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. 2020 Nov 5;9(11):1089. doi: 10.3390/antiox9111089

HR-LCMS-Based Metabolite Profiling, Antioxidant, and Anticancer Properties of Teucrium polium L. Methanolic Extract: Computational and In Vitro Study

Emira Noumi 1,2, Mejdi Snoussi 1,3,*, El Hassane Anouar 4, Mousa Alreshidi 1, Vajid N Veettil 1, Salem Elkahoui 1,5, Mohd Adnan 1, Mitesh Patel 6, Adel Kadri 7,8, Kaïss Aouadi 9,10, Vincenzo De Feo 11,*, Riadh Badraoui 1,12,13
PMCID: PMC7694502  PMID: 33167507

Abstract

In this study, we investigate the phytochemical profile, anticancer, and antioxidant activities of Teucrium polium methanolic extract using both in vitro and in silico approaches. The results showed the identification of 29 phytochemical compounds belonging to 13 classes of compounds and 20 tripeptides using High Resolution-Liquid Chromatography Mass Spectrometry (HR-LCMS). 13R-hydroxy-9E,11Z octadecadienoic acid, dihydrosamidin, valtratum, and cepharantine were the main compounds identified. The tested extract showed promising antioxidant activities (ABTS-IC50 = 0.042 mg/mL; 1,1-diphenyl-2-picrylhydrazyl (DPPH)-IC50 = 0.087 mg/mL, β-carotene-IC50 = 0.101 mg/mL and FRAP-IC50 = 0.292 mg/mL). Using both malignant Walker 256/B and MatLyLu cell lines, T. polium methanolic extract showed a dose/time-dependent antitumor activity. The molecular docking approach revealed that most of the identified molecules were specifically binding with human peroxiredoxin 5, human androgen, and human progesterone receptors with high binding affinity scores. The obtained results confirmed that T. polium is a rich source of bioactive molecules with antioxidant and antitumor potential.

Keywords: Teucrium polium L., HR-LCMS, phytochemistry, antioxidant, anticancer, Walker 256/B, MatLyLu, molecular docking

1. Introduction

Despite the arid and extra arid climate, the flora of Saudi Arabia is complex and contains more than 2285 species belonging to 855 genera [1,2]. In fact, 71.02% of these plants are herbs (1620 species), and many of them are classified as medicinal/aromatic ones. In the Hail region (northern central part of Saudi Arabia that extends between 250°29′ N and 380°42′ E Page: 2), the vegetation is influenced by those of the Mediterranean countries in the mountains and the Saharo-Arabian and Irano-Turranean phyto-geographical regions in An Nafud sand seas, open plains, and wadis [3]. A large majority of these plants have aromatic and medicinal virtues for the aromas they give off, their essential oil, and their rich content in polyphenols. In Saudi Arabia, more than 1200 (over 50%) of the total flowering plants (2250) are expected to be of medicinal importance.

The genus Teucrium includes more than 100 species and is largely distributed in Europe, North Africa, Asia, and especially in the Mediterranean region [4,5]. The Saudi Arabia flora comprises six Teucrium species: T. hijazicum Hedge & R.A. King, T. leucocladum Boiss, T. oliverianum Ging. exBenth, T. polium L., T. popovii R.A. King, and T. yemense Defl [6]. Most plant extracts and their bioactive molecules have been shown to be scavengers of free radicals, which form the basis of their therapeutic potential [7,8,9,10,11]. Their antioxidant nature has been closely linked with the cancer-preventing property of a plant-derived compound due to the fact that the inhibition of oxidative stress reduces mutations and chromosomal aberrations, which initiate carcinogenesis [12]. The antioxidant and anticancer properties of T. polium have been extensively studied over the years [13,14], which are attributed to certain identified polyphenolic compounds identified.

In fact, Teucrium members have been shown to contain different classes of compounds such as fatty acid esters, diterpenes, monoterpenes, sesquiterpenes, flavonoids, and polyphenolics [15,16,17]. Flavonoids that have been isolated from T. polium species include cirsimaritin, cirsiliol, cirsilineol, 5-hydroxy-6,7,30,40-tetramethoxyflavone, salvigenin, apigenin 5-galloylglucoside, apigenin-7-glucoside, vicenin-2-glucoside, and luteolin-7-glucoside [18,19,20,21]. In addition to their antioxidant activities, polyphenols have a wide range of biological activities [22,23,24,25,26]. Flavonoids also are known to protect the plant against ultraviolet radiation and possess anticancer [27,28], antioxidant, and anti-acetylcholinesterase activities [13,29,30,31,32]. It is also known that flavonoids possess antiviral, antifungal, and antibacterial properties [28,33]. To date, more than 134 bioactive compounds have been identified from different part of T. polium subspecies [34]. Teucrium species are used in folk medicine for treating many diseases such as abdominal pain, indigestion, common cold, diabetes, and urogenital diseases, and this plant has been reported to have hypolipidemic, antinociceptive, and anti-inflammatory effects [18,21]. It has been demonstrated that T. polium phytocompounds possess anti-diabetic, antiprofilative, pro-apoptotic, and anticancer activities [34,35,36,37,38].

The main objective of the present study was to investigate the phytochemical composition of T. polium methanolic extract using the HR-LCMS technique, the bioactive class of compounds (polyphenols, flavonoids, tannins…), and its antioxidant properties using four test systems. The antitumor effect was tested against two malignant cell lines: MatLyLu and Walker 256/B. The computational approach was used to confirm the antioxidant and anticancer activities of the identified compounds targeting the human peroxiredoxin 5 enzyme and human androgen/progesterone receptors.

2. Materials and Methods

2.1. Plant Material Sampling and Extract Preparation

The plant material was collected in October 2019 from a plant nursery in the Hail region (Saudi Arabia). The fresh aerial flowering parts (Figure 1) were dried at room temperature for ten days and then ground to a fine powder. Extracts were prepared according to Snoussi et al. [39]. Briefly, 40 g of the plant powder material were macerated in 400 mL of absolute methanol at room temperature for 48 h and re-extracted three times using the same procedure. Methanolic extracts were pooled, filtered, and the solvent was removed at 60 °C in the incubator chamber. The dried extracts were stored until further use. The yield was calculated using the following Formula (1):

Yield (%) = (W1 × 100)/W2, (1)

where W1 was the weight of extract after the evaporation of solvent, and W2 was the dry weight of the sample.

