarreridj (Annemer et al., 2022). The extraction yield of rosemary essential oil from plants cultivated in the Laghouat region in the present study was higher compared to plants cultivated in other regions of Algeria, with yields of 1.1% from the municipality of Maadid in eastern M’Sila and 0.7% in the province of Oran (Ain Turk) (Hendel et al., 2019). In Morocco, essential oil from plants of the Fez area was obtained with a yield of 1.4% (Elyemni et al., 2019), while plants from Oujda provided yields ranging from 0.6% to 1.7% (Sabbahi et al., 2020). The yield of essential oils can vary depending on several factors, such as the variety and type of plant material, maturity stage, and extraction conditions, all of which affect essential oil production (Abbas et al., 2022).

We used GC-MS to analyze the chemical composition of the essential oils. Based on the results presented in Figures 1 and 2 for both essential oils, the relevant molecules were identified, along with their percentages and retention indices. For both plant species, we identified 100% of the compounds (Table 1).

Figure 1.

GC-MS chromatogram of M. × piperita essential oil.

Figure 2.

GC-MS chromatogram of S. rosmarinus essential oil.

Table 1.

Chemical composition of essential oils of peppermint and rosemary.

Numbers	Retention index	Names of compounds	M. piperita (area%)	S. rosmarinus (area%)
1	914	Tricyclene	-	0.1
2	919	α -Thujene	-	0.18
3	924	α-Pinene	3.39	10.16
4	938	Heptanone <5-methyl-3->	0.33	-
5	938	Camphene	-	3.74
6	963	Sabinene	0.44	4.62
7	967	β-Pinene	2.92	1.11
8	983	Myrcene	0.27	-
9	988	3-Octanol	0.17	-
10	1003	δ-Carene	-	0.27
11	1009	α -Terpinene	-	0.23
12	1016	ρ-Cymene	0.28	2.65
13	1021	Limonene	5.33	-
14	1022	Eucalyptol	1.22	51.6
15	1051	γ-Terpinene	-	0.26
16	1080	Terpinolene	-	0.22
17	1092	Linalool	-	0.76
18	1134	Camphor	-	11.28
19	1147	Menthone	16.43	-
20	1148	Citronellal	0.27	-
21	1156	Isoborneol	13.13	-
22	1165	Borneol	-	3.66
23	1167	Menthol	42.73
24	1168	Terpinen-4-ol	-	0.74
25	1172	cis-Linalool oxide (pyranoid)	0.46	-
26	1175	Cymen-8-ol	0.8	-
27	1181	Isomenthol	2.63	-
28	1181	α-Terpineol	-	2.65
29	1183	neo-Verbanol	0.35	-
30	1203	Verbenone	0.44	-
31	1230	Pulegone	0.47	-
32	1244	Carvacrol, methyl ether	0.55	-
33	1277	Bornyl acetate	-	0.32
34	1286	Menthyl acetate	5.17	-
35	1293	Carvacrol	0.21	-
36	1297	(Z)-Methyl cinnamate	0.16	-
37	1367	α-Copaene	-	0.24
38	1376	(Z)-Ethyl cinnamate	0.17	-
39	1410	Caryophyllene	1.07	3.88
40	1420	ρ-Menth-1-en-9-ol acetate	0.19	-
41	1444	α-Humulene	0.27	0.6
42	1468	γ-Muurolene	-	0.19
43	1488	Viridiflorene	0.17	-
44	1514	δ-Cadinene	-	0.29
45	1572	Caryophyllenyl alcohol	-	0.27
		Total	100%	100%

