Skip to main content
Download PDF

Phytochemical profile, comparative evaluation of Satureja montana alcoholic extract for antioxidants, anti-inflammatory and molecular docking studies

BMC Complementary Medicine and Therapies volume 23, Article number: 108 (2023) Cite this article

Abstract

Background

The most common species in the Satureja genus is Satureja montana (family Lamiaceae). The present work aims to use the molecular docking study to predict the isolated constituents against an immune system immunomodulator and tested alcoholic extract as an in-vitro immunomodulatory agent.

Methods

All isolated compound's structures were determined using various chromatographic and spectroscopic techniques. Anti-inflammatory and antioxidant profiles were studied for an alcoholic extract of the plant; the molecular docking study was performed for the isolated compounds (1–4).

Results

In this work, four recognized compounds were extracted from the ethyl acetate fraction of S. montana (Sm) methanolic extract and identified as two triterpenes ursolic acid (1) and oleanolic acid (2), one phenolic acid as ellagic acid (3), and one flavonoidal compound as isoscutellarein (4).

The total alcoholic extract exhibited good in vitro anti-inflammatory, antioxidant, and apoptotic activity. Its IC50 was 10.12 compared to 15.1 μg/ml of standard celecoxib. It also showed potent antioxidant activity with IC50 10.4, 11.3, 14.6, and 22.3 IU/ml for SOD, MDA, catalase, and TAC, respectively. According to the drug similarity and ADMET tests, their ligands may have favorable pharmacokinetic characteristics with minimal carcinogenic influence. The molecular docking study was performed for the isolated compounds (1–4).

Conclusions

The alcoholic extract of the plant showed promising anti-inflammatory, antioxidant, and antiapoptotic properties. The theoretical studies for the isolated compounds showed promising binding affinity against all the examined enzymes.

Peer Review reports

Background

The Lamiaceae family is an important medicinal plant family. It includes about 236 genera and more than 6000 species. This family has great variations which are widely distributed all over the world [1]. Satureja (family Lamiaceae) is a genus of over 200 aromatic herbs and shrubs endemic to the Middle East, with more than 30 species found in the eastern Mediterranean. They are utilized as culinary herbs as well as in traditional medicine to treat a wide range of ailments, like antimicrobial, spasmolytic, hepatoprotective, antiviral, and anticarcinogenic effects [2]. Also, antioxidative efficacy can affect the etiology of chronic diseases and the aging process [3]. It was reported from the previous phytochemical studies on some species of this genus, the presence of phenolic acids, flavones, anthocyanins, sterols, diterpenes, and triterpenes [4,5,6,7,8]. The most widespread species in this genus, Satureja montana, has been the focus of much research that has revealed the existence of numerous chemical components and biological activity. The existence of luteolin-7-rhamnoside-4'-O-glucopyranoside, quercetin-3-O-L-rhamnopyranoside, and quercetin7-O-glucopyranoside antibacterial activity against all tested microbes was demonstrated in recent research on S. montana by the authors [9]. Also, the previous study showed higher antioxidant activity (87.7%) for the alcoholic extract than that for the hexane fraction. Furthermore, the immunomodulator term is the substance that helps in regulating the immune system of an organism. Immunomodulators are divided into two types; those that increase the immune response (immunostimulants) and those that decrease the immune response (immuno-suppressants). Aside from the various agents that can aid in the regulation of an overactive or underactive immune system. Immunomodulators are frequently used to prevent organ rejection in autoimmune diseases such as rheumatoid arthritis and organ transplantation. In traditional systems of medicine, several plants used for vitality and long-term sickness prevention have been discovered to have immune system effects [10,11,12]. The present work aims to complete the isolation of other chemical constituents and biological activity of S. montana. Then use the molecular docking study to predict the isolated constituents against the immune system immunomodulator and tested alcoholic extract as an in-vitro immunomodulatory agent.

Material and methods

General experimental procedures

UV spectra were recorded on Shimadzu model UV-240 and 2401 PC spectrophotometer (Shimadzu Inc., Tokyo, Japan). NMR experiments were recorded on Bruker spectrometer (Switzerland) 600 (1H NMR spectra: 600 MHz; 13C NMR spectra: 150 MHz). The chemical shifts are given in δ (ppm) relative to tetramethylsilane (Me4Si) TMS. Column chromatography (CC) was carried out on silica gel, 60–200 mesh, Fluca, India) and Sephadex LH-20 (Pharmazia, Uppsala, Sweden). Paper chromatography (PC, descending) Whatman No. 1 and 3 mm papers, silica gel aluminum sheets (20 × 20) using solvent systems hexane: chloroform: methanol 90:8:2 (S-1), 15% HOAc(S-2), BAW (n-BuOH: HOAc: H2O) 3:1:1(S-3), and BAW 4:1:5, the upper layer (S-4).

Plant material

S. montana L. was cultivated and grown on the farm of the National Research Center (NRC), Giza, Egypt, and it was collected in April 2020. The authentication of plant samples was achieved by Dr. Mohammed Elgibali a taxonomist at NRC. A voucher specimen was deposited in the herbarium of the National Research Centre (accession number SC-1510). The aerial parts were air-dried for 2 weeks under laboratory conditions at 28 ± 2 °C. The dried material was ground using a domestic blender to a fine powder [9].

