Skip to main content
Download PDF

Isolation and structure elucidation of the compounds from Teucrium hyrcanicum L. and the investigation of cytotoxicity, antioxidant activity, and protective effect on hydrogen peroxide-induced oxidative stress

BMC Complementary Medicine and Therapies volume 23, Article number: 447 (2023) Cite this article

Abstract

Background

Teucrium hyrcanicum L. (family Lamiaceae) is widely distributed in the North and Northwest of Iran. It has been used in the form of tea, tonic, and tincture for the treatment of various diseases such as cough, rheumatism, and fever.

Methods

In this study, the total phenolic and flavonoid contents, antioxidant and cytotoxic activities of methanol extract and different fractions of T. hyrcanicum were measured. Furthermore, the potential ability of T. hyrcanicum to protect against H2O2-induced oxidative stress was tested on the NIH3T3 cell line. Then, the isolation and structure elucidation of the compounds were performed on the most potent fractions. Finally, the quantification of isolated compounds in methanol extract (ME) was done by the HPLC method. Isolated phytochemicals were assessed for the cytotoxic and antioxidant activities.

Results

The results indicated that the methanol fraction (MF) had the highest amount of phenolic and flavonoid contents (69.36 mg GAE/g extract and 68.95 mg QE/g extract). The highest radical scavenging activities were observed from MF and ME (IC50 44.32 and 61.12 μg.ml−1, respectively). The best cytotoxicity was obtained by ethyl acetate fraction (EF) against A431 and MCF7 cell lines (IC50 values of 235.4and 326.6 μg.ml−1, respectively). The pretreatment with MF exerts the highest reduction in malondialdehyde (MDA) formation (IC50 2.51 μM, p < 0.001) compared to the H2O2 group (5.77 μM). Also, MF significantly inhibited H2O2-induced Glutathione (GSH) oxidation (p < 0.001). Furthermore, two phenolic compounds, acteoside and quercetin, were isolated and identified in MF and EF, respectively. The IC50 values of acteoside and quercetin in the DPPH assay were 7.19 and 5.56 µg.ml−1, respectively. Both quercetin and acteoside significantly reduced the MDA formation and inhibited GSH oxidation, which was comparable with BHA (as a standard antioxidant) (p < 0.05). Acteoside demonstrated significant cytotoxicity against all tested cell lines (IC50 = 32 to 145 μg.ml−1). The HPLC quantification of isolated compounds revealed that the quantity of acteoside and quercetin in ME were 93.31 and 16.87 μg.mg−1, respectively.

Conclusion

The isolated compounds (quercetin and acteoside) had significant antioxidant activities and revealed a protective effect on H2O2-induced oxidative stress which was comparable with BHA.

Peer Review reports

Background

Oxidative stress is one of the main reason for free radical formation in food, drugs, and living organisms [1, 2]. It was demonstrated that exposure to free radicals such as superoxide anion radical (O•2−), hydroxyl radical (•OH), and peroxyl radical (ROO•) can lead to lipid peroxidation, protein denaturation, DNA oxidation, strand breakage, and platelet dysfunction [1,2,3,4]. These destructive effects can potentially lead to cancer, cardiovascular abnormalities, Alzheimer’s disease, fibrosis, kidney and liver disease, arthritis, atherosclerosis, neurodegenerative disorders, and aging [1]. Also, lipid peroxidation is the primary cause of food deterioration and production of unwanted compounds during processing and storage [5]. The natural antioxidants, found in medicinal and dietary plants, fruits, and vegetables, can prevent oxidative damage and provide health benefits by scavenging reactive free radicals [2]. Various bioactive phytochemicals, such as vitamins, polyphenols, terpenoids, polysaccharides, and alkaloids, have been proven to have potential antioxidant effects [6,7,8,9,10]. The growing interest in using natural antioxidants was even more highlighted when researchers indicated that the most commonly used synthetic antioxidants like butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) were suspected to have genotoxic and carcinogenic effects [1, 2]. Phenolic phytochemicals (e.g., phenolic acids, flavonoids, quinones, coumarins, lignans, stilbenes, and tannins) can inhibit the oxidative process by scavenging free radicals and reacting oxygen species (ROS). The mechanism of this action is related to their aromatic structures and highly conjugated system with several hydroxyl groups, which can act as good electron or hydrogen atom donors [11]. There are pieces of evidence that indicate phenolic compounds such as flavonoids (as free radical scavengers) can exert antitumor activities by inducing apoptosis as well as inhibiting invasion and metastasis in various cancer cell lines [12, 13].

The genus Teucrium (family Lamiaceae) is a widespread genus with about 340 different species [14,15,16]. Many of them are well-known for valuable therapeutic properties and have been used in the form of tea, tonic, and tincture for the treatment of diseases such as cough, rheumatism, diarrhea, fever, diabetes, antiseptic, anthelmintic, and flatulence [14, 15, 17]. So far, different phytochemicals have been reported from species of this genus, including monoterpenes, polyphenols, flavonoids (such as apigenin and rutin), sesquiterpenes, fatty acids, steroids (such as β-sitosterol and stigmasterol), and iridoids [14, 18]. Moreover, numerous biological activities like antimicrobial, antioxidant, antipyretic, anti-inflammatory, antiulcer, antimalarial, anti-allergic, anthelmintic, and cytotoxic activities have been demonstrated [14]. This genus comprises 12 species in the flora of Iran [19], and among them, Teucrium hyrcanicum L. is wildly distributed in the North and Northwest of the country. Until now, anti-inflammatory, anti-nociceptive, antibacterial, and anti-acetylcholine esterase activities of T. hyrcanicum have been reported [20, 21]. The investigation of the essential oil of this species revealed the presence of (E)-β-farnesene, hexahydrofarnesyl acetone, linalool, dihydroedulane, and ar-curcumene, as the major compounds [21]. Besides, flavonoids (pedalitin and luteolin 7-O-B-D-glucopyranoside), and diterpenes furolactones have been isolated from T. hyrcanicum [22, 23].

To find natural antioxidants from edible plants, in this study, the antioxidant activities of methanol extract and different fractions of T. hyrcanicum were measured by DPPH radical scavenging assay and phosphomolybdenum reduction test. Furthermore, the potential ability of T. hyrcanicum for protection against H2O2-induced oxidative stress was tested on NIH 3T3 cell line. Also, the total phenolic and flavonoid contents were measured. Likewise, the cytotoxicity was tested against three cancerous cell lines, including A431 (epidermoid carcinoma), MCF7 (breast cancer), HepG2 (liver hepatocellular carcinoma), as well as HU02 (Foreskin fibroblast) as a normal cell line using MTT assay. Also, the isolation and structural elucidation of compounds were reported from the fractions with the highest biological activities. The quantitative HPLC analysis of methanol extract was performed. Finally, two pure compounds were tested for their antioxidant and cytotoxic activities.

Methods

Reagents and chemicals

Gallic acid, quercetin, 2,2-Diphenyl-1-picrylhydrazyl (DPPH•), and BHA (Butylated hydroxyanisole) were obtained from Sigma chemical Company (USA). Aluminum trichloride (AlCl3), Folin–Ciocalteu’s phenol reagent, ammonium molybdate tetrahydrate, thiobarbituric acid, dithionitrobenzoic acid, and sodium bicarbonate were purchased from Merck, Germany. Dulbecco's Modified Eagle Medium (DMEM) and fetal bovine serum were obtained from Gibco and Invitrogen, respectively. HPLC-grade acetonitrile and acetic acid were purchased from DUKSAN, Korea. The three cancerous cell lines (including MCF7, HepG2, and A431) were purchased from the Iranian Biological Resource Center. NIH3T3 and Hu02 cell lines were purchased from the National Cell Bank of Pasteur Institute (Tehran, Iran). Other used reagents and solvents were of analytical grade.