The nine major compounds of the essential oil of M. × piperita were menthol (42.73%), menthone (16.43%), isoborneol (13.13%), limonene (5.33%), menthyl acetate (5.17%), α-pinene (3.39%), β-pinene (2.92%), isomenthol (2.63%), and eucalyptol (1.22%). A comparison with the literature data revealed quantitative and qualitative discrepancies between the chemical composition of M. × piperita essential oil from the Laghouat region and those obtained in other areas of Algeria. The essential oil of M. × piperita from Chiffa, Algeria, contained some of these compounds, including 32.93% menthol, 24.41% menthone, 8.08% cis-carane, and 7.89% eucalyptol (1,8-cineole) (Benzaid et al., 2019). In the National Park of El-Kala in El-Tarf, Algeria, the essential oil composition was characterized by menthol (49.89%), menthone (20.84%), isomenthone (7.25%), and 1,8-cineole (6.73%) (Benabdallah et al., 2018). The study conducted by Abbas et al. (2024) in Soon Valley, District Khushab, Punjab, Pakistan, corroborated these results with quantity fluctuations being observed, particularly for menthol (52.85%) and menthone (25.93%). The results also varied for the Ouezzane region of Morocco, where the major components were pulegone (17.56%), mintlactone (10.62%), D-carvone (9.24%), eucalyptol (7.53%), and thymol (6.06%) (El Omari et al., 2024).

The major compounds in the essential oil of S. rosmarinus were eucalyptol (1,8-cineole) (51.6%), camphor (11.28%), α-pinene (10.16%), sabinene (4.62%), caryophyllene (3.88%), camphene (3.74%), and borneol (3.66%). Another recently published study similarly revealed the presence of 1,8-cineole (28.6%–51.1%), α-pinene (9.9%–16.2%), camphor (5.3%–16.8%), β-pinene (2.2%–8.0%), and camphene (2.3%–7.7%) for all samples during all periods in essential oil obtained from S. rosmarinus (Annemer et al., 2022). Another study indicated that the major compounds of S. rosmarinus were 1,8-cineole (60.1% and 53.1 %), linalool (9.6% and 5.7%), α-terpineol (4.8% and 4.6%), camphor (1.1% and 11.6%), α-pinene (5.8% and 7.1%), and β-pinene (4.2% and 3.7%) from the Béchar region of Algeria, while camphor (23.2% and 22.9%), 1,8-cineole (13.2% and 14.4%), borneol (10.5% and 8.8%), verbenone (7.6% and 10.2%), campholenol (1.3% and 1.4%), α-pinene (10.0% and 9.0%), and camphene (4.7% and 3.2%) were reported from the essential oil of S. rosmarinus from the Adrar region (Bekhechi et al., 2024). In northern India, the major constituents of this essential oil were camphor (23.9%–35.8%), 1,8-cineole (18.0%–23.9%), α-pinene (4.5%–14.4%), verbenone (6.5%–12.4%), camphene (2.5%–6.9%), limonene (2.1%–2.8%), bornyl acetate (1.1%–4.1%), α-terpineol (1.9%–3.6%), and β-pinene (2.1%–3.3%) (Verma et al., 2020).

The numbers of components of oils fluctuate according to the isolation technique employed. The chemical compositions of M. × piperita and S. rosmarinus are particularly sensitive. According to the literature, several factors influence the quality and quantity of oil components, including environmental conditions (Li et al., 2020), the extraction process (Sadeh et al., 2019), the collection site (Riabov et al., 2020), the plant’s genotype (Ben Ayed et al., 2022), and the harvest season and stage (Li et al., 2020; Verma et al., 2020).

3.2. Antioxidant activity

We evaluated the antioxidant activities of M. × piperita and S. rosmarinus essential oils using the DPPH and ABTS radical scavenging assays and compared the results to those of quercetin and Trolox as positive controls (Table 2). Both essential oils exhibited significant antioxidant activity, as indicated by their IC₅₀ values, which were comparable to those of the positive controls (Figure S1). Specifically, M. × piperita had IC₅₀ values of 67 μg/mL for DPPH and 61 μg/mL for ABTS, while S. rosmarinus had IC₅₀ values of 73 μg/mL and 63 μg/mL for DPPH and ABTS, respectively. These results are in line with those of previous studies reporting the powerful antioxidant properties of S. rosmarinus and M. × piperita essential oils (Chraibi et al., 2020; El Omari et al., 2024). M. × piperita essential oil had IC₅₀ values of 68 μg/mL for DPPH radical scavenging and 76 μg/mL for ABTS (El Omari et al., 2024). A different study showed that S. rosmarinus essential oil had strong antioxidant properties due to its composition involving various phenolic aromatic rings, with an IC₅₀ value of 2770 μg/mL for DPPH radical scavenging (Chraibi et al., 2020). These findings suggest that the essential oils of M. × piperita and S. rosmarinus could serve as natural sources of antioxidants for various applications, including food preservation, medicine manufacturing, and cosmetic formulations (Diniz do Nascimento et al., 2020).

