Inhibition of enzymes linked to type-2 diabetes and hypertension by essential oils from peels of orange and lemon

ABSTRACT

This study was designed to evaluate and compare the interactions of essential oils from orange (Citrus sinensis [L.] Osbeck) and lemon (Citrus limon) peels on enzymes linked to type-2 diabetes (α-amylase and α-glucosidase) and hypertension (angiotensin-I-converting enzyme [ACE]). The essential oils were obtained from the peels by hydrodistillation and were passed over anhydrous sodium sulphate. The interaction of the oil on α-amylase, α-glucosidase, and ACE activities was subsequently determined. The results revealed that the essential oils inhibited α-amylase and α-glucosidase activities. However, lemon peel (IC₅₀ = 8.16 µg/mL and 7.56 µg/mL) essential oil exhibited stronger inhibitory activity on α-amylase and α-glucosidase activities compared to orange peels (IC₅₀ = 11.51 µg/mL and 11.53 µg/mL), respectively. Similarly, both essential oils reduced the activity of ACE. Moreover, lemon peel (IC₅₀ = 26.17 µg/mL) essential oil showed higher inhibitory effect compared to orange peel (IC₅₀ = 31.79 µg/mL). Our findings revealed that essential oils from lemon and orange peels are potential antidiabetic and antihypertensive agents. Moreover, essential oil from lemon peels was more potent than orange peels.

KEYWORDS:

• Essential oil
• α-Amylase
• α-Glucosidase
• Angiotensin-I-converting enzyme
• Diabetes
• Hypertension

Introduction

Essential oils are aromatic oily liquids which are volatile in nature and are present in different parts of plants. These volatile liquids are known to contain different classes of compounds such as monoterpenes, sesquiterpenes, diterpenes, triterpenes, and terpenoids. Research into the use of essential oils has gained much interest recently and many of these oils are used as food preservatives, flavourants, and alternative therapy in traditional medicine for the treatment of some diseases.^[1,2] Essential oils have been reported to possess several biological activities which include antioxidative, antimicrobial, anti-inflammatory, antiaflatoxigenic, anticancer, and antidiabetic activities.^[3–5] Furthermore, essential oils have been extracted from the leaves, stem, roots, and peels of different plants.

Citrus sp. have been identified as excellent sources of essential oils. Citrus essential oil is largely present in the peels compared to other parts and it has a wide application in food industries as additives in the formulation of syrup, drinks, cakes, and ice cream.^[6,7] The yield of essential oils and their chemical constituents vary between species and their origin. The oils are present within the glands of the external layer of the fruit skin.^[7] Several reports have revealed the chemical composition of essential oils from some Citrus sp.^[8–10] Previous reports from our laboratory have shown the chemical composition, anticholinesterase, and antioxidant activities of essential oils from orange and lemon peels.^[11,12] However, we observed that there is dearth of information on the antidiabetic and antihypertensive activities of essential oils from orange and lemon peels.

Diabetes mellitus is a major public health problem affecting millions of people all over the world.^[13,14] The onset and development of type-2 diabetes is characterized by consistent hyperglycaemia due to enzymatic degradation of carbohydrates and lipids in the cell which affects glucose metabolism.^[15] Common therapeutic strategy to reduce hyperglycaemia in diabetic patients involves the inhibition of carbohydrate hydrolysing enzymes. These enzymes (α-amylase and α-glucosidase) break down complex carbohydrates to glucose which is rapidly absorbed into the bloodstream leading to hyperglycaemia.^[16] Previous reports have revealed that diabetes and hypertension are interrelated as 75% of diabetic patients are known to be hypertensive.^[17,18] Type-2 diabetic patients are more prone to develop hypertension which is a major risk factor of cardiac, renal, and cerebral dysfunction.^[19] Angiotensin-converting enzyme (ACE) plays a major role in the development of hypertension. Increase in ACE activity leads to elevated levels of angiotensin II which is a potent vasoconstrictor that can raise blood pressure.^[20] However, ACE inhibitors can reduce blood pressure in hypertensive patients by limiting the conversion of angiotensin I to angiotensin II. Therefore, in this study, we report for the first time a comparative study on the modulatory effects of essential oils from orange peels on α-amylase, α-glucosidase, and ACE activities.

