Metabolite Profiling, Antioxidant and Key Enzymes Linked to Hyperglycemia Inhibitory Activities of Satureja hispidula: An Underexplored Species from Algeria

In the present study, two extracts from the aerial parts of the endemic species Satureja hispidula were analyzed for the first time by ultra-high-performance liquid chromatography coupled with a diode array detector and an electrospray mass spectrometer (UHPLC-DAD-ESI/MS) method in order to identify and quantify their phenolic compounds. These extracts’ antioxidant, α-glucosidase and α-amylase inhibitory activities were also evaluated. UHPLC-DAD-ESI/MS allowed the identification of 28 and 20 compounds in the ethanolic and aqueous extracts, respectively; among them, 5-O-caffeoylquinic acid was the most abundant in both extracts. The biological assay results indicate that the species S. hispidula, besides its high antioxidant power, is also potentially useful for inhibiting the α-glucosidase enzyme. In both antioxidant and α-glucosidase inhibitory assays, the aqueous extract exhibited the most promising results, significantly better than the standards used as positive controls.


Introduction
Diabetes mellitus is a metabolic disorder characterized by high blood glucose levels and insufficient insulin production by the pancreas. Numerous studies have shown that free radicals and other oxidants have been substantially recognized to play a significant role in the development and complication of certain pathological disorders, including diabetes mellitus [1]. On the other hand, phenolic compounds have strong antioxidant properties and demonstrate promising potential in managing type 2 diabetes. Several antidiabetic medications are in use, but more efficient and safer agents are still needed. This explains the extensive research to find new active agents, particularly those that block important enzymes such as α-glucosidase and α-amylase.
Some compounds derived from plants exhibit this function, and the Algerian flora is a crucial source for researchers looking for new molecules. Among the Algerian species is Satureja hispidula Briq. (Lamiaceae), traditionally named summer savory and considered a medicinal plant for which several uses were reported [2], although not scientifically confirmed. Although its use in traditional Algerian medicine is not reported [3,4], there is information concerning S. hispidula aerial parts used in Turkey and Iran to treat several illnesses, including hyperglycemia [5]. Interesting biological properties were reported, mainly for S. hispidula essential oil [6], but considering these biological properties and the ones also established for other Satureja species [5,7]. S. hispidula is still considered an underexplored species. Moreover, its antihyperglycemic activity needs to be established. Thus, this study was conducted to assess for the first time the phytochemical composition as well as the antioxidant and α-glucosidase and α-amylase inhibitory properties of the aqueous and ethanolic extracts from Satureja hispidula growing in Algeria, for which no prior phytochemical and biological data have been described. Table 1 shows the values obtained for the total content of phenolic compounds and flavonoids. According to the findings, the plant contains significant amounts of both phenolic and flavonoid contents. The total phenolic content (TPC) of S. hispidula aqueous extract was reported to be 131.24 ± 0.81 mg GAE/g extract. TPC levels were substantially greater than in the ethanol extract (85.7 ± 0.46 GAE/g extract). However, in terms of total flavonoid content (TFC), no significant difference was found between the ethanol extract (22.9 ± 0.21 mg QE/g of extract) and the aqueous (22.5 ± 1.55 mg QE/g extract) extract.

UHPLC-DAD-ESI/MS Characterization of S. hispidula Extracts
The phenolic content of S. Hispidula ethanolic and aqueous extracts was analyzed using the UHPLC-ESI-MS/MS technique ( Figure 1). As stated in Table 2, a total of 27 and 17 phenolic compounds were detected for the first time in the ethanol and aqueous extracts, respectively. Overall, both chromatograms were characterized by the presence of several phenolic acids and flavonoids tentatively identified based on their fragmentation patterns and the data reported in Table 2. Even though UHPLC-ESI-MS/MS analysis of the ethanolic and aqueous extracts of S. hispidula revealed that their phenolic composition was quite different, the major compound in both cases was 5-O-caffeoylquinic acid (5-O-CQA) ( Figure 1; Table 2).   Seven phenolic acid derivatives (peaks 1, 3, 5, 6, 9, 17, and 21) were identified in both extracts. Based on their fragmentation patterns, compared with standards injected in the same conditions and data in the literature [8][9][10], these compounds were assigned to 3-O-CQA, 5-O-CQA, 4-O-CQA, caffeic acid, 1-O-CQA, 4,5-O-CQA and rosmarinic acid. Except for caffeic acid, the other derivatives were found in high quantities, especially in the aqueous extract (Table 2).
Other compounds were also present in the ethanolic extract, albeit in lower concentrations, namely oxodihydroxy octadecenoic acid, trihydroxyoctadecenoic acid, and dihydroxyoctadecanoic acid ( Table 2).
Although the extracts were quite different, several compounds occurred in both extracts, but in different amounts ( Table 2). It should be noted that, compared to the ethanolic extract, the aqueous extract was particularly rich in caffeoylquinic acids, occurring at 67.13 and 27.57 µg/mg of extract, respectively. Furthermore, the aqueous extract also showed higher flavonoid glycoside content (46.92 µg/mg of extract) than the ethanolic extract (30.28 µg/mg of extract).