Figure 1.

Figure 1

Vegetative growth (a), budding (b), and full bloom (c) of T. polium collected from Hail region.

2.2. Phytochemical Profile of T. polium Extract

2.2.1. Phytochemical Analysis

The methanolic extract from the aerial part of the felty germander was qualitatively tested for the presence of alkaloids, flavonoids, terpenoids, tannins, saponins, steroids, proteins, amino acids, and cardiac glucosides by following the protocol described by Sofowora [40], Trease and Evans [41], and Adetuyi and Popoola [42].

2.2.2. Identification of Bioactive by High Resolution-Liquid Chromatography Mass Spectroscopy

The phytochemical profile of the obtained crude methanolic extract from T. polium L. aerial parts was analyzed using a UHPLC-PDA-Detector 323 Mass Spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). Compounds were identified via their mass spectra and their unique mass fragmentation patterns. Compound Discoverer 2.1, ChemSpider, and PubChem were used as the main tools for the identification of the phytochemical constituents [43].

2.3. Biological Activities

2.3.1. Antioxidant Activities

DPPH Radical–Scavenging Activity

The ability to scavenge the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was calculated using the following Formula (2) as described by Chakraborty and Paulraj [44]:

DPPH scavenging activity (%) = (A0 − A1)/A0 × 100, (2)

where A0 is the absorbance of the control and A1 is the absorbance of the sample. The antioxidant activity was expressed as IC50 (mg/mL), which represented the extract concentrations scavenging 50% of DPPH radicals [45].

ABTS Radical Scavenging Activity Assay

The radical scavenging activity against ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) radical cations was measured using the method of Chakraborty and Paulraj [44]. The antiradical activity was expressed as IC50 (mg/mL), which represented the extract concentrations scavenging 50% of ABTS radicals [45]. The inhibition percentage of ABTS radical was calculated using the following Formula (3):

ABTS scavenging activity (%) = (A0 − A1)/A0 × 100, (3)

where A0 is the absorbance of the control, and A1 is the absorbance of the sample.

Reducing Power Capability Assay

The reducing power was determined using the method of Bi et al. (2013). The extract concentration providing 0.5 of absorbance (IC50) was calculated from the graph of absorbance at 700 nm against sample concentration [46]. Ascorbic acid was used as a standard.

β-carotene/Linoleic Acid Method

The β-carotene method was carried out according to Ikram et al. [47]. Antioxidant activity (inhibition percentage, PI%) was evaluated using the following Formula (4):

PI% = (A β-carotene T120/A β-carotene t0) × 100, (4)

where A β-carotene t0 and A β-carotene T120 refer to the corresponding absorbance values of the test sample standard and control measured before and after incubation for 2 h, respectively. All tests were performed in triplicate, and ascorbic acid (standard) was used for comparison.

2.4. In Vitro Anticancer Assessment Using Malignant MatLyLu and Walker 256/B Cell Lines and MTT Assay

Malignant MatLyLu (R33327) prostate cancer and Walker 256/B (W256) mammary gland cancer cells were used to test the in vitro anticancer activity of T. polium extract. These two cell lines were kindly provided by Prof. D. Chappard (Angers, France) to Dr. R. Badraoui (Sfax, Tunisia). MatLyLu and Walker 256/B malignant cells have a high osteolylic potential and are commonly used to induce osteosclerotic or osteolytic tumor lesions following the protocols previously described by Badraoui et al. [48,49].

MTT (3-[4จC-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) assay was performed by the quantitative colorimetric method. Malignant Walker 256/B and MatLyLu (5 × 102 cells/well) were seeded on 96-well plates with or without T. polium extract. The effect on the viability of the used cells was realized by using the following growing concentrations: 0–200 μg/mL. Pure ethanol was used as positive control. After 24 or 48 h, cells were incubated with MTT solution for 2 h. Then, the percentage of viability and inhibition was recorded by measuring the absorbance at 490 nm.

2.5. In Silico Study

The antioxidant activity of T. polium methanolic extract was confirmed by molecular docking of the identified phytochemical compounds from T. polium methanolic extract into the active site of the human peroxiredoxin 5 enzyme, the human progesterone (PR) enzyme, and human androgen receptor.

The intermolecular interactions between metabolites extracts and the active residues of peroxiredoxin 5 have been investigated using the AutoDock package [50]. The starting geometries of peroxiredoxin 5 and the original docked ligand benzoic acid were downloaded from the RCSB data bank web site: human peroxiredoxin 5 enzyme (PDB code 1HD2) [51], human progesterone (PDB code 4OAR) [46], human androgen receptor (PDB code 1E3G) [52].

The re-docking of the original ligand into the active site of the three tested target proteins are well reproduced with RMSD (root-mean-square deviation) values of 0.72, 1.14, and 0.651 Å, respectively, for peroxiredoxin 5 receptor, human progesterone receptor, and human androgen receptor. A stepwise molecular docking study was reported in previous study [48,53,54,55]. The docking calculations have been carried out using an Intel (R) Core (TM) i5-3770 CPU @ 3.40 GHz workstation.

2.6. Statistical Analysis

All measurements will be carried out in triplicate, and the results were presented as mean values ± SD (standard deviations).

3. Results

3.1. Phytochemical Composition

High Resolution-Liquid Chromatography Mass Spectrometry (HR-LCMS) was carried out the chemical composition of the active extract. This technique was performed in the separation and identification of the phytoconstituents based on their retention time, database difference (library), experimental m/z, MS/MS fragments, metabolite class, and proposed compounds. MS data were provided in negative and positive ionization mode. The majority of the m/z values in our extract were in the range from 133 to 742. In fact, HR-LCMS analysis identified peptide-like proteins in the methanolic extract of T. polium.

A total of 20 small peptides (tripeptides), with molecular weights ranging from 319 to 537 g/mol, were tentatively identified by comparison of spectrum data of the extract with that of known compounds. Details of identified peptides are given in Table 1.

Table 1.

Peptide-like proteins identified by the HR-LCMS technique in T. polium methanolic extract.