Table 2.

IC₅₀ values reflecting the antioxidant activities of M. × piperita and S. rosmarinus essential oils.

Essential oil	DPPH assay (μg/mL)	ABTS assay (μg/mL)
M. piperita	67±0.15	61±0.5
S. rosmarinus	73±0.3	63±0.2
Quercetin	7.3± 0.00	5.6±0.00
Trolox	5.8±0.00	0.95±0.00

3.3. Antilipase assay

We evaluated the inhibitory effects of peppermint and rosemary essential oils in comparison to quercetin and orlistat to quantify their enzymatic activities against CRL. IC₅₀ values were used to determine the inhibitory potency of these extracts, representing the concentration required to inhibit 50% of the enzyme’s activity. The results are presented in Table 3 (Figure S1).

Table 3.

Inhibitory activities of M. × piperita and S. rosmarinus essential oils against Candida rugosa lipase.

Essential oil	IC50 value (mg/mL)
M. piperita	0.56±0.005
S. rosmarinus	0.69±0.008
Quercetin	0.32±0.004
Orlistat	0.06±0.000

Table 3 reveals that all tested extracts exerted antilipase activity. M. × piperita essential oil, S. rosmarinus essential oil, quercetin, and orlistat had IC₅₀ values of 0.56 ± 0.005, 0.69 ± 0.008, 0.32 ± 0.004, and 0.06 ± 0.000 mg/mL, respectively. These results confirm the potent inhibitory activity of both peppermint and rosemary essential oils against CRL. Compared to quercetin, these essential oils can be considered among the most effective natural medicines for treating obesity today. The impact of the essential oils investigated here against CRL has not been previously reported in the literature except for one previous study in which peppermint essential oil demonstrated weak lipase inhibitory effects compared to our findings, with an IC₅₀ value of 1.09 mg/mL (Serseg et al., 2018). In a study of the inhibitory effect of S. rosmarinus essential oil against pancreatic lipase enzyme, the IC₅₀ value was 0.03225 mg/mL, confirming the importance of S. rosmarinus essential oil in treating obesity (Ali et al., 2014). Other studies have tested the inhibition of other types of lipase using essential oils from taxa of the family Lamiaceae. Lante et al. (2004) conducted a study of the activity of oregano essential oil against two types of lipase and found that this essential oil had an IC₅₀ value of 5.09 μg/mL against Candida antarctica lipase and an IC₅₀ value of 7.26 μg/mL against Pseudomonas fluorescens lipase. Other research has examined the effects of essential oils from different plant families against CRL. These oils had IC₅₀ values of 1.78 mg/mL for Cinnamomum zeylanicum (Lauraceae) and 1.13 mg/mL for Syzygium aromaticum (Myrtaceae) (Serseg et al., 2018).

3.4. Computational analysis

3.4.1. Biological activity prediction (PASS)

To further explore the possible use of the powerful inhibitors found in M. × piperita and S. rosmarinus essential oils in combating obesity, we used the online PASS server to construct their biological activity profiles. This computer program forecasts possible biological activities utilizing an extensive library of chemical compounds and their corresponding activities. Table 4 and Table S1 present the anticipated effects on obesity in terms of weight reduction for the most efficacious inhibitors, offering insights into their prospective therapeutic significance in tackling this widespread health issue. Table 4 presents the list of identified compounds and their potential activities against obesity based on the degrees of potential activity (Pa) and potential inactivity (Pi). Higher Pa scores indicate that a compound is more likely to exhibit antiobesity activity, although Pa scores are not absolute predictors of efficacy. Rather, they indicate potential efficacy. Table 4 reveals several promising compounds for the treatment of obesity, including carvacrol, ρ-cymene and 3-octanol, with Pa scores as high as 0.689, 0.626, and 0.583, respectively. This suggests that these compounds could be further investigated as potential antiobesity agents. However, we must keep in mind that the efficacy of these compounds may be affected by their bioavailability, metabolism, and specific targets within the complex human biological system. Moving forward, it is necessary to study these compounds and validate their efficacy in terms of lipase inhibition, which we address in the following section on molecular docking, to confirm their applicability in obesity management.

