Jentashapir J Cell Mol Biol
Jentashapir J Cell Mol Biol

article information

Jentashapir Journal of Cellular and Molecular Biology: Vol.13, issue 2; e128492
published online: August 22, 2022
article type: Research Article
received: May 27, 2022
revised: August 5, 2022
accepted: August 5, 2022
DOI: https://doi.org/10.5812/jjcmb-128492
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Chemical Constituents, Antioxidant and Cytotoxic Potential of Chloroform and Ethyl Acetate Extracts of Teucrium persicum

authors:

avatar Parvaneh Hajipour 1 , avatar Fatemeh Eizadifard 1 , avatar Majid Tafrihi ORCID 1 , *

Department of Molecular and Cell Biology, Faculty of Basic Sciences, University of Mazandaran, Babolsar, Iran

how to cite: Hajipour P, Eizadifard F, Tafrihi M. Chemical Constituents, Antioxidant and Cytotoxic Potential of Chloroform and Ethyl Acetate Extracts of Teucrium persicum. Jentashapir J Cell Mol Biol. 2022;13(2):e128492. https://doi.org/10.5812/jjcmb-128492.

Abstract

Background:

Teucrium persicum, a well-known Iranian endemic plant, has been used to treat various diseases in Iranian traditional medicine.

Objectives:

This study aimed to assess the antioxidant and cytotoxic effects of chloroform and ethyl acetate extracts of T. persicum on cancer cell lines.

Methods:

The total phenolic and flavonoid contents, as well as the antioxidant potential of chloroform and ethyl acetate extracts were assayed. The GC-MS analysis was used to detect the chemical constituents of these two extracts. The cytotoxicity effects of extracts on two cancer cell lines, PC-3, SW-480, and HEK-293, as normal cell lines were evaluated using the MTT assay.

Results:

The average total phenolic contents of chloroform and ethyl acetate extracts were found to be 48 ± 0.2 and 25 ± 0.4, respectively (P < 0.05). The total flavonoid contents of chloroform and ethyl acetate extracts were measured as 34 ± 0.6 and 36 ± 0.6 mg EQ/g of the dried extract, respectively (P < 0.05). The antioxidant potential of chloroform and ethyl acetate extracts were calculated as 2.5 ± 0.013 and 3 ± 0.0023 μg/mL, respectively (P < 0.05). According to the GC-MS analysis, 60 and 61 different bioactive compounds were detected in chloroform and ethyl acetate extracts, respectively. MTT results showed that the SW-480 cell line was more sensitive than PC-3 and HEK-293 cells.

Conclusions:

It was concluded that T. persicum had significant antioxidant and cytotoxic properties. Taking our study results into account, cancer inhibiting properties of T. persicum were strongly supported. However, it was recommended that further specific and detailed biochemical and molecular studies should be conducted in order to discover the effective compound(s) and mechanism(s) responsible for producing these effects.

1. Background

Teucrium is a large, polymorphic genus belonging to the Lamiaceae family that includes more than 300 species having been distributed worldwide (1). Several reports have indicated that Teucrium species have anti-microbial, anti-inflammatory, antioxidant, anti-ulcer, and anti-cancer properties (2-4). Chemical analyses of essential oils or extracts from Teucrium species have confirmed the existence of various flavonoids and phenolic compounds with biological activities (5-7).

In addition to Teucrium persicum Boiss, T. macrum Boiss et. Hausskn and T. melissoides Boiss et. Hausskn also are well-known Iranian endemic plants of this genus (8). Teucrium persicum Boiss grows in Fars province on the south of Iran and is called marv-e-talkh. As a traditional remedy, T. persicum has been used for treating headaches, abdominal pains, diabetes, hyperlipidemia, and inflammation (9). Many attempts have been made to identify the chemical compositions of the essential oils extracted from T. persicum. Furthermore, studies have shown that the essential oils of T. persicum possess valuable antioxidant properties (9-11). A few studies investigating the biological activities of T. persicum have also indicated that the aqueous extract of T. persicum exhibits anti-inflammatory activity (12), and T. persicum methanolic extract significantly impedes the proliferation of PC-3 cells (13).

2. Objectives

The antioxidant properties of ethyl acetate and chloroform extracts of T. persicum have been already evaluated by several studies. In the present study, therefore, the cytotoxicity effects of these two extracts on PC-3 (human prostate adenocarcinoma), SW-480 (human colorectal adenocarcinoma), and HEK-293T (human embryonic kidney) cell lines were evaluated.

