Background

Plants and their extracts, essential oils (EOs), and secondary metabolites have been widely used in the pharmaceutical, food, cosmetic, and perfume industries due to valuable medicinal, flavoring, and preserving properties. Among them, Salvia species have attracted a lot of attention [1]; for example, Salvia officinalis L. (sage) is used for food preservation, particularly meat and cheese [2]. In this respect, Salvia sclarea L., Salvia hispanica L., and Salvia divinorum Epling & Játiva are cultivated in many parts of the world due to their commercial interests [3, 4].

Salvia is one of the most important and largest genera of the Lamiaceae family containing approximately 900 distinct species worldwide (tropical, temperate, and arctic regions) [5] and widely distributed in Iran. Out of 58 different species existing in Iran, 17 are endemic [6]. The extracts and EOs of Salvia have depicted a wide range of biological activities including antibacterial, carminative, diuretic, spasmolytic, anti-inflammatory, antioxidant, anti-cancer, anti-diabetic, anxiolytic, and sedative properties. Also, the genus has long been considered in folk medicine to deal with different ailments such as epilepsy, cancer, malaria, bronchitis, tuberculosis, hepatitis, anti-diabetic, dementia, and dysmenorrhea [6-10]. Furthermore, Salvia plants have been extensively used for different therapeutic purposes in Persian medicine. For example, S. officinalis has been used as a diuretic, carminative, wound healing, and asthma-treating agent [11-13]. Today, the sale and production of sage have resulted in significant income benefits for several Asian nations due to its several applications in aromatherapy and promoting general health as well as food industry [14].

Diabetes Mellitus (DM) is an endocrine disease which can impair carbohydrate metabolism due to insulin deficiency or insulin resistance [15]. The global diabetes prevalence is worrying, and it is estimated that over 700 million people will suffer from the disease, by the year 2045 [16]. Sulfonylureas, biguanides, and other drugs possessing various mechanisms of action are used to treat type 2 diabetes mellitus (T2DM), which is the most common type of DM. Delaying the uptake of glucose by inhibiting α-glucosidase, has been an efficient therapeutic tool for the treatment of T2DM [17]. α-Glucosidase is the key enzyme which is located in the brush border of the intestine, catalyses dietary carbohydrates to glucose monomers and prepares them for absorption. Thus, post-prandial blood glucose can be controlled by inhibiting the enzyme [3]. However, continuous use of approved drugs causes adverse effects such as hypoglycemia, nausea, and dizziness [18, 19]. Recently, many studies have focused on plant-derived natural products which are safer and more affordable than the common medications. Also, various plants have been used as anti-diabetic agents in folk medicine, their EOs and extracts can be regarded as extremely valuable natural resources for the treatment of DM [20, 21]. In an in vivo study, S. officinalis EO exhibited anti-diabetic properties by reducing blood glucose up to 60% and elevating stored glycogen in the liver up to 43.7% [22]. Assaggaf et al. evaluated the chemical composition and α-glucosidase inhibitory activity of S. officinalis EO in the full flowering stage which inhibited the enzyme with an IC50 = 22.24 μg/mL, compared to acarbose as a standard (IC50 = 12.31 μg/mL) [23].

Salvia species have also been linked to neuroprotective characteristics. Generally, the small size and lipophilicity of EOs constituents allow them to easily pass the blood–brain barrier and therefore could be suggested as a potential strategy for the treatment of neurodegenerative disease [24]. Nowadays, approximately 50 million people suffer from Alzheimer’s disease (AD) worldwide and a three-fold increase in the incidence of the disease is estimated by 2050 [25]. The cholinesterases (ChEs) including AChE and BChE are responsible for the hydrolysis of acetylcholine (ACh) in the brain. Thus, reduction of the level of ACh can be terminated by the inhibition of ChEs. Currently, FDA-approved ChE inhibitors such as donepezil, rivastigmine, and galantamine are important medicines for controlling the symptoms of AD. The EOs of medicinal plants are rich sources of valuable phytochemicals which have been widely considered for the treatment of various neurodegenerative diseases such as AD [26]. In this regard, based on a clinical study, the EOs of Salvia lavandulifolia Vahl. and S. officinalis significantly improved memory performance and thereby, could be considered in aromatherapy [27]. Furthermore, S. lavandulifolia EO was found to be a selective AChE inhibitor with an IC50 value of 3 µg/mL while inhibiting BChE by 22% at 0.5 mg/mL [28]. Also, S. lavandulifolia EO reduced the activity of AChE in the striatum of rats, however, not in their hippocampus or cortex [29]. Moreover, in vitro evaluation of Salvia potentillifolia Boiss. & Heldr. ex Benth. EO indicated inhibitory activity against the BChE by 65.7% inhibition at the concentration of 200 µg/mL and the corresponding activity on AChE was obtained as 21.9% inhibition at the same concentration, compared with galantamine as a reference (75.5% and 81.4% inhibition at 200 μM, respectively) [30].