Table 4.

Predicted antiobesity effects of the constituents of peppermint and rosemary essential oils.

N	Compounds	Pa	Pi
1	Carvacrol	0.689	0.024
2	ρ-Cymene	0.626	0.035
3	3-Octanol	0.625	0.035
4	Sabinene	0.583	0.044
5	(Z)-Ethyl cinnamate	0.559	0.05
6	Carvacrol, methyl ether	0.544	0.055
7	Tricyclene	0.541	0.056
8	Terpinen-4-ol	0.54	0.056
9	Isoborneol	0.538	0.056
10	Borneol	0.538	0.056

3.4.2. Molecular docking

We analyzed all constituents of the obtained M. × piperita and S. rosmarinus essential oils to investigate the interactions between inhibitors and lipase in silico. This study aimed to identify the key compounds that inhibit the lipase enzyme and obstruct the substrate from accessing the enzyme by examining pancreatic lipase inhibitors and CRL and comparing the results with the inhibition effects of orlistat against the same enzymes. The best docking pose for each inhibitor was determined based on the number of repeat types observed from generated solutions (Serseg et al., 2018). The docking results (Table 5) indicated that all molecules exhibited significant inhibitory effects depending on the number of solutions produced, thus confirming the in vitro results. The type of inhibition can be predicted based on interactions between the inhibitors and the amino acids of active sites, namely His449, Gly123, and Gly124 (Serseg and Benarous, 2018). Other amino acids were not directly involved in the process but were crucial for the stability of the ligand–enzyme complex, such as Phe344 (Manetti et al., 2000), the first amino acid of the tunnel cavity in CRL. The amino acids implicated in the active site of pancreatic lipase are Ser152 (Hadváry et al., 1991; Egloff et al., 1995), His263, and Phe77 (Nguyen et al., 2020).

Table 5.

Molecular docking analysis of studied essential oil constituents against Candida rugosa lipase and human pancreatic lipase.

Enzyme-inhibitor	RR %	Energy (kcal/mol)	Closest residues	Hydrophobic interactions	Hydrogen bonds	Length (Å)
Human pancreatic lipase (1LPB)
Orlistat	100	−6.8±0.00	Ala259, Ala260, Leu264, Arg256, Pro180, Ile209, Leu213, Tyr114	Alkyl, Pi-Alkyl	Gly76 His151 Ser152 His263 Asp79	2.625 2.481 2.168 3.088 3.346
Menthol	100	−6.8±0.00	Tyr114, Phe215, Pro180, Ile209, Phe77, His263	Pi-Sigma, Alkyl, Pi-Alkyl	Ser152 Phe215	2.814 2.277
Menthone	100	−6.2±0.00	Ala178, Ile78, Pro180, Ile209, Phe77, Tyr114, Phe215, His263	Alkyl, Pi-Alkyl	-	-
Menthyl acetate	100	−6.8±0.00	Ala178, Ala259, Ala260, Phe77, Tyr114, Phe215	Alkyl, Pi-Alkyl	Phe77 Ser152	2.507 2.237
Caryophyllene	100	−7.4±0.00	Phe215, Tyr114, Ala260, Ile78, Phe77, His263	Pi-Sigma, Alkyl, Pi-Alkyl	-	-
Camphor	100	−5.6±0.00	Arg256, Ala259, Ala260, Leu264, Ile78	Alkyl	Arg256	2.759
α-Copaene	100	−7.9±0.00	Tyr114, Ala178, Ala260, Leu153, Phe77, Phe215, His263	Pi-Sigma, Alkyl, Pi-Alkyl	-	-
α-Humulene	100	−7.8±0.00	Phe215, Tyr114, Ala178, Ala260, Ile78, Leu153, Phe77, His263	Pi-Sigma Alkyl Pi-Alkyl	-	-
Candida rugosa lipase (1CRL)
Orlistat	100	−6.3±0.00	Gly128, Gly122, Ile453, Val127, Phe133, Phe344	Alkyl, Pi-Alkyl	Gly124	3.057
α-Copaene	100	−6.6±0.00	Leu297, Phe296, Phe344, Phe345, His449	Pi-Alkyl, Alkyl	-	-
α-Humulene	100	−6.7±0.00	Pro65, Val127, Leu297, Phe296, Phe344, Phe345	Alkyl, Pi-Alkyl	-	-
Menthone	80	−5.0±0.07	Phe344, Leu297, Phe296, Phe344, Phe345, His449	Pi-Sigma, Alkyl, Pi-Alkyl	-	-
Caryophyllene	80	−6.5±0.08	Pro65, Val127, Leu297, Phe296, Phe344, Phe345	Alkyl, Pi-Alkyl	-	-
Camphor	70	−5.2±0.14	Leu297, Phe296, Phe344, Phe345, His449	Alkyl, Pi-Alkyl	-	-