General procedures

Column chromatography was performed on Sephadex LH-20 (Fluka, Switzerland). Silica gel 60 F254 precoated plates (Merck, Germany) were used for TLC. HPLC separations were conducted on a Knauer system (Smart line system, Germany), Eurospher column (250 × 20 mm i.d.) and a PDA detector (λ: 210–400 nm). HPLC quantitative analysis was performed on a Waters Alliance e2695 HPLC apparatus equipped with Waters 2998 photodiode array (PDA) detector, and C18 reversed-phase column (Waters spherisorb, S5 ODS2, 4×250 mm, 5 μm). Data acquisition and quantitation were performed by Empower 3 software (Waters, USA). 1H and 13 C-NMR spectra were acquired using a Bruker NMR spectrometer (500 MHz for 1H and 125 MHz for 13C) with tetramethylsilane as an internal standard, and chemical shifts are given in δ (ppm). FT-IR spectra were obtained with a Spectrum Two FT-IR spectrometer with UATR accessory (PerkinElmer, USA) in the range of 400–4000 cm.−1

Plant material, extraction, and fractionation

Aerial parts of the plant were collected from Rostam Abad mountain, Guilan province, in July 2020. The plant collection complies with relevant guidelines and regulations of plant protection at Guilan University of Medical Sciences (GUMS). The plant was identified, and the scientific name was confirmed by Dr. Fatemeh Yousefbeyk, Associate Professor of Pharmacognosy, School of Pharmacy, GUMS. The voucher specimen (114-HGUM) was deposited in the herbarium of the School of Pharmacy, GUMS, where it is publicly available. The aerial parts of T. hyrcanicum (500 g) were dried at room temperature, powdered, and extracted with methanol by percolation method (7 days). The methanol extract of T. hyrcanicum was dried by a rotary vacuum evaporator (Heidolph, Germany) to give 92 g dried extract. A portion of 90 g of dried methanol extract was subjected to the fractionation using the liquid–liquid method [24]. The solvents, including hexane, ethyl acetate, and methanol, were added in the order of increasing polarity. First, 200 ml of hexane was added to the methanol extract and mixed using an ultrasonic bath (Elma, Germany) for 15 min. The supernatant was collected, and the process was repeated five times. Then, ethyl acetate and methanol solvents (200 ml × 5, each) were added successively, and the same procedure was repeated. Finally, all the fractions were dried using a rotary evaporator to obtain 10.68, 6.54, and 63.72 g of each fraction, respectively. The concentrated fractions were kept at 4°C for further investigation.

Isolation of compounds

Isolation of compounds was carried out on the most potent fractions, including ethyl acetate fraction (EF) and methanol fraction (MF). First, the methanol fraction (50 g) was dissolved in distilled water and extracted by n-BuOH (10,100× ml). The butanol fraction (6.5 g) was fractionated using a Sephadex LH20 column using MeOH as an eluent (5×30 cm, ֺflow rate 0.5 ml.min−1) to get 13 fractions (M1-M13). Fraction M2 (500 mg) was submitted to reversed phase semi-preparative HPLC. The mobile phase consisted of H2O/acetic acid (99:1) (solvent A) and acetonitrile (solvent B). The initial mobile phase, including 9:1 (A/B), was delivered at a flow rate 5 ml.min−1 for 10 min, before it was changed to 7:3 (A/B) until 20 min, and remained in this ratio for 10 min (30 min after starting point). Then, chromatography was carried on with the ratio 1:1 (A/B) for another 10 min (40 min after the starting point). Finally, a gradient elution was used to reach 100% B in 20 min (60 min after the starting point). It afforded compound 1 (Rt: 22.4 min). The separation elution curve is provided in Supplementary file.

The ethyl acetate fraction (5 g) was subjected to a Sephadex LH20 column with MeOH as an eluent (5×30 cm, flow rate 0.5 ml.min−1) and 20 fractions were collected (E1-E20). The sub-fraction E4 (170 mg) was loaded on a Sephadex LH20 column (2×40 cm, flow rate 0.5 ml.min−1) with MeOH for further isolation and the compound 2 was isolated.

Measurement of total phenolic content

The Folin-Ciocalteu colorimetric method was carried out to determine the concentration of total phenolics in methanol extract and fractions of this plant [25]. To begin, methanol extract and fractions (1 ml, each) were separately mixed with Folin-Ciocalteu reagent (5 ml, diluted tenfold with distilled water). The mixtures were allowed to stand for 10 min. A solution of sodium bicarbonate at concentration of 75 g.l−1 was also prepared and 4 ml was added to each sample. After 30 min in darkness, the absorbance was read by UV–Vis spectrophotometer at 765 nm. Different dilutions of gallic acid (GA), including 20, 50, 75, 100, and 150 µg.ml−1 were prepared for plotting the calibration curve. All the tests were repeated three times. Total phenolic contents were expressed as mg of gallic acid equivalents (GAE)/g extract [26].

Measurement of total flavonoid content

The concentration of the total flavonoids was determined using a method described by Saeidnia and Gohari [27, 28]. First, 5 ml of each sample was added to 5 ml of aluminum trichloride (AlCl3) (2% in methanol). After 10 min, the absorbance of the mixture was measured at 415 nm using a UV–Vis spectrophotometer. A standard curve was plotted for measuring the total flavonoid content using different concentrations of quercetin as the standard (10, 25, 50, 75, and 100 µg.ml−1). The tests were repeated three times for each sample. Finally, total flavonoid content was expressed as mg of quercetin as equivalents (QE)/g extract [26, 29].

In-vitro antioxidant activity

DPPH radical scavenging activity

The methanol extract, fractions, and isolated compounds were tested for their antioxidant activities using the 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. Briefly, 2 ml of DPPH solution (40 μg.ml−1) was added to 1 ml of each sample with different concentrations. The samples were kept at room temperature for 30 min before the absorbance was measured at 517 nm. The tests were repeated three times for each concentration. BHA was used as the positive control. The control comprised samples (1 ml) and water (2 ml). The blank contained distilled water (1 ml) and DPPH solution (2 ml). The inhibition activity was determined by the following formula [30]:

$$\%Inhibition=100-\left[\frac{(\mathrm{As}-\mathrm{Ac})}{\mathrm{Ab}}\right]\times 100$$

Where As was the absorbance of sample, Ac was the absorbance of control, and Ab was the absorbance of blank. The IC50 values were expressed as the concentration of each sample (µg.ml−1) resulting in 50% inhibition and calculated from the graph-plotted of inhibition (%) against samples concentration.

Determination of total antioxidant capacity by phosphomolybdenum reduction assay (PRA)

The antioxidant capacity of the methanol extract and fractions were determined by the phosphomolybdenum reduction assay (PRA) [31]. Generally, the samples reduce Mo(VI) to Mo(V), forming specific green phosphate-Mo(V) compounds in acidic pH. Briefly, 0.3 ml of each sample was mixed with 3 ml of a reagent mixture (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate) in a test tube. All samples were incubated in an oil bath at 95°C for 90 min. After cooling to room temperature, the absorbance of each mixture was measured at 695 nm [31]. The total antioxidant capacities are expressed as mg of α-tocopherol equivalents (αTE/g extract), using the following linear equation plotted by α-tocopherol as the standard: Y = 0.0024X + 0.004; R2 = 0.998. In this equation, Y is the absorbance at 695 nm, and X is the concentration of αTE (μg.ml−1).

Cellular antioxidant activity

Cell culture and treatment

NIH3T3 fibroblast cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS), supplemented with 100 U.ml−1 penicillin and streptomycin, and 2 mM L-glutamine in a humidified atmosphere of 95% air, 5% CO2 at 37 °C [32, 33]. The cell counts and viability were determined by trypan blue staining and a density of 1 × 106 cells/well were seeded into 6-well cell culture plates. The cells were treated with methanol extract (ME), methanol fraction (MF), ethyl acetate fraction (EF), two isolated compounds, including quercetin and acteoside, and BHA as the standard antioxidant (at concentrations equal to IC50 of DPPH assay), for 24 h. Then, cells were exposed to H2O2 (100 µM) for 2 h [34]. Finally, cell suspensions were homogenized and used for other experiments.

Measurement of lipid peroxidation

For the evaluation of lipid peroxidation, the end product of the reaction, malondialdehyde (MDA), was assessed using thiobarbituric acid (TBA) as the indicator [32, 33]. Briefly, 0.25 ml phosphoric acid (0.05 M) was added to 0.2 ml of cell homogenates with the addition of 0.3 ml TBA (0.2%). Later, the samples were placed in a boiling water bath for 30 min. Next, the tubes were transfered to an ice bath and n-butanol was added to each tube. Then, they were centrifuged at 3500 rpm for 10 min. The amount of malondialdehyde (MDA) formed in each sample was assessed by measuring the absorbance of the supernatant at 532 nm with an ELISA reader (Epoch™ Microplate Spectrophotometer, BioTek, USA).

