Chemical Composition, Anti-α-Glucosidase Activity, and Molecular Modelling Studies of Cleistocalyx operculatus Essential Oil

Abstract

In this study, chemical components, α-glucosidase inhibitory activities, and molecular modelling studies of the essential oil extracted from the Cleistocalyx operculatus leaves were investigated. In total, thirty compounds were identified using GC/MS, representing 98.3% of the oil. Of these, the two most dominant constituents of the essential oil were determined as (Z)-β-ocimene (30.4%) and allo-ocimene (31.6%). The α-glucosidase inhibitory experiments indicated that the essential oil exhibited potent α-glucosidase inhibitory activities, with IC₅₀ values of 61.82 ± 3.91 µg/mL. For further investigation into inhibitory mechanisms, molecular docking simulations were performed to investigate structural interactions between two dominant constituents and the α-glucosidase protein. The simulation revealed that allo-ocimene (31.6%) and (Z)-β-ocimene (30.4%) have protein binding affinities of −5.358 and −5.330 kcal/mol, respectively. Moreover, molecular dynamic simulation indicated that the complexes of two compounds and the target protein were stable over 100 ns. Overall, these findings suggest that the essential oil of C. operculatus leaves could be a natural source of potential α-glucosidase inhibitors.

1. Introduction

α-glucosidase is an enzyme that plays an essential role in the digestion and metabolism of complex carbohydrates [1]. Its primary function is to facilitate the hydrolysis of α-glycosidic linkages in oligosaccharides and disaccharides, leading to the breakdown of these molecules into individual glucose units [2]. The mechanism of α-glucosidase involves the interaction between the substrate, typically a carbohydrate molecule, and the enzyme’s active site. The active site comprises specific amino acid residues that facilitate the catalytic reaction by interacting with the substrate [3].

In patients diagnosed with type 2 diabetes, there is frequently a dysfunction in insulin activity and an elevated level of insulin resistance, which poses a significant challenge in effectively managing and controlling blood glucose levels [4]. Thus, developing a medication inhibiting the activity of α-glucosidase has been seen as a strategy to counteract diabetes type 2 by delaying the absorption of glucose from carbohydrates [5]. This delay helps prevent rapid spikes in blood sugar after meals, aiding in the management of blood glucose levels [5].

Inhibitors of α-glucosidase are compounds utilized to block or decrease the activity of the enzyme. They find applications in the treatment of conditions like type 2 diabetes, and serve as potential targets for drug design [6]. α-glucosidase inhibitors can bind to the enzyme’s active site either competitively or non-competitively, thereby preventing the substrate from binding or interfering with the catalytic mechanism [3]. Well-known inhibitors of α-glucosidase include acarbose, miglitol, and voglibose [7]. These inhibitors exhibit structural similarity to carbohydrates, enabling them to mimic the binding of carbohydrates to the active site. Understanding the mechanism, active site characteristics, and inhibitors of α-glucosidase provides valuable insights for the development of therapeutic strategies aimed at regulating carbohydrate metabolism and effectively managing conditions such as diabetes.

Cleistocalyx operculatus (Roxb.) Merr. et Perry (synonym: Syzygium nervosum), also known as “Voi” in Vietnamese, has been used in Vietnamese traditional medicine to treat a variety of ailments, such as bloating, diarrhea, acne, chronic colitis, and dysentery. In addition, this plant’s extracts and some isolated compounds demonstrated anti-diabetic, anti-obesity, anti-oxidant, anti-viral, anti-inflammatory, anti-microbial, neuroprotective, and cytotoxic activities [8]. Additionally, recent research has shown that the essential oil from the leaves or buds of C. operculatus has anti-inflammatory, anti-microbial, anti-oxidant, and cytotoxic activities [9,10,11,12]. As a part of our studies on the anti-diabetic effect of essential oil from herbs in Vietnam, we report herein the α-glucosidase enzyme inhibition of the essential oil of C. operculatus growing in Thua Thien Hue, Vietnam. This is the first report of the anti-diabetic activity of C. operculatus leaf essential oil.

2. Materials and Methods

2.1. Plant Materials

The Cleistocalyx operculatus leaves was collected in Nam Dong, Thua Thien Hue, Vietnam, in June 2022. The specimen (NCXS-H52) was identified by Dr. Do Van Truong (Centre for Life Sciences, VMMN, VAST) and was deposited at Mientrung Institute for Scientific Research, VNMN, VAST, Vietnam.

