Inhibitory effect of plant essential oils on α-glucosidase

Abstract

Diabetes mellitus, associated with α-glucosidase, has been considered as a chronic metabolic disorder, seriously affecting human health. Thus, searching natural α-glucosidase inhibitors and investigating their inhibition mechanism are urgently important. In this study, sixty-two essential oils (EOs), derived from aromatic plants, were found to exert different inhibition on α-glucosidase. The further study revealed that the most potent EOs against α-glucosidase were chuan-xiong, fructus cnidii, sacha inchi, aloe, ganoderma lucidum spore and ginger with IC₅₀ values of 3.02, 2.88, 7.37, 5.06, 5.32 and 7.40 μg/mL. Moreover, the inhibitory mechanism and kinetics studies found that chuan-xiong and sacha inchi were reversible and mixed-type inhibitors. Fructus cnidii, aloe, ganoderma lucidum spore and ginger were reversible and uncompetitive-type inhibitors. It is suggested that EOs, being of natural origin, would be promising anti-α-glucosidase agents.

Introduction

Diabetes mellitus, caused by deficiency in insulin secretion, was a common metabolic disease, severely affecting human health (Hu and Jia, 2019). Insulin secretion deficiency would lead to high blood glucose and serious complications, including high blood pressure, stroke, blindness and kidney disease (Matsui et al., 2007; Duckworth et al., 2009). It was well known that α-glucosidase was directly associated with postprandial hyperglycemia which played a key role in the development of type 2 diabetes mellitus (Priscilla et al., 2014). Thus, lowering postprandial hyperglycemia by inhibiting α-glucosidase was confirmed to be an effective strategy to treat type 2 diabetes mellitus (Bhandari et al., 2008; Kalra and Bhutani, 2014; Rhabasa-Lhoret and Chiasson, 2004). It was indicated that acarbose, metformin and sitagliptin had been used for glycemic control (Ali et al., 2006). However, long-term use of these synthetic drugs would lead to adverse effects, such as hypoglycemia, diarrhea and flatulence, which seriously affected the absorption of nutritious ingredient (Kumar and Sinha, 2012). Herein, there is an urgent need for searching safe α-glucosidase inhibitors.

Natural edible products, such as vegetables, fruits and Chinese traditional medicines, conducting various of bioactivities without possible adverse effects, were widely used to treat metabolic diseases (Ruxton, 2016). Essential oils (EOs), derived from natural edible products, were secondary metabolites comprising different bioactive components (Maffei et al., 2011). They were complex mixtures of different bioactive components, covering a broad range of functional properties, such as anti-oxidative, anti-diabetic, anti-microbial and anti-inflammatory properties (Raut and Karuppayil, 2014). Hence, EOs were considered an emerging repository of small molecules for drug discovery (Maffei et al., 2011). To date, several studies with plants essential oils were done to test their (and major components) inhibition on α-glucosidase (Yang et al., 2016; Zhao et al., 2017). Previous studies have found that the extract of tea could lower postprandial hyperglycemia through inhibiting the activities of α-amylase and α-glucosidase, which help to reduce the risk of diabetes mellitus (Goh et al., 2015). It has been reported that the EO of Origanum vulgare L. produced 57.7% α-glucosidase inhibition at concentration of 18 μg/mL (Salazar et al., 2020). In addition, the IC₅₀ values of EO derived from Thymus species for α-amylase and α-glucosidase were found to be 7.19 ± 0.14 and 1.08 ± 3.34 mg/mL (Oboh et al., 2013). As described above, due to their inadequate potency, most of EOs have not yet used for anti-α-glucosidase applications.

Considering the relationship between the main bioactivity components in EOs and the α-glucosidase inhibition properties, and the lack of systematic studies about natural α-glucosidase inhibitors, the present study was aimed to develop more potent α-glucosidase inhibitors from natural source. Sixty-two commercial EOs which extracted from plants were evaluated for their anti-α-glucosidase activities. Furthermore, the main components of potent EOs were analyzed by gas chromatograph–mass spectrometer (GC–MS). The kinetics and the anti-α-glucosidase mechanism of potent EOs were discussed in this paper.