Materials and methods

Sample collection and preparation

Orange fruits were purchased from Akure main market in Ondo State, while lemon fruits were obtained in a farm in Akute Ogun State both in Southwest Nigeria. The fruits were identified and authenticated at the Biology department, Federal University of Technology of Akure, Nigeria. The peels were sun dried to constant weight and pulverised into powder using a laboratory blender.

Extraction of essential oil

Essential oil was extracted from the powder of the orange peels (100 g) by hydrodistillation via a Clevenger apparatus.^[21] The essential oil layer was collected from the apparatus via the outlet and passed over anhydrous sodium sulphate. The oils were stored separately in sealed vials at 4°C for further analysis.

Gas chromatography analysis

Essential oils from orange and lemon peels were analysed using Hewlett-Packard 5890 gas chromatograph equipped with flame ionization detectors (FID) and DB-5 column. The sample 1 µL solution in hexane (1:100) was injected into the system. The following conditions were applied: (1) injection temperature: 290°C; (2) injection volume: 1.0 µL; (3) injection mode: split (1:50); (4) temperature program: 50°C for 4 min, rising at 3°C/min to 240°C, then rising at 15°C/min to 300°C, held at 300°C for 3 min; and (5) FID (290°C): (i) H₂ flow: 50 mL/min and (ii) air flow: 400 mL/min. The individual constituents of the essential oils were identified via Mass spectrometry (MS) and their identity was confirmed in comparison with mass spectra of authentic standards and that of the data that are already available in National institute for standards (NIST) and Wiley mass spectral libraries. The per cent composition of the essential oils was computed from the area of the peaks of the Gas Chromatography (GC). Kovats retention index (KI) was calculated using the KI formula.

α-Amylase inhibition assay

The reaction mixture that contains different concentration of each essential oil (0–16 µg/mL) and 500 µL of Hog pancreatic α-amylase (EC 3.2.1.1) (0.5 mg/mL) was incubated at 25°C for 10 min. In total, 500 µL of starch (1%) solution prepared with 0.02 M sodium phosphate buffer was added to the previous mixture and was incubated for 10 min at 25°C. Dinitrosalicyclic acid (1.0 mL) was added to the mixture to stop the reaction. The resultant solution was incubated for 5 min at 100°C. The solution was allowed to cool and distilled water was added to the mixture before the absorbance was measured at 540 nm.^[22] The α-amylase activity was calculated and expressed as percentage inhibition using the following formula:

(1)

α-Glucosidase inhibition assay

The enzyme (1.0 U/mL) was prepared in phosphate buffer (0.1 M). Different concentration of the essential oil (0–16 µg/mL) was added to 100 µL of the enzyme solution which was incubated at 25°C for 10 min. Moreover, 50 µL of p-nitrophenyl-α-D-glucopyranoside solution (5 mM) which was prepared in 0.1 M phosphate buffer (pH 6.9) was added. The solution was incubated at room temperature for 5 min. After the incubation, the absorbance was measured at 405 nm.^[13] The α-glucosidase inhibitory activity was expressed as percentage inhibition using Eq. (1).

Angiotensin-I-converting enzyme inhibition assay

The effect of the essential oils on ACE activity was evaluated using the method of Golbidi et al.^[19] ACE solution (50 µL, 4 mU) and the essential oils (0–60 µg/mL) were incubated at 37°C for 15 min. After the incubation, 150 μL of 8.33 mM of Bz–Gly–His–Leu in 125 mM Tris–HCl buffer (pH 8.3) was added to the solution. The mixture was incubated at 37°C for 30 min and 250 µL of 1 M HCl was added to stop the reaction. The Bz–Gly formed in the reaction was extracted with 1.5 mL ethyl acetate. After the extraction, the ethyl acetate fraction was separated by centrifugation and 1 mL of the fraction was allowed to evaporate in a test tube. The residue that was formed was dissolved with distilled water and the absorbance was measured at 228 nm.^[23] The ACE inhibitory activity was calculated and expressed as percentage inhibition using Eq. (1).