We observed that the peppermint essential oil consisted mainly of menthol (42.73%), menthone (16.43%), menthyl acetate (5.17%), caryophyllene (1.07%), and α-humulene (0.27%), which interact with the active site of lipase with several hydrophobic interactions. The terpenes menthol and menthyl acetate have a hydroxyl group and an oxygen atom, which may act as acceptors or proton donors, thus forming hydrogen bonds with the amino acids of the active site of 1LPB Ser152 with lengths of 2.81 and 2.23 Å. In addition, the five terpenes menthol, menthone, menthyl acetate, caryophyllene, and α-humulene have 100% repetition ratios and contain aromatic rings capable of hydrophobic interactions of pi-sigma, alkyl, and pi-alkyl with the hydrophobic amino acids His263, Phe215, Phe77, and Tyr114. The affinity energy of each terpene was −6.8, −6.2, −6.8, −7.4, and −7.8 kcal/mol, respectively. Among these five terpenes, menthone, caryophyllene, and α-humulene achieved binding energies of −5, −6.5, and −6.7 kcal/mol, respectively. These terpenes also form hydrophobic interactions of the pi-sigma, alkyl, and pi-alkyl types with the Phe344 and His449 amino acids of the active site of 1CRL.

The four main terpenes of rosemary essential oil, including camphor, caryophyllene, α-copaene, and α-humulene, exhibited 100% repetition ratios in their interactions with the active sites of both lipases, suggesting a robust and consistent binding pattern. Their aromatic ring structures facilitated hydrophobic interactions with key amino acids. Three terpenes interacted with His263, Phe215, Phe77, and Tyr114. These interactions were of the pi-sigma, pi-alkyl, and pi-sigma types. Camphor engaged in alkyl interactions with Arg256, Ala259, Ala260, Leu264, and Ile78. These hydrophobic interactions further stabilized the inhibitor–enzyme complexes, enhancing the inhibitory potential. The terpene camphor exhibited hydrogenic interaction with Arg256, which has a longer length of 2.75 Å. Notably, the binding energies of the four terpenes were −5.6, −7.4, −7.9, and −7.8 kcal/mol, indicating strong affinity for the lipase active site. These terpenes are interesting because they had binding energies close to that of CRL, reaching −5.2, −6.5, −6.6, and −6.7 kcal/mol. This suggests that these terpenes can exert significant inhibitory effects against pancreatic and fungal lipases.