2.2. Extraction of Essential Oil from Leaves of C. operculatus

The fresh leaves of C. operculatus were carefully washed with tap water to remove all the dust particles and then dried at room temperature for 24 h and cut into small pieces before distilling to obtain essential oil. The leaves (500 g) were subjected to hydro-distillation in a vessel (5 L), material/water ratio (1/7, w/v) for 4 h with a Clevenger-type apparatus, as described by the Vietnamese Pharmacopoeia. Extractions were performed in triplicate, and values were expressed as mean ± standard deviation (SD). The essential oil was treated with Na₂SO₄ to remove moisture and stored in a dark glass vial at 4 °C for chemical analysis and biological testing.

2.3. Gas Chromatography–Mass Spectrometry (GC/MS) Analysis of Essential Oil of C. operculatus

The chemical components of the essential oil were identified by GC/MS analysis using a Shimadzu GCMS-QP2010 Plus system (Shimadzu, Kyoto, Japan). An equity-5 capillary column (30 m × 0.25 mm, film thickness 0.25 µm, Supelco, Bellefonte, PA, USA) with helium as the carrier gas at a flow rate of 1.5 mL/min was utilized. The temperature of the GC oven was set to 60 °C for 2 min, then programmed to 240 °C at a rate of 4 °C/min and maintained at 240 °C for 10 min before being programmed to 280 °C at a rate of 5 °C/min. The essential oil was diluted in n-hexane solvent (Merck, Darmstadt, Germany) at a ratio of 1:30 (v/v). It was then carefully shaken for 3 min and allowed to stabilize for 5 min before injection. The essential oil sample was injected into the system using a splitless mode. The temperature of the injector was performed at 280 °C, and mass spectra were recorded at 70 eV. The mass was recorded in the range of 40 to 500 amu at a sampling rate of 0.5 scan/s. The chemical components of essential oil were identified by comparing their relative retention index (RI) to a series of n-alkanes (RIs were calculated using a homologous series of n-alkanes C₇–C₄₀, Sigma-Aldrich, MO, USA). Computer matching of MS and RI against the commercial databases (WILEY7 Library and NIST11 Library), as well as MS and RI data of known oils from the literature, were used for the identification [13,14].

2.4. Evaluation of α-Glucosidase Enzyme Inhibition of Essential Oil of C. operculatus

The α-glucosidase enzyme inhibitory experiments were performed according to the method reported by Ranilla et al. (2010), with slight modifications [15]. Briefly, 50 μL of the sample (the essential oil diluted in dimethyl sulfoxide to 20, 50, 100, 250, 500, and 1000 μL/mL) was placed in 96-well plates and incubated with 100 μL of 0.1 M PPB (potassium phosphate buffer at pH 6.8) containing α-glucosidase (0.5 U/mL) at 37 °C for 10 min. The reaction was initiated by supplementing 50 μL of 5 mM p-nitrophenyl-α-D-glucopyranoside (pNPG) to each well. After incubating at 37 °C for 30 min, the reaction was stopped by adding 50 μL of 0.2 M Na₂CO₃ into each well, and then the absorbance of the reactions was measured at 405 nm in a microplate reader (Biotek, Winooski, VT, USA). Acarbose (Sigma-Aldrich, MO, USA) was used as the positive control. The α-glucosidase inhibition was calculated by the equation α-glucosidase inhibition (%)  =  (1 − A/A₀) × 100, where A is the absorbance of the essential oil and A₀ is the absorbance of the blank, respectively. The IC₅₀ values were calculated by GraphPad Prism software v.8.0. Experiments were carried out in triplicate, and values were expressed as mean ± standard deviation (SD).

2.5. Molecular Docking Simulation

The crystal structures of α-glucosidase (PDB ID: 5NN8) were obtained from the Research Collaboratory for Structural Bioinformatics Protein Data Bank. To prepare the structure, we removed the attached hetatm and performed energy minimization using the standard optimization parameters in the Swiss PDB Viewer [16]. Next, we added polar hydrogen atoms and Kollman charges to the protein using AutoDock 1.5.6. The resulting macromolecule was then exported in a dockable pdbqt format for molecular docking.

The co-crystallized ligand, acarbose, has been seen as a commercial α-glucosidase inhibitor. In the crystal structure (PDB ID: 5NN8), acarbose binds to α-glucosidase at catalytic residues (from Val400 to Arg600). Therefore, we identified the docking site following the interactive site between acarbose and α-glucosidase (Figure 1). We set the grid box site dimensions at 18.75 × 18.75 × 18.75 for x, y, and z coordinates, respectively. To obtain the 3D structures of the candidate compounds, we downloaded them from the PubChem library. Subsequently, the compounds underwent geometry optimization and were subjected to the MMFF94 force field using Avogadro 1.2.0. For the molecular docking process, we utilized Autodock Vina version 1.2.0 [17]. Finally, the top nine conformations with the best docking score were reported in units of kcal/mol.