Materials and methods

Materials

α-Glucosidase (from Saccharomyces cerevisiae, EC 3.2.1.20) and acarbose were obtained from Sigma-Aldrich (St. Louis, MO). Sixty-two commercial EOs, pure without additive, were acquired from Jingjing Biotechnology Co. (Guangzhou, China). The information of these EOs was listed in Table 1. Other solvents and reagents which had analytical grade were obtained from Tansoole (Shanghai, China). α-Glucosidase solution was prepared as the following: α-glucosidase was dissolved with 0.05 M phosphate buffer solution (PBS, pH 6.81 ± 0.01), and then diluted to 0.3 U/mL. The final concentrations of α-glucosidase in PBS were kept in 15 U/L. Acarbose was additionally used as a standard compound.

Table 1.

The information of sixty-two EOs

Name of EOs	Scientific Name	Family	Extracted Part	Solvent
Angelica	Angelica sinensis (Oliv.)Diels	Apiaceae	Root	CO2
Angelica pubescence	Angelica pubescens Maxim.f. biserrata Shan et Yuan	Apiaceae	Root	CO2
Bupleurum	Bupleurum scorzonerifolium Willd	Apiaceae	Root	CO2
Celery seed	Apium graveolens L	Apiaceae	Seed	H2O
Chuan-xiong	Ligusticum chuanxiong hort	Apiaceae	Root	CO2
Fructus cnidii	Cnidium monnieri (L.) Cuss	Apiaceae	Fruit	H2O
Ligusticum	Ligusticum sinense Oliv	Apiaceae	Root	EtOH
Notopterygium	Rhizoma et Radix Notopterygii	Apiaceae	Root	CO2
Saposhnikovia divaricata	Saposhnikovia divaricata (Trucz.) Schischk	Apiaceae	Root	CO2
Gallnut	Rhus chinensis Mill	Anacardiaceae	Leaf	H2O
Ginseng	Pannx ginseng C.A. Meyer	Araliaceae	Root	H2O
Sabal	Serenoarepens (Bartr.) J.K.Small	Arecaceae	Fruit	CO2
Epimedium	Epimedium brevicornu Maxim	Berberidaceae	Whole	CO2
Olive	Olea europaea	Burseraceae	Fruit	CO2
Frankincense	Boswellia carterii Birdw	Burseraceae	Resin	CO2
Honeysuckle	Lonicera japonica Thunb	Caprifoliaceae	Flower	EtOH
Papaya	Carica papaya L	Caricaceae	Fruit	EtOH
Carnation	Dianthus ‘Carnation'	Caryophyllaceae	Flower	EtOH
Tripterygium wilfordii	Tripterygium wilfordii Hook. f	Celastraceae	Leaf	H2O
Burdock	Arctium lappa L	Compositae	Fruit	H2O
Chrysanthemum	Chrysanthemum indicum L	Compositae	Flower	EtOH
Costus	Aucklandia lappa Decne	Compositae	Root	H2O
Mugwort	Artemisia argyi Levl.et Vant	Compositae	Leaf	CO2
Cypevol	Cyperus rotundus L	Cyperaceae	Root	CO2
Sseabuckthorn	Hippophae rhamnoides Linn	Elaeagnaceae	Fruit	EtOH
Seabuckthorn seed	Hippophae rhamnoides Linn	Elaeagnaceae	Seed	EtOH
Sacha inchi	Plukenetia volubilis Linneo	Euphorbiaceae	Fruit	CO2
Oleum anisi stellati	Illicium verum	Illiciaceae	Fruit	H2O
Peppermint	Mentha canadensis Linnaeus	Labiatae	Whole	CO2
Patchouli	Pogostemon cablin (Blanco) Benth	Labiatae	Leaf	H2O
Schizonepeta tenuifolia	Schizonepeta tenusfolia Briq	Labiatae	Flower	CO2
Bay	Laurus nobilis	Lauraceae	Flower	EtOH
Cinnamon	Cinnamomum cassia Presl	Lauraceae	Skin	CO2
Linseed	Linum usitatissimum L	Linaceae	Seed	H2O
Aloe	Aloe vera (Haw.) Berg	Liliaceae	Leaf	CO2
Tulip	Tulipa gesneriana L	Liliaceae	Flower	H2O
Moringa leaf	Moringa oleifera Lam	Moringaceae	Leaf	CO2
Clove	Eugenia caryophμllataThunb	Myrtaceae	Flower	H2O
Rosewood	Rhodamnia dumetorum (Poir.) Merr. et Perry	Myrtaceae	Stem	EtOH
Dendrobe	Dendrobium nobile Lindl	Orchidaceae	Stem	EtOH
Licorice	Glycyrrhiza uralensis Fisch	Papilionaceae	Leaf	EtOH
Black pepper	Piper nigrum	Piperaceae	Fruit	EtOH
Citronella	Mosla chinensis Maxim	Poaceae	Leaf	H2O
Lemongrass	Cymbopogon citratus (DC.) Stapf	Poaceae	Leaf	CO2
Palmarosa	Cymbopogon martini	Poaceae	Leaf	EtOH
Ganoderma lucidum spore	Ganoderma Lucidum Karst	Polyporaceae	Spore	CO2
Agrimonia	Agrimonia pilosa Ldb	Rosaceae	Whole	EtOH
Lavender	Rosa"Lavande	Rosaceae	Flower	EtOH
Rose	Rosa rugosa Thunb	Rosaceae	Flower	EtOH
Rosehip	Rosa rugosa Thunb	Rosaceae	Fruit	EtOH
Sweet almond	Amygdalus Communis Vas	Rosaceae	Seed	EtOH
Bergamot	Citrus medica L. var.sarcodactylis Swingle	Rutaceae	Fruit	EtOH
Lemon	Citrus limon (L.) Burm. f	Rutaceae	Leaf	CO2
Pomelo	Citrus maxima (Burm) Merr	Rutaceae	Fruit	H2O
Zanthoxylum	Zanthoxylum bungeanum Maxim	Rutaceae	Fruit	EtOH
Moringa seed	Moringa oleifera Lam	Saxifragaceae	Seed	CO2
Black currant	Ribes nigrum L	Saxifragaceae	Fruit	CO2
Boxthorn seed	Lycium chinenseMill	Solanaceae	Seed	CO2
Schizandrae fructus	Schisandra chinensis	Schisandraceae	Fruit	EtOH
Camellia seed	Camellia sinensis (L.) O.Ktze	Theaceae	Seed	H2O
Ginger	Zingiber officinale Rosc	Zingiberaceae	Root	CO2
Zedoary turmeric	Curcuma phaeocaulis Valeton	Zingiberaceae	Root	EtOH

