Salvia mirzayanii and Salvia hypoleuca Essential Oils: Chemical Composition, Alpha-Glucosidase Inhibitory, and Synergistic Effects of Selected Components

Diabetes mellitus (DM) is a chronic metabolic disease in which hyperglycemia occurs due to the lack of insulin production or insensitivity of the related receptors (1). According to the estimations, there will be 700 million diabetic individuals globally by 2045 (2). Basically, delaying postprandial hyperglycemia in the gastrointestinal canal is one of the substantial therapeutic approaches for treating DM, particularly type 2, that is achieved by decreasing glucose absorption through the inhibition of carbohydrate-hydrolyzing enzymes. Alpha-amylase and α-glucosidase are two primary enzymes located in the brush border of the intestinal villi and are responsible for the breakdown of carbohydrates. Alpha-amylase degrades long-chain carbohydrates; however, starch and disaccharides are converted to glucose by α-glucosidase, leading to an increase in plasma glucose levels and postprandial hyperglycemia. As α-glucosidase is an essential enzyme that catalyzes the final stage of carbohydrate digestion, its inhibitors have been at the center of attention. Acarbose, miglitol, and voglibose are substantial medications for decreasing blood sugar levels in diabetic patients that inhibit α-glucosidase inhibitors (3). However, they have caused gastrointestinal side effects, notably flatulence, diarrhea, abdominal pain, and pneumatosis intestinalis in patients with DM (4). In this respect, the design and development of synthetic (5-7) and natural α-glucosidase inhibitors (8, 9) have received great attention.

Hyperglycemia is associated with various metabolic disorders and oxidative stress. An increase in the level of free radicals in the long term brings up some concerns, such as cancer, cardiovascular, and neurological disorders. Additionally, oxidative stress itself has been linked to the development and progression of diabetes (10). As a result, it is critical to find compounds possessing both antioxidant properties and hypoglycemic effects.

Medicinal herbs have been used traditionally to treat diabetes, and researchers are currently focusing on the bioactive compounds derived from plant extracts or their essential oils (EOs) to develop more potent and safer medications. On the other hand, EOs are high in a variety of biochemicals that have a wide range of activities, such as antioxidant properties (11).

Salvia, the largest genus of the Lamiaceae family, possesses over 900 species of herbaceous aromatic plants and can be found all over the world, especially in Asia. There are 60 species of Salvia in Iran, and among them, 17 species are endemic. Salvia species have been used to treat a variety of diseases, such as colds, aches, infections, bronchitis, Alzheimer’s disease, and hemorrhage (12, 13). Furthermore, extracts and EOs from various Salvia spp. have received great attention in modern medicine. The Salvia sclarea EOs from two different regions of Lebanon were tested for acute and subchronic anti-diabetic effects in alloxan-induced diabetic rats and indicated that the plant collected from Beirut reduced blood glucose levels by 51.70% and 52.00% in acute and subchronic tests (at the concentration of 200 mg/kg), respectively (14). In another study, the S. officinalis EO in the flowering stage was observed to have an α-glucosidase inhibitory activity (half-maximal inhibitory concentration [IC₅₀] = 22.24 ± 0.07 µg/mL, compared to acarbose as a standard [IC₅₀ = 12.31 ± 0.05 µg/mL]) (15).

Salvia mirzayanii (known as “Moor-e-Talkh” in Persian) is a plant native to the southern parts of Iran. Salvia hypoleuca (known as “Boland Maryam-goli” in Persian) is a tall perennial plant that is widespread in Iran, in the northern and central provinces, particularly in Alborz, Tehran, and Mazandaran provinces. They have been used for years in the treatment of various diseases by local individuals living in the aforementioned regions.

To the best of our knowledge, the α-glucosidase inhibitory activity of S. mirzayanii and S. hypoleuca EOs has not been reported in the literature, and this study aimed to identify the components of the EOs of the plants and evaluate their α-glucosidase inhibitory activity. An attempt was also made to study any synergistic or antagonistic interactions of selected main bioactive compounds on this enzyme.

In the present study, S. mirzayanii EO mainly contained monoterpenes (50.39%); however, sesquiterpenes accounted for 39.91% of the constituents. It also consisted of linalool (9.97%), α-terpineol (8.33%), α-terpinyl acetate (8.15%), 1,8-cineole (5.2%), and thymol (4.36%). In the S. hypoleuca EO, sesquiterpenes and monoterpenes accounted for 68.4% and 26.83%, respectively. Among different classes of compounds, sesquiterpene hydrocarbons were observed to be the most abundant components (45.76%). Moreover, it mainly included trans-β-caryophyllene (14.12%), caryophyllene oxide (12.17%), α-pinene (9.04%), germacrene D (8.69%), and β-pinene (8.47%). Comparing the EOs components (Figure 1) revealed that the amounts of monoterpenes and phenolic compounds in S. mirzayanii are higher than those of S. hypoleuca; however, it contained higher amounts of sesquiterpene hydrocarbons. It merits mentioning that sesquiterpene hydrocarbons constituted the larger portion of sesquiterpenes in both EOs (Figure 1).

