1. Introduction
Oxidative stress has been recognized as the main cause of many life-threatening diseases, comprising atherosclerosis, cardiovascular diseases, diabetes, obesity, cancer, and many inflammatory conditions [
1,
2]. The unregulated inflammatory responses further worsen the previously mentioned disorders that could be managed via the consumption of steroidal or non-steroid anti-inflammatory and immunosuppressant drugs. However, they trigger a plethora of side effects [
3,
4]. In addition, diabetes mellitus and Alzheimer’s disease (AD) are among the most debilitating disorders that greatly influence patients’ capabilities causing a significant reduction in their activities and affecting their well-being [
5].
Naturally occurring antioxidants of plant origin are highly popular for combatting oxidative stress and counteracting inflammation and its associated disorders, owing to their richness with bioactive secondary metabolites [
6,
7,
8,
9]. Furthermore, they have the potential to effectively inhibit oxidative stress that is associated with diseases such as diabetes and neurodegenerative disorders [
10]. As a result, they are highly welcomed by a large category of patients all over the globe and, thus, can be used as an alternative to synthetic drugs due to their lower adverse effects and price compared to synthetic ones [
11,
12]. The pronounced anti-inflammatory potential of medicinal plants is mainly attributed to their richness in natural antioxidants, such as flavonoids, polyphenols, tocopherols, carotenoids, and ascorbic acid [
1,
13].
Genus
Teucrium belonging to the family Lamiaceae includes about 300 species greatly spread throughout North Africa, Europe and Asian temperate regions [
14], where its different species showed many activities [
15,
16].
Teucrium polium L. is widely used in traditional medicine to treat hypertension and diabetes or as a wound-healing agent [
13].
T. polium is a deciduous shrub native to the Western Mediterranean region [
17]. It showed many biological activities, such as anti-inflammatory, antiviral, antifungal, antibacterial, cytotoxic, antioxidant, hypoglycemic, hypolipidemic, hepatoprotective, analgesic, antiulcer effects, in addition to anticonvulsant potential. These activities are highly attributed to plants’ bioactive secondary constituents, such as phenylethanoid glycosides, flavonoids, diterpenes, iridoids and essential oil [
13,
14,
17].
In this study, we aimed to evaluate the antioxidant, anti-hyperglycaemic, anti-Alzheimer and anti-inflammatory effects of Teucrium polium hydroalcoholic extract from the aerial parts. The antioxidant was assessed using 2-diphenyl-1-picrylhydrazyl (DPPH), β-carotene and Galvinoxyl radical (GOR) assays, whereas the anti-hyperglycemic was determined in vitro by the α-amylase inhibition method. The anti-inflammatory effect was determined in vitro by inhibiting denaturation of BSA (Bovine Serum Albumin) and in vivo by inhibiting mouse paw edema induced by carrageenan. However, the anti-Alzheimer activity of the extract of T. polium aerial parts was assessed in vitro via the determination of the anti-cholinesterase activity carried out by the acetylcholinesterase inhibitor method (AChE) and butyrylcholinesterase (BChE). Total phenol and flavonoid content, as well as mineral contents, were evaluated for the first time. Moreover, the correlation between the studied activities and its major previously identified metabolites was determined in silico using molecular docking within the active sites of human α-amylase (HA), acetylcholine esterase and 5-lipoxygenase using Discovery Studio 4.5 (Accelrys Inc., San Diego, CA, USA) with C-docker protocol to further consolidate the obtained results. This work is part of an overall program in our laboratory to use nuclear analytical techniques for studying natural food samples relevant to human health and nutrition.
4. Discussion
Teucrium polium L. belonging to the family Lamiaceae is widely used in traditional medicine to treat hypertension, diabetes, or as a wound-healing agent [
13]. Results illustrated in this study showed that
Teucrium polium aerial parts are rich in polyphenols with 36.35 ± 0.294 μg GAE/mg and in flavonoids with 24.30 ± 0.44 μg QE/mg. Variation in geographical origin undoubtedly influences the total content of phenol and flavonoids in the same species [
35]. The extract of
T. polium growing in Morocco demonstrated a higher polyphenol content estimated by 95.53 ± 1.65 mg GAE/g in addition to a higher flavonoid content reported to be 101.9 ± 1.97 mg RE/g (Rutin equivalent per gram of dry weight) [
36]. Furthermore, the extract of Tunisian
T. polium revealed a lower flavonoid content that was estimated to be 2.67 ± 0.05 mg RE/g [
37]. In addition,
T. polium L. aerial parts are highly rich in phenolic compounds, as evidenced by HPLC-UV-MS analysis previously conducted on the extract of Algerian
Teucrium polium aerial parts that revealed the existence of many phenolic compounds belonging mainly to flavonoids and caffeic acid derivatives [
14].