Isolation and purification of the chemical constituents

The powder of the aerial parts (2 kg) was extracted three times at room temperature with aqueous methanol (80%, 3 × 2.5 L). The methanolic extract was evaporated under reduced pressure to obtain a residue (250 g) which dissolved in hot distilled water (900 ml), and left-over night in the refrigerator and after then the precipitated matter was filtered off [9]. The aqueous filtrate was defatted with n-hexane (500 ml × 3), followed by a partition with ethyl acetate (400 ml × 3). The ethyl acetate extract (3.5 g) was chromatographed on a silica gel column (4 × 65). Elution was started up with n-hexane (2 L) with increasing the polarity by 20% stepwise addition of chloroform followed by methanol, 250 ml each were collected, and the elution was monitored using solvent S-1, the similar fractions were pooled together, and the solvents were evaporated under reduced pressure at 40◦C. Two main fractions were found promising, so they were subjected to further purifications as follows: Fraction -I (75 mg) eluted with hexane-chloroform (70–30) was found to contain two main compounds and further rechromatographed over a small silica gel column (1 × 25 cm) eluted with hexane-chloroform (80–20) to afford compounds 1 and 2 in pure form (18 mg and 24 mg, respectively). Fraction -II (80 mg, eluted with hexane-chloroform–methanol (50–50-10), was found to contain two main compounds (one flavonoidal compound and the other a phenolic acid) and was passed over Sephadex LH-20 column (1 × 25 cm) eluted with 100% methanol to afford compounds 3 and 4 together (Fraction -IIA). The fraction -IIA was subjected to preparative paper chromatography using Whatman 3 mm and solvent S-4 to give compounds 3 and 4 in pure form (20 mg and 25 mg, respectively).

Molecular modeling

Preparation of small molecule

The 3D structures for compounds (1:ursolicacid; CHEBI:9908), (2:oleanolic acid; CHEBI:37,659), (3: ellagic acid; CHEBI:5,281,855), (4: isoscutellarein; CHEBI:66,085), were gained from NCBI web server, USA (www.pubchem.ncbi.nlm.nih.gov). The chemical structures were optimized using the PM3 semi-empirical Hamiltonian molecular orbital calculation MOPAC16 package [13], then docked to receptors as implemented in MOE 2021 package [14].

Selection of proteins structures

A docking experiment was carried out for the target active sites of human immunomodulatory receptors such as NF-kappa B p52 (PDB:1A3Q) [15]. Proinflammatory cytokines such as interleukin-1 “IL-1”; (PDB:2NVH), interleukin-6 “IL-6”;(PDB:1P9M) [16], antioxidant activity as (PDB:1P9M). Viral interleukin-10 (PDB:1VLK), anti-inflammatory receptors such as cyclooxygenase-2 (PDB:1CX2) [17]. MOE, 2021 [14], was used for correcting errors of active sites by the structure preparation process in MOE. After the correction, hydrogens were added and partial charges (Amber12: EHT) were calculated. Energy minimization (AMBER12: EHT, root mean square gradient: 0.100) was performed.

Binding site analysis

The binding site of receptors was identified through the MOE. Site Finder program, which uses a geometric approach to calculate putative binding sites in a protein, starting from its tridimensional structure. This method is not based on energy models, but only on alpha spheres, which are a generalization of convex hulls. The prediction of the binding sites, performed by the MOE Site Finder module, confirmed the binding sites defined by the co-crystallized ligands in the holo-forms of the investigated proteins.

MOE stepwise docking method

The crystal structures of the enzymes were obtained. Water and inhibitors molecule was removed, and hydrogen atoms were added. The parameters and charges were assigned with MMFF94x force field. After alpha-site spheres were generated using the site finder module of MOE. The optimized 3D structures of molecules were subjected to generate different poses of ligands using the triangular matcher placement method, which generates poses by aligning ligand triplets of atoms on triplets of alpha spheres represented in the receptor site points, a random triplet of alpha sphere center was used to determine the pose during each iteration. The pose generated was rescored using London dG. scoring function. The poses generated were refined with MMFF94x force field, also, the solvation effects were treated. The Born solvation model (GB/VI) was used to calculate the final energy, and the final assigned poses were assigned a score based on the free energy in Kcal/mol. [18].

ADMET predictions

The ADMET in silico profile was applied using “MOE” and “admet SAR" tools to the prediction of pharmacokinetic and ADMET characters (absorption, distribution, metabolism, excretion, and toxicity).

In-vitro studies

In-vitro anti-arthritic study

The enzymatic assays of COXs, LOX, TAC, Catalase, MDA, GSH, SOD, TNF -α, and MPO were determined colorimetrically as mentioned previously in reports [19,20,21].

Lymphocyte proliferation activity using MTT reduction assay

The proliferation activity was determined by measuring mitochondrial activity using the MTT reduction method according to Mosaddegh et al., 2012 [22].

Statistical analysis

For the assays, linear regression was done for the calculation of IC50 (50% inhibitory concentration). Microsoft Excel 2010 program. All the data in Table 4 was represented as mean ± SD [23].

Results

All the isolated compounds were obtained from the fraction of ethyl acetate S.m aerial parts methanolic extract, twelve compounds were isolated and discovered; eight of them were isolated before by authors [9], and the other isolated four compounds were reported here and identified as follow (Fig. 1).