Inhibition assay of EOs against α-glucosidase

All the EOs and acarbose were dissolved in acetone to obtain varying concentrations as required. As previously described (Chen et al., 2020), inhibition of EOs at different concentrations against α-glucosidase was tested. Typically, the solution containing 35 μL PBS, 10 μL α-glucosidase, and 5 μL EOs (acetone in blank) were firstly added in the 96-well plates at 0 °C and then pre-incubated for 10 min at 37 °C. Secondly, 50 μL p-Nitrophenol glucoside (PNPG) was immediately added in the 96-well plates to initiate the enzyme reaction and incubated in the microplate reader (Multiskan GO, Thermo Scientific, USA) at 37 °C for 30 min. Finally, the change in absorbance at 405 nm during the 30 min reaction was determined and recorded. The assays were respectively conducted as triplicate and the concentration required for 50% inhibition of each EO was determined as IC₅₀ value. The inhibition rates of EOs were tested at more than 5 concentrations. The IC₅₀ values were calculated by Origin 9.0 software. Inhibitory effect of EOs the α-glucosidase reaction was calculated as follows:

$$\text{Inhibition Rate}/\text{\%}=\frac{(A-B)-(C-D)}{A-B}\times 100\text{\%}$$

where A represents the absorbance of samples (30 min); B represents the absorbance of samples (30 min); C represents the absorbance of the blank (30 min); D represents the absorbance of the blank (30 min).

Kinetic analysis of α-glucosidase inhibition

As previously reported (Chen et al., 2020), the inhibition mechanism of chuan-xiong, fructus cnidii, sacha inchi, aloe, ganoderma lucidum spore and ginger was investigated. In brief, a series of diluted EO solutions were firstly prepared and the PNPG solution was kept in a constant concentration (1 mM). Secondly, the inhibitory effect of chuan-xiong, fructus cnidii, sacha inchi, aloe, ganoderma lucidum spore and ginger on α-glucosidase was determined with different concentrations of α-glucosidase (0, 5, 10 and 10 U/L), respctively. In addition, the inhibition kinetics of α-glucosidase was measured by Lineweaver–Burk plots. Typically, a series of diluted EO solutions were obtained and the α-glucosidase solution was kept in a constant concentration (10 U/L). The inhibition rates were tested with different concentrations of PNPG (0.75, 0.60, 0.45, 0.30, and 0.15 mM) by using the above method.