Unlike synthetic substances, which are often based on a single active component, EOs include a variety of compounds that interact synergistically or additively with each other to either reduce the risk of drug resistance or boost the effectiveness of the treatment [31]. Several studies have reported the chemical composition of different species of Salvia genus EOs and α-pinene, β-pinene, germacrene D, spathulenol, bicyclogermacrene, 1,8-cineole, camphor, borneol, α-thujone and β-thujone, thymol, caryophyllene, and caryophyllene oxide have been commonly determined as the most prominent components [6, 32].

The scientific research of numerous plant species to find new natural bioactive agents is a time-consuming and resource-intensive procedure. As a result, researchers are now receiving assistance in their quest to identify active pharmaceutical ingredients in medicinal plants, which were previously used to treat illnesses in a simple and cost-effective manner. In this study, constituents of EOs of 14 plants from 12 Iranian native Salvia species were investigated, and they were evaluated for their α-glucosidase and ChE inhibitory activity to develop dual natural anti-diabetic and anti-AD agents as AD has been considered as type 3 diabetes and the role of insulin resistance in inducing impaired brain glucose metabolism, neurodegeneration, and cognitive impairment has been comprehensively discussed in the literature [33].

Methods

Chemicals

α-Glucosidase (from Saccharomyces cerevisiae; EC3.2.1.20, 20 U/mg), acetylcholinesterase (AChE, E.C. 3.1.1.7, Type V-S, lyophilized powder, from electric eel, 1000 unit), butyrylcholinesterase (BChE, E.C. 3.1.1.8, from equine serum), p-nitrophenyl α-D-glucopyranoside (p-NPG), 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB), acetylthiocholine iodide (ATCI), and butyrylthiocholine iodide (BTCI) were provided from Sigma-Aldrich.

Plant material

Aerial parts of 14 native Iranian medicinal plants from 12 distinct species of the Salvia genus were collected from different parts of Iran during the flowering stage. After verification by the botanist Sedighe Khademian at Shiraz University of Medical Sciences, voucher specimens were deposited in the herbarium of the Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran (Table 1, Fig. 1). Comparing the identified herbarium and flora specimens with the identified plant species enabled for reliable plant identification. Experimental research and field studies on plants (either cultivated or wild), including the collection of plant material, complies with relevant institutional, national, and international guidelines and legislation.

Table 1 EOs obtained from aerial parts of some Salvia spp., collected from different parts of Iran
Fig. 1
figure 1

Geographical distribution of  14 studied plants from Salvia genus

Extraction of the essential oils

The aerial parts of plants were collected and dried at room temperature away from direct sunlight. To isolate the corresponding EO, each milled dried plant (100 g) was hydro-distilled for 4 h using a Clevenger-type apparatus according to European Pharmacopoeia (2020). Anhydrous sodium sulfate was added to the isolated EO to remove the water. All EOs were stored in a dark sealed vial at 4 °C for further experiments. The yield of extraction was reported as w/w%.

GC–MS analysis

GC–MS analysis was performed on a 7890B Agilent gas chromatograph including a DB-5 column (60 cm, 0.25 µ) and a 5977A Agilent mass spectrometer. 1 μL of diluted samples (with ethyl acetate) was injected into the injection site. The temperature program was scheduled as follows: the initial temperature of the oven was 40˚C (held for 7 min) and programmed to reach 140˚C with a rate of 10 ˚C /min, eventually reached 250˚C with a rate of 3˚C /min and held for 7 min at this temperature. Helium with 99.99% purity was utilized as a carrier gas (flow rate: 1 mL/min). Also, the ionization voltage of the detector was set at 70 eV. To determine the components, normal alkanes (C7–C21) were injected in the same manner to compare calculated retention indices with those in authentic references. For more accurate identification, the mass spectra of each compound were reconciled with the NIST database [34] and Adams's book [35].

α-Glucosidase inhibition assay

The α-glucosidase inhibitory activity of EOs was evaluated based on the previously described method [20] using α-glucosidase (from Saccharomyces cerevisiae).

Cholinesterase inhibition assay

Inhibitory activity against AChE (E.C. 3.1.1.7, Type V-S, lyophilized powder, from electric eel, 1000 unit) and BChE (E.C. 3.1.1.8, from equine serum) was performed using modified Ellman's method [36].