Small Peptides Retention Time (mn) Molecular Weight Formula [m/z]− [m/z]+
Asn Asn Asn 0.945 360.1384 C12H20N6O7 341.1201 -
His Phe Gln 3.988 430.1976 C20H26N6O5 411.1797 -
Gln His Phe 4.05 430.1978 C20H26N6O5 447.1566 -
Thr Leu Trp 6.593 418.2222 C21H30N4O5 435.1808 -
Arg Glu Trp 7.029 489.2233 C22H31N7O6 - 512.2123
Gln Phe Tyr 7.241 456.202 C23H28N4O6 491.1714 -
Trp Phe Trp 7.837 537.2444 C31H31N5O4 - 373.1268
Phe Tyr Gln 8.417 456.2011 C23H28N4O6 - 183.1149
Gln Phe Phe 8.42 440.2065 C23H28N4O5 - 174.1475
Gln Tyr Trp 9.304 495.2108 C25H29N5O6 - 341.1374
Thr Leu Ser 9.441 319.1731 C13H25N3O6 - 342.1705
Tyr Glu Trp 9.572 496.1978 C25H28N4O7 - 95.0482
Pro Trp Pro 9.663 398.1964 C21H26N4O4 - 95.0479
Trp Tyr Gln 9.76 495.2113 C25H29N5O6 - 95.0486
Asn His Met 9.986 400.1527 C15H24N6O5S - 95.0490
Thr Trp Phe 10.036 452.2084 C24H28N4O5 - 95.0499
Lys His Cys 10.229 386.1734 C15H26N6O4S - 149.0968
Lys Phe Cys 10.598 396.1822 C18H28N4O4S - 95.0491
Trp Ser Tyr 10.78 454.187 C23H26N4O6 - 95.0489
Trp Pro Ile 12.677 414.2271 C22H30N4O4 - 299.1620

Figure 2 summarizes the most dominant chemical compounds identified in T. polium methanolic extract by using HR-LCMS techniques.

Figure 2.

Figure 2

Chemical structures of the most dominant identified compounds in T. polium methanolic extract by using High Resolution-Liquid Chromatography Mass Spectrometry (HR-LCMS). (A). 13R-hydroxy-9E,11Z-octadecadienoic acid, (B). Rhoifolin, (C). Sericetin diacetate, (D). Selinidin, (E). Harpagoside, (F). Valtratum, (G). Triptonide, (H). Koparin 2′-Methyl Ether, (I). Dihydrosamidin (J). 10S,11R-Epoxy-punaglandin, (K). 4, 16alpha, 17beta-Estriol 3-(beta-D-glucuronide), (L). Khayanthone. (M). 10-Hydroxyloganin, (N). 7-Epiloganin tetraacetate, (O). Cepharanthine, (P). Deoxyloganin tetraacetate, (Q). Carapin-8 (9)-Ene, (R). 1-dodecanoyl-sn-glycerol.

Peptides of T. polium extract were composed of a majority of essential and non-essential amino acids distributed unevenly. Aromatic amino acids were predominant in 85% of the peptides with tryptophan (Trp) being the most commonly occurring amino acid (18.3%). Tryptophan along with phenylalanine and tyrosine accounted for 43.3% of the total amino acids, followed by glutamine, asparagine, and histidine (25%). Leucine, lysine, serine, glutamic acid, and cysteine together accounted for 16.7%; and proline and threonine made up 10% of the total amino acids. Arginine, isoleucine, and methionine were amongst the least abundant amino acids, which accounted for 5% of the amino acids in the identified peptides. Aromatic rings were the most abundant side chains followed by amide side chains. Sulfur-containing side chains were rare in the identified peptides.

The repertoire of peptides of T. polium extract appeared to be more hydrophobic than hydrophilic from the amino acid composition, with hydrophobic amino acids accounting for 51.7% of the total amino acids. In addition, the majority of the peptides were composed solely of hydrophobic amino acids. Neutral amino acids made up of 88% of the amino acids followed by basic amino acids (8%). Consequently, 14 of the 20 peptides were neutral in nature. Glutamic acid was the only acidic amino acid detected and was present in two of the 20 peptides identified. As seen in Table 2 (m/z values), most of the peptides had a net positive charge as determined by spectrophotometric data.

Table 2.

Phytochemical composition of T. polium methanolic extract using the HR-LCMS technique.

Identified Compound Name Class of Compounds RT [min] Formula [M + H]+ (m/z) [M + H] (m/z)
1 10-Hydroxyloganin Isoprenoid 0.945 C17H26O11 - 406.1439
2 13R-Hydroxy-9E,11Z octadecadienoic acid Octadecanoid 1.046 C18H32O3 296.232 -
3 Bis (2-hydroxypropyl) amine Amino Alcohol 1.062 C6H15NO2 133.1111 -
4 9-Aminononanoic acid Amino Fatty Acid 1.447 C9H19NO2 173.1401 -
5 10-Aminodecanoic acid Amino Fatty Acid C10H21NO2 187.156 -
6 7-Epiloganin tetraacetate Isoprenoid 4.739 C25H34O14 - 558.1987
7 b-D-Glucopyranoside uronic acid, 6-(3-oxobutyl)-2- naphthalenyl Organic Acid, Phenol 5.342 C20H22O8 - 390.1284
8 Cepharanthine Alkaloid 5.948 C37H38N2O6 606.2582
9 Rhoifolin Flavonoid 6.376 C27H30O14 578.1634 -
10 Sericetin diacetate Flavonol 7.838 C29H28O7 488.191 -
11 Troxerutin Flavonol 5.96 C33H42O19 - 742.2379
12 Deoxyloganin tetraacetate Isoprenoid 6.319 C25H34O13 - 542.2048
13 CMP-N-acetylneuraminic acid Amino Sugar 6.329 C20H31N4 O16P - 614.1563
14 Carapin-8 (9)-Ene Limonoid 8.198 C27H30O7 - 466.1996
15 Selinidin Coumarin Derivative 8.848 C19H20O5 328.1303 -
16 Harpagoside Iridoid Glycoside 9.1 C24H30O11 494.1782 -
17 8-Epiiridodial glucoside tetraacetate Isoprenoid 9.126 C24H34O11 498.2126 -
18 Larixol Acetate - 9.262 C22H36O3 348.2635 -
19 Valtratum Terpene 9.525 C22H30O8 422.1935 -
20 Triptonide Diterpene triepoxide 9.807 C20H22O6 358.1419 -
21 Koparin 2′-Methyl Ether Isoflavonoid 10.036 C17H14O6 314.0792 -
22 Dihydrosamidin Coumarins 10.779 C21H24O7 388.1524 -
23 10S,11R-Epoxy-punaglandin 4 Eicosanoid 11.022 C25H35ClO9 514.1895 -
24 16Alpha,17beta-Estriol 3-(beta-D-glucuronide) Steroidal glycosides 11.279 C24H32O9 464.2051 -
25 16-Hydroxy-4-carboxyretinoic Acid Isoprenoid 11.28 C20H24O5 344.1621 -
26 Isotectorigenin, 7- Methyl ether IsoFlavonoid 12.149 C18H16O6 328.0939 -
27 3-hydroxy-3′,4′- Dimethoxyflavone Flavonoid 13.274 C17H14O5 298.0829 -
28 Khayanthone Limonoid 18.427 C32H42O9 570.2854 -
29 1-Dodecanoyl-sn-glycerol Glycerolipid 20.37 C14H22N2O3 - 266.1651