Type	Mechanism	Characteristic
Competitive-type	Only bound free enzyme and not the enzyme–substrate complex	1. Ki ≈ Kis; 2. The lines of Lineweaver–Burk are crossed on a 1/V axis
Noncompetitive-type	Only bound the enzyme–substrate complex	1. Ki ≈ Kis; 2. The lines of Lineweaver–Burk crossed on a 1/[S] axis
Mixed-type	Bound free enzyme and the enzyme–substrate complex	1. The lines of Lineweaver–Burk crossed on crossed on the second or third quadrant; 2. Ki < Kis, competitive-type; 3. Ki > Kis, noncompetitive-type

The Inhibition kinetics, inhibition constants (Ki) and slope inhibition constant (Kis) were analyzed by Lineweaver–Burk plots. The specific type, mechanism and characteristic were show as following.

Analysis of the most potential EOs with GC–MS

EOs were analyzed with GC (TRACE 1300E, Thermo Scientific Corporation, USA) coupled to MS (ISQ Qd, Thermo Scientific Corporation, USA) using a TG-5 MS silica column (30 m × 0.25 mm; film thickness 0.25 μm). The procedure for analyzing GC–MS was showed as follow: helium was firstly used as the carrier gas, flowing with a rate of 1.0 mL/min. The oven temperature was secondly programmed at 60 °C, with an increase of 5 °C/min to 160 °C (isotherm at 2 min), and then 10 °C/min to 260 °C and held for 20 min. The mass spectra were recorded at 70 eV with a scanning range from 35 to 450 m/z. Composition (%) of EOs was calculated in software by using the peak normalization method. Comparing to Kovats retention indices and relative to a C₈–C₄₀ n-alkanes standard, the peak identification of different constituents in EOs was determined. Identification of the EOs constituents was performed by comparing the acquired mass spectrum with NIST mass spectral library.

Results and discussion

Inhibitory effect of sixty-two EOs against α-glucosidase

It has been indicated that EOs derived from natural products and their active constituents were promising α-glucosidase inhibitors (Zhao et al., 2017). However, systematic research in plant derived EOs as natural α-glucosidase inhibitors was still lacking. Hence, we determined the effect of sixty-two EOs on α-glucosidase in vitro. As shown in Table 2, all of EOs at 25 μg/mL showed stronger inhibitory activity against α-glucosidase than that of acarbose which was used as the positive control. Furthermore, most of EOs exhibited more effectively inhibition than acarbose (475.6 ± 24.1 μg/mL). The results indicated that the inhibitory effect of plant derived EOs from the family Apiaceae on α-glucosidase was in the descending order as follow: fructus cnidii > chuan-xiong > angelica > saposhnikovia divaricata = ligusticum > 50 μg/mL. In the family Burseraceae, olive EO (11.8 ± 0.1 μg/mL) showed the strongest inhibition against α-glucosidase. Moreover, costus EO with IC₅₀ value of 16.3 ± 0.4 μg/mL showed most significant inhibition in the family Compositae. Aloe EO (5.06 ± 0.18 μg/mL) exhibited more effective inhibiton than that of other EO derive from the family Liliaceae. In the family Rosaceae, sweet almond EO (11.4 ± 3.7 μg/mL) showed the strongest inhibition against α-glucosidase activity. Among these EOs, the most potent EOs against α-glucosidase were chuan-xiong, fructus cnidii, sacha inchi, aloe, ganoderma lucidum spore and ginger with IC₅₀ values of 3.02, 2.88, 7.37, 5.06, 5.32 and 7.40 μg/mL, respectively. Taken together, chuan-xiong, fructus cnidii, sacha inchi, aloe, ganoderma lucidum spore and ginger were selected for further investigation of the anti-α-glucosidase mechanism of action.

Table 2.