Measurement of glutathione (GSH) concentration

GSH concentration of the cell homogenates was determined by dithionitro benzoic acid (DTNB) [35]. The method is based on the formation of yellow color when DTNB reacts with compounds containing sulfhydryl groups. To begin the reaction, 2.3 ml of potassium phosphate buffer (0.2 M, pH 7.6) was added to 0.2 ml of cell lysate supernatant. Then, 0.5 ml of DTNB (0.001 M) was added to the solution. The absorbance of the products was observed after 5 min at 412 nm.

Cell cultures and cytotoxicity assay

The methanol extract, fractions, and isolated compounds were tested for cytotoxic activities against three cancerous cell lines (including MCF-7, HepG2, and A431) and the normal cell line (Hu02) by methyl thiazol tetrazolium (MTT) assay. Briefly, for each cell line, 104 cells per well were seeded in a 96-well plate in complete DMEM and incubated for 24 h at 37°C in a humidified atmosphere containing 5% CO2. Next, non-adherent cells were removed, and adherent cells were treated with different concentrations of samples (62.5, 125, 250, 500, and 1000 μg.ml−1). The treated cell lines were incubated in the same condition for 48 h. Afterward, the MTT solution (20 µl, 5 mg.ml−1 in PBS) and DMEM (180 µl) were added to seeded cells and incubated for 4 h. Then, the supernatants were removed, DMSO (150 µl) was added to each well, and the samples were shaken for 10 min to dissolve formazan crystals [36, 37]. The amount of purple formazan produced is related to the number of viable cells [38]. The solution optical density (OD) was measured by an absorbance microplate reader (BioTek) at 490 nm, using a reference wavelength of 630 nm. Each MTT assay was repeated three times. The percentage of viable cells in each treated group was calculated based on the following equation:

$$\mathrm{\%\ Viability}=(\frac{\left[\mathrm{OD\ treated\ group}-\mathrm{OD\ background}\right]}{\left[\mathrm{OD\ control}-\mathrm{OD\ background}\right]})\times 100$$

IC50 of samples against each cell line was defined as the concentration in which only 50% of cells were alive and computed using GraphPad Prism (Version 8, GraphPad Software, USA) [39, 40].

Quantitative HPLC analysis of T. hyrcanicum methanol extract

The quantitative analysis of acteoside and quercetin, as major compounds, was performed on the methanol extract of T. hyrcanicum using a Waters Alliance e2695 HPLC apparatus equipped with the Waters 2998 photodiode array (PDA) detector and C18 reversed-phase column (Waters spherisorb, S5 ODS2, 4×250 mm, 5 μm). The absorbance changes were monitored at 210 nm to 400 nm. Data acquisition and quantitation were performed using Empower 3 software (Waters, USA). The mobile phase consisted of H2O/acetic acid (99:1) (solvent A) and acetonitrile (solvent B). The flow rate of 1 ml.min−1 was used for a total run time of 60 min. Elution was performed using the following gradient program: 1–10 min: 10% B, 10–20 min: linear gradient from 10 to 30% B, 20–60 min: linear gradient from 30 to 100% B. The peak related to each compound was identified by comparison of DAD absorbance spectra with external standards. Acteoside and quercetin, as standard compounds, were used to prepare two different concentration levels (100 to 1000 μg.ml−1). The peak areas of each standard were plotted against the corresponding standard concentrations (μg.ml−1) using linear regression to create standard curves. The determination coefficient (R2) was calculated by means of the least-square analysis. Limits of detections (LODs) and limits of quantitations (LOQs) were determined using the expressions 3.3σ/s and 10σ/s, respectively, in which σ is the intercept standard deviation, and s is the slope of the calibration curve.

Statistical analysis

All the results were expressed as the mean ± SD (standard deviation of the mean). The statistical analyses were done using the SPSS software, version 13 (SPSS, Chicago, IL). All the statistical significance was calculated using the one-way analysis of variance (ANOVA) test, followed by the post-hoc Tukey test. Statistical significance was set at p < 0.05.

Results and discussion

Total phenolic and flavonoid contents

The total phenolic content of methanol extract and fractions were measured by the Folin-Ciocalteu method. As is presented in Table 1, the highest content of phenolic compounds was reported from MF (69.36 ± 0.02 mg of GAE/g extract), followed by ME (58.13 ± 0.02 mg of GAE/g extract), using the standard curve of gallic acid (y = 0.00096x − 0.0365, R2 = 0.995). Moreover, the total flavonoid content was measured, by standard curve of flavonoid quercetin (y = 0.0187x—0.0254, R2 = 0.975) and reported in the range of 68.95 ± 0.05 to 18.48 ± 0.01 mg QE/g extract. The MF and ME had the highest amount of flavonoids (68.95 ± 0.05 and 53.53 ± 0.01 mg of QE/g extract).

Table 1 Total phenolic and flavonoid contents of methanol extract and different fractions of T. hyrcanicum
Full size table

A literature survey showed that the total phenolic content of different species of Teucrium has been reported. In Table 2, the reported amounts of total phenols in methanol extract of different Teucrium species were summarized. As is presented, the phenolic content of T. hyrcanicum is in the range of previous studies.

Table 2 The comparison of total phenolic content of methanol extract of T. hyrcanicum with other Teucrium species
Full size table

Isolation and purification of compounds

The methanol and ethyl acetate fractions were subjected to phytochemical investigation to purify the main bioactive compounds. For the first time, acteoside and quercetin were isolated, and identified in MF and EF of T. hyrcanicum, respectively (Fig. 1).

Fig. 1
figure 1

Structures of acteoside (1) and quercetin (2), isolated from T. hyrcanicum

Full size image

The identification of compounds was performed by comparison of their spectral data (FT-IR, 1 H and 13C-NMR) with those published in the literature [47, 48]. The peak assignments of the detected compounds are reported as follows:

Compound 1: FT-IR (νmax): 3290 (OH), 1659 (C = O), 1605 (Alkene C = C), 1442 and 1558 (aromatic C = C), 1158 (C–O–C); 1H NMR (DMSO-d6, 500 MHz): δ ppm 7.47(d, 1H, J = 15.5 Hz), 7.04 (s, 1H), 6.99 (d, 1H, J = 7.5 Hz), 6.77 (d, 1H, J = 7 Hz), 6.64 (s, 1H), 6.64 (s, 1H), 6.51 (d, 1H, J = 7.5 Hz), 6.21 (d, 1H, J = 16 Hz), 5.04 (s, 1H), 4.36 (d, 1H, J = 8 Hz), 3.6 (dd, 2H, J = 9 Hz, J = 15.75 Hz), 2.7 (t, 2H, J = 7.5 Hz), 0.97 (d, 3H, J = 6 Hz). 13C NMR (DMSO-d6, 125 MHz) δ ppm 166.18 (C9'''), 148.98 (C4'''), 146.05 (C3'''), 145.46 (C3), 145.46 (C7'''), 144.01 (C4), 129.60 (C1), 125.97 (C1'''), 121.92 (C6'''), 120.02 (C6), 116.78 (C2), 116.27 (C5'''), 115.94 (C5), 115.15 (C2'''), 114.02 (C8'''), 102.74 (C1'),101.69 (C1''), 79.56 (C3'), 74.98 (C2'), 74.96 (C5'), 72.13 (C4''), 70.99 (C2''), 70.85 (C3''), 70.73 (C8), 69.59 (C4'), 69.21(C5''), 61.20 (C6'), 35.66 (C7), 18.62 (C6'').

Compound 2: FT-IR (νmax): 3298 (OH), 1688 (C = O), 1606 (Alkene C = C), 1439 and 1576 (aromatic C = C), 1038 and 1266 (phenyl alkyl C–O–C); 1H NMR (DMSO-d6, 500 MHz): δ ppm 12.5 (s, 1H), 10.70 (brs, 1H), 9.34 (brs, 3H), 7.69 (s, 1H), 7.55 (d, 1H, J = 8Hz), 6.9 (d, 1H, J = 8Hz), 6.41 (s, 1H), 6.19 (s, 1H). 13C NMR (DMSO-d6, 125 MHz) δ ppm 176.3 (C4), 164.35 (C7), 161.19 (C9), 156.60 (C5), 148.16 (C2), 147.26 (C4´), 145.51 (C3´), 136.20 (C3), 122.42 (C6´ and C1´), 116.10 (C5´), 115.54 (C2´), 115.52 (C10), 98.64 (C6), 93.82 (C8).