Fig. 1
figure 1

Compounds isolated from aerial parts of S.m

Full size image

Ursolic acid (1) The compound was obtained from a silica gel column after further purification as an off-white powder (18 mg), The UV spectrum displayed λ max (MeOH) at 438, and 419 nm and EI-MS gives a peak of molecular ion M+ at m/z = 456 for this formula C30H48O3. The 1H-NMR spectrum was recorded at 600 MHz in CDCl3 which gave different signals at d in ppm = 0.73, 0.78, 0.79, 0.93, 0.94, 0.95, and 1.15 (7 s, 21H, due to CH3 groups), The other protons have appeared as 1.38 (m, 2H, H-21), 1.39 (m, 2H, H-16), 1.44 (m, 2H, H-20), 1.52 (m, 4H, H-18, H-19, and H-15), 2.10 (m, 3H, H-1, and H-9), 2.14 (m, 2H, H-14), 3.18 (t, 2H, J = 7 Hz, H-2), 3.39 (s, 2H, H-7), 4.57 (s, 2H, H-11), 4.59 (s, 1H, H-12). While the 13C-NMR data (150 MHz, CDCl3): δ (ppm) at (from C-1 to C-30) 39.3, 28.2, 78.2, 38.3, 55.9, 18.7, 33.8, 40.1, 48.2, 37.6, 23.7, 125.7, 139.4, 42.6, 28.8, 25.0, 48.2, 53.6, 39.5, 39.5, 31.2, 37.5, 28.8, 16.5, 15.6, 17.6, 24.1, 179.7, 17.6, 21.5. all these data were followed and reported for a triterpene compound which was identified as ursolic acid [24].

Oleanolic acid (2) The compound was found as a white powder (24 mg), UV λ max (MeOH) 209 nm. The 1H -NMR spectrum (600 MHz, CDCl3) proved the presence of the following signals: 0.79, 0.89, 0.91, 0.92, 0.97, 1.08, and 1.34 (7 s, 21H, all-CH3), 1.37 (m, 2H, H-21), 1.37 (m, 2H, H-16), 1.52 (m, 5H, H-18, H-19, and H-15), 2.08 (m, 3H, H-1, and H-9), 3.16 (t, 1H, J = 7 Hz, H-2), 3.36 (s, 2H, H-7), 4.58 (s, 2H, H-11), 4.61 (s, 1H, H-12). The 13C-NMR spectral data (150 MHz, CDCl3): at δ (ppm) were assigned as follows: from C-1 to C-30 appeared at 39.4, 28.4, 78.3, 39.4, 55.9, 18.7, 33.3, 39.9, 48.3, 37.5, 23.8, 122.7, 144.9, 42.8, 28.5, 23.8, 46.8, 42.2, 46.6, 31.2, 34.2, 33.4, 28.8, 16.6, 15.7, 17.6, 26.4, 180.3, 33.4, 23.9. There is a good agreement between the obtained data and the data reported by Werner et al., 2003 [25], for the compound which was identified as oleanolic acid.

Ellagic acid (3) The compound was obtained as a white amorphous powder (20 mg) after multi-purification Sephadex -LH20 column. The UV spectral data λ max (MeOH) 250 nm, the 1H-NMR spectrum (600 MHz, CDCl3) showed one signal at δ (ppm) at: 7.39 (2H, s, H-4,9) and other signals at 10.53 (s, 4H, -OH groups). The 13C-NMR (150 MHz, DMSO-d6) data showed signals at δ (ppm) as 158.5 (C = O, 5, 10), 147.9 (C-3, 8), 139.7(C-2,7), 136.9 (C-1a, 6a), 112.6 (C-4b,9b), 110.9 (C-4, 9), 107.5 (4a, 9a). So, this compound was identified as ellagic acid [26].

Isoscutellarein (4) The compound was found as a yellow powder (25 mg) and it is an aglycone in nature according to its chromatographic behavior on paper in different solvents. The UV spectral data λmax(nm) in MeOH: 269, 338 indicate the flavone type of this compound. The bathochromic shift in band-I with increasing intensity on the addition of NaOMe: (255, 401) proves the presence of a free OH group at C-4'; (AlCl3): 267, 287, 401; (AlCl3/HCl): 267, 307, 355; while the bathochromic shift in band-II (5 nm) on the addition of NaOAc (274, 369) substantiate the occurrence of a group of free OH at C-7 and (NaOAc/H3BO3): 274,374 [27].

The 1H-NMR (600 MHz, DMSO-d6,) spectrum displayed signals at δ (ppm)7.62 (2H, d, J = 8.5 Hz, H-2', 6'), 6.81 (2H, d, J = 8.5 Hz, H-3', 5'), The two aromatic singlets at 6.74 and 6.52 were assigned to H-3 and H-6, respectively. The appearance of a single signal at δ 6.62 ppm of H-6 more downfield than normally indicated that the carbon (C-8) is oxygenated. While the 13C-NMR (150 MHz, DMSO-d6) at δ in ppm 182.3 (C-4), 163.8 (C-2), 162.3 (C-4), 161 (C-9), 155 (C-5), 161.4 (C-7), 128.9 (C-2, 6), 128.7 (C-8), 121.5 (C-1), 116.8 (C-3, 5), 103.4 (C-10), 103.8 (C-3), 98.7 (C-6). These data supported the identification of compound 4 as isoscutellarein [28].