The half maximal inhibitory concentration of sixty-two EOs and acarbose

NO	EOs	Inhibition rate at 50 μg/mL (%)	IC50 (μg/mL)
1	Angelica	99.24 ± 0.17	11.0 ± 0.1
2	Angelica pubescence	26.99 ± 2.01	101.7 ± 0.1
3	Bupleurum	30.22 ± 3.42	67.6 ± 2.8
4	Celery seed	72.48 ± 6.66	37.3 ± 2.4
5	Chuan-xiong	99.87 ± 0.05	3.02 ± 0.06
6	Fructus cnidii	99.71 ± 0.15	2.88 ± 0.04
7	Ligusticum	89.80 ± 4.61	25.6 ± 1.1
8	Notopterygium	38.25 ± 6.74	157.0 ± 1.1
9	Saposhnikovia divaricata	98.33 ± 0.06	25.6 ± 0.3
10	Gallnut	63.62 ± 10.21	42.5 ± 2.4
11	Ginseng	53.39 ± 4.86	46.7 ± 10.7
12	Sabal	65.69 ± 3.19	26.2 ± 3.1
13	Epimedium	98.89 ± 0.11	22.5 ± 5.0
14	Olive	95.54 ± 0.12	11.8 ± 0.1
15	Frankincense	28.78 ± 4.73	301.5 ± 33.6
16	Honeysuckle	47.20 ± 2.34	61.4 ± 0.8
17	Papaya	46.70 ± 1.49	62.7 ± 0.7
18	Carnation	99.79 ± 0.08	10.1 ± 0.4
19	Tripterygium wilfordii	32.65 ± 5.49	198.8 ± 37.6
20	Burdock	92.71 ± 4.47	28.1 ± 1.1
21	Chrysanthemum	99.16 ± 0.93	17.8 ± 1.4
22	Costus	98.89 ± 0.38	16.3 ± 0.4
23	Mugwort	34.55 ± 2.74	91.4 ± 16.2
24	Cypevol	67.24 ± 0.49	18.9 ± 0.1
25	Seabuckthorn	98.79 ± 0.69	17.5 ± 1.1
26	Seabuckthorn seed	99.23 ± 0.54	10.6 ± 1.2
27	Sacha inchi	99.50 ± 0.21	7.37 ± 0.47
28	Oleum anisi stellati	31.48 ± 2.94	81.9 ± 9.7
29	Peppermint	46.95 ± 1.19	40.9 ± 4.1
30	Patchouli	98.56 ± 1.02	17.5 ± 0.4
31	Schizonepeta tenuifolia	57.91 ± 1.86	46.8 ± 1.0
32	Bay	97.95 ± 0.08	20.4 ± 2.4
33	Cinnamon	51.61 ± 1.54	30.6 ± 4.3
34	Linseed	24.22 ± 1.64	253.7 ± 22.5
35	Aloe	98.77 ± 1.08	5.06 ± 0.18
36	Tulip	22.44 ± 2.32	83.4 ± 0.3
37	Moringa leaf	70.65 ± 4.24	23.1 ± 0.4
38	Clove	49.80 ± 8.01	45.1 ± 13.0
39	Rosewood	63.73 ± 5.30	33.3 ± 7.6
40	Dendrobe	35.53 ± 6.13	105.8 ± 13.0
41	Licorice	83.32 ± 2.86	14.3 ± 1.1
42	Black pepper	84.27 ± 0.34	28.0 ± 0.5
43	Citronella	30.25 ± 1.81	230.4 ± 38.8
44	Lemongrass	54.70 ± 1.32	44.7 ± 3.7
45	Palmarosa	83.92 ± 1.14	17.6 ± 0.3
46	Ganoderma lucidum spore	99.31 ± 0.12	5.32 ± 0.13
47	Agrimonia	42.02 ± 0.12	85.0 ± 4.7
48	Lavender	57.85 ± 1.47	52.4 ± 28.3
49	Rose	99.16 ± 0.93	17.8 ± 0.6
50	Rosehip	99.62 ± 0.13	12.9 ± 1.6
51	Sweet almond	98.52 ± 1.05	11.4 ± 3.7
52	Bergamot	75.58 ± 2.34	31.0 ± 2.3
53	Lemon	53.56 ± 1.69	53.9 ± 3.4
54	Pomelo	36.42 ± 2.45	39.3 ± 16.7
55	Zanthoxylum	90.63 ± 1.38	21.9 ± 0.4
56	Moringa seed	10.63 ± 2.15	218.2 ± 82.7
57	Black Currant	9.47 ± 3.42	1074.7 ± 17.1
58	Boxthorn seed	26.70 ± 1.61	95.8 ± 1.4
59	Schizandrae fructus	98.99 ± 0.27	23.0 ± 0.1
60	Camellia seed	9.01 ± 2.11	1095.3 ± 45.0
61	Ginger	99.55 ± 0.04	7.40 ± 0.18
62	Zedoary turmeric	65.56 ± 1.73	32.1 ± 1.1
63	Acarbose	8.61 ± 0.21	475.6 ± 24.1