It is important to note that all compounds were first reported in this study for T. polium aerial parts methanolic extract analyzed with HR-LC/MS. The complete list of identified chemical bioactive compounds is summarized in Table 2.

3.2. Phytoconstituents and Antioxidant Activities

Phytochemical analysis showed the presence of diverse bioactive constituents such as saponins, cardiac glucosides, anthocyanin, terpenes, tannins, sterols, flavonols/flavanones, quinones, alkaloids, and coumarines. The results are summarized in Table 3.

Table 3.

Qualitative analysis of phytochemicals in methanolic extract of T. polium aerial parts.

Phytochemical Compounds SAP CAG ANT TER FLAV TAN ST QUI COU FLA FAT ALK
T. polium extract (++) (++) (+) (++) (+) (+) (++) (+) (+) (+) (+) (+)

SAP: saponins, CAG: cardiac glucosides, ANT: anthocyanin, TER: terpenes, FLAV: flavonols and flavanones, TAN: tannins, ST: sterols, QUI: quinones, COU: coumarines, FL: flavonoids, FAT: fatty acids, ALK: alkaloids. (+): presence, (-): absent, (++): abundant.

The quantitative determination of phytochemical compounds specifies that the T. polium methanolic extract was rich in flavonoids (725 ± 0.001 mg QE/g extract), tannins (239 ± 0.006 mg QE/g extract), and phenols (72 ± 0.011 mg QE/g extract). The obtained T. polium methanolic extract was evaluated for its antioxidant potentiality using four methods. The IC50 of each test was calculated and determined (Table 4). As it is shown, this extract had the strong radical inhibition of ABTS (IC50 = 0.042 mg/mL) followed by DPPH (IC50 = 0.087 mg/mL), β-carotene/linoleic acid (IC50 = 0.101 mg/mL), and FRAP (IC50 = 0.292 mg/mL).

Table 4.

Antioxidant activities of T. polium + methanolic extract as compared to ascorbic acid and BHT.

Test Systems T. polium Methanolic Extract (BHT) (AA)
Phytochemical Composition
Total Flavonoids Content (mg QE/g Extract) 725 ± 0.001 - -
Total Tannins Content (mg TAE/g Extract) 239 ± 0.006 - -
Total Phenols Content (mg GAE/g Extract) 72 ± 0.011 - -
Antioxidant Activities
DPPH IC50 (mg/mL) 0.087 ± 0.001 b 0.023 ± 3 × 10−4 a 0.022 ± 5 × 10−4 a
ABTS IC50 (mg/mL) 0.042 ± 0.014 b 0.018 ± 4 × 10−4 a 0.021 ± 1 × 10−3 a
β-carotene IC50 (mg/mL) 0.101 ± 0.020 c 0.042 ± 3.5 × 10−3 b 0.017 ± 1 × 10−3
FRAP IC50 (mg/mL) 0.292 ± 0.042 c 0.05 ± 0.003 a 0.09 ± 0.007 b

BHT: butylated hydroxytoluene, AA: ascorbic acid. The letters (a–c) indicate a significant difference between the different antioxidant methods according to the Duncan test (p < 0.05).

3.3. Anticancer Activities

The anticancer effect of the T. polium extract was investigated by MTT assay on two malignant lineages: mammary gland carcinoma (Walker 256/B cells) and prostate cancer (MatLyLu). Both cell lines have high metastatic potential and are commonly used to induce bone metastases [48,49]. The cells were treated with the plant extract at different concentrations 0, 50, 100, and 200 µg/mL for 24 or 48 h.

Our findings, validated by the antiproliferative effects on malignant Walker 256/B and MatLyLu cells, suggested that the methanolic extract of T. polium possess an anticancer effect. Its phytochemical profile might act as chemopreventive agents against both Walker 256/B and MatLyLu. In fact, the extract suppressed the growth of the two malignant lines once compared with the controls (0 µg/mL) (Figure 3). The lowest viability was noticed with 200 µg/mL of T. polium extract for both cell lines. Overall, the effect was accentuated with the dose increase (dose-dependent), and it was more prominent after 48 h of treatment; the effect was dose and time-dependent.

Figure 3.

Figure 3

Anticancer effect of T. polium methanolic extract on malignant Walker 256/B mammary gland carcinoma cells (White) and MatLyLu prostate cancer cells (gray) in 24 (A) and 48 h (B). Legend: * p < 0.05, ** p < 0.01, *** p < 0.001.

3.4. In Silico Study

Overall, the in silico approach showed that for antioxidant and antitumor tests, the activity differs from one compound to another. These differences can be explained by the structural geometry of its basic skeleton, and to the presence of different specific substituted groups and heteroatoms in the studied bioactive compounds. In an attempt to rationalize the observed antioxidant activity of the identified molecules in T. polium extract, a molecular docking study has been carried out to determine their binding modes from one side and from another site of the active residues of human peroxiredoxin 5. The results of the number of conventional intermolecular hydrogen bonding established between the docked compounds and active site residues of human peroxiredoxin 5 are summarized in Table 5.