Toxicological study in silico

Pharmacokinetic profile in silico

In silico cytotoxicity screening utilizing ADMET parameters is a critical step in the creation of therapeutic bioactive compounds. The descriptors for 1–4 were generated using MOE, Swiss ADMET [29], and the admit-SAR model [30], as shown in Table 1. Compounds 1–4's physicochemical and ADMET properties revealed that they met Lipinski's criteria, with molecular weight as an exception. Furthermore, the tested compounds fail when the Ghose Veber, Egan, and Muegge criteria are used. Consequently, the tested compounds exhibited a range of bioavailability scores (0.85- 0.17) with biodegradation range values (0.78 to 0.96). These data revealed that the compounds tested had a high oral bioavailability but little biodegradability. The Bioavailability Radar planner created a lead-likeness profile for the medication under consideration (Fig. 2). Pink denotes the ideal range for each feature (polarity, size, lipophilicity, solubility, saturation, and flexibility).

Table 1 ADMET data for compounds 1–4
Full size table
Fig. 2
figure 2

Bioavailability Radar plot of 1–4, respectively. Each attribute's optimum range is represented by the pink area (Lipophilicity: 0.7 XLOGP3 + 5.0, size: 150 MW 500 g/mol, polarity: TPSA 140 2, solubility: log S 6, and flexibility: rotatable bonds 9. The white zone represents very probable HIA (GI) absorption, whereas the yellow zone shows very likely BBB permeability. The outer grey region is made up of compounds that are expected to have poor absorption but no brain penetration. Furthermore, the blue point indicates P-gp substrate (PGP +), whereas the red point shows P-gp non-substrate (PGP-)

Full size image

Prediction of oral toxicity

The possible toxicity was predicted [31] by utilizing data from the Chemical European Biology Laboratory (ChEMBL) database to calculate an estimate of rodent oral toxicity. Furthermore, we determined the median fatal dosages (LD50) in rats, and our ligands had a high LD50. This database's calculated toxicity is based on the highest endpoints, which comprise thirty-three models (Table 1). The estimated toxicity was divided into different stages of toxicity as “toxicity, toxicological endpoints (mutagenicity, carcinotoxicity, organ toxicity (hepatotoxicity), cytotoxicity and immunotoxicity). Toxicological pathways and toxicity targets thereby, providing an appropriate vision into the probable mode of action including the toxic rejoinder. These compounds 1–4 are categorized as low toxicity classes III and do not include any dangerous components or bind to any toxic objectives. In general, the medicines investigated, exhibited no main health effects, and high oral bioavailability, high BBB transport capacity for rodent toxicity profiles.

In silco detection of immunomodulatory targets

To identify the biological activity of the isolated compounds we targeted active sites of human immunomodulatory receptors such as NF-kappaB p52 (PDB:1A3Q) at 2.1 A° resolution, Proinflammatory cytokines such as interleukin-1 “IL-1”; (PDB:2NVH [32],) at 1.35 A° resolution, viral interleukin-10 (PDB:1VLK [33]) at 1.09 A° resolution, anti-inflammatory receptor such as cyclooxygenase-2 (PDB:1CX2) at 3.00 A° resolution and antioxidant activity as (PDB:1P9M(at 3.65 A° resolution). The gold score function, which was included in MOE 2021, was used to calculate the behavior of the ligand-protein interaction. All docking experiment calculations were shown in (Table 2). The crystal structures were derived from the protein databank. The ligands under consideration docked into the active site for these enzymes.

Table 2 Docking energy scores (Kcal/mol) for isolated compounds (1–4)
Full size table

The docking protocol was affirmed before estimating docking calculations by the self-docking of identified compounds with targeted enzymes in their original binding pocket viaX-ray. The obtained pose was subsequently equated to that of the X-ray structures obtained experimentally. For the isolated compounds, the root means square deviation (RMSD) was lower than 2Å. This viable docking protocol was then used to expeled docking calculations for the test ligands. Furthermore, calculation of the molecular dynamic as with ordinary procedures using MOE (Table 2). All enzymes by compared with molecular docking displayed more healthier molecular dynamic free energy. In addition, the applied molecular dynamics had no effect on the bond formation.

Biological study

In-vitro anti-inflammatory effect of extracts on (COX-1, COX-2, 5-LOX, and MPO)

To investigate the effects on COX-1, COX-2, and 5-LOX, the plant material was extracted with 80% aqueous methanol. The extract was evaluated for anti-inflammatory activity using COX-1, COX-2, 5-LOX, and MPO inhibition assays, which are the major mediators of inflammation (Table 3). The total extract showed the higher anti-inflammatory potency against reference drug with the lower IC50 value for COX-2 (0.17 μg/ml) and LOX (9.25 μg/ml), MPO (9.50 ng/ml), followed by COX-1 (10.21 μg/ml).

Table 3 IC50 values of the anti-inflammatory activity of Satureja montana alcoholic extract
Full size table

In-vitro antioxidant assay of extracts on (TAC, Catalase, MDA, and SOD)

Total antioxidant capacity is the measure of the number of free radicals scavenged by a test solution, being used to evaluate the antioxidant capacity of biological samples. The assay showed that the lowest IC50 values are 10.4 IU/ml and 11.3 ng/ml for SOD and MDA, respectively, followed by Catalase (14.6 IU/ml), then TAC (22.3 IU/ml). The standard ascorbic acid results were (19.7, 17.2 and 13.1 IU/ml) for TAC, Catalase and SOD, respectively.