Statistical analysis

The GraphPad Prism software was used to carry out statistical analysis. Data comparisons were performed by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons as the post-hoc test. P values < 0.05 were considered statistically significant.

Results

Yield of isolation of essential oils

The yield of isolation of EOs from each species was reported in Table 1. They were obtained in the range of 0.06–0.92 w/w%. The lowest and highest values were related to S. reuterana and S. mirzayanii (Jahrom region), respectively.

Chemical composition of essential oils

According to the GC–MS analysis, 139 components were identified in the isolated EOs from 14 plants as recorded in Table 2. They were categorized into monoterpenes, sesquiterpenes, oxygenated diterpenes, carbonyl compounds, alcohols, acids, esters, alkanes, and phenolic compounds. As reported in Table 2, sesquiterpenes were the major compounds in the series of Salvia spp. EOs. However, in the case of S. multicaulis, monoterpenes were the most abundant components.

Table 2 Chemical composition of EOs (%) of some native Iranian Salvia spp.a

The GC–MS analysis also revealed that among all EOs components (Table 2), sclareol oxide (24.56%), and trans-β-caryophyllene (24.12%) (entries 137 and 66, respectively) were the highest in value. It is worth mentioning that caryophyllene oxide was detected as the main compound in 7 Salvia spp. S. sharifii and S. sclarea EOs contained the corresponding compound as 21.11 and 22.26%, respectively, which were more significant than the others. Moreover, linalool, α-terpineol, trans-β-caryophyllene, spathulenol, and caryophyllene oxide were ubiquitous in all isolated EOs.

Biological activity of essential oils

The percentage inhibition values for α-glucosidase and ChE inhibitory activity of the Salvia EOs were reported in Table 3.

Table 3 Enzyme inhibitory activity of Salvia spp. EOs at the concentration of 500 μg/mLa

α-Glucosidase inhibitory activity

It was perceived that 8 EOs expressed a notable inhibitory effect toward α-glucosidase at 500 μg/mL, compared with acarbose. More specifically, S. spinosa EO exhibited the strongest activity (90.5% inhibition) and EOs of S. virgata and S. reuterana were also able to block the enzyme with high percentage inhibition (89.7% inhibition). Additionally, the EOs of S. mirzayanii collected from Darab and S. hypoleuca showed a weak α-glucosidase inhibitory activity with percentage inhibition values of 25.5%, and 22.7%, respectively, while S. mirzayanii from Jahrom displayed no activity.

Cholinesterase inhibitory activity

In vitro ChE inhibitory assay of the 14 investigated EOs indicated moderate to remarkable activity toward both AChE and BChE at 500 μg/mL, however, they were less active than donepezil (Table 3).

S. mirzayanii collected from Shiraz revealed the highest inhibitory effect on AChE and BChE (40.6% and 72.68%, respectively), and S. mirzayanii collected from Jahrom (30.81% and 64.76%) and Darab (23.86% and 63.02%), as well as S. syriaca (15.8% and 52.1%), showed good activity. S. verticillata and S. multicaulis had a negligible effect toward AChE (0.5% and 1.1% respectively) and S. palaestina and S. hypoleuca had no significant inhibitory effect on BChE (7% and 11.3%, respectively). Furthermore, S. sharifii (42%), S. santolinifolia (44.1%), and S. multicaulis (44.1%) were found to be moderate inhibitors of BChE.

Finally, the selectivity index (SI) of the tested EOs in the inhibition of BChE over AChE showed that S. multicaulis EO was the most selective BChE inhibitor (SI = 88.2). Meanwhile, the three populations of S. mirzayanii EOs exhibited no noticeable selectivity in the inhibition of ChEs.

Discussion

GC–MS analysis indicated that monoterpenes and sesquiterpenes were the predominant components of EOs. Even though significant components were similar in the isolated EOs, some constituents such as viridiflorene, trans-γ-bisabolene, β-copaen-4-α-ol, and valeranone were detected only in the specific species. However, caryophyllene oxide, spathulenol, linalool, α-terpineol, and trans-β-caryophyllene were detected in all EOs. Additionally, caryophyllene oxide (0.38–22.26%), trans-β-caryophyllene (0.72–24.12%), spathulenol (0.94–17.62%), germacrene D (0–16.33%), and δ-cadinene (0–11.72%) were generally found to be the main components in this series of EOs. Comparing the major components of the studied Salvia spp. with those reported in the literature (Table 4), revealed the significant variation in the EOs components of different Salvia species. Genetic composition, environmental and climate factors, and developmental stages are important reasons that may contribute to this diversity [4, 37].