Table 5.

Docking binding energies, conventional hydrogen bonding, and the number of closest residues to the docked compounds into the active site of human peroxiredoxin 5.

Compound No. Class of Compounds Free Binding Energy (kcal/mol) Conventional H-Bonds (HBs) Number of Closest Residues to the Docked Ligand in the Active Site
1 Isoprenoid −3.82 6 7
2 Octadecanoid −5.35 6 5
3 Amino Alcohol −2.15 3 2
4 Amino Fatty Acid −4.54 6 6
5 Amino Fatty Acid −4.60 5 5
6 Isoprenoid −4.66 5 6
7 Organic Acid, Phenol −5.05 3 6
8 Alkaloid −5.07 1 6
9 Flavonoid −5.09 8 11
10 Flavonol −6.00 4 9
11 Flavonol −2.01 8 9
12 Isoprenoid −5.13 5 8
13 Amino Sugar −8.06 5 6
14 Limonoid −7.09 3 8
15 Coumarin Derivative −5.94 3 7
16 Iridoid Glycoside −4.78 6 6
17 Isoprenoid −5.25 5 6
18 - −6.03 2 7
19 Terpene −5.17 5 8
20 diterpene triepoxide −5.70 5 5
21 Isoflavonoid −4.70 5 6
22 Coumarins −5.37 4 6
23 Eicosanoid −3.18 2 6
24 Steroidal Glycosides −5.45 5 6
25 Isoprenoid −5.15 3 7
26 IsoFlavonoid - - -
27 Flavonoid −5.11 2 7
28 Limonoid −6.14 4 8
29 Glycerolipid −2.68 6 5

All the complexes formed between the composition of T. polium methanolic extract (Table 5) and the active residues of Peroxiredoxin 5 have negative binding energies, which may explain the potent antioxidant activity of T. polium methanolic extract. The band energies of the stable complexes range from −8.06 to −2.01 kcal mol−1. According to the molecular docking results, the amino sugar 13 showed the lowest binding energy (−8.06 kcal mol−1), and thus, the highest antioxidant activity was well fitted into the binding cavity of human peroxiredoxin 5. This amino sugar forms five strong hydrogen bonding with amino acids ALA A42, THR A44, THR A147, GLY A46, and CYS A47 at distances of 2.04, 2.88, 2.54, 3.11, and 2.81Å, respectively (Figure 4), as well as a carbon hydrogen bond with PRO A45.

Figure 4.

Figure 4

Three-dimensional (right) and two-dimensional (left) closest interactions between active site residues of peroxiredoxin 5 and some molecules belonging to different class compounds with the best score result. Legend: (A): Compound 13 (CMP-N-acetylneuraminic acid, Class: Amino Sugar), (B): Compound 14 (Carapin-8 (9)-Ene, Class: Limnoid), (C): Compound 10 (Sericetin diacetate, Class: Flavonol), and (D): Compound 8 (Cepharantine, Class: Alkaloid).

It appears from the docking outputs in Table 5 that the antioxidant activity varies with subclass family. For instance, lemonoids 14 (−7.09 kcal mol−1) and 28 (−6.14 kcal mol−1) showed higher binding affinity compared with other subclasses (Table 5).

Table 6 summarized the calculated binding energies of the stable complexes ligand– progesterone, the number of conventional intermolecular hydrogen bonding established between the docked compounds, and the active site residues of progesterone.

Table 6.

Docking binding energies, conventional hydrogen bonding, and the number of closest residues to the docked compounds into the active site of the human progesterone.

No. Class of Compounds Free Binding Energy (kcal/mol) Conventional H-Bonds (HBs) Number of Closest Residues to the Docked Ligand in the Active Site
1 Isoprenoid −7.10 8 8
2 Octadecanoid −6.84 3 4
3 Amino Alcohol −3.61 3 4
4 Amino Fatty Acid −3.69 5 3
5 Amino Fatty Acid −4.78 4 4
6 Isoprenoid −7.83 3 7
7 Organic Acid, Phenol −7.61 4 8
8 Alkaloid −8.56 0 9
9 Flavonoid −8.46 4 12
10 Flavonol −9.48 0 5
11 Flavonol −6.41 7 10
12 Isoprenoid −7.97 3 9
13 Amino Sugar −3.90 4 9
14 Limonoid −10.45 0 10
15 Coumarin Derivative −8.38 1 8
16 Iridoid Glycoside −7.41 5 10
17 Isoprenoid −8.07 2 10
18 - −9.01 2 9
19 Terpene −8.13 3 8
20 Diterpene triepoxide −9.28 2 9
21 Isoflavonoid −7.70 3 8
22 Coumarins −8.78 2 12
23 Eicosanoid −6.89 2 5
24 Steroidal Glycosides −10.06 7 12
25 Isoprenoid −8.63 4 11
26 IsoFlavonoid −6.69 2 8
27 Flavonoid −7.03 2 9
28 Limonoid −10.83 2 11
29 Glycerolipid −5.56 3 2

All the complexes formed between the composition of T. polium methanolic extract (Table 6) and the active residues of progesterone showed negative binding energies, which may explain the observed anticancer activity of T. polium methanolic extract. The bond energies of the stable complexes range from −10.83 to −3.61 kcal mol−1. According to molecular docking results, limonoids 28 and 14 showed the higher anticancer activity with lowest binding energies of −10.83 and −10.45 kcal mol−1. The higher activity of 28 compared with 14 may be due to the extra intermolecular types of hydrogen bonding, and π-sulfur types appear in the former compared with the latter (Figure 5). Indeed, in the stable complex 28-progesterone, two strong hydrogen bonds were formed between 28 and the progesterone receptor. The first one is formed between the lone pair of oxygen atoms of the furan ring and the amino acid GLN A725 at a distance of 3.15 Å, and the second one at a distance of 3.06 Å is established between the lone pair of an oxygen atom of cyclopentanone and VAL A760. π-Sulfor intermolecular interactions are formed between the π bonds of furan ring and the amino acids MET A759 and MET A801 (Figure 5) of distances 5.44 and 5.29 Å, respectively.

Figure 5.