Mechanism study

In order to investigate the inhibitory mechanism of chuan-xiong, fructus cnidii, sacha inchi, aloe, ganoderma lucidum spore and ginger on α-glucosidase, the inhibition constants and inhibition types of chuan-xiong, fructus cnidii, sacha inchi, aloe, ganoderma lucidum spore and ginger on α-glucosidase activity were tested by using PNPG as the substate. As described in Fig. 1, the four lines were obtained from three different concentrations of chuan-xiong, fructus cnidii, sacha inchi, aloe, ganoderma lucidum spore and ginger, respectively. The four lines were crossed on the origin. In addition, the slope of the line was gradually decreased with increasing EOs (chuan-xiong, fructus cnidii, sacha inchi, aloe, ganoderma lucidum spore and ginger) concentrations. These results indicated that the effect of chuan-xiong, fructus cnidii, sacha inchi, aloe, ganoderma lucidum spore and ginger on α-glucosidase activity was reversible.

Fig. 1.

The inhibitory mechanism of chuan-xiong, fructus cnidii, sacha inchi, aloe, ganoderma lucidum spore and ginger against α-glucosidase

As shown in Fig. 2, the inhibition kinetics of chuan-xiong, fructus cnidii, sacha inchi, aloe, ganoderma lucidum spore and ginger were analyzed by Lineweaver–Burk plots. The four lines were obtained from five different concentrations of fructus cnidii, aloe, ganoderma lucidum spore and ginger (Fig. 2B, 2D, 2E and 2F). These lines of fructus cnidii, aloe, ganoderma lucidum spore and ginger were crossed on a 1/V axis, indicating that fructus cnidii, aloe, ganoderma lucidum spore and ginger exhibited competitive-type inhibition on α-glucosidase, respectively. These results indicated that fructus cnidii, aloe, ganoderma lucidum spore and ginger only bound free enzyme and not the enzyme–substrate complex. In addition, the four lines with different slopes were obtained from four different concentrations of chuan-xiong and sacha inchi (Fig. 2A and 2C). These lines were respectively crossed on the second or third quadrant, indicating that chuan-xiong and sacha inchi showed mixed-type inhibition.

Fig. 2.

Lineweaver-Burk plots for chuan-xiong, fructus cnidii, sacha inchi, aloe, ganoderma lucidum spore and ginger against α-glucosidase. (A), (B), (C), (D), (E) and (F) represents the plot of slope versus the concentration of chuan-xiong, fructus cnidii, sacha inchi, aloe, ganoderma lucidum spore and ginger. (G) and (H) represents the plot of intercept versus the concentration of chuan-xiong and sacha inchi for the determination of K_i. (I), (J), (K), (L), (M) and (N) represents the plot of intercept versus the concentration of chuan-xiong, fructus cnidii, sacha inchi, aloe, ganoderma lucidum spore and ginger for the determination of K_is

To investigate whether chuan-xiong, fructus cnidii, sacha inchi, aloe, ganoderma lucidum spore and ginger inhibited α-glucosidase activity by competitively forming enzyme-inhibitor (EI) complex or interrupting enzyme–substrate-inhibitor (ESI) complex in non-competitive manner, we determined EI dissociation constants K_i of chuan-xiong and sacha inchi, and the ESI dissociation constants K_is of chuan-xiong, fructus cnidii, sacha inchi, aloe, ganoderma lucidum spore and ginger. Inhibition type, K_i and K_is values of chuan-xiong, fructus cnidii, sacha inchi, aloe, ganoderma lucidum spore and ginger, were showed in Table 3. K_i values of chuan-xiong and sacha inchi were 3.90 ± 0.25 and 21.32 ± 2.36 μg/mL, respectively. Furthermore, a lower value of K_i in comparison with K_is indicated that there was stronger binding between enzyme and chuan-xiong, suggesting that it preferred competitive over noncompetitive manner. A lower value of K_is in comparison with K_i revealed that there was weaker binding between enzyme and sacha inchi, suggesting that it preferred noncompetitive over competitive manner. It was concluded that chuan-xiong, fructus cnidii, sacha inchi, aloe, ganoderma lucidum spore and ginger had the significant inhibition on α-glucosidase activities, indicating that they might be potent α-glucosidase inhibitors.