In-vitro apoptotic Assay of extract (GSH and TNFα)

Excessive apoptosis in the testis has been related to Leydig cell damage and a decrease in testosterone levels in the blood. Furthermore, an increase in the death of Leydig cells results in a decrease in testosterone. The apoptotic extract's assays (GSH & TNFα) were done. The extract showed good IC50 values of 16.41 pg/ml for TNFα in comparison to 23.1 pg/ml for (GSH). These findings indicate that the extract possesses antiapoptotic properties.

Cell viability measurement

The MTT assay was performed to evaluate the effect of the extract on cell viability in mouse peritoneal macrophage cells in vitro. At whatever dosage of the treatment, no significant difference in the percent lives cell population was seen as compared to normal cells (Table 4).

Table 4 Immuno-modulatory using MTT results, data expressed as mean ± SD
Full size table

Discussion

The first look of 1–4 molecules showed variations such as saturation with Csp = 0.9 for 1, solubility and lipophilicity for 2 and 3; and polarity for 4, respectively (Fig. 2). Both compounds (2 and 3) exhibited a high saturation degree in the area (Fraction Csp3 = 0.9- 0.29), which allowed them to pass the Fraction Csp30.25 filter [34]. When applied Logs [35], these compounds exhibited excellent solubility in H2O, which is a critical factor in absorption. Lipophilicity criteria in medicinal chemistry were also used to detect hazardous regions in bioactive compounds as a lead-likeness filter, depending on two structural warnings known as PAINS and Brenk's filters. No structural warnings for compounds 1–3 against the PAINS filter, but compound 4 showed one alert.

Using admet-sar pharmacokinetic characteristics, mutagenicity, tumorigenicity, reproductive efficacy, irritability, and human intestine absorption were investigated in silico [36]. In addition, the other (SVM) method was used to determine the substrate or non-substrate of skin permeability (Log Kp), Caco-2, blood–brain barrier (BBB), and p-glycoprotein (P-gp), as well as to discover inhibitory influence on the main cytochromes P450 isoenzymes (CYP1A2, CYP2C19, CYP2C9, CYP2D6, CYP3A4). The obtained results of (Table 1) indicated that the isolated 1–4 inhibited both CYP2C9 and CYP3A4 but had no inhibitory effect against any other P450. The BOILED-Egg model is presented in (Fig. 2) as a connection for WLOGP versus TPSA, indicating that, the human Ether-à-go-go-Related Gene (hERG) against High BBB permeability, high GI absorption, high brain penetration, and ambiguous inhibitory action. Furthermore, the compounds (1–4) presented P-gp activation (multidrug resistance protein 1). Furthermore, negative skin permeability (Kp) values for isolated compounds revealed that all compounds exhibited low skin permeability. The carcinogenic behavior of 1–4 compounds was investigated and compared with 981 other carcinogenic chemical structures selected from the “Carcinogenic Potency Database (CPDB) [37], with the findings indicating that the tested compounds had no carcinogenicity, mutagenicity, or tumorigenicity effects.

The enzymes' active sites were identified as residues within a 3.5 radius of the atoms of reference medications. The molecular docking was carried out with the use of an (AMBER12:EHT) force field with a gradient convergence range of 0.05 kcal/mol, which was utilized to decrease the energy for the produced ligand-enzyme complexes. To evaluate the tested ligand binding affinity, the highest MOE scoring function was employed (Table 2).

In the case of 1VLK

The 1–4 ligands revealed MOE score arranged as 2>4>3>1(-6.1, -5.8, -5.7, and -4.6 kcal/mol), respectively (Table 2). The 1–4 hybrids occupied the binding pocket by the formation of H-bond interactions with important amino acids Gly61 and Phe111 (Fig. 3). It was hypothesized that hydrophobicity and membrane permeability are important pharmacokinetic characteristics for absorption compounds in biological systems due to their strong interaction with hydrophobic residues of the binding site.

Fig. 3
figure 3

Interaction between the tested compounds (1–4) with binding sites of tested enzymes

Full size image

In the case of 1A3Q

The binding affinities were arranged as 4>1>3>2 (-8.1, -6.1, -3.4, and -2.4 kcal/mol), respectively (Table 2). These clusters localized in the binding site through H-bond interactions with important amino acids Lys252 & Lys283. The interaction mode of tested compounds with nuclear factor NF-kappa B p52 demonstrated that, the vital role of hydrophilicity in binding interaction.

In the case of 2NVH

Compound 4 has the highest binding affinity(-6.28 kcal/mole) when compared with all tested compounds 2–4. That showed nearly the same binding scores (-5.4, -5.01, and -5.4 kcal/mol), respectively for 2–3 (Table 2). These compounds formed H-bond interactions with important amino acids Gly25 Lys74 The 79, phe 133, and Gly138.

In the case of 1CX2

The binding affinities were arranged as 4> 3 > 2>1 These clusters localized in the binding site through H-bond interactions with important amino acids Ala52 Arg120 Tyr355 and Ser530.

In the case of 1P9M

The 1–4 ligands revealed MOE scores arranged as 4>3>2>1 respectively (Table 2). All compounds showed nearly the same binding affinity of about -6 kcal. /mol. The 1–4 hybrids occupied binding pockets by formation H-bond interactions with Lys66, Gln135, Arg168, and Gln 135, Fig. 3.