Figure 5

Three-dimensional (right) and two-dimensional (left) closest interactions between active site residues of the human progesterone receptor and some molecules belonging to different class compounds with the best score result. Legend: (A): Compound 8 (Cepharantine, Class: Alkaloid), (B): Compound 20 (Triptonide, Class: Diterpene triepoxide), (C): Compound 10 (Sericetin diacetate, Class: Flavonol), (D): Compound 24 (16alpha,17beta-Estriol 3-(beta-D-glucuronide, Class: Steroidal glycosides), (E): Compound 14 (Carapin-8 (9)-Ene, Class: Limnoid), (F): Compound 28 (Khayanthone, Class: Limnoid).

Table 7 summarizes the calculated binding energies of the stable ligand–progesterone complexes, the number of conventional intermolecular hydrogen bonds established between the docked compounds, and the active site residues of the human androgen receptor. The complexes formed between the composition of T. polium methanolic extract (Table 7) and the active residues of androgen showed negative and positive binding energies. On one hand, the complexes that show negative binding energies may explain the anticancer activity of T. polium methanolic extract. On the other hand, the positive binding energies may indicate that the corresponding metabolites are not active; i.e., they have no anticancer activity. The bond energies of the stable complexes range −11.01 to −3.49 kcal mol−1. According to the binding energies, compound 18 showed the higher anticancer activity with the lowest binding energy of 11.01 kcal mol−1 (Table 7 and Figure 6).

Table 7.

Docking binding energies, conventional hydrogen bonding, and the number of closest residues to the docked compounds into the active site of the human androgen receptor.

No. Class of Compounds Free Binding Energy (kcal/mol) Conventional H-Bonds (HBs) Number of Closest Residues to the Docked Ligand in the Active Site
1 Isoprenoid −7.61 5 7
2 Octadecanoid −8.01 3 4
3 Amino Alcohol −3.51 3 3
4 Amino Fatty Acid −5.74 5 3
5 Amino Fatty Acid −6.13 5 3
6 Isoprenoid −7.18 6 8
7 Organic Acid, Phenol −8.31 2 7
8 Alkaloid +134.65 0 5
9 Flavonoid +19.33 1 10
10 Flavonol −3.49 0 11
11 Flavonol +29.13 4 11
12 Isoprenoid −5.95 0 9
13 Amino Sugar +2.64 4 8
14 Limonoid −4.94 3 13
15 Coumarin Derivative −9.70 0 9
16 Iridoid Glycoside −8.07 3 9
17 Isoprenoid −7.73 2 14
18 - −11.01 2 13
19 Terpene −9.66 1 10
20 Diterpene Triepoxide −9.61 1 14
21 Isoflavonoid −8.96 4 9
22 Coumarins −9.81 0 9
23 Eicosanoid −4.42 2 7
24 Steroidal Glycosides +17.12 4 11
25 Isoprenoid −4.96 3 12
26 IsoFlavonoid −8.29 3 9
27 Flavonoid −8.37 1 7
28 Limonoid +9.39 0 7
29 Glycerolipid −5.89 2 2

Figure 6.

Figure 6

Three-dimensional (right) and 2D (left) closest interactions between the active site residues of the human androgen and some molecules belonging to different class compounds with the best score result. Legend: (A): Compound 15 (Selinidin, Class: coumarin derivative), (B): Compound 18 (larixol acetate), (C): Compound 19 (Valtratum, Class: Terpene) and (D): Compound 22 (dihydrosamidin, Class: coumarins).

The stability of the 18-androgen complex may refer to the strong interactions formed between 18 and amino acids of androgen (Figure 5). Indeed, two strong hydrogen bonds were formed between the acetyl groups of 18 and amino acids THR A877 and ARG 752 of the androgen receptor at distances of 3.32 and 3.40 Å, respectively (Figure 6). Furthermore, a strong sulfur-X bond is formed between the oxygen atom of the acetyl group of 18 and the sulfur atom of the methylthiol moiety of MET A780 at a distance of 3.17 Å (Figure 6).

4. Discussion

4.1. Phytochemical Composition of T. polium Extract

According to our result, terpenoids, also known as isoprenoids, are the most abundant compound class in the methanolic extract of T. polium aerial parts as defined by the HR-LCMS technique. Among the identified compounds (Table 1), many secondary metabolites with known antioxidants and anticancer activities belong to different classes of bioactive molecules, including isoprenoid, fatty acids, amino fatty acids, amino alcohols, glycerolipids, amino sugars, phenol, alkaloids, flavanol, small peptides, etc. [55,56]. It has been demonstrated that Liquid Chromatography Mass Spectrometry is a very sensitive method, which can identify many new compounds. Indeed, Aghakhani et al. [57] isolated twenty-two new flavonoid compounds that were first reported for Phlomis species. Our results are in agreement with previous studies that indicated that the Teucrium genus contains different classes of phytoconstituents such as monoterpenes, sesquiterpenes [58], polyphenols, flavonoids [59,60], and fatty acid esters [61,62]. Moreover, flavonoids are polyphenols that are detected in medicinal plants with a wide variety of biological activities [63]. Many previous studies have described the presence of various flavonoids such as apigenin, luteolin, rutin, cirsiliol, cirsimaritin, salvigenin, and eupatorin in the roots, aerial parts, and inflorescences of the plant [17,59,60,61,62,63,64,65,66]. Furthermore, one intermediate in the biosynthetic pathways of alkaloids, iridoid glycoside (Harpagoside), was also detected [60]. This compound has been detected in a wide variety of plants and in some animals. Furthermore, two iridoid glycosides, teucardoside and teuhircoside, from the hydrophilic fraction of T. polium var. pilosum and T. polium var. Alba were isolated [34]. Elmasri et al. [37] isolated iridoid and phenylethanol glycosides and a monoterpenoid from the areal part of T. polium. It was reported that 3′,4′,5trihydroxy-6,7-dimethoxy-flavone exhibited an inhibition of the biofilm-forming strain Staphylococcus aureus [37]. It was reported by Sharififar et al. [65] that the methanolic extract of the areal part of T. polium contains four major flavonoids where rutin and apigenin were found to be the most active fractions as radical scavengers. In 2018, Özer et al. [66] used the LC-MS/MS method to analyze compounds and to investigate the antioxidant activity of T. polium L. in decoction and infusion. Among the secondary metabolites, polyphenols represent an interesting class that have biological activities such as antioxidant and anticancer [67,68]. Moreover, flavonoids, another secondary metabolites class, are widely produced by plants to fight against biotic and abiotic aggression. It has been well demonstrated that this compounds exhibited antioxidant and anticancer activities [69,70]. The degree of polymerization and the differences arising from hydroxyl group substitutions makes this class of metabolites very large, with about 4000 different compounds [69].