Table 3.

Type of mechanism, K_i and K_is value of chuan-xiong, fructus cnidii, sacha inchi, aloe, ganoderma lucidum spore and ginger

EOs	Inhibition mechanism	Ki value (μg/mL)	Kis value (μg/mL)
chuan-xiong	Mixed type	3.90 ± 0.25	6.84 ± 0.55
fructus cnidii	Uncompetitive type	–	4.50 ± 0.60
sacha inchi	Mixed type	21.32 ± 2.36	14.25 ± 1.90
aloe	Uncompetitive type	–	7.87 ± 0.85
ganoderma lucidum spore	Uncompetitive type	–	12.68 ± 1.10
ginger	Uncompetitive type	–	7.28 ± 0.50

Main constituents of chuan-xiong, fructus cnidii, sacha inchi, aloe, ganoderma lucidum spore and ginger

To characterize the constituents of the potential EOs that contribute to the anti-α-glucosidase activity, six EOs (chuan-xiong, fructus cnidii, sacha inchi, aloe, ganoderma lucidum spore and ginger) were analyzed by GC–MS. As listed in Table 4, the main constituent of chuan-xiong EO was (E)-ligustilide (43.89%), followed by senkyunolide (20.78%), diacetone alcohol (12.0%), linoleic acid (4.05%) and neocnidilide (3.68%), etc. In fructus cnidii EO, osthole was the most abundant compound (40.56%), followed by oleic acid (6.70%), D, L-isobornylacetate (8.52%), myrtenyl isovalerate benzeneacetaldehyde (5.37%) and linoleic acid (3.35%), etc. Sacha inchi EO was characterized by the highest diacetone alcohol and (Z)-13-docosenamide content (67.84% and 16.06% of the total EO, respectively), followed by chlorbutanol (4.82%), o-phenyl carbamate (4.56%), dioctyl terephthalate (3.82%), methylcarbamic acid phenyl ester (2.90%). In addition, the main constituent of aloe EO was diacetone alcohol (68.25%), followed by (Z)-13-docosenamide (15.57%), chlorbutanol (4.89%), phenol (4.57%), linoleic acid (4.02%) and methylcarbamic acid phenyl ester (2.69%). In ganoderma lucidum spore EO, diacetone alcohol was reported to be the most abundant compound (70.74%), followed by (Z)-13-docosenamide (13.71%), chlorbutanol (4.94%), phenol (4.19%), bis(2-ethylhexyl)iosphthalate (3.60%) and methylcarbamic acid phenyl ester (2.82%). Finally, ginger EO was characterized by the highest α-zingiberene and diacetone alcohol content (25.62% and 11.50 of the total EO, respectively), followed by (-)-Sabinene (10.39%), β-Bisabolene (6.31%), 6-shogaol (6.27%), α-Curcumene (6.26%), etc.

Table 4.

Chemical components of chuan-xiong (CX), fructus cnidii (FC), sacha inchi (SI), aloe (A), ganoderma lucidum spore (GLS) and ginger (G)