Acteoside, also named verbascoside, is a phenylethanoid glycoside, which is among the most prevalent disaccharide caffeoyl esters [49]. It comprises caffeic acid, 3′,4′-dihydroxyphenylethanol, glucose, and rhamnose [50]. It has been isolated from several plants species, such as Verbascum species, Buddleja brasiliensis, Striga asiatica, Olea europea, Paulownia tomentosa var. tomentosa, Lippia javanica, Lantana camara, Plantago lanceolate, and Cistanche deserticola [49, 51,52,53]. Biological activities, such as anti-inflammatory and regulation of cell apoptosis, have been reported from this compound [49, 54, 55]. This is the first report of acteoside from T. hyrcanicum. Previously, Sadeghi et al. reported the presence of phenylethanoid glycosides such as verbascoside and rabinosyl-verbascoside in T. chamaedrys and Teucrium sub spinosum, respectively [56].

The flavonol quercetin (3,31,41,5,7-pentahydroxyflavone) is among the most abundant flavonoids [57]. It exists in numerous types of vegetables and fruits, such as onions, apples, tea, and grapes [57, 58]. Several pharmacological properties, including anti-obesity, anti-inflammatory, cardioprotective, antihypertensive, antidiabetic, anti-hypercholesterolemic, and anti-atherosclerotic activities, have been reported from this compound [59, 60].

Antioxidant activity

The methanol extract, different fractions, and isolated compounds from T. hyrcanicum were investigated for antioxidant activities by the DPPH radical scavenging assay. The IC50 results of DPPH assay varied from 44.32 to 205.40 µg.ml−1 (Table 3). Also, the highest radical scavenging activities were found in MF and ME (IC50 of 44.32 and 62.12 µg.ml−1, respectively). The IC50 values of two isolated compounds, acteoside and quercetin, were 7.19 ± 0.8 and 5.56 ± 0.9 µg.ml−1, respectively. BHA, the standard antioxidant, showed the IC50 value of 7.81 µg.ml−1.

Table 3 The antioxidant activities of T. hyrcanicum and isolated compounds
Full size table

Furthermore, the total antioxidant capacity was determined by phosphomolybdate reduction assay (PRA) (Table 3). The results revealed that EF, ME, and MF had the relatively close reducing ability (363.6 ± 0.04, 354 ± 0.02, and 347.5 ± 0.01 mg αTE/g extract).

Until now, different Teucrium species have been investigated for their antioxidant activities. Sharififar et al. indicated that the antioxidant activities of different fractions prepared from T. polium, growing in Kerman, Iran, were between 20.1 µg.ml−1 (methanol fraction) and 85.4 µg.ml−1 (chloroform fraction) [2]. Stankovic et al. showed that the antioxidant activities of different extracts of T. chamaerdys L. var. glanduliferum varied from 341.08 µg.ml−1 (petroleum ether extract) to 29.46 µg.ml−1 (methanol extract) [43]. In another study, Stankovic et al. investigated the antioxidant activities of various extracts from T. montanum var. nontanum and obtained results ranged from 29.41 (water extract) to 1388.03 µg.ml−1 (petroleum ether extract) [1]. The results of a study by Mazhangara et al. revealed that the antioxidant activities of methanol and ethanol extracts from T. trifidum were 17 and 12 µg.ml−1, respectively [46]. Furthermore, Vlase et al. reported that the hydroalcoholic extract from T. chamaedrys had an IC50 of 26.70 µg.ml−1 [41]. Another study conducted by Mukarram Shah et al. displayed that methanol extract of T. stocksianum exerted high antioxidant activity (IC50 = 12.5 µg.ml−1) [44]. Also, the antioxidant activities of different extracts from T. barbeyanum were tested and the IC50 ranged from 5.39 to 77 µg.ml−1. The best antioxidant activity was reported from ethyl acetate fraction [61]. The results of the present study exhibited that two isolated compounds, quercetin and acteoside, had the highest antioxidant effects in the DPPH radical scavenging assay. Therefore, T. hyrcanicum can potentially be used as a source of these natural antioxidants.

Effects of T. hyrcanicum methanol extract, fractions, and isolated compounds on lipid peroxidation induced by H2O2

A significant increase (p < 0.001) in MDA concentration (5.77 μM) was observed in NIH3T3 fibroblast cells treated with 100 mM H2O2, after 24 h, compared to the control group (1.8 μM) (Fig. 2). Interestingly, pretreatment by T. hyrcanicum methanol extract, fractions, and isolated compounds significantly (p < 0.001) prevented MDA elevation in NIH3T3 fibroblast cells treated with H2O2, for 24 h. BHA, as a standard antioxidant, showed potential antioxidant activity by decreasing the MDA formation (2.86 μM, p < 0.01). Moreover, methanol fraction, quercetin, and acteoside exert the highest reduction in MDA formation (2.51, 2.55, and 2.66 μM, respectively), compared to the ME and EF.

Fig. 2
figure 2

The effects of methanol extract (ME), methanol fraction (MF), ethyl acetate fraction (EF), acteoside, quercetin, and BHA on the malondialdehyde (MDA) level in NIH3T3 fibroblast cells treated with 100 mM H2O2 after 24 h. Values are the mean ± S.E.M. for six experiments; *: significantly different (p < 0.05) from the control; **: significantly different (p < 0.01) from the control; *** Significantly different (p < 0.001) from the control; ###: significantly different (p < 0.001) from H2O2 group

Full size image

Lipid peroxidation is associated with aging and various chronic health diseases, such as cancer and atherosclerosis (41). MDA is a crucial degradation product of lipid peroxidation and acts as an indicator for measuring the degree of lipid peroxidation (42). In this study, the MF, quercetin, and acteoside showed better protective activities than BHA. They showed significant antioxidant properties, which lead to a lower degree of lipid peroxidation and higher hydroxyl radical scavenging abilities.

Effects of T. hyrcanicum methanol extract, fractions, and isolated compounds on GSH oxidation induced by H2O2

As shown in Fig. 3, exposure to H2O2 leads to a significant decrease in GSH level compared to the control group (p < 0.001). BHA showed significant antioxidant activity by elevating GSH level (142.69 μM, p < 0.05). The results showed that pretreatment with MF, quercetin, and acteoside considerably inhibited the H2O2-induced GSH oxidation in NIH3T3 fibroblast cells (p < 0.001, p < 0.01, and p < 0.001). Pretreatment with ME and EF could not significantly increase the GSH level related to H2O2 group.

Fig. 3
figure 3

The effects of methanol extract (ME), methanol fraction (MF), ethylacetate fraction (EF), acteoside, quercetin, and BHA on GSH oxidation induced by H2O2 in NIH3T3 fibroblast cells treated with 100 mM H2O2, after 24 h. Values are the mean ± S.E.M. for six experiments; *: significantly different (p < 0.05) from the control; **: significantly different (p < 0.01) from the control; *** Significantly different (p < 0.001) from the control;# significantly different (p < 0.05) from H2O2 group; ## significantly different (p < 0.01) from H2O2 group; ###: significantly different (p < 0.001) from H2O2 group

Full size image

The GSH is a vital antioxidant defense agent against free radicals. GSH (ɣ-glutamylcysteinylglycine) is a ubiquitous low molecular weight thiol in eukaryotes and is the most abundant antioxidant within all cells. It acts to remove ROS and therefore, reduce the lifetime of the oxidative signals [62, 63]. Reactive oxygen species, such as superoxide anion, hydroxyl radical, and H2O2, are regularly produced intracellularly because of usual metabolic activities, and can cause cellular oxidative damage [64]. In this study, hydrogen peroxide was used to induce oxidative stress. It is one of the most frequently generated ROS. It can easily pass through a lipid membrane, reacting with metal ions to produce highly toxic hydroxyl radicals, resulting in oxidative stress. Hydrogen peroxide can deplete GSH levels in cells [65]. According to the results, the MF fraction of T. hyrcanicum can be considered a significant antioxidant, even more potent than BHA in increasing GSH level (169.69 μM).

