Comparative study of the essential oil composition of Salvia urmiensis and its enzyme inhibitory activities linked to diabetes mellitus and Alzheimer’s disease

ABSTRACT

The genus Salvia has economic importance due to its broad uses in traditional medicine, perfume, food, and pharmaceutical industries. In the present work, various extracts and essential oils of Salvia urmiensis Bunge., were screened for their inhibitory activity against acetylcholinesterase and butyrylcholinesterase, the enzymes linked to neurodegeneration, and against α-amylase and α-glucosidase (involved in diabetes mellitus; DM). Chemical compositions of the essential oils of leaves and flowers of the plant were also determined. The tested samples exhibited moderate to high anti-diabetic potential (IC₅₀ = 8–145 µg/mL) and moderate anticholinesterase activity (IC₅₀ = 44–892 µg/mL). Essential oil of leaves was rich in ester compounds such as ethyl linoleate (19%), methyl hexadecanoate (17%), and methyl linoleate (7.5%). The major compound of essential oil of flowers was 6,10,14-trimethyl-2-pentadecanone (55.7%). This is the first report on the enzyme inhibitory activity of S. urmiensis and also the chemical composition of its leaves and flowers in essential oils. The results indicated that S. urmiensis could be considered a valuable source for functional foods and pharmaceuticals.

KEYWORDS:

• Salvia
• Essential oil
• Neuroprotection
• Enzyme inhibition
• Diabetes mellitus
• Alzheimer’s disease

Introduction

Lamiaceae family comprises more than 4000 species in which the genus Salvia is the largest member of the family with about 1000 species distributed all around the world.^[1] Salvia species are ethnobotanically used for the treatment of several diseases such as colds, aches, infections, bronchitis, Alzheimer’s disease (AD), and inflammation.^[2,3] Moreover, this genus has economic importance because of its uses in pharmaceutical, food, and perfume industries.^[4,5] For example, S. officinalis has wide uses as flavour and preservative in the preparation of food products and S. sclarea is used in wine production.^[5] Other Salvia species such as S. hispanica, S. yunnanensis, and S. divinorum are cultivated for their utilization in food, perfume, and pharmaceutical industries.^[6] Salvia species are rich in essential oils, flavonoids, and terpenoids,^[7–9] which are responsible for wide bioactivities of the genus such as antimalarial, antiviral, antinociceptive, cytotoxicity, antimicrobial, antioxidant, anti-inflammation, and neuroprotection.^[10–13]

Salvia urmiensis is an endemic species of Iranian flora growing wild in the northwest of the country. Previous studies on this species led to the isolation of several new E-seco-ursane-type triterpenoids with rare carbon skeleton and new polyhydroxylated triterpenoids with antitrypanosomal and antiproliferative activities.^[14–17] Moreover, several extracts of the plant exhibited considerable antioxidant and cytotoxic activities.^[18] A literature survey showed that there is just one report on the essential oil components of S. urmiensis (Lorestan province of Iran).^[19]

AD is a chronic neurodegenerative disorder related to age, which is characterized by memory impairment, cognitive dysfunction, and limitation in daily activities. Inhibition of acetylcholinesterase and butyrylcholinesterase is considered an important strategy for the treatment of AD. Salvia species are used in European folk medicine for memory-enhancing purposes.

In recent years there has been an increase in the occurrence of diabetes mellitus (DM) because of changes in human lifestyle. DM affects hundreds of millions of people all around the world. Natural or synthetic drugs are used for lowering the blood glucose level. Intestinal α-glucosidase and pancreatic α-amylase are key enzymes that catalyse carbohydrates to glucose monomers. In this case, α-glucosidase and α-amylase inhibitor drugs reduce the level of glucose in blood and are used for the treatment of DM.

In this work, we aimed to investigate the volatile oil composition of the leaves and flowers of the plant for the first time (collected from Takab, West Azerbaijan province, Northwest Iran). In addition, we evaluated the anti-diabetic and anti-Alzheimer’s potential (based on enzyme inhibitory assays) of the oils and several extracts of S. urmiensis. To the best of our knowledge, this is the first report on its enzyme inhibitory activities.