Chemical Composition a		Content/%
CAS No	Name	RIb	RSIc	CX	FC	SI	A	GLS	G
1943-79-9	Methylcarbamic acid phenyl ester	759	760			2.90	2.69	2.82
112-84-5	(Z)-13-docosenamide	803	809	3.62		16.06	15.57	13.71	3.88
33900-84-4	Myrtenyl isovalerate	817	824		5.37
137-89-3	Bis(2-ethylhexyl)iosphthalate	830	852					3.60
484-12-8	Osthole	853	864		40.56
108-95-2	Phenol	876	876				4.57	4.19
4567-33-3	Neocnidilide	881	885	3.68
57-15-8	Chlorbutanol	882	886			4.82	4.89	4.94
112-80-1	Oleic acid	882	897		6.70
622-46-8	o-phenyl carbamate	883	891			4.56
6422-86-2	Dioctyl terephthalate	883	893			3.82
60-33-3	Linoleic acid	888	904	4.05	3.35		4.02
15111-96-3	p-mentha-1, 8-dien-7-ylacetate	902	942		3.27
10408-16-9	(−)-sabinene	915	920						10.39
555-66-8	6-shogaol	916	924						6.27
123-42-2	Diacetone alcohol	920	920	12.00	4.25	67.84	68.25	70.74	11.50
63038-10-8	Senkyunolide	927	933	20.78
303187–89-5	4-(3-hydroxy-2-methoxyphenyl) -butan-2-one	930	937						4.11
495-62-5	β-bisabolene	930	937						6.31
495-60-3	α-zingiberene	932	942						25.62
92618-89-8	d, l-isobornylacetate	933	933		8.52
644-30-4	α-curcumene	942	942						6.26
81944-08-3	(E)-ligustilide	944	957	43.89
	Others	945	963	11.98	27.98	0.00	0.00	0.00	25.66

^aMajor components (content > 1%), listed in the order of RI value, are listed in the table

^bLinear retention index is obtained from https://webbook.nist.gov/chemistry/

It has been indicated that α-glucosidase, a key digestive enzyme, was responsible for the hydrolysis of α(1–4) glucosidic bonds to control the amount of absorbable monosaccharides which released from oligosaccharides and glycoconjugates (Yue et al., 2017). Previous studies reported that α-glucosidase inhibitors could effectively control postprandial glucose level imbalance and caused serious side effects (Rosas-Ramírez et al., 2020; Kumar and Sinha, 2012). Therefore, the finding of safe α-glucosidase inhibitors would be urgently needed.

It was revealed that EO had a wild range of bioactivities (Raut and Karuppayil, 2014). Hence, we investigated whether the EOs derived from natural products had effect on α-glucosidase in present study. Our results showed that among these EOs, the most potent EOs against α-glucosidase were chuan-xiong, fructus cnidii, sacha inchi, aloe, ganoderma lucidum spore and ginger with IC₅₀ values of 3.02, 2.88, 7.37, 5.06, 5.32 and 7.40 μg/mL, respectively (Fig. 1). Interesting, our further study indicated that diacetone alcohol, a main constituent of sacha inchi, aloe and ganoderma lucidum spore EO, had an inhibitory effect on α-glucosidase with IC₅₀ values of 22.89 mg/mL. Previous studies revealed that (E)-ligustilide, osthole and α-zingiberene had anti-α-glucosidase activity (Chen et al., 2021; Karakaya et al., 2018; Asraoui et al., 2021). Thus, chuan-xiong EO, fructus cnidii EO, and ginger EO might be responsible for anti-α-glucosidase activity of these compounds. These results indicated that EO and their main constituents may be valuable α-glucosidase inhibitor.

As shown in Fig. 2, our results suggested that there was stronger binding between enzyme and chuan-xiong, suggesting that it preferred competitive over noncompetitive manner. Furthermore, there was weaker binding between enzyme and sacha inchi, suggesting that it preferred noncompetitive over competitive manner. Above all, the results concluded that chuan-xiong, fructus cnidii, sacha inchi, aloe, ganoderma lucidum spore and ginger had significantly anti-α-glucosidase activity, suggesting that they would be used as natural α-glucosidase inhibitors.

In summary, EOs (chuan-xiong, fructus cnidii, sacha inchi, aloe, ganoderma lucidum spore, and ginger), isolated from edible products, were safe and have potent anti-α-glucosidase activity. The in vitro experiments indicated that chuan-xiong and sacha inchi were reversible and mixed-type inhibitors, and fructus cnidii, aloe, ganoderma lucidum spore and ginger were reversible and uncompetitive-type inhibitors, revealing the mechanism of these EOs against α-glucosidase. Collectively, the results have provided new insights into potential application of EOs from natural products as α-glucosidase inhibitors to treat diabetes.

Author contributions

WL designed the experiments; ZY and YL conducted the experiments; WL and XZ analyzed the data; WL wrote the manuscript; while KZ, XZ and VKWW revised the manuscript.

Declarations

Conflict of interest

The authors have declared no conflict of interest.