2.6. Molecular Dynamic Simulation

The simulation protocol followed in this study was based on our previous work [6]. Molecular dynamics simulations were conducted using GROMACS 2020.4 to assess the stability of the complexes between ligands and α-glucosidase. The topology of the complex was prepared using the CHARMM36 force field, and the system was solvated in a truncated octahedral box with TIP3P water molecules using the CHARMM-GUI server [18]. Then, the system was neutralized using sodium chloride ions. To ensure a fully stable system, the simulation was equilibrated to reach objective values of 300 K temperature and 1 bar pressure. Finally, a molecular dynamics simulation was run for 100 ns to study the behavior of the complex.

3. Result and Discussion

3.1. Percentage Yield and Chemical Composition of C. operculatus Essential Oil

The hydrolytic distillation of fresh leaves of C. operculatus resulted in the isolation of the pale yellow oil. The percentage of essential oil obtained was 0.18 ± 0.03% (v/v), much higher than that obtained from the C. operculatus leaf collected in Nghe An, Vietnam (0.10%, fresh leaves) of Dung et al. (1994) [19]. Different geographical circumstances and extraction techniques can be blamed for variations in the amount of essential oil. The GC-MS experiment showed that the C. operculatus leaf essential oil contained 30 volatile compounds, representing 98.3% of the oil content. The essential oil of leaf C. operculatus showed the presence of main groups, including monoterpene hydrocarbons (88.3%), sesquiterpene hydrocarbons (8.3%), oxygenated sesquiterpenes (0.9%), and oxygenated monoterpenes (0.6%) (

Table 1. Chemical constituents of the essential oil from C. operculatus leaves.

No	Compounds	RT (min)	SI (%)	RIa	RIb	Content (%)
1	α-Pinene	6.70	97	932	932	5.6
2	β-Pinene	8.11	96	975	974	0.4
3	β-Myrcene	8.58	96	990	988	1.1
4	Limonene	10.02	90	1028	1024	2.7
5	(Z)-β-ocimene	10.38	97	1036	1032	30.4
6	(E)-β-ocimene	10.92	97	1050	1044	14.0
7	Terpinolene	12.41	94	1087	1086	1.1
8	Linalool	12.98	94	1100	1095	0.3
9	Allo-ocimene	14.44	96	1134	1128	31.6
10	Veratrole	14.63	82	1138	1141	0.2
11	Neo-allo-ocimene	14.81	97	1142	1140	1.4
12	cis-Chrysanthenyl acetate	20.03	84	1260	1261	0.1
13	α-Copaene	25.09	88	1377	1374	0.1
14	Geranyl acetate	25.43	94	1385	1379	0.2
15	β-caryophyllene	26.96	97	1421	1422	4.6
16	β-Copaene	27.33	89	1430	1430	0.1
17	γ-Elemene	27.52	89	1435	1434	0.1
18	Aromadendrene	27.73	93	1440	1439	0.2
19	α-Humulene	28.34	97	1455	1452	1.0
20	γ-Muurolene	29.28	92	1478	1478	0.5
21	Germacrene-D	29.47	92	1482	1480	0.3
22	β-Selinene	29.68	93	1487	1489	0.2
23	Viridiflorene	30.04	92	1496	1496	0.4
24	α-Bulnesene	30.54	84	1509	1509	0.1
25	δ-Amorphene	30.80	90	1515	1511	0.2
26	δ-Cadinene	31.18	94	1525	1522	0.4
27	(E)-Nerolidol	32.74	95	1565	1561	0.3
28	Caryophyllene oxide	33.51	91	1584	1582	0.4
29	Eremoligenol	35.14	87	1627	1629	0.2
30	α-Cadinol	36.23	81	1657	1652	0.1
	Monoterpene hydrocarbons					88.3
	Sesquiterpene hydrocarbons					8.3
	Oxygenated sesquiterpenes					0.6
	Oxygenated monoterpenes					0.9
	Non-terpenes					0.2
	Total					98.3

RT: Retention time; SI: similarity index; RI^a: Retention indices relative to C₇–C₄₀ n-alkanes calculated on Equity-5 capillary column; RI^b: Literature retention indices [13,14,21].