Antioxidant Activity
The antioxidant potential of both ethanolic and aqueous extracts was estimated through three different antioxidant assays (DPPH • , ABTS •+ and FRAP). The results are shown in Table 3. The aqueous extract showed higher DPPH and ABTS radical scavenging activities compared to the ethanolic extract. The IC 50 of DPPH and ABTS of the aqueous extract were 2.43 ± 0.11 and 2.16 ± 0.1 µg/mL, respectively, and were more than 1.5 times greater than that of the standard ascorbic acid used as control (IC 50 = 3.86 ± 0.23 and 4.01 ± 0.09 µg/mL in DPPH and ABTS assays, respectively). Although its activity was lower than that of the aqueous extract, the ethanolic extract showed very good antioxidant activity, as revealed by the DPPH and ABTS tests with IC 50 values of 5.67 ± 0.07 and 5.95 ± 0.82 µg/mL, respectively. On the other hand, the same tendency was also observed in the FRAP assay, in which the aqueous extract (A 0.5 = 5.82 ± 0.17 µg/mL) displayed better activity compared to the ethanolic extract (A 0.5 = 10.77 ± 0.12 µg/mL) and the standard ascorbic acid (A 0.5 = 10.03 ± 0.22 µg/mL).

Inhibition of Enzymatic Activities
The key metabolic enzymes linked to hyperglycemia are commonly employed to assess a substance's antidiabetic potential. In the present study, the ability of the ethanolic and aqueous extracts of S. hispidula to inhibit the α-glucosidase and α-amylase enzymes was assessed. The results are shown in Table 4. Table 4. Enzyme inhibitory activities of S. hispidula extracts against the activity of α-glucosidase and α-amylase enzymes.

Sample
Enzyme Inhibitory Activity The statistical analysis performed for the results obtained in the α-glucosidase inhibition assay method noted significant differences (with p < 0.05) in the inhibitory activities of the aqueous and ethanol extracts of S. hispidula against this enzyme. The aqueous extract displayed the highest inhibitory activity against the α-glucosidase enzyme, with IC 50 values in mg/mL of 23.52 ± 6.33 µg/mL when compared to the ethanolic extract showing IC 50 = 106.94 ± 1.55 µg/mL. Still, both extracts proved to have more potent α-glucosidase inhibitory activity than the positive control, Acarbose (IC 50 = 405.77 ± 34.83 µg/mL). However, with regard to α-amylase inhibitory activity, the aqueous extract (4.86 ± 0.004%) was shown to be less potent than the ethanol extract (30.34 ± 4.58%) in inhibiting the enzymatic activity of α-amylase tested at the concentration of extract equal to 10 mg/mL (Table 4).

Compound Identification
The caffeoylquinic acid derivatives presented the expected fragmentation pattern. The monocaffeoyl showed a pseudomolecular ion [M-H] − at m/z 353, and the dicaffeoyl a pseudomolecular ion [M-H] − ion at m/z 515. Typically, in dicaffeoylquinic acids, the fragment ion at m/z 353 is observed, which is produced by the loss of a caffeoyl moiety [8]. The remaining fragments are similar to the ones produced by the monocaffeoylquinic acid derivatives (Table 2), which comprise the fragments at m/z 191 and m/z 179, confirming the presence of the quinic and caffeic acid moieties, respectively [8][9][10]. Due to the loss of water or carbon dioxide, other interesting fragments could also be detected, as in the case of 5-O-CQA (Figure 2), the major metabolite found in the plant extracts (Table 2).
In the case of rosmarinic acid, with a pseudomolecular ion [M-H] − at m/z 359, the fragmentation was quite different because the caffeoyl moiety is not linked to a quinic moiety but is instead a dimer of two caffeoyl moieties. Naturally, the fragment at m/z 179, confirming the presence of the caffeic acid moiety, was detected, as well as the fragment at m/z 181, confirming the saturated form (Table 2) (Figure 3), these compounds were identified as the flavonoid ophiopogonanone E and an isomer [11][12][13].