Materials and methods

Plant material

The aerial parts of S. urmiensis were collected in May 2013 from Takab (36°27′N 47°04′E), West Azarbaijan province, Iran, at the flowering stage. The plant was identified by Dr Sonboli in the herbarium of Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, Iran. The herbarium code of the plant was MPH-1220.

Preparation of solvent extracts

The powdered aerial parts of S. urmiensis (20 g) were extracted successively with n-hexane, chloroform, and methanol (200 mL) by shaking at room temperature during 48 h. The solvent of the extracts was removed using a rotary evaporator at 40 °C to afford crude n-hexane (1.1 g), chloroform (0.7 g), and methanol (2.5 g) extracts.

Isolation of essential oils

The essential oil samples were isolated by hydrodistillation using a Clevenger-type apparatus in 3 h. The oils were dried using anhydrous sodium sulphate and stored at 4 °C in dark until analysis.

GC-FID and GC-MS analysis

The essential oil analysis was performed using an Agilent instrument (model 7890A) equipped with a flame ionization detector (FID) and a DB-5 capillary column (30 m × 0.32 mm i.d., film thickness 0.25 µm). Helium was used as the carrier gas (1.1 mL/min, in constant linear velocity mode). Temperatures of the injector and detector were set at 240 and 250 °C, respectively. The oven temperature was programed from 35 to 180 °C at the rate of 4 °C/min, then raised to 250 °C at 17 °C/min, and held at this temperature for 10 min. The injection volume was 1 µL in split mode (1:100). GC-MS analysis was carried out using a Thermoquest Finnigan instrument (model Trace GC, Trace MS) equipped with a fused silica capillary DB-5 column. Essential oil was diluted in n-hexane (1/100, v/v) and 0.5 µL was injected manually. The temperature program of the column was as mentioned above for the GC-FID. Spectra were obtained in the electron ionization (EI) mode. Identification of individual compounds was carried out by calculating their retention indices (RIs) using n-alkanes (C₆–C₂₄) under the same GC condition. The constituents of the oil were identified by comparison of their RIs and mass spectra with those published in the literature and with NIST, Wiley, and Adams Mass Spectral libraries.

α-Amylase and α-glucosidase inhibition assays

The α-amylase (ex-porcine pancreas, EC 3.2.1.1, Sigma) inhibitory activities of the extracts and essential oils of S. urmiensis were evaluated.^[20] The sample solutions (25 µL) were mixed with the enzyme solution (50 µL) in phosphate buffer (pH = 6.9 with 6 mM sodium chloride) in 96-well microplates and incubated for 10 min at 37 °C. The reaction was initiated with the addition of starch solution (50 µL, 0.05%). Similarly, a blank was prepared by adding sample solution to all the reaction reagents without an enzyme solution. The mixture was incubated for 10 min at 37 °C. The reaction was then stopped with the addition of HCl (25 µL, 1 M). This was followed by addition of the iodine-potassium iodide solution (100 µL). The sample and blank absorbances were read at 630 nm. The absorbance of the blank was subtracted from that of the sample and the α-amylase inhibitory activity was expressed as IC₅₀ values (µg/mL). α-glucosidase (from Saccharomyces cerevisiae, EC 3.2.1.20, Sigma) inhibitory activities of the extracts and essential oils of S. urmiensis were also evaluated according to a previously published method.^[21] In brief, 20 μL of enzyme solution was mixed with 120 μL of 100 mM potassium phosphate buffer (pH = 6.9) and 10 μL of the extract/EO/reference samples. The mixture was incubated at 37 °C for 15 min and then enzymatic reaction was initiated by adding 20 μL of 5 mM 4-nitrophenyl-α-D-glucopyranoside (pNPG). The mixture was incubated at 37 °C for another 15 min. Later, the reaction was stopped by the addition of 80 μL sodium carbonate solution (0.2 M). Finally, the absorbance of 4-nitrophenol released from pNPG was measured at 405 nm. The system without α-glucosidase was used as a blank for correcting the background absorbance. The increasing of absorbance was compared with that of the control (buffer instead of sample solution) to calculate the inhibitory activity. Acarbose was used as a positive control. Analyses were run in triplicate and the results were expressed as average ± standard error mean (SEM).