Cytotoxicity

The anti-proliferative activities of methanol extract, different fractions, and isolated compounds were evaluated on three cancerous cell lines, including the human breast adenocarcinoma (MCF-7), hepatocellular carcinoma (HepG2), and epidermoid carcinoma (A431) cell lines as well as a normal cell line (Hu02) by MTT assay and IC50s were demonstrated in Table 4. The cell lines were subjected to the increasing doses of each sample ranging from 62.5 to 1000 μg.ml−1 for 48 h, which reduced the viability of these cell lines.

Table 4 The cytotoxic activities of methanol extract, fractions, and isolated compounds on cancerous and normal cell lines by MTT assay
Full size table

Among different fractions, the best cytotoxicity was achieved by EF against A431 and MCF7 (235.4 and 326.6 μg.ml−1, respectively). Also, ME had cytotoxic activity on MCF7 with IC50 of 392 μg.ml−1. Other fractions exhibited lower cytotoxicity on tested cell lines (> 500 μg.ml−1). The isolated compound, acteoside, demonstrated significant cytotoxicity against all tested cell lines (ranging from 32 to 194.6 μg.ml−1). Quercetin represented its best antiproliferative activity against A431 (132.32 μg.ml−1).

So far, the genus Teucrium has been introduced as a natural source of anticancer compounds [42]. A literature survey demonstrated that the extracts of T. polium potentiate the cytotoxic and proapoptotic effects of anticancer drugs like doxorubicin, vincristine, and vinblastine against breast, epidermoid, bladder, and oral cavity epidermal cell lines [66]. In another study, the cytotoxic activity of aqueous extract from T. stocksianum was evaluated on MCF7 cell lines, and the IC50 value was 199.99 µg.ml−1 [67]. The methanol extracts of T. polium, T. chamaedrys, T. botrys, and T. montanum showed cytotoxic activities against HCT-116 (human colon cancer cell line) with the IC50 of 253.39, 190.07, 183.15, and 75.73 µg.ml−1, respectively [42].

So far, the extracts or isolated compounds have been investigated for anti-tumor activities. These diverse and complicated structures can trigger anti-inflammatory, anti-tumor, and/or anti-metastatic reactions and reduce multidrug resistance [54, 68]. In our study, acteoside exhibited the best cytotoxic activity against MCF7, followed by A431 cell lines. In another study, acteoside showed antiproliferative effect against HL-60 (human promyelocytic leukemia cells) with IC50 of 30 μM [69]. Acteoside at dose of 10 µM meaningfully decreased the proliferation of gastric adenocarcinoma (AGS) cells by 19%, and it was inactive on the colorectal adenocarcinoma (HT-29) cell line [70]. Acteoside efficiently inhibits the growth of oral squamous cell carcinoma (OSCC) cells [71]. This compound showed cytotoxic activity on HCT-116 (colon adenocarcinoma cell line) with IC50 of 32.49 μg.ml−1 [72]. The IC50 values of this compound on HEP-2 (human larynx epidermoid carcinoma) and RD (human rhabdomyosarcoma) cells were 55.6 and 36.24μg.ml−1, respectively [73].

Former studies proved that polyphenol antioxidants can cause antiproliferative effects on specific cancer cells by acting as prooxidant agents [42, 74, 75]. The prooxidant activities of phenolic compounds are mediated by forming phenoxyl radicals that can react with oxygen to produce oxygen radical (O2●) and quinone-semiquinones complex. These compounds can cause DNA damage and apoptosis in normal and cancer cells [74, 75]. It is suggested that the significant antioxidant activity of acteoside can be due to its prooxidant activity.

Flavonoids have recently attracted a lot of attention due to their potential role in cancer prevention and cancer therapy. They can regulate cell proliferation and pathways leading to cancer [76]. Quercetin is an important flavonoid that exists in many plants. Quercetin has potential anticancer effect due to antiproliferative, growth factor suppression, and antioxidant activities [77]. In this study, quercetin showed its best cytotoxic activity on A431 cell lines (IC50 of 132.32 μg.ml−1).

The quantitative HPLC analysis of quercetin and acteoside in methanol extract

In this study, the quantitative analyses of acteoside and quercetin in methanol extract of T. hyrcanicum were performed by developing an HPLC method. Figure 4 depicted a typical HPLC chromatogram of T. hyrcanicum methanol extract. The acteoside and quercetin were detected at Rt 15 and 54 min, respectively. The quantities of these compounds in ME were 93.31 and 16.87 μg.mg−1 extract.

Fig. 4
figure 4

HPLC chromatogram of methanol extract from T. hyrcanicum, (1) acteoside (Rt 15 min), and (2) quercetin (Rt 54 min)

Full size image

The results of method validation for analysis of quercetin and acteoside according to linearity, LOD, and LOQ revealed that the developed method was valid (Table 5). Outstanding linearity was obtained in the range of 10–1000 μg.ml−1, with R2 = 0.999 and 0.986 for quercetin and acteoside, respectively.

Table 5 Linear range, LOD, and LOQ parameters of quercetin and acteoside
Full size table

Conclusion

This study focused on the investigation of total phenolic and flavonoid contents, antioxidant, and cytotoxic activities of methanol extract and different fractions from T. hyrcanicum (family Lamiaceae). Among prepared samples, MF showed the highest amount of phenolic and flavonoid contents, and the best antioxidant activity. Also, MF significantly protected the NIH3T3 cell line against H2O2-induced oxidative stress by reducing MDA formation and inhibition of GSH oxidation. The results of the MTT assay demonstrated that EF had the highest cytotoxic activity. The phytochemical analysis resulted in the isolation and identification of two phenolic compounds (acteoside and quercetin). The isolated compounds had significant antioxidant activities and revealed protective effects on H2O2-induced oxidative stress, which was comparable with BHA (standard antioxidant). The acteoside had the best cytotoxicity against the A431 cancerous cell line. The HPLC quantification revealed that the methanol extract of this plant contained considerable amounts of acteoside and quercetin.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

BHA:

Butylated hydroxyanisole

BHT:

Butylated hydroxytoluene

ROS:

Reacting oxygen species

DPPH:

2,2-Diphenyl-1-picrylhydrazyl

GA:

Gallic acid

DMEM:

Dulbecco's Modified Eagle Medium:

MTT:

Methyl thiazol tetrazolium

ME:

Methanol extract

EF:

Ethyl acetate fraction

MF:

Methanol fraction

HF:

Hexane fraction

GAE:

Gallic acid equivalent

QE:

Quercetin equivalent

PRA:

Phosphomolybdenum reduction assay

αTE:

α-Tocopherol equivalent

TBA:

Thiobarbituric acid

MDA:

Malondialdehyde

GSH:

Glutathione

DTNB:

Dithionitrobenzoic acid

LOD:

Limit of detection

LOQ:

Limit of quantitation

References

  1. Stankovic MS, Niciforovic N, Topuzovic M, Solujic S. Total phenolic content, flavonoid concentrations and antioxidant activity, of the whole plant and plant parts extracts from Teucrium montanum L. var. montanum, f. supinum (L.) Reichenb. Biotechnology Biotechnolog Equip. 2011;25(1):2222–7.

    Article  Google Scholar 

  2. Sharififar F, Dehghn-Nudeh G, Mirtajaldini M. Major flavonoids with antioxidant activity from Teucrium polium L. Food Chem. 2009;112(4):885–8.

    Article  CAS  Google Scholar 

  3. Salehi F, Behboudi H, Kavoosi G, Ardestani SK. Oxidative DNA damage induced by ROS-modulating agents with the ability to target DNA: A comparison of the biological characteristics of citrus pectin and apple pectin. Sci Rep. 2018;8(1):1–16.

    Article  Google Scholar 

  4. Rashid S, Rather MA, Shah WA, Bhat BA. Chemical composition, antimicrobial, cytotoxic and antioxidant activities of the essential oil of Artemisia indica Willd. Food Chem. 2013;138(1):693–700.

    Article  CAS  PubMed  Google Scholar 

  5. Čanadanović-Brunet JM, Djilas SM, Ćetković GS, Tumbas VT, Mandić AI, Čanadanović VM. Antioxidant activities of different Teucrium montanum L. extracts. Int J Food Sci Technol. 2006;41(6):667–73.