Data analysis

All the experiments were performed in triplicates and the data were expressed as mean ± standard deviation (SD). Statistical analysis was conducted by one-way analysis of variance using Graph pad prism 5.0. The concentration of the samples that caused 50% inhibition was calculated using nonlinear regression analysis.

Results and discussion

Chemical constituents of essential oils

Table 1 presents the components of essential oils from orange and lemon peels which were identified via GC–MS. The essential oil constituents comprise of monoterpene hydrocarbons, oxygenated monoterpenes, and sesquiterpene hydrocarbons and were listed in their order of their elution with their KI. The most abundant compound in orange peel essential oil is limonene (92.14%). Moreover, β-myrcene (2.70%) was also present in appreciable levels compared to other compounds. However, lemon peel had lower levels of limonene (53.07%) compared to orange peels. Other constituents such as β-pinene (9.53), borneol (5.57%), neral (4.70%), sabinene (4.18%), α-pinene (3.82%), linalool (3.7), 1,8-cineole (3.38%), β-myrcene (3.33%), geranial (3.33%), β-caryophyllene (1.49%), and linalyl acetate (1.48%) were present in appreciable levels compared to orange peels.

Table 1. Chemical composition of essential oil.

	Orange peels		Lemon peels
Compounds^a	KI^b	%^c	KI^b	%^c
α-Phenandrene	1088	0.14	–	–
Sabinene	1149	0.11	1094	4.18
Limonene	–	–	1188	53.07
α-Pinene	1225	0.70	1228	3.82
β-Pinene^d	1267	0.02	1306	9.53
Limonene	1309	92.14	–	–
Cis-ocimene	1356	0.12	1385	0.01
β-Myrcene	1382	2.7	1391	3.33
Allo-ocimene	1400	0.07	1396	0.04
Pinene-2-ol	–	–	1429	–
α-Thujene	1424	0.19	1450	0.05
Decanal	1448	0.22	–	–
γ-Terpinene	1461	0.23	1483	0.11
Geijerene	1468	0.12	–	–
Fenchone	1514	0.13	–	–
Neral	1529	0.09	1502	4.70
Geranial	1539	0.07	1516	3.33
Isoartemisia	1550	0.05	1559	0.04
1,8-cineole	1569	0.33	0.33	0.33
Geraniol	1599	0.06	–	–
Nerol	1620	0.08	–	–
Linalool	1639	0.05	1622	3.70
Borneol	1662	0.17	1628	5.57
α-Terpineol	1689	0.17	1677	0.25
Terpinen-4-ol	1703	0.09	1681	0.23
Thymyl methyl ether	1731	0.16	1634	0.06
Ascaridole	1749	0.08	1741	0.06
Linalyl acetate	1652	0.13	–	–
Ethyl cinnamate	1694	0.14	1709	0.10
Borneol acetate	1688	0.12	1689	0.13
Neryl acetate	1697	0.06	–	–
β-Bisabolene	–	–	1715	0.11
Linalyl acetate	–	–	1705	–
Geranyl acetate	1703	0.15
β-Caryophyllene	1668	0.12	1731	1.49
Trans-α-bergamotene	1760	0.12	–	–
Cardinene	1777	0.08	–	–
β-Elemene	1789	0.03	1778	0.61
Germacrene D	–	–	1800	0.10
Bicyclogermacrene	1811	0.19	1800	0.08
α-Copane	1850	0.03	–	–
Germacrene D	1886	0.09	–	–
Acetyleugenol	1891	0.05	–	–
Elemicin	1912	0.02	–	–
Benzyl benzoate	1959	0.09	–	–
α-Humulene	2010	0.09	–	–
Viridiflorol	–	–	2042	0.05
Torreyol	–	–	2049	0.04
Tetradecanoic acid	–	–	2039	0.08
Total identified		99.84		99.76
Grouped components (%)
Monoterpene hydrocarbons		58.00		74.10
Oxygenated monoterpenes		32.10		21.28
Sesquiterpene hydrocarbons		9.70		2.48

^aCompounds are listed in order of their elution from a DB-5 FID column.