Inhibitory assays against cholinesterases

The acetylcholinesterase (Electric ell acetylcholinesterase, Type-VI-S, EC 3.1.1.7, Sigma) and butyrylcholinesterase (horse serum butyrylcholinesterase, EC 3.1.1.8, Sigma) inhibitory potential of the plant samples was evaluated using the Ellman method with some modification.^[22] Briefly, 50 µL of sample solutions was mixed with 125 µL 5, 5'-dithio-bis-(2-nitrobenzoic acid) and 25 µL enzyme solution in Tris–HCl buffer (pH 8.0) and incubated for 15 min at 25 °C. A sample of 25 µL of butyrylthiocholine chloride (BTCl)/acetylthiocholine iodide (ATCI) was added to initiate the reaction. A mixture of the reagents without enzymes was used as the blank sample. After a 10 min incubation, absorbance values were read at 405 nm. The enzyme inhibitory activities were expressed as IC₅₀ values (µg/mL). Galanthamine was used as the reference anticholinesterase drug. Results were expressed as mean ± SEM.

Results and discussion

Essential oils composition

The present work is the first comparative study of the chemical compositions of leaves (EO-L) and flowers (EO-F) of S. urmiensis (Table 1). The chemical structure of the major compounds is shown in Fig. 1. Yields of EO-L and EO-F were 0.2% and 0.3% v/w, respectively. Isolated oils were yellow viscose liquids with strong odours. In total, 23 compounds comprising 90.8% of the total oil were identified in EO-F, of which 6,10,14-trimethyl-2-pentadecanone (55.7%), 1,8-cineol (6.5%), and β-pinene (6.4%) were the major components. Moreover, 43 compounds were identified in EO-L, comprising 91.5% oil with ethyl linoleate (19%), methyl hexadecanoate (17%), and 6,10,14-trimethyl-2-pentadecanone (13%) as the most abundant constituents (Table 1). Generally, essential oils obtained from Salvia species are rich in mono and sesquiterpenoids, ^[23,24] while EO of S. urmiensis is rich in ketones and esters. These results are thoroughly different from the previous report on the essential oil of S. urmiensis.^[19] There is no information about the presence of S. urmiensis in Lorestan province (the previous report on EO of this plant). Moreover, reported volatile compounds in the previous paper (benzyl benzoate and its derivatives) are very rare in plant essential oils and more specifically in Salvia species. Hence, we think the author of that paper has made a mistake in taxonomic identification and presumably in determination of the chemical composition of the studied plant.

Table 1. Chemical composition of the essential oils from flowers and leaves of Salvia urmiensis.