Inhibition of the remarkable proinflammatory mediators with the plant secondary metabolites are significant in recent years. The mediators act simultaneously, to initiate and endure the inflammatory cascade [38]. From plants, the pure compounds and crude extracts, aim these mediators and flagged the way for evolution of a new therapeutic accession [39]. The high phenolic content of S. montana, exhibited reducing power and high free radical-scavenging properties [3, 9].

The in vitro biological activity of S. montana alcoholic extract, displayed a good antioxidant and anti-inflammatory activity in addition to apoptotic activity. IC50 for COX1 & 2 were 10.21 and 0.17 µg/ml in comparison to 15.1 and 11.6 µg/ml for celecoxib (standard). TAC of the total alcoholic extract in our results was 22.3 IU/ml compared to 19.7 IU/ml of the standard ascorbic acid.

Catalase is a unique enzyme, which protect cells from oxidative damage and play a vital role, with catalyzing the breakdown of hydrogen peroxide into water and oxygen, forming noticed, efficient and specific complex chemical reactions. Catalase IC50 value was (14.6 IU/ml), compared to 17,2 IU/ml of the standard ascorbic acid.

Superoxide dismutase is also an enzyme, has a special role in catalyzing the dismutation of superoxide radicals into oxygen and hydrogen peroxide for protecting cells from oxidative stress. In additition to keep normal physiological processes, so its deficiency led to different pathological conditions, such as neurodegenerative disorders, cancer, and aging. SOD IC50 value was (10.4 IU/ml), compared to 13,1 IU/ml of the standard ascorbic acid. These results coincide with previous studies that reported, the presence of flavonoids, phenolic acids, alkaloids, and terpenoids in plants modulate SOD activity relying on their concentration and chemical structure [40].

The reduced glutathione is a potent antioxidant, that neutralize ROS, in addition to other free radicals, which produced through metabolic processes. GSH act as a cofactor for different enzymes such as glutathione peroxidase and glutathione S-transferase in detoxification pathways. GSH IC50 value was (23.1 pg/ml), which indicating that the extract possesses antiapoptotic properties. These results indicated that Satureja montana alcoholic extract possess a remarkable antioxidant effect. In addition, it also lowers MDA level to 11.3ug/ml compared to 15.3 for standard ascorbic acid [41].

Conclusions

The most common species in the Satureja genus is Satureja montana (family Lamiaceae). Four recognized substances were extracted and identified from the ethyl acetate fraction of S. montana methanolic extract in this investigation. The anti-inflammatory and antioxidant profiles of the alcoholic extract, as well as the molecular docking of the isolated compounds, were investigated, and it was discovered that they had potential anti-inflammatory and antioxidant capabilities. The drug investigations, such as similarity and ADMET, suggested that their ligands may have a favorable pharmacokinetic profile with no carcinogenic impact. The isolated compounds 1–4 were subjected to a molecular docking analysis, which revealed promising binding affinity against all enzymes. As a result, these compounds show promise as antioxidants, anti-inflammatory agents, and anticancer agents. Theoretical investigations were supplemented with experimental data.

Availability of data and materials

All data generated or analysed during this study are included in this published article.

References

  1. Duggan KC, Hermanson DJ, Musee J, Prusakiewicz JJ, Scheib JL, Carter BD, Banerjee S, Oates JA, Marnett LJ. (R)-Profens are substrate-selective inhibitors of endocannabinoid oxygenation by COX-2. Nat Chem Biol. 2011;7(11):803–9.

    Article  PubMed  PubMed Central  Google Scholar 

  2. Ćavar S, Šolić ME, Maksimović M. Chemical composition and antioxidant activity of two Satureja species from Mt. Biokovo Botanica Serbica. 2013;37(2):159–65.

    Google Scholar 

  3. Hassanein HD, Said-AlAhl HAH, Abdelmohsen MM. Antioxidant polyphenolic constituents of Satureja montana L. growing in Egypt. Int J Pharm Pharm Sci. 2014;6(4):578–81.

    Google Scholar 

  4. Zgórka G, Głowniak K. Variation of free phenolic acids in medicinal plants belonging to the Lamiaceae family. J Pharm Biomed Anal. 2001;26(1):79–87.

    Article  PubMed  Google Scholar 

  5. Pedersen JA. Distribution and taxonomic implications of some phenolics in the family Lamiaceae determined by ESR spectroscopy. Biochem Syst Ecol. 2000;28(3):229–53.

    Article  CAS  Google Scholar 

  6. Ferreres F, Barberán FAT, Tomas F. Free flavone aglycones and flavonoid glycosides from Satureja obovata. Int J Crude Drug Res. 1987;25(4):246–50.

    Article  CAS  Google Scholar 

  7. Alvarenga SAV, Gastmans JP, do Vale Rodrigues G, Moreno PRH, Emerenciano V. A computer-assisted approach for chemotaxonomic studies - diterpenes in Lamiaceae. Phytochemistry. 2001;56(6):583–95.

    Article  Google Scholar 

  8. Gouhari AR, Haji AA, Saeidinia S, Shafiei A, Ebrahimi ES. Cytotoxic terpenoids from Satureja macrantha CA-Mey. Daru. 2005;13(4):177–80.

    Google Scholar 

  9. El-Hagrassi AM, Abdallah WE, Osman AF, Abdelshafeek KA. Phytochemical study of bioactive constituents from Satureja montana L. growing in Egypt and their antimicrobial and antioxidant activities. Asian J Pharm Clin Res. 2018;11:142–8.