^bKovat Index.

^cPercentages obtained by FID peak-area normalization [Sources: Oboh et al. (2014) and Oboh et al. (2016)].

^d Main compounds in bold font.

– Not determined.

Effect of essential oil on α-amylase and α-glucosidase activities

The antidiabetic activity of essential oils from orange and lemon peels was evaluated through different methods. The result obtained from the enzyme assays revealed that essential oils from orange peels (IC₅₀ = 11.51 µg/mL) exhibited lower capacity to inhibit α-amylase activity than lemon peels (IC₅₀ = 8.16 µg/mL) as shown in Fig. 1 and the IC₅₀ values in Table 2. Moreover, both essential oils showed lower inhibitory effects on α-amylase activity compared to acarbose (IC₅₀ = 7.45 µg/mL). Similarly, orange peel (IC₅₀ = 11.53 µg/mL) essential oils exhibited lower inhibitory effects on α-glucosidase activity than lemon peel oil (IC₅₀ = 7.56 µg/mL) as shown in Fig. 2 and the IC₅₀ values in Table 2. However, essential oils from lemon peels exhibited higher inhibitory capacity on α-glucosidase activity than acarbose (IC₅₀ = 8.44 µg/mL). Previous experimental investigation has shown that α-amylase and α-glucosidase are important therapeutic targets in the management and/or treatment of type-2 diabetes.^[24] α-amylase hydrolyses starch by degrading the internal glycosidic bonds to release oligosaccharides which are further hydrolysed by α-glucosidase into monosaccharides and are absorbed into the bloodstream.^[25] The accumulation of these monosaccharides especially glucose in the bloodstream leads to postprandial hyperglycaemia, a major risk factor in the pathogenesis of diabetes mellitus.^[26] The essential oils from orange and lemon peels which were used in this study inhibited α-amylase and α-glucosidase activities. Our findings revealed that lemon peel essential oil exhibited higher inhibitory effects on α-amylase and α-glucosidase activities compared to orange peels. More importantly, our findings revealed that lemon peel essential oil showed higher inhibitory effects on acarbose. Moreover, the α-glucosidase inhibitory activity of lemon peels was higher than that of α-amylase inhibitory activity. Previous report has shown that high α-glucosidase inhibition is more effective in the management of type-2 diabetes.^[25] This is due to the fact that antidiabetic drugs such as acarbose inhibits α-amylase excessively and this may lead to several side effects. The inhibition of α-amylase activity will limit the hydrolysis of the internal glycosidic linkages of polysaccharides, while decrease in α-glucosidase activity will limit the breakdown of disaccharides and delay glucose absorption into the blood from the small intestine.^[27] The observed inhibitory effects of the essential oils give credence to the fact that both essential oils could be promising antidiabetic agents. However, our findings suggest that lemon peel essential oil is more potent than orange peel oil. The observed higher inhibitory capacity of essential oil from lemon is not fully understood; however, our findings suggest that the antidiabetic activities of both essential oils could be due to the synergistic effects of monoterpenes and sesquiterpene hydrocarbons which were identified in the oils. An in vivo investigation on the antidiabetic effect of limonene revealed that this monoterpene exhibited hypoglycaemic effects in streptozotocin-induced diabetic rats.^[28] Furthermore, previous report from our laboratory on the antidiabetic activity of clove bud showed that its essential oil inhibited α-amylase and α-glucosidase activities in vitro and the inhibitory effects were attributed to the chemical components.^[29] Similarly, some reports have also revealed that terpenes are capable of exerting blood glucose lowering effects and monoterpenes such as α-pinene, β-pinene, myrcene, 1,8-cineole, and sabinene have been identified as potent inhibitors of α-amylase and α-glucosidase activities.^[28–31]

Table 2. IC₅₀ values of α-amylase, α-glucosidase, and ACE inhibition.