    Article  Google Scholar 

  6. Cai Y, Luo Q, Sun M, Corke H. Antioxidant activity and phenolic compounds of 112 traditional Chinese medicinal plants associated with anticancer. Life Sci. 2004;74(17):2157–84.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Yang W, Huang G. Preparation and analysis of polysaccharide from Solanum tuberdsm. Ultrason Sonochem. 2023;98:106520.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Lin B, Wang S, Zhou A, Hu Q, Huang G. Ultrasound-assisted enzyme extraction and properties of Shatian pomelo peel polysaccharide. Ultrason Sonochem. 2023;98:106507.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Zhang W, Huang G. Preparation, structural characteristics, and application of taro polysaccharides in food. J Sci Food Agric. 2022;102(14):6193–201.

    Article  CAS  PubMed  Google Scholar 

  10. Tang Z, Huang G. Extraction, structure, and activity of polysaccharide from Radix astragali. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie. 2022;150:113015.

    Article  CAS  Google Scholar 

  11. Zhang H, Tsao R. Dietary polyphenols, oxidative stress and antioxidant and anti-inflammatory effects. Curr Opin Food Sci. 2016;8:33–42.

    Article  Google Scholar 

  12. Leopoldini M, Russo N, Toscano M. The molecular basis of working mechanism of natural polyphenolic antioxidants. Food Chem. 2011;125(2):288–306.

    Article  CAS  Google Scholar 

  13. Fresco P, Borges F, Diniz C, Marques MP. New insights on the anticancer properties of dietary polyphenols. Med Res Rev. 2006;26(6):747–66.

    Article  CAS  PubMed  Google Scholar 

  14. Milutinović MG, Maksimović VM, Cvetković DM, Nikodijević DD, Stanković MS, Pešić M, Marković SD. Potential of Teucrium chamaedrys L. to modulate apoptosis and biotransformation in colorectal carcinoma cells. J Ethnopharmacol. 2019;240:111951.

    Article  PubMed  Google Scholar 

  15. Rizvi T, Hussain I, Ali L, Mabood F, Khan A, Shujah S, Rehman N, Al-Harrasi A, Hussain J, Khan A. New gorgonane sesquiterpenoid from Teucrium mascatense Boiss, as α-glucosidase inhibitor. S Afr J Bot. 2019;124:218–22.

    Article  CAS  Google Scholar 

  16. Özkan G, Kuleaşan H, Çelik S, Göktürk RS, Ünal O. Screening of Turkish endemic Teucrium montbretii subsp. pamphylicum extracts for antioxidant and antibacterial activities. Food Control. 2007;18(5):509–12.

    Article  Google Scholar 

  17. Grujičić D, Marković A, Vukajlović JT, Stanković M, Jakovljević MR, Ćirić A, Djordjević K, Planojević N, Milutinović M, Milošević-Djordjević O. Genotoxic and cytotoxic properties of two medical plants (Teucrium arduini L. and Teucrium flavum L.) in relation to their polyphenolic contents. Mutat Res Genet Toxicol Environ Mutagen. 2020;852:503168.

    Article  PubMed  Google Scholar 

  18. Boghrati Z, Naseri M, Rezaie M, Pham N, Quinn RJ, Tayarani-Najaran Z, Iranshahi M. Tyrosinase inhibitory properties of phenylpropanoid glycosides and flavonoids from Teucrium polium L. var. gnaphalodes. Iran J Basic Med Sci. 2016;19(8):804.

    PubMed  PubMed Central  Google Scholar 

  19. Jamzad Z. Flora of Iran, vol. 76 Research Institute of Forests and Rangelands. 2012.

    Google Scholar 

  20. Golfakhrabadi F, Yousefbeyk F, Mirnezami T, Laghaei P, Hajimahmoodi M, Khanavi M. Antioxidant and antiacetylcholinesterase activity of Teucrium hyrcanicum. Pharmacognosy Res. 2015;7(Suppl 1):S15.

    PubMed  PubMed Central  Google Scholar 

  21. Morteza-Semnani K, Saeedi M, Akbarzadeh M. Chemical composition and antimicrobial activity of essential oil of Teucrium hyrcanicum L. J Essent Oil Bearing Plants. 2011;14(6):770–5.

    Article  CAS  Google Scholar 

  22. Gács-Baitz E, Radics L, Oganessian G, Mnatsakanian VA. Teucrins H1–H4, novel clerodane-type diterpenes from Teucrium hyrcanicum. Phytochemistry. 1978;17(11):1967–73.

    Article  Google Scholar 

  23. Oganesyan G, Mnatsakanyan V. Flavonoids of Teucrium hircanicum. Chem Nat Compd. 1987;23(6):764–5.

    Article  Google Scholar 

  24. Abubakar AR, Haque M. Preparation of Medicinal Plants: Basic Extraction and Fractionation Procedures for Experimental Purposes. J Pharm Bioallied Sci. 2020;12(1):1–10.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Golfakhrabadi F, Shams Ardekani MR, Saeidnia S, Yousefbeyk F, Jamalifar H, Ramezani N, Akbarzadeh T, Khanavi M. Phytochemical analysis, antimicrobial, antioxidant activities and total phenols of Ferulago carduchorum in two vegetative stages (flower and fruit). Pakistan J Pharm Sci. 2016;29(2):623–8.

    CAS  Google Scholar 

  26. Hamidi M, Ghasemi S, BavafaBighdilou B, EghbaliKoohi D. Evaluation of antioxidant, antibacterial and cytotoxic activity of methanol extract from leaves and fruits of Iranian squirting cucumber (Ecballium elaterium (L.) A. Rich). Res J Pharmacognosy. 2020;7(1):23–9.

    Google Scholar 

  27. Soodabeh Saeidnia ARG. Pharmacognosy andmolecular pharmacognosy in prectice. Berlin: LAP LAMBERT Academic Publishing; 2012.

    Google Scholar 

  28. Yousefbeyk F, Ebrahimi-Najafabadi H, Dabirian S, Salimi S, Baniardalani F, Azmian Moghadam F, Ghasemi S. Phytochemical Analysis and Antioxidant Activity of Eight Cultivars of Tea Camellia sinensis and Rapid Discrimination with FTIR Spectroscopy and Pattern Recognition Techniques. Pharm Sci. 2023;29(1):100–10.

    CAS  Google Scholar 

  29. Yousefbeyk F, Soltani Tehrani B, Zeinoddini Z, Aghajani Torshkooh F, Andalib S. Evaluation of Anti-depressant and Anti-anxiety Activity of Methanol Extract of Stachys annua L. Mice. Res J Pharmacognosy. 2021;8(1):29–38.

    Google Scholar 

  30. Tofighi Z, Alipour F, Hadavinia H, Abdollahi M, Hadjiakhoondi A, Yassa N. Effective antidiabetic and antioxidant fractions of Otostegia persica extract and their constituents. Pharm Biol. 2014;52(8):961–6.

    Article  CAS  PubMed  Google Scholar 

  31. Yousefbeyk F, Ghasemi S, Evazalipour M, Dabirian S, Schubert C, Hekmatnia S, Habibi Y, Eghbali Koohi D, Böhm V. Phytochemical analysis, antioxidant, antibacterial, and cytotoxic activities of leaves and roots of Rubus hyrcanus Juz. Eur Food Res Technol. 2022;248(1):141–52.

    Article  CAS  Google Scholar 

  32. Linden A, Gülden M, Martin H-J, Maser E, Seibert H. Peroxide-induced cell death and lipid peroxidation in C6 glioma cells. Toxicol In Vitro. 2008;22(5):1371–6.

    Article  CAS  PubMed  Google Scholar 

  33. Azari A, Shokrzadeh M, Zamani E, Amani N, Shaki F. Cerium oxide nanoparticles protects against acrylamide induced toxicity in HepG2 cells through modulation of oxidative stress. Drug Chem Toxicol. 2019;42(1):54–9.

    Article  CAS  PubMed  Google Scholar 

  34. Coyle CH, Kader KN. Mechanisms of H2O2-induced oxidative stress in endothelial cells exposed to physiologic shear stress. ASAIO J. 2007;53(1):17–22.