    Article  Google Scholar 

  10. Fedson DS. Confronting the next influenza pandemic with anti-inflammatory and immunomodulatory agents: why they are needed and how they might work. Influenza Other Respir Viruses. 2009;3(4):129–42.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Chandrashekar PM, Prashanth KVH, Venkatesh YP. Isolation, structural elucidation, and immunomodulatory activity of fructans from aged garlic extract. Phytochemistry. 2011;72(2–3):255–64.

    Article  CAS  PubMed  Google Scholar 

  12. Ragupathi G, Yeung KS, Leung PC, Lee M, San Lau CB, Vickers A, Hood C, Deng G, Cheung NK, Cassileth B. Evaluation of widely consumed botanicals as immunological adjuvants. Vaccine. 2008;26(37):4860–5.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Stewart JJ. Optimization of parameters for semiempirical methods VI: more modifications to the NDDO approximations and re-optimization of parameters. J Mol Model. 2013;19(1):1–32.

    Article  CAS  PubMed  Google Scholar 

  14. Molecular Operating Environment (MOE), 2019.01; Chemical Computing Group ULC, 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2021.

  15. Sobieszczanski-Sobieski J, Kodiyalam S, Yang RY. Optimization of car body under constraints of noise, vibration, and harshness (NVH), and crash. Struct Multidiscip Optim. 2001;22(4):295–306.

    Article  Google Scholar 

  16. Boulanger MJ, Chow DC, Brevnova EE, Garcia KC. Hexameric structure and assembly of the interleukin-6/IL-6 α-receptor/gp130 complex. Science. 2003;300(5628):2101–4.

    Article  CAS  PubMed  Google Scholar 

  17. Kurumbail RG, Kiefer JR, Marnett LJ. Cyclooxygenase enzymes: Catalysis and inhibition. Curr Opin Struc Biol. 2001;11(6):752–60.

    Article  CAS  Google Scholar 

  18. Molecular operating environment (MOE), Chemical Computing Group Inc 1010 Sherbooke St. West, Suite# 910, Montreal, 2016.

  19. Copeland RA, Williams JM, Giannaras J, Nurnberg S, Covington M, Pinto D, Pick S, Trzaskos JM. Mechanism of selective inhibition of the inducible isoform of prostaglandin G/H synthase. Proc Natl Acad Sci. 1994;91(23):11202–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Egan RW, Paxton J, Kuehl FA. Mechanism for irreversible self-deactivation of prostaglandin synthetase. J Biol Chem. 1976;251(23):7329–35.

    Article  CAS  PubMed  Google Scholar 

  21. Reddanna P, Whelan J, Maddipati KR, Reddy CC. Purification of arachidonate 5-lipoxygenase from potato tubers. Methods Enzymol. 1990;187:268–77.

    Article  CAS  PubMed  Google Scholar 

  22. Mosaddegh M, Esmaeili S, Naghibi F, Hamzeloo Moghadam M, Haeri A, Pirani A, Moazzeni H. Ethnomedical survey and cytotoxic activity of medicinal plant extracts used in Kohgiluyeh and Boyerahmad Province in Iran. J Herbs Spices Med Plants. 2012;18(3):211–21.

    Article  Google Scholar 

  23. Casanova E, Valdes AE, Zuker A, Fernandez B, Vainstein A, Trillas MI, Moysset L. Plant Sci. 2004;176:551–60.

    Article  Google Scholar 

  24. Babalola IT, Shode FO. Ubiquitous ursolic acid: a potential pentacyclic triterpene natural product. J Pharmacogn Phytochem. 2013;2(2):214–22.

    CAS  Google Scholar 

  25. Werner S, Nebojsa S, Robert W, Robert S, Olaf K. Complete assignments of 1H and 13C-NMR resonances of oleanolic acid, 18α-oleanolic acid, ursolic acid and their 11-oxo derivatives. Magn Reson Chem. 2003;41:636–8.

    Article  Google Scholar 

  26. Rashed K, Ono L. Phytochemical, cytotoxic, anti-HSV-1 (Herpes Simplex Virus type-1) and anti bacterial studies of Terminalia laxiflora Engl. and, Diels. Hygeia J D Med. 2013;5(2):76–86.

    Google Scholar 

  27. Markham KR. Techniques of flavonoid identification. London: Academic press; 1982.

    Google Scholar 

  28. Graham TL. Flavonoid and flavonol glycoside metabolism in Arabidopsis. Plant Physiol Biochem. 1998;36(1–2):135–44.

    Article  CAS  Google Scholar 

  29. Yang H, Lou C, Sun L, Li J, Cai Y, Wang Z, Li W, Liu G, Tang YJB. admetSAR 2.0: web-service for prediction and optimization of chemical ADMET properties. Bioinformatics. 2019;35(6):1067–9.

    Article  CAS  PubMed  Google Scholar 

  30. Ritchie TJ, Ertl P, Lewis R. The graphical representation of ADMET-related molecule properties for medicinal chemists. Drug Discov Today. 2011;16(1–2):65–72.

    Article  CAS  PubMed  Google Scholar 

  31. Zhao YH, Abraham MH, Le J, Hersey A, Luscombe CN, Beck G, Sherborne B, Cooper I. Rate-limited steps of human oral absorption and QSAR studies. Pharm Res. 2002;19(10):1446–57.