Additionally, during our investigation, we identified 20 small peptide fragments with varying amino acid sequences via HR-LCMS analysis. Therefore, it was of interest to analyze the role of these peptides in the antioxidant and anticancer properties of the plant extracts. Peptides and peptide-like proteins have been found to be integral components of various plant species and have therapeutic applications due to their broad spectrum of biological activities such as antimicrobial, antioxidation, antihypertensive, immunomodulatory, and anticancer properties [71]. Although peptides from different plant species vary greatly with respect to amino acid composition and sequence, bioactive peptides possess certain common features such as small size, hydrophobicity, large percentage of aromatic amino acids, and amphiphatic nature [72].

4.2. Antioxidant Activities

The antioxidants of T. polium extracts have been widely studied [73,74,75]. For instance, using DPPH and β-carotene/linoleic acid assays, respectively, the petroleum ether (IC50 = 73.2; 9.2 µg/mL), chloroform (IC50 = 85.4; 5.1 µg/mL), methanol (IC50 = 20.1; 25.8 µg/mL), and water (IC50 = 40.6; 19.2 µg/mL) extracts of this plant which were collected from Kerman (Iran) showed strong antioxidant activity compared to our findings [65]. The aqueous extract of T. polium, collected from Israel, was evaluated for its antioxidant potentiality using various tests [76]. It inhibited superoxide radical (IC50 = 12.0 µg/mL), hydroxyl radical (IC50 = 66.0 µg/mL), β-carotene (inhibition percentage IP = 60% at 100µg/mL), iron-induced lipid peroxidation (IC50 = 7.0µg/mL), 2,20-azobis (2-amidinopropan) dihydrochloride (AAPH)-induced plasma oxidation (IP = 84% at 667µg/mL). It had also the capacity to bind iron (IC50 = 79.0 µg/mL). At 1 mg/mL, this aqueous extract had the ability to increase intracellular GSH (glutathione) levels in cultured HepG2 cells [76]. The 80% ethanolic extract of T. polium, collected from Iran, was determined for its in vivo antiradical activity using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical (78.6 and 90.7%), total antioxidant power (23.6 and 37.5%), and thiobarbituric acid reactive substances (24.7 and 31.8%) in serum at 50 and 100 mg/kg, respectively [77].

Our study showed the strong antioxidant capacity of the methanolic extract. This activity could not be related to the total phenolic, (0.072 mg GAE/g extract), flavonoid (0.725 mg QE/g extract), or tannin contents (0,239 mg TAE/g extract), and the extract was analyzed by HR-LCMS in order to identify the major active compounds (Table 1). The results showed the presence of various chemical compounds, such as fatty acids, terpenes, alkaloids, coumarines, and flavonoids. These classes are already known by their antioxidant abilities [78,79], and thus, they may explain the present activity. The analyzed peptides showed in Table 2 can also explain in part the antioxidant activity [80,81,82].

Furthermore, a previous study showed that the isoprenoid 10-hydroxyloganin had an insecticidal effect [83]. The limonoid khayanthone was detected in Punica granatum methanolic extract, which exhibited antioxidant potentiality [84]. The compounds 13R-hydroxy-9E,11Z-octadecadienoic, acid bis (2-hydroxypropyl) amine, 9-aminononanoic acid, and 10-aminodecanoic acid found in this plant, which belong to fatty acids, amino alcohols, and amino acid derivatives, were proved in the literature to have antiradical activity [85,86]. Cepharanthine is widely known in several clinical uses. It was used for the treatment of various diseases such as the inhibition of free radicals, radiation-induced leukopenia, venomous snakebites, etc. [87,88]. At 30 μg/mL, cepharanthine had the ability of 94.6% inhibition on the peroxidation of linoleic acid [89]. It also exhibited activity using DPPH, ABTS, superoxide anion, ferrous ion chelating, total antioxidant activity, hydrogen peroxide, reducing power, and N,N-dimethyl-p-phenylenediamine dihydrochloride radicals scavenging [89]. Moreover, harpagoside inhibited free radicals at 1 mg/mL [90].

The relationship between the structure and activity of natural peptides with antioxidant properties has been vividly elucidated in a report by Zou et al. [91] where certain aspects of the chemical structure, namely the small size, presence of certain amino acids in large amounts, and hydrophobicity, are described as important factors that influence the antioxidant nature of peptides. Similar findings have been reported in several plant peptides such as those from Sphenostylis stenocarpa [92], hemp seed [93], phaseolin, bean [94], and Jatropha curcas [95] using several in vitro antioxidant evaluation systems such as diphenyl-1-picryhydradzyl (DPPH) and linoleic acid oxidation. In our study, many of the above-mentioned characteristics (small size, hydrophobicity, and high occurrence of aromatic amino acids) were seen in the peptides from T. polium, which further confirms the potential of the plant extract as a therapeutic agent.

4.3. Anticancer Activities of T. polium Extract

The anticancer effect of the Teucrium polium extract was investigated by MTT assay on two malignant lineages: mammary gland carcinoma (Walker 256/B cells) and prostate cancer (MatLyLu). Both cell lines have high metastatic potential and are commonly used to induce bone metastases [42,43]. The cells were treated with the plant extract at different concentrations 0, 50, 100 and 200 µg/mL for 24 or 48 h.