    Article  CAS  PubMed  Google Scholar 

  35. Zamani E, Shokrzadeh M, Ziar A, Abedian-Kenari S, Shaki F. Acrylamide attenuated immune tissues’ function via induction of apoptosis and oxidative stress: Protection by l-carnitine. Hum Exp Toxicol. 2018;37(8):859–69.

    Article  CAS  PubMed  Google Scholar 

  36. Yousefbeyk F, Tabaside J, Ostad SN, Salehi Sourmaghi MH, Amin GR. Investigation of chemical composition and cytotoxic activity of aerial parts of Ziziphora clinopodioides Lam. Res J Pharmacognosy. 2016;3(2):47–51.

    CAS  Google Scholar 

  37. Azmian Moghadam F, Evazalipour M, Kefayati H, Ghasemi S. 6,7-Disubstituted-4-anilinoquinazoline: Design, Synthesis and Anticancer Activity as a Novel Series of Potent Anticancer Agents. Pharm Sci. 2021;27(2):209–18.

    Article  Google Scholar 

  38. Ghasemi S, Davaran S, Sharifi S. Comparison of cytotoxic activity of L778123 as a farnesyltranferase inhibitor and doxorubicin against A549 and HT-29 cell lines. Adv Pharm Bull. 2013;3(1):73.

    PubMed  PubMed Central  Google Scholar 

  39. Azmian Moghadam F, Evazalipour M, Ghasemi S, Kefayati H. Design, synthesis, biological evaluation and docking study of novel 4-anilinoquinazolines derivatives as anticancer agents. Iran J Chem Chemical Eng (IJCCE). 2020;41(2):353–67.

    Google Scholar 

  40. Moghadam FA, Evazalipour M, Kefayati H, Ghasemi S. 6, 7-disubstituted-4-anilinoquinazoline: design, synthesis and anticancer activity as a novel series of potent anticancer agents. Pharm Sci. 2020;27(2):209–18.

    Article  Google Scholar 

  41. Vlase L, Benedec D, Hanganu D, Damian G, Csillag I, Sevastre B, Mot AC, Silaghi-Dumitrescu R, Tilea I. Evaluation of antioxidant and antimicrobial activities and phenolic profile for Hyssopus officinalis Ocimum basilicum and Teucrium chamaedrys. Molecules. 2014;19(5):5490–507.

    Article  PubMed  PubMed Central  Google Scholar 

  42. Stankovic MS, Curcic MG, Zizic JB, Topuzovic MD, Solujic SR, Markovic SD. Teucrium plant species as natural sources of novel anticancer compounds: antiproliferative, proapoptotic and antioxidant properties. Int J Mol Sci. 2011;12(7):4190–205.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Stankovic MS, Topuzovic M, Solujic S, Mihailovic V. Antioxidant activity and concentration of phenols and flavonoids in the whole plant and plant parts of Teucrium chamaerdys L. var. glanduliferum Haussk. J Med Plants Res. 2010;4(20):2092–8.

    CAS  Google Scholar 

  44. Shah SMM, Sadiq A, Shah SMH, Ullah F. Antioxidant, total phenolic contents and antinociceptive potential of Teucrium stocksianum methanolic extract in different animal models. BMC Complement Altern Med. 2014;14(1):1–7.

    Article  Google Scholar 

  45. Stankovic M, Jakovljevic D, Topuzovic M, Zlatkovic B. Antioxidant activity and contents of phenolics and flavonoids in the whole plant and plant parts. Oxid Commun. 2014;37(2):522–32.

    CAS  Google Scholar 

  46. Mazhangara IR, Idamokoro EM, Chivandi E, Afolayan AJ. Phytochemical screening and in vitro evaluation of antioxidant and antibacterial activities of Teucrium trifidum crude extracts. Heliyon. 2020;6(9):e04395.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Pan W-g, Jiang S-p. Luo P, Gao P, Chen B, Bu H-t: Isolation, purification and structure identification of two phenolic glycosides from the roots of Incarvillea younghusbandii Sprague and their antioxidant activities. Acta Pharmaceutica Sinica. 2011;46(4):422–7.

    CAS  PubMed  Google Scholar 

  48. Ahmedova A, Paradowska K, Wawer I. 1H, 13C MAS NMR and DFT GIAO study of quercetin and its complex with Al(III) in solid state. J Inorg Biochem. 2012;110:27–35.

    Article  CAS  PubMed  Google Scholar 

  49. Alipieva K, Korkina L, Orhan IE, Georgiev MI. Verbascoside — A review of its occurrence, (bio)synthesis and pharmacological significance. Biotechnol Adv. 2014;32(6):1065–76.

    Article  CAS  PubMed  Google Scholar 

  50. Liu Y-H, Lu Y-L, Han C-H, Hou W-C. Inhibitory activities of acteoside, isoacteoside, and its structural constituents against protein glycation in vitro. Bot Stud. 2013;54(1):6.

    Article  PubMed  PubMed Central  Google Scholar 

  51. Lee KY, Jeong EJ, Lee H-S, Kim YC. Acteoside of Callicarpa dichotoma attenuates scopolamine-induced memory impairments. Biol Pharm Bull. 2006;29(1):71–4.

    Article  CAS  PubMed  Google Scholar 

  52. Peng XM, Gao L, Huo SX, Liu XM, Yan M. The mechanism of memory enhancement of acteoside (Verbascoside) in the senescent mouse model induced by a combination of d-gal and AlCl3. Phytother Res. 2015;29(8):1137–44.

    Article  CAS  PubMed  Google Scholar 

  53. Li L, Tsao R, Liu Z, Liu S, Yang R, Young JC, Zhu H, Deng Z, Xie M, Fu Z. Isolation and purification of acteoside and isoacteoside from Plantago psyllium L. by high-speed counter-current chromatography. J Chromatogr A. 2005;1063(1–2):161–9.

    CAS  PubMed  Google Scholar 

  54. Cheimonidi C, Samara P, Polychronopoulos P, Tsakiri EN, Nikou T, Myrianthopoulos V, Sakellaropoulos T, Zoumpourlis V, Mikros E, Papassideri I, et al. Selective cytotoxicity of the herbal substance acteoside against tumor cells and its mechanistic insights. Redox Biol. 2018;16:169–78.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Reinke D, Kritas S, Polychronopoulos P, Skaltsounis AL, Aligiannis N, Tran CD. Herbal substance, acteoside, alleviates intestinal mucositis in mice. Gastroenterol Res Pract. 2015;2015:327872.

    Article  PubMed  PubMed Central  Google Scholar 

  56. Sadeghi Z, Yang J-L, Venditti A, Moridi Farimani M. A review of the phytochemistry, ethnopharmacology and biological activities of Teucrium genus (Germander). Nat Prod Res. 2022;36(21):5647–64.

    Article  CAS  PubMed  Google Scholar 

  57. Mlcek J, Jurikova T, Skrovankova S, Sochor J. Quercetin and its anti-allergic immune response. Molecules. 2016;21(5):623.

    Article  PubMed  PubMed Central  Google Scholar 

  58. Nickel T, Hanssen H, Sisic Z, Pfeiler S, Summo C, Schmauss D, Hoster E, Weis M. Immunoregulatory effects of the flavonol quercetin in vitro and in vivo. Eur J Nutr. 2011;50(3):163–72.

    Article  CAS  PubMed  Google Scholar 

  59. Chen S, Jiang H, Wu X, Fang J. Therapeutic Effects of Quercetin on Inflammation, Obesity, and Type 2 Diabetes. Mediators Inflamm. 2016;2016:9340637–9340637.

    Article  PubMed  PubMed Central  Google Scholar 

  60. Eisvand F, Tajbakhsh A, Seidel V, Zirak MR, Tabeshpour J, Shakeri A. Quercetin and its role in modulating endoplasmic reticulum stress: A review. Phytother Res. 2021;36(1):73–84.

    Article  PubMed  Google Scholar 

  61. Alwahsh MAA, Khairuddean M, Chong WK. Chemical constituents and antioxidant activity of Teucrium barbeyanum Aschers. Rec Nat Prod. 2015;9(1):159–63.

    Google Scholar 

  62. Masella R, Di Benedetto R, Varì R, Filesi C, Giovannini C. Novel mechanisms of natural antioxidant compounds in biological systems: involvement of glutathione and glutathione-related enzymes. J Nutr Biochem. 2005;16(10):577–86.