3. Methods

3.1. Preparation of Extracts

Teucrium persicum plants were collected from the southern parts of Iran and confirmed by Dr. Alireza Naqinezhad from the Department of Biology, Faculty of Sciences, University of Mazandaran (9008-HUMZ). Then, 50 gr of the powdered aerial parts of T. persicum were soaked in 100 mL of ethyl acetate and chloroform separately and shaken for 48 hours. The concentrated supernatants were dried using a freeze dryer (Christ, Germany), dissolved in DMSO (25 µg/mL), and stored at -20ºC for later application.

3.2. Determination of Total Phenolic Contents

The total phenolic contents (TPC) of chloroform and ethyl acetate extracts were calculated according to the Singleton and Rossi method with minor modifications (14). Briefly, 50 and 150 µg/mL of the gum were added to 1.6 mL of deionized-distilled water, and 100 µL of Folin-Ciocalteu phenol reagent was added and preserved for 3 min at 37°C. Then, 300 µL of saturated Na2CO3 (7%) was added, and the mixture was transferred to a 10 mL tube with the addition of deionized-distilled water. The concoction was held in a dark room for 120 min, and the absorbance was quantified at 765 nm by an ELISA reader (BioTek, USA). The gallic acid was used as a reference standard, and the TPC was expressed as mg of gallic acid equivalents per gram of the gum on a dry basis.

3.3. Total Flavonoid Contents Determination

In this study, the aluminum chloride colorimetric method was applied in order to determine the total flavonoid contents (TFC) of chloroform and ethyl acetate extracts (14). Then the absorbance was measured at 510 nm by using an ELISA reader (BioTek, USA). The quercetin was applied as a standard reference, and the TFC was expressed as a milligram of quercetin equivalents per gram of extracts on a dry basis. All assays were performed in triplicates.

3.4. Evaluation of DPPH Radical Scavenging Activity

One mL of 0.1 mM solution of DPPH radical in methanol was added to 20, 50, and 100 µg/mL of chloroform and ethyl acetate extracts. After performing vigorous vortextion and 30 min incubation in the dark room, the absorbance was measured at 517 nm by an ELISA reader (BioTek, USA), and the scavenging activity (SDPPH) in percent was calculated adopting the following formula:

SDPPH [%] = 100 × [(Ablank-Asample)/Ablank]

where Ablank indicates the absorbance of the control, and Asample shows the absorbance of the extract. Then, 50% of DPPH- radical scavenging activity (IC50) was calculated from a graph by plotting SDPPH against the concentrations of extracts. All assays were performed in triplicates.

3.5. Ferric Reducing Antioxidant Power (FRAP) Assay

Concentrations of 25, 75, and 125 µg/mL of chloroform and ethyl acetate extracts were added to a mix containing 2 mL of distilled water and 3 mL of the FRAP reagent. After performing a vigorous vortex and 10 min of incubation at 37°C, the absorbance was measured at 593 nm using an ELISA reader (BioTek, USA). The quercetin and aqueous solution of FeSO4 were applied as positive control and standard, respectively (15). All assays were performed in triplicates.

3.6. Gas Chromatography

Analytical GC-MS assay was carried out on an Agilent 5975C MSD, which was coupled to an Agilent 7890A (Agilent Technologies, USA) and equipped with a flame ionization detector, FID. The GC separation was performed on DB5 column of 60 m × 0.25 mm i.d. and 0.25 µm film thickness in temperature program: initial temperature of 50°C was maintained for 4 min, then increased to 120°C at 5°C/1 min, and the final temperature of 260°C was maintained for 13 min with a constant helium flow rate of 1 mL min-1. Taking into account the library data from Wiley 9th ed. & NIST 2008 Lib. SW (Version 2010) or other data from the literature, the individual compounds were recognized by matching their mass spectra, studying their retention times, and corresponding mass spectra with those of reliable standards.

3.7. Cell lines and Cell Culture

HEK-293, PC-3, and SW-480 cell lines were obtained from the National Cell Bank of Iran (NCBI, Pasteur Institute, Tehran, Iran). The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (Sigma) and 1% penicillin/streptomycin (Sigma) and incubated in a 5% CO2 humidified incubator at 37°C.

3.8. MTT Assay

To perform MTT assay, 4 × 103 cells were seeded in 150 µL of growth medium in each well of a 96-well plate and allowed to grow to 70% confluency. The cells were then treated with different concentrations of chloroform and ethyl acetate extracts (1 - 200 µg/mL) for 48 hours. After removing the medium, 100 µL of MTT solution (3- [4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) (5 mg/mL in PBS) was added to each well. The MTT solution was changed to 100 µL of DMSO three hours later, and the absorbance was measured at 590 nm using an ELISA reader (BioTek, USA). Cell survival rate was calculated as (%) = (a-c)/(b-c) x100, which a = absorbance at every concentration of the test reagent, b = absorbance at 0 μM of the test reagent, and c = absorbance of the blank. The IC50 values were calculated by adopting approximate linear regression of the percentage survival versus the drug concentration (16). All assays were conducted in triplicates.