Orlistat, a powerful drug used to treat obesity (Padwal et al., 2003), had hydrophobic interactions with the amino acid in the active site of 1CRL, Phe344, and the neighboring amino acids Gly128, Gly122, Ile453, Val127, and Phe133 with alkyl and pi-alkyl bonds (Figure 3). The amino acid of the active site, Gly124, forms hydrogen bonds with it, with length of 3.05 Å and estimated binding energy of −6.3 kcal/mol. It also had several interactions with the 1PLB enzyme (Figure 4), including hydrophobic interactions with several amino acids of the active site including Ala259, Ala260, Leu264, Arg256, and Tyr114, with alkyl and pi-alkyl bonds. The active site amino acids Ser152 and His263 exhibited hydrogen bonds with lengths of 2.16 and 3.08 Å, respectively. Orlistat had the same binding energy of −6.8 kcal/mol.

Figure 3.

Molecular docking results for orlistat (a) and the most active terpenes of peppermint and rosemary essential oils, including α-humulene (b), menthone (c), and camphor (d), against Candida rugosa lipase.

Figure 4.

Molecular docking results for orlistat (a) and the most active terpenes of peppermint and rosemary essential oils, including α-humulene (b), menthone (c), and camphor (d), against human pancreatic lipase.

Overall, studying the different compounds of M. × piperita and S. rosmarinus essential oil showed that they could effectively inhibit lipase activity. They engaged with several amino acids in the enzyme active site by utilizing hydrogen bonds and hydrophobic interactions. These results provide significant insights into the molecular mechanisms that enable their functions as inhibitors, and they also have significant potential as therapeutic agents for the treatment of obesity and associated metabolic diseases.

3.4.3. ADMET evaluations

ADMET studies were performed for the lipase inhibitors found in the M. × piperita and S. rosmarinus essential oils to determine whether they are safe for human use. We adopted a multipronged approach using online software, which provided initial insights into the potential ADMET properties of the key components. The results of all of these tests offered a full picture of the ADMET profiles of the inhibitors, allowing us to judge whether they could be used in therapeutic settings to treat obesity safely and effectively. The results are presented in Table 6, which shows how seven compounds of interest are absorbed, distributed, broken down, and eliminated and how dangerous they are. These compounds are menthol, menthone, methyl acetate, caryophyllene, camphor, α-copaene, α-methyl acetate, and α-humulene. The data indicated that all compounds had good oral absorption (>99%), with menthol acetate showing the highest value. Several compounds, including menthol, menthone, and camphor, exhibited excellent penetration of the blood–brain barrier, indicating potential central nervous system activity. Of note, α-copaene showed the highest binding to plasma protein, suggesting that it may have a longer half-life in the body. Interestingly, α-humulene blocked several cytochrome P450 enzymes, such as 2D6, 2C19, and 2C9. This suggests that the compound might interact with other drugs. This compound also had a lower clearance rate and longer half-life.

Table 6.

Pharmacokinetic parameters of the constituents of the studied essential oils.

Pharmacokinetics	Menthol	Menthone	Menthyl acetate	Caryophyllene	Camphor	α-Copaene	α-Humulene
Molecular weight (Da)	156.150	154.140	198.160	204.190	152.120	204.19	204.190
Absorption
Caco-2 cell permeability (nm/s)	0.8127	0.8127	0.8219	0.6327	0.8084	0.6705	0.6771
Human intestinal absorption (HIA %)	0.9944	0.9944	0.9970	0.9926	0.9971	1	0.9972
P-glycoprotein inhibition	None	None	None	None	None	None	None
Blood–brain barrier penetration (C.brain/C.blood)	0.9408	0.9408	0.9490	0.9536	0.983	0.9455	0.9733
MDCK cell permeability (nm/s)	2.7e-05	2.4e-05	2.2e-05	2.3e-05	2.2e-05	1.4e-05	1.6e-05
Plasma protein binding (%)	82.256	80.782	87.271	92.166	79.215	97.259	95.718
Metabolism
Cytochrome P450 2D6 inhibition	None	None	None	None	None	None	Yes
Cytochrome P450 2D6 substrate	None	None	None	Yes	Yes	None	Yes
Cytochrome P450 3A4 inhibition	None	None	None	None	None	None	None
Cytochrome P450 3A4 substrate	None	None	None	None	None	None	None
Cytochrome P450 2C19 inhibition	None	None	None	None	None	None	Yes
Cytochrome P450 2C19 substrate	Yes	Yes	Yes	Yes	Yes	Yes	None
Cytochrome P450 2C9 inhibition	None	None	None	None	None	None	None
Cytochrome P450 2C9 substrate	Yes	Yes	Yes	Yes	Yes	None	Yes
Excretion
Clearance	14.327	15.445	6.670	5.458	13.808	19.832	3.400
T1/2	0.354	0.585	0.318	0.100	0.701	0.059	0.403
Toxicity
Ames test	None	None	None	None	None	None	None
Carcinogencity	None	Yes	None	None	None	None	None
HERG-inhibition	None	None	None	None	None	None	None
Eye Sensitization	Yes	Yes	Yes	Yes	Yes	None	Yes
Skin Sensitization	None	None	Yes	Yes	None	None	Yes
Hepatotoxicity	None	None	None	None	None	None	None
Respiratory toxicity	None	Yes	None	None	Yes	Yes	None