Table 4 Comparison of major components of studied Salvia spp. with those reported in the literature

According to the literature review, α-glucosidase inhibitory activity of S. syriaca, S. spinosa, S. virgata, and S. sclarea EOs have been previously recorded. Bahadori et al. reported that the α-glucosidase inhibitory activity of S. syriaca EO (IC50 = 1.2 mg/mL) was more potent than acarbose (IC50 = 9.6 mg/mL) [58] which is in contrary with our findings. In another study, the α-glucosidase inhibitory activity of S. spinosa EO was evaluated, which demonstrated moderate inhibitory activity with an IC50 value of 43.79 µg/mL, compared with acarbose (IC50 = 17.1 µg/mL) [49]. On the contrary, another study indicated that neither S. virgata nor S. sclarea EOs were active toward α-glucosidase [7].

EOs possessing high amounts of p-cymene, borneol, γ-terpinene, and phytol have shown good inhibitory effect on α-glucosidase. For example, Carum carvi L. and Coriandrum sativum L. EOs containing high amounts of these compounds showed very potent α-glucosidase inhibitory activity, compared with acarbose [59] which is in alignment with our results. Although no study was reported on the inhibitory activity of β-eudesmol, α-humulene, and camphene, which are present in high concentration in the S. santolinifolia, S. verticillata and S. multicaulis EOs, respectively; they may be effective in inducing α-glucosidase inhibitory activity.

AChE accounts for roughly 80% of cholinesterase activity in the normal brain, with BChE making up the remaining 20%. However, in severe AD, AChE activity may decline to 55–67% of baseline levels in particular brain regions, whereas BChE activity rises. Also, BChE may contribute to the accumulation of β-amyloid plaques that occur in the early phases of AD progression [60]. Therefore, nowadays there is a considerable deal of interest in discovering compounds that can precisely inhibit the BChE.

To the best of our knowledge, cholinesterase inhibitory activity of the S. sharifii, S. santolinifolia, S. reuterana, S. spinosa, S. palaestina, S. virgata, S. hypoleuca, and S. mirzayanii EOs are reported for the first time and those of S. sclarea, S. verticillata, S. multicaulis, and S. syriaca EOs were previously investigated [58, 61-63].

Orhan et al. [63] studied the BChE inhibitory activity of two populations of cultivated S. sclarea which were exposed to different fertilizers. The EOs of plants treated with organic and chemical fertilizers were able to inhibit the enzyme with the percentage inhibition of 76.0% and 45.1%, respectively at the concentration of 1 mg/mL. However, S. sclarea EO exhibited 32.8% inhibition toward BChE at the concentration of 500 μg/mL, in our study. On the other hand, only the plant EO treated with chemical fertilizers were able to inhibit the AChE (11.6%, at 1 mg/mL), while in our study, S. sclarea EO inhibited this enzyme less than 10% at 500 μg/mL.

Kunduhoglu et al. reported the cholinesterase inhibitory activity of S. verticillata and S. wiedemannii EOs. In agreement with our results, S. verticillata EO had a weak inhibitory effect on the ChEs (20.4% and 1.8% against AChE and BChE, respectively), however, inconsistent with our results, the EO inhibited the AChE more strongly than the BChE [62].

S. syriaca EO demonstrated AChE and BChE inhibitory activity stronger than the reference (galantamine) [58], while compared to other understudied EOs in our survey, S. syriaca EO showed significant inhibition on the ChEs, but they couldn't exceed donepezil as the standard.

As reported by Akdeniz et al., S. multicaulis EO was able to inhibit AChE and BChE by 25.6% and 71.2%, respectively, at the concentration of 100 μg/mL [61], but the EO inhibited BChE by 44.1% with no inhibitory activity on AChE, in our study (at 500 μg/mL).

According to the study of Loizzo et al. on the EO of S. leriifolia, α-pinene and 1,8-cineole showed BChE inhibitory activity with IC50 values of 0.87 and 0.93 mM, respectively [64]. Also, good AChE inhibitory activity has been reported for α-pinene, however, 1,8-cineole has not been active [63]. Although these results were in good agreement with ours in some cases, the α-pinene content was not found to be important for inducing desired AChE inhibitory activity in isolated EOs.