). Out of 30 compounds, allo-ocimene (31.6%), (Z)-β-ocimene (30.4%), (E)-β-ocimene (14.0%), α-pinene (5.6%), β-caryophyllene (4.6%), and limonene (2.7%) were found to be major components. These results appear to differ from other locations’ oil chemical compositions when compared to the literature. Indeed, the major components of essential oils from Phong Dien, Thua Thien Hue (Vietnam) were (E)-β–ocimene (52.9%), (Z)-β-ocimene (10.9%), caryophyllene (8.1%), humulene (2.5%), γ-muurolene (2.0%), and epiglobulol (2.1%); those of Vinh, Nghe An (Vietnam) were (Z)-β-ocimene (32.1%), (E)-β-ocimene (9.4%), myrcene (24.6%), and β-caryophyllene (14.5%); and those of Nepal were myrcene (69.70%), (E)-β-ocimene (12.24%), (Z)-β-ocimene (4.79%), linalool (4.08%), p-cymene (2.71%), (E)-caryophyllene (1.11%), and (E)-Nerolidol (1.57%) [12,19,20]. The observed dissimilarity in the quality and content of these essential oils might be attributed to the distribution of species, as well as differences in analysis and identification methods. However, compounds (Z)-β-ocimene and (E)-β-ocimene were always present in the major components of this essential oil. Therefore, compounds (Z)-β-ocimene and (E)-β-ocimene could be the marker compounds of the essential oil.

3.2. In Vitro α-Glucosidase Inhibitory Activity of C. operculatus Essential Oil

In this study, the essential oil of C. operculatus leaves was demonstrated to have α-glucosidase inhibitory effects three times stronger than those of acarbose, with IC₅₀ values of 61.82 ± 3.91 and 201.4 ± 8.46 µg/mL, respectively.

These outcomes are also in line with earlier investigations on C. operculatus’s anti-diabetic effects. The leaf extract of C. operculatus has shown α-glucosidase inhibitory action with an IC₅₀ value of 0.11 mg/g [22]. Furthermore, Chukiasiri et al. (2022) revealed that the plant’s fruit extract has a good ability to inhibit α-amylase and α-glucosidase, with IC₅₀ values of 0.42 and 0.23 g/mL, respectively [23]. In addition, pharmacological studies showed that the C. operculatus extracts had positive effects on streptozotocin-diabetic rats, such as reduced blood glucose levels and protective activities on the β-cells of pancreatic islets [24,25]. The α-glucosidase inhibitory activity of the essential oil of C. operculatus leaves has never been documented before this investigation. However, both earlier investigations and the findings of the current study support the notion that C. operculatus may be a source of natural compounds with anti-diabetic properties.

3.3. Modeling the Structures of α-Glucosidase Complexes with Main Essential Oil Compounds

Based on the GC-MS results, the two compounds with the highest percentage in the essential oil composition of C. operculatus leaves were selected for interaction analysis with α-glucosidase. Two compounds, (Z)-β-ocimene and allo-ocimene, were docked at the active site between acarbose and α-glucosidase.

The docking conformations of the two compounds were shown in

Table 2. Molecular docking results of two main essential oil compounds with α-glucosidase.

Compound	Docking Score (kcal/mol)	Residues Interaction
allo-ocimene	−5.358	Trp376, Phe649, His674, Trp613, Trp516, and Ile441
(Z)-β-ocimene	−5.330	Phe649, Trp376, Leu405, His674, Ile441, Trp481, and Trp516

and Figure 1 and Figure 2. Allo-ocimene had docking score of −5.358 kcal/mol and was found to interact with the enzyme, with six hydrophobic interactions at Trp376, Phe649, His674, Trp613, Trp516, and Ile441 (Figure 2A). Meanwhile, the results revealed (Z)-β-ocimene had docking score of −5.330 kcal/mol, with seven Alkyl and Pi-Alkyl interactions at Phe649, Trp376, Leu405, His674, Ile441, Trp481, and Trp516 (Figure 2B). For further analysis, the best conformation of two candidates was selected for molecular dynamic simulation over 100 ns.

Small molecules that interact with protein surfaces have the potential to induce significant changes in the protein’s tertiary structure, thereby offering opportunities for improving drug design. Molecular dynamics modeling stands out for its capability to assess the flexibility of protein-ligand complexes. This approach was employed to precisely evaluate the thermodynamics and kinetics of drug–enzyme interactions. To assess the stability of the ligands and protein, 100 ns trajectories of the complex formed between two primary compounds and α-glucosidase were analyzed. The simulation results reveal that the complex involving allo-ocimene and α-glucosidase remained stable throughout the simulation, with fluctuations within a range of 2 Å (Figure 3A). Furthermore, analysis of the root mean square fluctuation (RMSF) indicated that the ligand stabilized specific interacting residues at the active site (Figure 3B). The radius of gyration (Rg), which characterizes the distribution of atoms around the protein’s axis, was also examined (Figure 3C). Additionally, the solvent-accessible surface area (SASA) (Figure 3D) aided in visualizing conformational changes in the protein following ligand binding.