3.5. Conclusion

This study demonstrated that peppermint and rosemary essential oils possess significant capacity to inhibit the activity of lipase. This is interesting because it suggests that these essential oils may be useful in treating disorders associated with lipid metabolism and could be used as effective natural solutions to treat obesity. The results obtained from in silico studies confirmed that these essential oils are effective in inhibiting the lipase enzyme’s function with the possibility of occupation of the active site by their components.

Supplementary materials

Table S1.

Predicted antiobesity effects of the constituents of peppermint and rosemary essential oils.

N	Compounds	Pa	Pi	N	Compounds	Pa	Pi
1	Carvacrol	0.689	0.024	24	α -Thujene	0.431	0.102
2	ρ-Cymene	0.626	0.035	25	(Z)-Methyl cinnamate	0.425	0.106
3	3-Octanol	0.625	0.035	26	Camphor	0.421	0.108
4	Sabinene	0.583	0.044	27	neo-Verbanol	0.421	0.108
5	(Z)-Ethyl cinnamate	0.559	0.05	28	Terpinolene	0.411	0.114
6	Carvacrol, methyl ether	0.544	0.055	29	γ-Terpinene	0.41	0.115
7	Tricyclene	0.541	0.056	30	Verbenone	0.402	0.12
8	Terpinen-4-ol	0.54	0.056	31	α-Pinene	0.363	0.144
9	Isoborneol	0.538	0.056	32	Limonene	0.355	0.149
10	Borneol	0.538	0.056	33	Eucalyptol	0.345	0.155
11	α -Terpinene	0.535	0.057	34	γ-Muurolene	0.32	0.172
12	Heptanone <5-methyl-3->	0.529	0.059	35	α-Humulene	0.308	0.179
13	Menthol	0.514	0.064	36	Viridiflorene	0.308	0.18
14	Isomenthol	0.514	0.064	37	Caryophyllene	0.307	0.18
15	Menthyl acetate	0.512	0.065	38	(+)-delta-Cadinene	0.302	0.184
16	β-Pinene	0.501	0.069	39	Caryophyllenyl alcohol	0.266	0.213
17	Cymen-8-ol	0.498	0.07	40	Myrcene	0	0
18	Bornyl acetate	0.49	0.073	41	δ-Carene	0	0
19	Camphene	0.47	0.082	42	Linalool	0	0
20	ρ-Menth-1-en-9-ol acetate	0.466	0.084	43	Citronellal	0	0
21	Menthone	0.453	0.09	44	α-Terpineol	0	0
22	cis-Linalool oxide (pyranoid)	0.45	0.092	45	α-Copaene	0	0
23	Pulegone	0.445	0.094

Figure S1

In vitro experimental results. (a) IC50 histogram of different extracts studied. (b) IC50 histogram of DPPH assay for Mentha piperita essential oil. (c) IC50 histogram of ABTS assay for Mentha piperita essential oil. (d) IC50 histogram of DPPH assay for Salvia rosmarinus essential oil. (e) IC50 histogram of ABTS assay for Salvia rosmarinus essential oil.