Parameter (unit)	Orange	Lemon	Acarbose	Lisinopril
α-Amylase (µg/mL)	11.51 ± 0.83^a	8.16 ± 0.43^b	7.45 ± 0.43^b	–
α-Glucosidase (µg/mL)	11.53 ± 0.41^a	7.56 ± 0.84^b	8.44 ± 0.43^b	–
ACE (µg/mL)	31.79 ± 0.52^a	26.17 ± 0.52^b	–	24 ± 0.43^b

Values represent means of triplicate reading. Values with different letters along the rows are significant different at p < 0.05. – Not determined. Acarbose and lisinopril were used as standards.

Table 3. R² values of α-amylase, α-glucosidase, and ACE inhibition.

Parameter	Orange	Lemon	Acarbose	Lisinopril
α-Amylase	0.98	0.95	0.76	–
α-Glucosidase	0.96	0.81	0.86	–
ACE	0.29	0.22	–	0.56

– Not determined.

Antihypertensive activity of essential oils from orange and lemon peels

Figure 3 shows the interaction of orange and lemon peel essential oils and lisinopril on ACE activity. Lemon peel oil (IC₅₀ = 26.17 µg/mL) showed significant inhibitory capacity on ACE activity than orange peel oil (IC₅₀ = 31.79 µg/mL). The IC₅₀ in Table 2 also revealed that lisinopril (IC₅₀ = 24.03 µg/mL) was more potent than essential oils from orange and lemon peels. The inhibition of ACE is an important therapeutic intervention in the management/treatment of hypertension. The decrease in ACE activity hinders the conversion of angiotensin I to angiotensin II, a vasoconstrictor which has been implicated in elevated blood pressure.^[32] In addition, inhibition of angiotensin-II production is could also prevent the progression of type-2 diabetes as elevated levels angiotensin II may induce insulin insensitivity in the peripheral tissues, which is critical in the development of this degenerative condition.^[33] In this study, we observed that the essential oil from orange and lemon peels reduced ACE activity. Moreover, the ACE inhibitory of the essential oils agrees with previous studies which revealed that essential oils from Periploca laevigata, Thymus algeriensis, and Aframomum danielli inhibited ACE activity in vitro.^[10,34,35] Lemon peels exhibited stronger inhibitory capacity on ACE compared to orange peels. However, lisinopril exhibited stronger inhibitory effects than the essential oils from lemon and orange peels. Previous studies have linked the ACE inhibitory activities of bioactive compounds such as terpenoids and flavonoids to their ability to compete with the substrate of the enzyme and chelation of zinc metal ions at the active site.^[10] Furthermore, the ACE inhibitory activity of the essential oils can also prevent the generation of reactive oxygen species which is induced by elevated levels of angiotensin II. The terpenoids, monoterpenes, and sesquiterpenes which are present in the essential oils could contribute to their inhibitory effect on ACE. Our findings revealed that essential oils from orange and lemon peels are potential antihypertensive agents, although essential oil from lemon peels was more potent than orange peels.

Figure 1. α-Amylase inhibitory activity of essential oils from orange and peels.

Figure 2. α-Glucosidase inhibitory activity of essential oils from orange and lemon peels.

Figure 3. Angiotensin-I-converting enzyme inhibitory activity of essential oils from orange and lemon peels.

Conclusion

This study revealed that essential oils from orange and lemon peels show in vitro inhibitory effects on enzymes linked to type-2 diabetes (α-amylase and α-glucosidase) and hypertension (ACE). Moreover, lemon peel essential oil exhibited higher antidiabetic and antihypertensive activities compared to orange peels. Our findings suggest that these essential oils are potential antidiabetic and antihypertensive agents.