    Article  CAS  PubMed  Google Scholar 

  32. Zhao Y, Zheng X, Zhang H, Zhai J, Zhang L, Li C, Zeng K, Chen Y, Li Q, Hu X. In vitro inhibition of AKR1Cs by sulphonylureas and the structural basis. Chem Biol Interact. 2015;240:310–5.

    Article  CAS  PubMed  Google Scholar 

  33. Banerjee P, Eckert AO, Schrey AK, Preissner R. ProTox-II: a webserver for the prediction of toxicity of chemicals. Nucleic Acids Res. 2018;46(W1):257–63.

    Article  Google Scholar 

  34. Cheng F, Li W, Zhou Y, Shen J, Wu Z, Liu G, Lee PW, Tang Y. admetSAR: a comprehensive source and free tool for assessment of chemical ADMET properties. J Chem Inf Model. 2012;52(11):3099–105.

    Article  CAS  PubMed  Google Scholar 

  35. Bento AP, Gaulton A, Hersey A, Bellis LJ, Chambers J, Davies M, Kruger FA, Light Y, Mak L, McGlinchey S, Nowotka M, Papadatos G, Santos R, Overington JP. The CHEMBL bioactivity database: an update. Nucleic Acids Res. 2014; 42(Database issue):D1083-90.

  36. Quillin ML, Wingfield PT, Matthews BW. "Determination of solvent content in cavities in IL-1β using experimentally phased electron density. Proc Natl Acad Sci. 2006;103(52):19749–53.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Zdanov A, Schalk-Hihi C, Menon S, Moore KW, Wlodawer A. Crystal structure of Epstein-Barr virus protein BCRF1, a homolog of cellular interleukin-10. J Mol Biol. 1997;268(2):460–7.

    Article  CAS  PubMed  Google Scholar 

  38. Lee J, Ree J, Kim HJ, Kim HJ, Kim WJ, Choi TG, Lee S, Hong YK, Hong SB, Park Y. Anti-apoptotic and Anti-inflammatory effects of an ethanolic extract of Lycium chinense root against particulate matter 10-induced cell death and inflammation in RBL-2H3 basophil cells and BALB/c mice. Plants. 2022;11:2485.

    Article  PubMed  PubMed Central  Google Scholar 

  39. Deghima A, Righi N, Daoud I, Ansorena D, Astiasar′an I, Bedjou F. Fatty acid composition, acute toxicity and anti-inflammatory activity of the n-hexane extract from Ranunculus macrophyllus Desf. roots. S Afr J Bot. 2022;148:315–25.

    Article  CAS  Google Scholar 

  40. Wang Y, Branicky R, Noë A, Hekimi S. Superoxide dismutases: dual roles in controlling ROS damage and regulating ROS signaling. J Cell Biol. 2018;217:1915–28.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Cullen JJ, Mitros FA, Oberley LW. Expression of anti-oxidant enzymes in diseases of the human pancreas: Another link between chronic pancreatitis and pancreatic cancer. Pancreas. 2003;26:23–7.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors are thankful to National Research Centre, Egypt, for providing the appropriate facility to conduct the research activities.

Funding

Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). This work was supported by own.

Author information

Authors and Affiliations

  1. Chemistry of Medicinal Plants Department, Pharmaceutical and Drug Industries Research Institute, National Research Centre, 33 El Buhouth St. (Former El Tahrir St.), 12622-Dokki, Giza, Egypt

    Khaled Abdelhady Abdelshafeek & Walid Elsayed Abdallah

  2. Chemistry of Natural Compounds Department, Pharmaceutical and Drug Industries Research Institute, National Research Centre, 33 El Buhouth St. (Former El Tahrir St.), 12622-Dokki, Giza, Egypt

    Abeer Fouad Osman

  3. Department of Pharmacology, Faculty of Veterinary Medicine, Cairo University, Cairo, 12211, Egypt

    Samar Mohamed Mouneir

  4. Chemistry Department, Faculty of Science, Al-Azhar University, 11884 Nasr City, Cairo, Egypt

    Ahmed Abdelmonem Elhenawy

Authors
  1. Khaled Abdelhady Abdelshafeek
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Abeer Fouad Osman
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Samar Mohamed Mouneir
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ahmed Abdelmonem Elhenawy
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Walid Elsayed Abdallah
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

K.A. designed the study, and then reviewed the manuscript. A.F. investigation, analyzed and interpreted the data, edited the manuscript and contributed to actual laboratory work. S.M. investigation and performed the biological studies, drafted manuscript. A.A. conduct the theoretical study of chemical compounds. W.E. contributed to actual laboratory work, analyzed data, reviewed and edited the manuscript. All the authors have read and approved the final manuscript.

Corresponding author

Correspondence to Walid Elsayed Abdallah.

Ethics declarations

Ethics approval and consent to participate

All methods were performed in accordance with the relevant guidelines and regulations by the Institutional Ethics Committee, National Research Centre in Egypt.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing of interests.

Additional information

Publisherʼs Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Abdelshafeek, K.A., Osman, A.F., Mouneir, S.M. et al. Phytochemical profile, comparative evaluation of Satureja montana alcoholic extract for antioxidants, anti-inflammatory and molecular docking studies. BMC Complement Med Ther 23, 108 (2023). https://doi.org/10.1186/s12906-023-03913-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12906-023-03913-0

Keywords

BMC Complementary Medicine and Therapies

ISSN: 2662-7671

Contact us