Meanwhile, the complex between (Z)-β-ocimene and α-glucosidase also remained stable throughout the simulation time, with fluctuations within the range of 2 Å (Figure 4A). The RMSF, radius of gyration (Rg), and solvent-accessible surface area (SASA) results also demonstrated a similar interaction between allo-ocimene and α-glucosidase (Figure 4B–D). This can be explained by the similar structures of the two compounds. Interestingly, there are no hydrogen bonds created from compounds and protein; however, the complexes were quite stable because of hydrophobic interactions between double-bonds and amino acids. Although the effects of the constituents in C. operculatus essential oil are believed to be synergistic in terms of α-glucosidase activity, we would expect that the two main components (comprising approximately 70%) are believed to have the most impact on the α-glucosidase activity of essential oils. However, further studies regarding the structure of the protein complex are needed to elucidate the hypotheses proposed in this study. From there, it could serve as evidence to develop C. operculatus essential oil as a treatment in the future.

The bioactivities of C. operculatus’ essential oils, including anti-inflammatory, anti-microbial, anti-oxidant, and cytotoxicity bioactivities, were investigated [9,10,11]. The antimicrobial activities of bud essential oil were evaluated for inhibiting skin pathogens such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE), and multiantibiotic-resistant bacteria (MARB), with MIC and MBC values in the range of 1–20 μM/mL [9]. In another study, the buds’ essential oil also exhibited significant anti-inflammatory activities by suppressing the expression of pro-inflammatory cytokines such as tumor necrosis factor-a (TNF-a) and interleukin-1b (IL-1b) in RAW 264.7 cells via blocking NF-κB activation [10]. In addition, the antioxidant activity of C. operculatus bud essential oil was reported [11]. In particular, Tran Gia Buu et al. (2018) reported the antibacterial and anti-inflammatory properties of C. operculatus leaf essential oil. Particularly, the essential oil also had a supportive effect on the burn wound healing process, with a contraction rate of 100%, obviously higher than that of tamanu-oil-treated mice (79%) and control mice (71%) after 20 days of application [26]. In summary, essential oils extracted from C. operculatus had notable antimicrobial and anti-inflammatory effects, and could be exploited as topical dermatological agents. So far, many studies have investigated the antihyperglycemic activities of compounds and extracts from C. operculatus with promising results [24,27]. However, to the best of our knowledge, there has not been any published paper reporting on the antidiabetic potential of C. operculatus essential oil. Meanwhile, several publications have reported this effect of essential oils from other Syzygium species. In 2016, Aisha Ashraf et al. reported that Syzygium aromaticum essential oil could inhibit α-amylase as the emulsion of 25% v/v of essential oil exhibited α-amylase inhibitory activity of 95.30% inhibition, and two major components of the essential oil were identified as eugenol (18.7%) and α-pinene (15.6%) [28].

In conclusion, C. operculatus leaf essential oil is a promising substance to treat diabetes. However, the in vivo antihyperglycemic benefits of this essential oil need to be further researched.

4. Conclusions

Our study investigated the chemical composition, α-glucosidase inhibitory properties, and molecular modelling studies of the essential oil of C. operculatus leaves collected in Thua Thien Hue, Vietnam. In total, thirty compounds were identified using GC/MS, representing 98.3% of the oil. Of these, the two most dominant constituents of the essential oil were determined as (Z)-β-ocimene (30.4%) and allo-ocimene (31.6%). The α-glucosidase inhibitory experiments indicated that the essential oil displayed potent α-glucosidase inhibitory activities, with an IC₅₀ of 61.82 ± 3.91 µg/mL. A molecular docking simulation was utilized to assess the molecular interaction of the main components with the α-glucosidase protein to further illustrate the mechanism. We observed that allo-ocimene (31.6%) and (Z)-β-ocimene (30.4%) have protein-binding affinities of −5.358 and −5.330 kcal/mol, respectively. Molecular dynamic simulation also demonstrated that the complexes of two compounds and the target protein were stable over 100 ns. Overall, these findings suggest that the essential oil of C. operculatus leaves could be a natural source of potential α-glucosidase inhibitors.