Biological Activities
The term "oxidative stress" refers to an attack on cells by free radicals, also known as "reactive oxygen species" (ROS). The accumulation of ROS in the body results in significant tissue damage. Many illnesses, most notably cancer, diabetes, and neurological disorders, are known to be linked to oxidative stress. When ROS accumulate in the cell, they can be neutralized by antioxidant molecules. There are two types of antioxidants: natural and synthetic. The latter has been linked to many harmful side effects. Thus, the search for nontoxic, natural antioxidant compounds has increased recently. Plants are an essential source of natural antioxidants due to the secondary metabolites they contain. These plants can be screened by assessing their antioxidant activity using various in vitro assays. Because numerous processes are involved in neutralizing free radicals, using one single test makes it impossible to determine the real antioxidant potential [25]. As a result, the S. hispidula extracts herein studied were analyzed through three different tests (DPPH, ABTS, and FRAP) to further measure the overall antioxidant potential.
The results for the antioxidant activity of both aqueous and ethanolic extracts of S. hispidula species could be related to their high content of hydroxycinnamic acids, since it was previously reported that such substances displayed strong antioxidant activities [26][27][28]. It was not possible to compare the obtained results to the literature. Indeed, we found no information on the biological activity of S. hispidula aerial parts in the literature. This work is, therefore, the first contribution to the study of the biological potential of this plant.
Diabetes mellitus is a chronic non-transmissible metabolic disorder characterized by hyperglycemia. Diabetes affects 422 million people across the world [29]. This number is continually increasing, with 642 million people predicted to be affected by the disease in 2040 [30]. Inhibiting the activity of digestive enzymes such as α-glucosidase and α-amylase, which limit the absorption of glucose derived from starch, is among the current strategies standing out for controlling hyperglycemia [31]. Many diabetes medications, such as acarbose, voglibose, and miglitol, act as enzymatic antagonists of α-glucosidase, reducing intestinal glucose absorption and postprandial glycemia. However, these inhibitors produce harmful side effects such as flatulence, diarrhea, and abdominal pain [31]. Thus, the search for new compounds with antidiabetic potential has increased recently. Medicinal plants with little toxicity and few side effects are critical therapeutic possibilities for treating diabetes [32]. The therapeutic effects of these plants have been linked to the presence of phytochemicals such as alkaloids, terpenoids, flavonoids, carotenoids, and β-glycans. These bioactive constituents provide therapeutic effects by combating reactive oxygen species or acting as hypoglycemics.
The aqueous extract had shown the highest content of rosmarinic acid (Table 2), which suggests that this compound may play an essential role in the inhibitory activity against the α-glucosidase enzyme. Hence, rosmarinic acid isolated from the leaf extract from Portulaca oleracea species exhibited a strong α-glucosidase inhibitory effect [33]. The flavonoid glycosides, highly present in ethanolic and aqueous extracts, may also contribute to their strong α-glucosidase inhibitory activity herein recorded. In the present work, both aqueous and ethanol extracts showed more potent inhibition of α-glucosidase enzyme than α-amylase. This behavior is beneficial because it is known that a strong inhibition of the α-glucosidase enzyme and a weak inhibition of the α-amylase enzyme has the advantage of having fewer adverse effects compared to those caused by an excessive inhibition of the α-amylase enzyme, as this results in an abnormal bacterial fermentation of undigested carbohydrates in the colon and increases abdominal discomfort [34,35].
The dual antioxidant and α-glucosidase inhibitor potential recorded for the two S. hispidula extracts suggest that this plant could be used as a natural remedy to control type 2 diabetes or as an excellent source of new molecules for the development of antidiabetic drugs. Indeed, several experimental and clinical studies have shown that diabetes is associated with a high level of oxidative stress [36][37][38]. Some of the processes involved in oxidative stress in diabetic patients include oxygen free radicals created by glycosylation reactions and changes in the content of the endogenous antioxidant defense systems [39]. On the other hand, hyperglycemia could also be the consequence of a significant increase in oxidative stress.
In such a context, it appears important to control both hyperglycemia and oxidative stress in diabetic patients by using medications that jointly target these two pathological disorders.
Separate extractions were performed using two solvents: water and ethanol. To prepare the aqueous extract, 10 g of the plant powder was mixed with 200 mL of distilled water and boiled for 15 min before filtering. The resultant solution was frozen, freeze-dried, and kept at 4 • C until needed. In turn, an ethanol extract was prepared by macerating 10 g of powder in 200 mL of ethanol at room temperature for 48 h, followed by solvent replacement for two additional times. The ethanol was then removed at reduced pressure.