    Article  CAS  PubMed  Google Scholar 

  63. Harris Isaac S, Treloar Aislinn E, Inoue S, Sasaki M, Gorrini C, Lee Kim C, Yung Ka Y, Brenner D, Knobbe-Thomsen Christiane B, Cox Maureen A, et al. Glutathione and Thioredoxin Antioxidant Pathways Synergize to Drive Cancer Initiation and Progression. Cancer Cell. 2015;27(2):211–22.

    Article  CAS  PubMed  Google Scholar 

  64. Dudek EJ, Shang F, Taylor A. H2O2-mediated oxidative stress activates NF-κB in lens epithelial cells. Free Radical Biol Med. 2001;31(5):651–8.

    Article  CAS  Google Scholar 

  65. Salla S, Sunkara R, Ogutu S, Walker LT, Verghese M. Antioxidant activity of papaya seed extracts against H2O2 induced oxidative stress in HepG2 cells. LWT-Food Sci Technol. 2016;66:293–7.

    Article  CAS  Google Scholar 

  66. Rajabalian S. Methanolic extract of Teucrium polium L potentiates the cytotoxic and apoptotic effects of anticancer drugs of vincristine, vinblastine and doxorubicin against a panel of cancerous cell lines. Exp Oncol. 2008;30(2):133–8.

    CAS  PubMed  Google Scholar 

  67. Ullah I, Abamor EŞ, Bağirova M, Shinwari ZK, Allahverdiyev AM. Biomimetic production, characterisation, in vitro cytotoxic and anticancer assessment of aqueous extract-mediated AgNPs of Teucrium stocksianum Boiss. IET Nanobiotechnol. 2017;12(3):270–6.

    Article  PubMed Central  Google Scholar 

  68. Long S, Sousa E, Kijjoa A, Pinto MM. Marine natural products as models to circumvent multidrug resistance. Molecules. 2016;21(7):892.

    Article  PubMed  PubMed Central  Google Scholar 

  69. Lee K-W, Kim HJ, Lee YS, Park H-J, Choi J-W, Ha J, Lee K-T. Acteoside inhibits human promyelocytic HL-60 leukemia cell proliferation via inducing cell cycle arrest at G 0 /G 1 phase and differentiation into monocyte. Carcinogenesis. 2007;28(9):1928–36.

    Article  CAS  PubMed  Google Scholar 

  70. Obied HK, Prenzler PD, Konczak I. Rehman A-u, Robards K: Chemistry and bioactivity of olive biophenols in some antioxidant and antiproliferative in vitro bioassays. Chem Res Toxicol. 2009;22(1):227–34.

    Article  CAS  PubMed  Google Scholar 

  71. Zhang Y, Yuan Y, Wu H, Xie Z, Wu Y, Song X, Wang J, Shu W, Xu J, Liu B. Effect of verbascoside on apoptosis and metastasis in human oral squamous cell carcinoma. Int J Cancer. 2018;143(4):980–91.

    Article  CAS  PubMed  Google Scholar 

  72. Encalada MA, Rehecho S, Ansorena D, Astiasarán I, Cavero RY, Calvo MI. Antiproliferative effect of phenylethanoid glycosides from Verbena officinalis L. on colon cancer cell lines. LWT-Food Sci Technol. 2015;63(2):1016–22.

    Article  CAS  Google Scholar 

  73. Harput US, Genc Y, Saracoglu I. Cytotoxic and antioxidative activities of Plantago lagopus L and characterization of its bioactive compounds. Food Chem Toxicol. 2012;50(5):1554–9.

    Article  CAS  PubMed  Google Scholar 

  74. Eghbaliferiz S, Iranshahi M. Prooxidant activity of polyphenols, flavonoids, anthocyanins and carotenoids: updated review of mechanisms and catalyzing metals. Phytother Res. 2016;30(9):1379–91.

    Article  CAS  PubMed  Google Scholar 

  75. Zheng LF, Dai F, Zhou B, Yang L, Liu ZL. Prooxidant activity of hydroxycinnamic acids on DNA damage in the presence of Cu(II) ions: mechanism and structure-activity relationship. Food Chem Toxicol. 2008;46(1):149–56.

    Article  CAS  PubMed  Google Scholar 

  76. Wang B, Zhang X. Inhibitory effects of Broccolini leaf flavonoids on human cancer cells. Scanning. 2012;34(1):1–5.

    Article  PubMed  Google Scholar 

  77. Anand David AV, Arulmoli R, Parasuraman S. Overviews of Biological Importance of Quercetin: A Bioactive Flavonoid. Pharmacogn Rev. 2016;10(20):84–9.

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by Guilan University of Medical Sciences.

Funding

This work was supported by Guilan University of Medical Sciences (grant number 98040903).

Author information

Authors and Affiliations

  1. Department of Medicinal Chemistry, School of Pharmacy, Guilan University of Medical Sciences, Rasht, Iran

    Saeed Ghasemi

  2. Department of Pharmaceutical Biotechnology, School of Pharmacy, Guilan University of Medical Sciences, Rasht, Iran

    Mehdi Evazalipour

  3. Department of Pharmacognosy, School of Pharmacy, Guilan University of Medical Sciences, Rasht, Iran

    Nastaran Peyghanbari, Mahan Molaee, Diba Eghbali Koohi & Fatemeh Yousefbeyk

  4. Department of Pharmacology and Toxicology, School of Pharmacy, Guilan University of Medical Sciences, Rasht, Iran

    Ehsan Zamani

  5. Institute of Clinical Chemistry, University of Zurich & University Hospital Zurich, Zurich, Switzerland

    Peter Bellstedt

Authors
  1. Saeed Ghasemi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mehdi Evazalipour
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nastaran Peyghanbari
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ehsan Zamani
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Peter Bellstedt
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mahan Molaee
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Diba Eghbali Koohi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Fatemeh Yousefbeyk
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

SGH managed the research project, supervised the study and interpreted the HPLC and NMR data. ME performed cell culture and the acquisition of cytotoxicity data. EZ designed the oxidative stress assays and analysis the data. NP, MM, and DEK contributed in the acquisition of data. FY designed and supervised the study, managed the methods of phytochemical analysis and antioxidant assays, data analysis, and drafted the manuscript. PB contributed in the acquisition of NMR data. All authors reviewed the manuscript.

Corresponding author

Correspondence to Fatemeh Yousefbeyk.

Ethics declarations

Ethics approval and consent to participate

This study was approved by the Ethical Committee of Guilan University of Medical Sciences (ID: IR.GUMS.REC.1398.224). The collection of plant material complies with relevant institutional and national guidelines and regulations of plant protection.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s Note

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

Supplementary Information

Additional file 1: Figure S1.

The separation elution curve for compound 1(acteoside): compound 1 was separated in Rt=22.4 min, using a semi-preparative HPLC. Figure S2-1. H-NMR spectra of compound 1 (acteoside). Figure S2-2. The expanded H-NMR spectra of compound 1 (acteoside). Figure S2-3. The expanded H-NMR spectra of compound 1 (acteoside). Figure S3-1. 13C-NMR spectra of compound 1 (acteoside). Figure S3-2. The expanded 13C-NMR spectra of compound 1 (acteoside). Figure S3-3. The expanded 13C-NMR spectra of compound 1 (acteoside). Figure S3-4. The expanded 13C-NMR spectra of compound 1 (acteoside). Figure S3-5. The expanded 13C-NMR spectra of compound 1 (acteoside). Figure S4. FT-IR spectra of compound 1 (acteoside). Figure S5. H-NMR spectra of compound 2 (quercetin). Figure S6. C-NMR spectra of compound 2 (quercetin). Figure S7: FT-IR spectra of compound 2 (quercetin).

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

Ghasemi, S., Evazalipour, M., Peyghanbari, N. et al. Isolation and structure elucidation of the compounds from Teucrium hyrcanicum L. and the investigation of cytotoxicity, antioxidant activity, and protective effect on hydrogen peroxide-induced oxidative stress. BMC Complement Med Ther 23, 447 (2023). https://doi.org/10.1186/s12906-023-04262-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12906-023-04262-8

Keywords

BMC Complementary Medicine and Therapies

ISSN: 2662-7671

Contact us