As shown in Figure 3, T. polium extract seems to possess an anticancer effect. In fact, it inhibits the proliferation of both malignant Walker 256/B and MatLyLu cells in a concentration- and time-dependent manner. It has been previously reported that T. polium inhibited the proliferation of prostate cancer cells [96]. Similarly, several medicinal plant extracts inhibit the invasion, cancer evolution, and metastases. A recent study expected that in the near future, T. polium extract may be a novel anticancer agent [97]. This possibility is certainly related to the promising effects of the plant extract and could explain the ethno-pharmacological applications and the traditional use of T. polium. In fact, as shown in Table 1 and Table 2, the plant methanolic extract exhibits a relevant and promising phytochemical composition following HR-LCMS assay. It includes, but is not limited to, flavonoid and isoflavonoid, isoprenoid, diterpene triepoxide (such as triptonide), and terpene (such as valtratum). All these chemical compounds possess pharmacological activities. Basically, these natural compounds, such as the triptonide, were effective in inhibiting tumorigenicity and tumor growth in a wide variety of cancers, including pancreatic cancer and thyroid induced metastases by activating the tumor-suppressive MAPK (mitogen-activated protein kinase) signaling pathway and via astrocyte elevated gene-1, respectively [98,99]. These phytochemical compounds might have better pharmacological properties together rather than separated. In fact, it has been reported that the effect of the whole plant is usually much better than that of its active phytochemical compounds [100].

Previous studies with T. polium extract reported potential anticancer effects by the inhibition of cell invasion and motility of human prostate cancer [96]. The mechanism includes E-caderin/catenin complex restoration. In this study, an anticancer effect has been revealed on the prostate MatLyLu cell line. Similarly, an in vivo study of prostate cancer and its lymph node, lung, and bone metastases complication due to MatLyLu cells could inhibit the invasion and metastatic potential via E-caderin/catenin complex restoration.

The effect of related plants showed efficient effects against breast and prostate cancer. In fact, extract from T. persicum was reported to inhibit PC-3 prostate cancer cells proliferation [101]. It has been demonstrated that both T. capitatum and T. creticum were effective against MCF-7 breast cancer cells [102]. Moreover, T. romasissimun and has an anticancer potential by inhibiting K562 proliferation [103]. Nevertheless, the reported IC50 values were different, and that could be related to the area from which the plant has been collected and/or the cancer cell types. However, it is well known that T. polium extract may also potentiate the apoptotic effects of some anticancer drugs such as doxorubicin and vinblastine on several cancer cell lines, including Saos-2, A431, SW480, and Skmel-3 [104]. Further preclinical investigations concerning the in vivo anticancer effects on Walker 256/B, MatLyLu, and other cancer types might be of potential interests to confirm the protective effect of T. polium and its possibility to contribute to the discovery of new drug products to treat cancer.

4.4. In Silico Study

Our molecular docking results with antioxidant human peroxiredoxin 5 were perfectly correlated with those of Eze et al. [105], confirming the same and highest binding pose of 1-(phenylsulphonyl)-N-propylpyrrolidine-2-carboxamide in the binding cavity of human peroxiredoxin 5 with a binding energy of (−13.86 kcal mol−1) and interactions with THR44, PRO40, PRO 45, GLY46, ARG127, THR147, and CYS A47 residues. Similarly, α-tocopherol (−7.2 kcal mol−1) Cymbopogon citratus essential oil was perfectly fitted into the cavity of peroxiredoxin 5 establishing H-bonding with Arg 127(A) and non-covalent interactions with ASP A113, THR A147, LEU A116, SER A115, LEU A112, PRO A40, THR A44, GLY A46, CYS A47, PHE A120, and ASP A145. Additionally, caryophyllene oxide (−7.1 kcal mol−1) from the same essential oil was interacting with non-covalent interactions with PRO A40, THR A147, THR A44, PHE A120, PRO A45, LEU A116, and ILE A119 with amino of peroxiredoxin 5 amino acids residues, confirming therefore the high potency T. polium methanolic extract.

The obtained molecular docking interactions (Figure 5) of T. polium methanolic extract into the active site of the human progesterone enzyme were in good agreement with those reported by Acharya et al. [46]. In fact, these authors demonstrated that furanocoumarins, xanthotoxol, bergapten, angelicin, psoralen, and imperatonin are potent anti-breast cancer agents against progesterone. These molecules were buried into the active site of the human progesterone enzyme via the following molecular interactions: LEU718, GLN725, MET 759, LEU763, ARG766, PHE778 (xanthotoxol), GLN725, LEU721, LEU718, MET756, MET759, LEU763, ARG766, PHE778, MET801(bergapten), LEU718, LEU721, GLY722, GLN725, MET759, LEU763, LEU763, ARG766, PHE778 (angelicin), LEU718, LEU721, GLN725, MET756, MET759, LEU763, ARG766, PHE778, MET801 (psoralen) and LEU718, LEU721, GLY722, GLN725, MET756, LEU763, ARG768, PHE778, LEU797, MET801, and LEU887 (imperatonin).

Our results indicated that our selected compounds displaying the lowest binding energies share a higher number of common residues with the active sites of receptor 1E3G having the corresponding interacting residues, LEU 701, LEU 704, ASN 705, LEU 707, GLY 708, GLN 711, TRP 741, MET742, MET 745, VAL 746, MET 749, ARG 752, PHE 764, MET 780, MET787, LEU 873, PHE 876, THR 877, LEU 880, MET 895, and ILE 899. The docking results were well supported by those in vitro showing that T. polium methanolic extract can be a new potential resource of natural antioxidant and anticancer compounds.

5. Conclusions

The overall hydrophobic nature of peptides identified in T. polium methanolic extracts coupled with the small size of peptides and high concentration of aromatic amino acids ascertain its antioxidant and anticancer characteristics. Further in vivo studies are necessary to confirm the pharmacological properties of the identified molecules in vivo as promising antioxidant and antitumor agents.

Author Contributions

Conceptualization, M.S., M.A. (Mousa Alreshidi), and V.D.F.; methodology, E.N., M.P., A.K., E.H.A., and S.E.; software, E.H.A., M.S.; validation, all authors; formal analysis, all authors; investigation, E.N., V.N.V., K.A., and A.K.; writing—original draft preparation, R.B., V.N.V., E.N., and S.E.; writing—review and editing, all authors; visualization, M.S. and V.D.F.; supervision, M.A. (Mousa Alreshidi), M.S.; project administration, M.A. (Mousa Alreshidi), M.S., and V.D.F.; funding acquisition, M.A. (Mousa Alreshidi). All authors have read and agreed to the published version of the manuscript.

Funding

This research has been funded by Scientific Research Deanship at University of Ha’il-Saudi Arabia through project number RG-191311.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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