Total Bioactive Content
The Folin-Ciocalteu method, previously described by [40], was used to determine the total phenolic content (TPC) [40]. In brief, 15 µL from each plant extract (1 mg/mL) were combined with 15 µL of Folin's reagent and 60 µL of water. After incubating the mixture for 5 min, 150 µL of Na 2 CO 3 solution (20% w/v) was added. The mixture was incubated in the dark for another 60 min, and absorbance at 760 nm was measured. The total phenolic content was reported as mg gallic acid equivalents per gram of extract (mg GAE g of extract).
The total flavonoid content (TFC) was assessed using a slightly modified technique reported by [41]. In brief, 100 µL of each plant extract was combined with 100 µL of a 2% solution of aluminum chloride (AlCl 3 ). After incubating the mixture at room temperature for 10 min, the absorbance at 415 nm was measured. The total phenolic content was reported as mg of quercetin equivalents per gram of extract (mg QE/g of extract).

UHPLC-DAD-ESI/MS Characterization of S. hispidula Extracts
The phenolic composition of S. hispidula ethanolic and aqueous extracts was determined using an analytical liquid chromatograph, the Dionex Ultimate 3000 (Dionex Co., San Jose, CA, USA) apparatus equipped with a diode array detector (Dionex Co. San Jose, CA, USA) coupled to a Thermo LTQ XL mass spectrometer (Thermo Scientific, San Jose, CA, USA) with an electrospray ionization source operating in negative mode. The column used was a Hypersil Gold (Thermo Scientific, USA) C18 column (100 mm length; 2.1 mm i.d.; 1.9 µm particle diameter, end-capped). The eluents used were a mixture of formic acid [0.1%(v/v)] in water (A) and acetonitrile (B) with an injection volume of 10 µL and a flow rate of 0.2 mL/min. The elution gradient was 5% (solvent A) for 14 min, 40% (solvent A) over 2 min, 100% (solvent A) over 7 min, and the re-equilibration of the column with 5% of solvent A for 10 min. The complete scan encompassed the mass range from m/z 100 to 2000. UV-Vis spectral data were collected between 200 and 500 nm, while chromatogram profiles were obtained at 280 nm. A Thermo Xcalibur Qual Browser data system (Thermo Scientific, USA) was used to analyze the spectra, and chemicals were tentatively identified by comparing their mass spectra and retention durations to what had previously been described in the literature.
Stock solutions of standard references were prepared in the same conditions. Quantification was carried out using the external standard method. The identification of peaks was confirmed by comparing their retention times and MS and DAD spectra with those of reference, or by comparing with MS and UV-Vis data previously reported in the literature. The antioxidant activity was tested by inhibiting the free 1,1-diphenyl-2-picrylhydrazyl radical (DPPH • ), as described by [42]. In brief, 250 µL of DPPH solution (8.66 × 10 −5 M) were mixed with 50 µL of each extract concentration. The mixtures were incubated at room temperature in the dark for 30 min. The absorbance was then observed at 734 nm. The negative control was prepared in the same way, but with the extract replaced by its solvent. The following formula was used to measure the percentage of inhibition: DPPH Scavenging effect = (Ac − As)/Ac 100% where the absorbance of the control is represented by Ac, and the absorbance of the sample is represented by As.
The sample concentration required to inhibit 50% of the reaction (IC 50 ) was measured. The results were then compared to the ascorbic acid used as reference standard.