Additionally, the evaluation of elemental mineral concentrations in
Teucrium polium L. growing in Algeria was conducted for the first time. Mineral elements, particularly the micro and macro-nutrients are essential to various human metabolic processes and significantly contribute to human health [
38]. The essential elements of Na, Fe and K were detected as the highest level among the other elements, where K showed the highest level, estimated at 7619 mg/kg, followed by Fe (984 mg/kg) and then Na (803 mg/kg). In addition, the concentration of essential elements acting as micronutrients, such as Zinc, Chromium and Cobalt, ranged from 24 to 0.48 mg/kg. Although two potential toxic elements were detected, their levels lie below the tolerance limits compared with the recommended values (RDA) [
39].
Moreover,
Teucrium polium L. showed pronounced antioxidant, antihyperglycaemic, anti-Alzheimer activity and anti-inflammatory activity exceeding the used standards. This is mainly attributed to its richness with phenolic compounds, particularly flavonoids highlighted by the detected total flavonoid contents and previously isolated compounds. Flavonoids and phenolics are greatly popular as the largest phytochemical entities with pronounced antioxidant properties from plants [
40,
41]. Regarding the antioxidant activity, the obtained results follow what was previously reported by Sharififar et al. [
42], which reported the antioxidant potential of the Iranian
T. polium hydroalcoholic extract owing to its free radicle scavenging properties. However, herein, the reported results from the Algerian species showed better antioxidant potential using DPPH assay from the previously reported the Iranian species that showed the IC
50 value of 20.1 ± 1.7 µg/mL. The variation in the obtained results reflected the effect of geographical origin in altering to some extent the biological activity that is, in turn, influenced by the secondary metabolites. Moreover, flavonoids significantly prohibit oxidative stress-related diseases via scavenging of reactive oxygen species (ROS) directly by different mechanisms comprising antioxidant enzymes stimulation, inhibition of nitric oxide-induced oxidative stress in addition to metal-chelating activity [
43], whereas scavenging of free radicals is considered the most important mode of antioxidant action of flavonoids as the polyphenol groups interrupt the free radical chain reaction [
44]. Regarding the structure–activity relationship, for efficient radical scavenging behavior, the critical structural features are manifested by the presence of an ortho-dihydroxy structure in ring B, which is critical for electron delocalization; the existence of a 2,3 double bond in conjugation with a 4-keto function that allows the electron delocalization from the B ring in addition to the presence of hydroxyl groups at positions 3 and 5 provides hydrogen bonding to the keto groups that are present in most of the identified compounds in [
44]. It is noteworthy to highlight that the antioxidant potential of phenolic compounds and flavonoids is greatly influenced by functional group arrangement, substitution, configuration, the number of hydroxyl groups that, in turn, affect metal ion chelation ability and radical scavenging activity [
45,
46].
Furthermore, the pronounced antihyperglycaemic activity of
Teucrium polium aerial parts evidenced by an in vitro study and further supported by in silico studies greatly relied upon the richness of
Teucrium polium L. with flavonoids. It is worth highlighting that
T. polium hydroalcoholic extract showed one hundred and thirty times higher activity than acarbose, which could be attributed to the richness of the plant with various phytochemicals such as flavonoids, tannins, and saponins [
47]. Previous studies on α-amylase inhibitors identified from medicinal herbs revealed that several potent inhibitors belong to the flavonoid class [
32]. The obtained results further consolidated what was previously reported by Dastjerdi et al. [
48], showing that Iranian
T. polium possesses a very good activity in α-amylase inhibitory assay with IC
50 values of 3.01 and 1.64 mg/mL for the dichloromethane extract and with ethyl acetate extracts, respectively. Flavonoids elicited an antihyperglycemic effect via suppressing α-glucosidase and α-amylase activities, attenuating insulin resistance and promoting pancreatic cell proliferation [
5,
49]. Moreover, many studies further supported the results of in silico studies, where luteolin revealed a potent antihyperglycemic effect via inhibition of α-amylase [
50]. In addition, luteolin showed an improved insulin action by direct activation of the PPAR pathway, by acting at the insulin signaling pathway, and GLUT4 expression in addition to the up-regulation of synaptic proteins expression and improving endothelial insulin resistance responsible for inflammation [
51]. Flavonoid antihyperglycemic activity may also elicit α-glucosidase inhibition. This inhibition was greatly influenced by the hydroxylation and galloylation of flavonoids that improved the inhibitory activity. On the contrary, the glycosylation of the hydroxyl group and hydrogenation of the C2=C3 double bond on flavonoids weaken the inhibition; however, caffeoylquinic acids showed strong prohibition of α-glucosidase [
52].