	Percentage			RI	Identification
Compounds	Flowers	Leaves	RI^a	lit^b	method
3-Methyl-pentanol	2.4	1.9	838	833	MS
α-Pinene	3.1	1.5	939	932	RI, MS
Camphene	0.7	-	956	946	RI, MS
β-Pinene	6.4	0.2	984	974	RI, MS
Limonene	0.2	-	1033	1024	RI, MS
1,8-Cineol	6.5	1.1	1036	1026	RI, MS
2-Nonanal	-	0.5	1100	1097	RI, MS
n-Decanal	-	0.3	1202	1201	RI, MS
β-Cyclocitral	-	0.1	1225	1217	RI, MS
Pulegone	-	0.3	1244	1233	RI, MS
Bornyl acetate	-	0.2	1291	1287	RI, MS
α-Copaen	-	0.1	1397	1374	RI, MS
β-Bourbonene	-	1.2	1400	1387	RI, MS
α-Gurjunene	0.4	-	1426	1409	RI, MS
E-Caryophyllene	1.0	3.4	1436	1417	RI, MS
γ-Elemene	-	0.3	1443	1434	RI, MS
2,6,10-Trimethyl-Dodecane	4.0	1.3	1461	1450	MS
α-Humulene	-	0.2	1471	1452	RI, MS
Allo-aromadendrene	-	0.2	1479	1458	RI, MS
E-β-Ionone	0.2	0.8	1492	1487	RI, MS
n-Pentadecane	-	0.3	1497	1500	RI, MS
α-Muurolene	0.4	-	1512	1500	RI, MS
δ-Cadinene	1.0	0.2	1534	1522	RI, MS
Caryophyllene oxide	1.6	6.3	1605	1582	RI, MS
Humulene epoxide	-	0.2	1630	1608	RI, MS
Pentadecanone	0.6	0.7	1697	1697	RI, MS
Methyl tetradecanoate	-	1.3	1720	1716	RI, MS
12-Methy, methyl tetradecanoate	-	0.1	1791	1776	MS
Octadecane	0.6	0.8	1795	1800	RI, MS
9-Mctadecenoic acid (Z), methyl ester	-	0.5	1803	1806	MS
Methyl pentadecanoate	-	0.6	1819	1823	MS
6,10,14-Trimethyl-2-pentadecanone	55.7	13.0	1845	1846	RI, MS
14-Methyl, methyl pentadecanoate	-	0.1	1884	1886	MS
n-Nonadecane	0.7	0.7	1897	1900	RI, MS
9-Hexadecenoic acid (Z), methyl ester	-	0.6	1902	1907	MS
Methyl hexadecanoate	-	17.0	1925	1926	RI, MS
Hexadecanoic acid	1.4	2.6	1964	1968	RI, MS
Eicosane	0.6	0.8	1997	2000	RI, MS
14-Methyl, methyl hexadecanoate	0.2	0.2	2011	2015	MS
16-Octadecenoic acid, methyl ester	-	0.1	2031	2035	MS
Abietatriene	-	0.1	2042	2055	RI, MS
Methyl linoleate	-	7.5	2047	2061	RI, MS
Ethyl linoleate	-	19.0	2051	2065	RI, MS
Methyl octadecanoate	-	3.1	2061	2098	RI, MS
Heneicosane	0.5	0.3	2099	2100	RI, MS
Docosane	2.4	1.4	2199	2200	RI, MS
Tricosane	0.3	0.4	2298	2300	RI, MS
Monoterpene hydrocarbons	16.9	2.8
Sesquiterpene hydrocarbons	6.8	6.9
Oxygenated sesquiterpenes	1.6	6.5
Aliphatic ketones	56.5	14.5
Alkanes	5.1	4.8
Esters	0.2	50.1
Others	3.6	5.9
Total identified	90.8	91.5

^c Linear retention index on DB-5 column, experimentally determined using homologous series of C₆-C₂₄ alkanes. ^d Linear retention index taken from Adams (2007) and/or NIST 08 (2008).

Figure 1. Chemical structures of the major volatile compounds in the essential oils of S. urmiensis.

Enzyme inhibitory activity

DM is considered as one of the major global health problems of the 21st century. Today, the prevalence of DM is rising and it is estimated to increase significantly over future decades. The number of people affected by DM worldwide is about 400 million. At this point, many therapeutic strategies have been developed for this health problem and the key enzyme inhibitory theory is the most effective between the common strategies. α-amylase and α-glucosidase are the most important enzymes in the catabolism of carbohydrates and the inhibition of their activity has a vital role in decreasing the blood glucose level in DM patients.^[25] As shown in Table 2, the MeOH extract has the strongest α-amylase and α-glucosidase inhibitory activities (IC₅₀ = 24 and 8.3 µg/mL, respectively). There are several reports in the literature that show that phenolic compounds and flavonoids have strong inhibitory activity against α-amylase and α-glucosidase.^[26,27] The trend of anti-diabetic activity of S. urmiensis extracts is methanol> chloroform> n-hexane, which is matching with their total phenolics and total flavonoid contents.^[18] EOs were inactive in these assays at test concentrations (Table 2). The results indicate that esters, ketones, and alkane compounds in EOs composition could not be strong inhibitors of α-amylase and α-glucosidase. Comparison of our results with those in the literature shows that the anti-diabetic activity of S. urmiensis methanolic extract is higher than many species reported previously, ^[28,29] while EOs and other extracts of S. urmiensis showed weak enzyme inhibitory activities.