According to the Chowdhury and Kumar report, α-terpinyl acetate was introduced as a natural monoterpenoid with potent ChE inhibitory activity (IC50 values of 54.7 and 47.5 µM against AChE and BChE, compared with donepezil with IC50 values of 0.15 and 5.8 µM, respectively) [65]. These results were also supported by ours indicating that S. mirzayanii and S. syriaca EOs have α-terpinyl acetate in higher amounts than other EOs, which induced higher ChEs inhibitory properties particularly more potent BChE inhibitory activity.

Orhan et al. showed that geraniol inhibited the BChE by 55.4% over 15.3% for AChE [63]. However, the amount of geraniol was not found to be a remarkable factor associated with the ChE inhibitory activity, in our investigations.

It seems that the presence of δ-cadinene is also important for inducing BChE inhibitory activity as S. mirzayanii and S. syriaca EOs containing high levels of that compound, showed good activity against BChE. Also, the amount of mesitylene as one of the main components of S. syriaca EO (9.13%) was found to be important for higher BChE inhibitory activity. However, further study is needed to determine whether these compounds are responsible for BChE inhibitory property.

On the other hand, no correlation was detected between the ChE inhibitory activity of the EOs and limonene, trans-β-caryophyllene, spathulenol, caryophyllene oxide, and linalool. Bonesi et al. studied the ChE inhibitory activity of Cordia gilletii De Wild. EO suggesting that trans-β-caryophyllene is playing an important role in BChE inhibitory activity [66]. Moreover, Sadaoui et al. suggested limonene as a strong BChE inhibitor (IC50 values of 51.6 and 66.7 µg/mL against AChE and BChE, respectively) [67]. However, the amount of these compounds was not found to be effective on the desired ChE inhibitory activity, in this study. As EOs of plants are complex mixtures, their inhibitory effects depend on the synergistic or antagonistic interactions.

According to the literature, AChE and BChE have approximately 65% amino acid sequence identity [68]. However, the substrate selectivity and sensitivity to the inhibitors of these enzymes, which are encoded by different genes, clearly vary. The primary distinction between the AChE and BChE relates to the acyl bonds and peripheral anionic sites where the substrate is linked to the enzyme. AChE has two aromatic amino acids, Phe295 and Phe297 in the acyl binding site whereas these amino acids have been replaced by linear ones, Leu286 and Val288 in the BChE [69]. The structural difference between these two enzyme active sites can explain why various compounds have different inhibitory activity. As stated earlier, AChE plays a greater role than BChE in the normal brain. However, in severe AD, the ratio between these two enzymes rises from 0.5 to 11 as the BChE effect grows [60]. Therefore, it is crucial to find a compound that could more efficiently inhibit the BChE. The acquired SI (selectivity index) from this study indicated that all the investigated EOs were able to inhibit the BChE more than the AChE, which makes them remarkable in treating AD in the advanced stages. Herein, the highest selectivity for BChE over AChE was obtained by S. multicaulis EO possessing high amounts of trans-β-caryophyllene (19.02%), borneol (12.52%), α-pinene (9.43%), and carvacrol (5.66%).

Comparing wild plants with cultivated ones, demonstrated that cultivated counterparts offer numerous advantages to the pharmaceutical industry, including fewer chemical changes, a more manageable supply chain, minimized batch variations, and stable raw material prices, albeit still higher than those for wild plants [70]. Because of factors such as changing climate conditions and depleting natural resources, traditional methods of growing aromatic plants do not always produce EOs with the appropriate quantity and quality of secondary metabolites. As a result, it is possible to optimize plant growth, EO efficiency, and EO constituents for cultivated herbs by using methods such as fertilizer or programmed temperature conditions and water supply.

In this work, the EO of S. mirzayanii cultivated in Shiraz, showed the strongest activity toward ChEs while the S. spinosa EO was the most potent inhibitor of α-glucosidase. It seems that they can be a potential and guaranteed source for the industrial production of new medicinal agents to control T2DM and AD.

Conclusions

This study aimed to find natural and safe resources for the treatment of two common diseases; T2DM and AD, as many people with Type 2 diabetes have shown a higher risk of developing AD and anti-diabetic agents have been recently found to be active in the treatment of AD. Evaluation of 14 Salvia species EOs by GC–MS led to the identification of 139 compounds. The S. spinosa EO showed high inhibitory activity toward α-glucosidase, while three S. mirzayanii EOs exhibited the strongest inhibitory effects on ChEs. Plants in the genus Salvia, especially S. mirzayanii and S. spinosa which were cultivated in Shiraz, can be considered as natural resources for industrial production of important supplements to be effective as a treatment for AD and T2DM. However, it should be mentioned that further studies are required to determine the responsible bioactive components for desired biological activities. Also, the toxicology and bioavailability profile of EOs are in high demand.