Concerning the anti-Alzheimer activity of
Teucrium polium aerial parts, it can be concluded that
T. polium could be used in the alleviation of Alzheimer’s evidenced by its promising BChE and AChE inhibitory potential. This activity is perhaps due to its richness in polyphenols since the members of Lamiaceae were found to be rich in phenolic acids as active constituents that significantly contribute to their neuroprotective properties [
53]. Moreover, a study by Valdimir-Knežević et al. [
54] on the ethanol extract of several Lamiaceae cultivated in Croatia showed that
T. polium was among the most potent plant extracts, with significant AchE inhibitory rates exceeding 75% at 1 mg/mL concentration. Alzheimer’s disease is a form of dementia that is characterized by the presence of senile plaques, neurofibrillary tangles with concomitant synaptic loss and neuronal death leading to gradual memory loss, a decrease in language skills in addition to cognitive impairments [
55]. The neurotransmitter acetylcholine has a crucial role in learning process and memory in the hippocampus. Two enzymes, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), are involved in the of acetylcholine hydrolysis, decreasing its level in Alzheimer’s disease, and thus, prohibition of both enzymes is a well-established strategy for the alleviation of Alzheimer’s disease [
56]. Flavonoids are promising natural products with neuroprotective potential that prevent or slow the progression of Alzheimer disease via the inhibition of key enzymes such as AChE, BChE and BACE-1 (Beta-site APP Cleaving Enzyme-1) [
57]. Moreover, Shimmyo et al. showed that flavonols and flavones are capable of inhibiting BACE-1 where the presence of OH groups of C3′ and C4′ stabilized the binding poses of flavonoids within the BACE-1 active center via hydrogen bond formation. Additionally, the existence of OH at C3 interacted in a direct manner with the Asp catalytic residue, causing a notable enhancement of the BACE-1 inhibitory activity [
58]. Moreover, previous studies reported the effectiveness of cirsiliol as a neuroprotective agent and sedative acting as CNS depressants at the GABA chloride channel and/or at glutamate binding sites that further supported the in silico studies [
59].
Concerning the anti-inflammatory activity of
Teucrium polium aerial parts, its pronounced activity evidenced by BSA denaturation inhibitory potential estimated as 97.53% at 2 mg/mL mainly relied upon its richness with flavonoids. It is worth highlighting that water/ethanol is an effective solvent in extracting phytochemicals from the plant as it combines polar and medium polarity properties [
60]. Thus, it appears that the anti-inflammatory effect of the extract may be due to the presence of flavonoids and phenolic compounds in the plant [
54,
61]. Cirsiliol has been reported to possess a potent and selective 5-lipoxygenase inhibitory potential that further consolidated the in silico studies [
62]. Several mechanisms of action have been suggested in an effort to interpret the mode of action of flavonoids, such as their antioxidant potential, their ability to modulate the production of proinflammatory cytokines and the expression of genes [
63,
64]. Thus, flavonoids hinder the inflammation process by a reduction in cytokines and inflammatory markers expression and via interaction with proteins incorporated in the incidence of inflammation. Additionally, they modulate arachidonic acid (AA)-metabolizing enzymes activity, such as COX, phospholipase A2 (PLA2), lipoxygenase (LOX), as well as the NO-producing enzyme nitric oxide synthase (NOS) and thus reduce the production of AA, PG, leukotriene, and NO, which are critical mediators of inflammation accounting for cellular mechanisms of anti-inflammation [
65]. Luteolin was previously shown to inhibit the rat renal medulla COX with an IC
50 of 100–130 µM, whereas other flavonoids exhibited a significant inhibition of LOX where the reduction in the C-2, 3-double bond and glycosylation reduced the flavonoids inhibitory activities [
63]. Thus,
T. polium can act as a good candidate for alleviating many health-debilitating problems and can be highly beneficial in the pharmaceutical industry and medical research.