Figure 1.

Classification of Saliva mirzayanii and S. hypoleuca EOs components

As reported in Table 5, the components of two EOs were compared to those documented in the literature. In a study reported by Hasheminya and Dehghannya, EO constituents of S. mirzayanii collected from Kerman, Iran, were identified, indicating the presence of monoterpene hydrocarbons (5.48%), and the main compounds also belonged to the oxygenated monoterpenes (34.34%) (21). In another study, the EO components of S. mirzayanii from Lamerd, Fars province, Iran, were classified as sesquiterpene hydrocarbons (26.10%), oxygenated monoterpenes (18.40%), oxygenated sesquiterpenes (11.00%), and monoterpene hydrocarbons (1.00%) (22). Furthermore, the results from the analysis of S. mirzayanii EO collected from Jahrom in Fars province, Iran, are in line with the data of the current study as oxygenated monoterpenes (56.50%) and monoterpene hydrocarbons (7.00%) were observed to be the highest and lowest abundant components (23).

According to the study of Nickavar et al., sesquiterpene hydrocarbons (44.90%) were identified as the most abundant components in the S. hypoleuca EO, collected from Haraz, Tehran province, Iran, in comparison to the other compounds (oxygenated sesquiterpenes [28.20%], hydrocarbon monoterpenes [15.50%], and oxygenated monoterpenes [3.40%]) (24). The EO components of S. hypoleuca were collected from Mazandaran province, Iran, in three different vegetative, flowering, and fruiting stages. More specifically, in the flowering stage, sesquiterpene hydrocarbons and oxygenated sesquiterpenes were the most abundant compounds, with 32.90% and 28.80%, respectively (25). In another study, the analysis of EOs components of 21 populations of S. hypoleuca collected from different sites of Alborz, Tehran, and Mazandaran provinces in Iran demonstrated that sesquiterpene hydrocarbons constituted 39.70%; nevertheless, oxygenated monoterpenes comprised 3.00% of the EO compounds of the sample collected from Alborz province, Chalos Road, Dizin, that are in alignment with two previous studies and the present study (26).

It is critical to identify efficient compounds in inducing desired biological activity. It is important to answer the question of whether major compounds are responsible for the biological activity (27) or whether a synergistic effect occurs. In this regard, the synergistic interactions of three major constituents of S. mirzayanii and S. hypoleuca EOs were investigated, as reported in Table 6. These combinations were provided based on their natural proportions in Salvia spp. EOs. The mixture of linalool, α-terpineol, and 1,8-cineole (40:35:25) exhibited α-glucosidase inhibitory activity with percentage inhibition of 72.5 ± 0.8; nonetheless, the mixture of caryophyllene oxide, α-pinene, and β-pinene (50:25:25) demonstrated a weaker inhibition (14.0 ± 1.8%).

The combination effect of components can be characterized by the combination index (CI), which is defined as:

Where C_x,n is the concentration of the compound_n alone that inhibits the enzyme (x%). C_n is the concentration of the compound_n in combination with other compounds which inhibits the enzyme (x%). When the CI is equal to, less than, or greater than 1, the combination effect would be additive, synergistic, or antagonistic, respectively (28). According to the calculated CI values, the constituents of the first mixture seem to interact with one another synergistically (CI < 1); in the meantime, the components of the second mixture have an antagonistic impact on one another (CI > 1).

The S. mirzayanii EO was also observed to have higher antioxidant activity than the S. hypoleuca EO. To the best of our knowledge, no study reported the DPPH radical scavenging capacity of S. mirzayanii EO. However, in a study conducted in 2016, the antioxidant activity of S. hypoleuca EO at different growth stages was assessed by DPPH assay. The EO at the flowering stage showed the best radical scavenging capacity with IC₅₀ = 25 mg/mL among the two other stages (positive control: Butylated hydroxytoluene [BHT] with an IC₅₀ value of 0.4 mg/mL) (25). Various compounds, such as 1,8-cineole, α-pinene, camphor (29), carvacrol, and thymol (30), were reported to have antioxidant activity. Because the S. mirzayanii EO contains substantially more of the compounds than that of S. hypoleuca, it has shown greater antioxidant activity. Additionally, the results of the present study showed that S. mirzayanii EO has a higher phenolic content (TPC) (78.3 ± 1.3 mg GAE/g EO) than S. hypoleuca EO (49.4 ± 1.1 mg GAE/g EO) (Table 4). Total phenolic content and the antioxidant activity based on the DPPH assay were observed to be directly related, and the higher TPC of S. mirzayanii EO resulted in a lower DPPH IC₅₀ value that can be explained by the fact that phenolic compounds neutralize free radicals by transferring the hydrogen of their hydroxyl group to them (31).