Table 2. Enzyme inhibitory activity of the extracts and essential oils of Salvia urmiensis. ^a

Samples	α-Amylase (IC50:µg/mL)	α-Glucosidase (IC50:µg/mL)	Acetylcholinesterase (IC50:µg/mL)	Butyrylcholinesterase (IC50:µg/mL)
Essential oil-Leaves	>1000	>1000	211.3 ± 10.5	422.0 ± 0.1
Essential oil-Flowers	>1000	>1000	44.6 ± 1.8	86.6 ± 4.1
n-hexane	145.3 ± 10.6	85.3 ± 4.8	366.3 ± 23.1	422.3 ± 30.4
Chloroform	97.3 ± 5.3	46.6 ± 2.3	186.6 ± 12.1	212.0 ± 15.5
Methanol	24.0 ± 1.3	8.3 ± 0.5	892.0 ± 25.4	>1000
Reference	33.3 ± 2.2	10.6 ± 0.8	20.3 ± 0.9	25.0 ± 1.1

^a Values expressed are mean ± SEM of three independent experiments.

Cholinesterases inhibition is a common strategy for the treatment of neurodegenerative diseases such as AD.^[30] Physostigmine, rivastigmine, galanthamine, and huperzine are common cholinesterase inhibitor drugs in the market but they have several negative effects on human health.^[31] Recently, remarkable studies have been performed regarding the acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity of extracts, fractions, and purified compounds from medicinal plants. The genus Salvia is considered a rich natural source of AChE and BChE inhibitors for the treatment of AD.^[32–34] In this direction, in the present work, the anti-AD potential of S. urmiensis based on its enzyme inhibitory activity was evaluated. EO-F showed the highest acetylcholinesterase and butyrylcholinesterase inhibitory potential (IC₅₀ = 44 and 86 µg/mL, respectively), followed by chloroform extract (IC₅₀ = 186 and 212 µg/mL, respectively). This is in agreement with the traditional uses of Salvia species in European folkloric medicine as memory enhancers.^[35] IC₅₀ values for acetylcholinesterase inhibitory activity of several EOs and extracts of S. nemorosa ranged from 223 to 488 µg/mL.^[6] Acetone extract of S. syriaca roots showed moderate activity with IC₅₀ of 499 µg/mL.^[31] EOs of S. fruticosa and S. officinalis showed anti-butyrylcholinesterase activity with 150 and 140 µg/mL, respectively.^[36] The n-hexane extract of S. leriifolia exhibited anticholinesterase activity with IC₅₀ values of 590 and 210 µg/mL, for AChE and BChE, respectively.^[37] In comparison to previous studies, the anticholinesterase activity of S. urmiensis was similar to or higher than other Salvia species reported in the literature. There is no information in the literature about cholinesterase inhibitory activities of major compounds present in EOs of S. urmiensis (ethyl linoleate, methyl hexadecanoate, and methyl linoleate in EO-L and 6,10,14-trimethyl-2-pentadecanone in E,O-F). Nevertheless, these esters and ketones could not be the compounds responsible for the observed activities because the inhibitory effects of essential oils could not be interpreted with their ester and/or ketone profile. However, the cholinesterase inhibitory activities of EOs agree with their α-pinene, β-pinene, and 1,8-cineol compositions. EO-F with higher inhibitory activity than EO-L has higher amounts of the mentioned monoterpenoids (16% versus 2.8%) which are well known for their cholinesterase-inhibitory effects.

Conclusıon

Salvia species have great potential to produce structurally and biologically interesting metabolites. This genus is known in European traditional medicine as a memory-enhancer herb. Moreover, DM is one of the important health problems in the world. In this direction, in the present study, the chemical composition of essential oils obtained from leaves and flowers of S. urmiensis together with their solvents extracts were evaluated against enzymes involved in AD and DM for the first time. The methanol extract exhibited remarkable anti-diabetic potential and the EO of flowers showed promising cholinesterase inhibitory activities. EOs analysis revealed that S. urmiensis is rich in aliphatic esters and ketones. Results showed that this species contains pharmacologically important natural agents. In addition, the anti-diabetic and neuroprotective properties of S. urmiensis indicated that this species has potential for possible uses as food supplements and pharmaceutical ingredients.

Acknowledgments

Financial support by the Shahid Beheshti University Research Council is gratefully acknowledged.