Determination of 2,2 -Azinobis(3-Ethylbenzothiazoline-6-Sulfonic Acid) Scavenging Activity
The S. hispidula extracts' free radical scavenging ability was assessed using an ABTS •+ decolorization test developed by [43]. In brief, 250 µL of ABTS •+ were mixed with 50 µL of each extract concentration. The mixture was incubated at room temperature in the dark for 20 min before the absorbance at 734 nm was measured. The following equation was used to determine ABTS + scavenging ability: ABTS •+ Scavenging effect = (Ac − As)/Ac 100% where Ac represents the absorbance of the control, and As represents the absorbance of the sample. The sample concentration capable of inhibiting 50% of the reaction (IC 50 ) was measured. The results were then compared to the ascorbic acid used as reference standard.

Ferric Reducing Antioxidant Power Assay
Total antioxidant potential of S. hispidula was measured by the ferric reducing antioxidant power assay (FRAP) according to the technique described by [44]. In a redox-linked colorimetric process, the colorless ferric iron (Fe 3+) is reduced to bright blue ferrous iron (Fe 2+ ) using antioxidants present in the samples as reductants. A 200 µL amount of sample/standard and 200 µL of potassium ferrocyanide (1% w/v) were mixed concurrently. After 20 min at 50 • C, 200 µL of TCA (10% w/v) were added. The mixture was then transferred to a 96-well plate, and 75 µL of deionized water and 15 µL of iron(III) chloride (0.1% w/v) were added at the same time. Following ferric iron reduction, a blue color appeared that could be measured colorimetrically at 690 nm. The results were then compared to the ascorbic acid used as reference standard.
4.6. Inhibition of Enzymatic Activities 4.6.1. Inhibition of α-Glucosidase Activity The α-glycosidase test was carried out using a slightly modified version of the technique reported by [45]. The p-nitrophenyl glycopyranoside (pNPG) substrate solution was produced in 20 mM phosphate buffer, pH 6.9. The extract (50 µL) was combined with a 50 µL solution of 4-nitrophenyl-D-glucopyranoside (PNPG). The reaction was then started by adding 100 µL of the α-glucosidase enzyme solution to the mixture. At 37 • C, the absorbance was measured at 405 nm every minute for 20 min. The negative control was prepared in the same way, but with the extract replaced by its solvent.
The following formula was used to compute the % inhibition: (Ac − As)/Ac × 100 = Inhibition% where Ac denotes the absorbance of the control (enzyme and buffer), and As denotes the absorbance of the sample (enzyme and buffer).

Inhibition of α-Amylase Activity
The α-amylase inhibition assay was carried out with minor changes to the technique previously published by [45]. A 200 µL amount of each extract concentration was combined with 400 µL of a 0.8% (w/v) starch solution on a 96-well plate and incubated for 5 min at 37 • C. The reaction was initiated by the addition of 200 µL of α-amylase. After that, 200 µL of the mixture was combined with 600 mL of 3,5-dinitrosalicylic acid (DNS) reagent. A second aliquot of 200 µL of the first mixture was taken and mixed with DNS reagent around 15 min later. Then, 250 µL of each combination was heated at 100 • C for 10 min before being transferred to a 96-well microplate, and the absorbance at 450 nm was measured. The inhibiting action against α-amylase was estimated as inhibition percentage using the following formula: (Ac − As)/Ac × 100 = Inhibition% where Ac represents the absorbance of the control (enzyme and buffer), and As represents the absorbance of the sample (enzyme and inhibitor). The 50% inhibitory concentration (IC 50 ) of a sample was measured. The results were then compared to acarbose, a commercial standard.

Statistical Analysis
All calculations were performed in MINITAB software (version 16, State College, PA, USA). Data from three separate assays were reported as the mean standard deviation (m SD). The data recorded as the mean ± standard deviation (m ± SD) were submitted to analysis of variance (ANOVA), followed by a post hoc honestly significant difference (HSD) Tukey's test at p < 0.05.

Conclusions
The present study is the first report devoted to the identification and quantification of the phytochemical constituents of aerial parts of the endemic species S. hispidula growing in Algeria, as well as the assessment of their antioxidant and antidiabetic effects. The results indicate that aqueous and ethanolic extracts exhibited high antioxidant and α-glucosidase inhibitory activities; the aqueous extract was the most efficient. Finally, the current findings support the use of S. hispidula in Algerian folk medicine to treat hyperglycemia, suggesting it is an essential source for developing new antidiabetic medications.