3.9. Statistical Analysis

The data from all three replicates were reported as means ± standard deviations. Statistical analyses were carried out using SPSS (IBM Statistics 25.0). A P-value < 0.05 was found to be statistically significant. The IC50 value was computed using Excel (Microsoft office 2016) and GraphPad Prism software version 8 (California, USA).

4. Results

4.1. Total Phenolic Contents of Chloroform and Ethyl Acetate Extracts of T. persicum

The average total phenolic content of chloroform and ethyl acetate extracts of T. persicum were calculated by adopting the Folin-Ciocalteu method, and it was found that the chloroform extract contained more phenolic contents than the ethyl acetate extract (Table 1) (P < 0.05).

Table 1.

Total Phenolic, Flavonoids, and Radical Scavenger Activities of Chloroform and Ethyl Acetate Extracts of Teucrium persicum

VariablesChloroform ExtractEthyl Acetate Extract
Total phenolic contents a48 ± 0.225 ± 0.4
Total flavonoid contents b34 ± 0.636 ± 0.6
Free radical scavenging activity (IC50) (μg/mL)2.5 ± 0.0133 ± 0.0023
Antioxidant activity c3.5 ± 0.0034.5 ± 0.01

a The phenolic contents are expressed as µg gallic acid equivalent per 100 µg extract.

b mg EQ/g of dried extract.

c Total antioxidant capacity (phosphomolybdenum assay) was expressed in mg ascorbic acid equivalents/mg extract).

4.2. Total Flavonoid Contents of Chloroform and Ethyl Acetate Extracts of Teucrium persicum

The average total flavonoid content of T. persicum chloroform and ethyl acetate extracts was calculated, and no significant difference was observed between the total flavonoid contents of these two extracts (Table 1) (P < 0.05).

4.3. Radical Scavenging Activity Chloroform and Ethyl Acetate Extracts of Teucrium persicum

The radical scavenging activity of the chloroform and ethyl acetate extracts was evaluated by adopting the DPPH method. The DPPH and ascorbic acid were used as a stable free radical and as a standard, respectively. As Table 1 shows, the concentrations responsible for 50% inhibition for both extracts were not significantly different (Table 1). These concentrations were detected to be 2.5 ± 0.013 and 3 ± 0.0023 μg/mL for the chloroform and ethyl acetate extracts, respectively. The IC50 value of ascorbic acid was calculated as 8 μg/mL.

4.4. Antioxidant Potential of Chloroform and ethyl Acetate Extracts of Teucrium persicum

The antioxidant potential of the extracts was assessed by performing the FRAP analysis. The average ferric reducing antioxidant power of the chloroform and ethyl acetate extracts and quercetin were 3.5 ± 0.003, 4.5 ± 0.01 µM of FeSO4, respectively; while the power of quercetin was found to be 1.2 µM of FeSO4.

4.5. Composition of Chloroform and Ethyl Acetate Extracts

The results from phytochemical screening of chloroform and ethyl acetate extracts of T. persicum are presented in Tables 1 and 2. (-)-Spathulenol (17.87%), (-)-Neoclovene-(I), dihydro (9.98), isoaromadendrene epoxide (7.39%), culmorin (6.6%), oxiranecarboxaldehyde, 3-methyl-3-(4-methyl-3-pentenyl)- (5.44%), and caryophyllene oxide (5.31%) were the most abundant components of the chloroform extract (Table 2). Longifolenaldehyde (11.5%), culmorin (6.42%), α-Farnesene (5%), alloaromadendrene oxide-(1) (4.65%), 1-Heptatriacotanol (4.38%) were the most abundant components of the ethyl acetate extract (Table 3).

Table 2.

The Chemical Composition of Chloroform Extract of Teucrium persicum

Compound NameExtract (%)Retention TimeChemical Formula
1. 1-Pentanol0.177.074C5H12O
2. Eucalyptol0.3211.180C10H18O
3. Culmorin6.6026.737C15H26O2
4. Santolina triene0.1412.190C10H16
5. Caryophyleine-(I3)0.3536.229C15H24
6. α-Farnesene3.2726.498C15H24
7. Globulol0.5422.878C15H26O
8. Isocaryophillene0.4021.435C15H24
9. Octadecanoic acid0.5133.732C17H35CO2H
10. Ferruginol0.2536.550C20H30O
11. Ledol0.3236.731C15H26O
12. Isomyocorene1.5930.280C10H16
13. 3-Octadecyne0.2015.975C18H34
14. β-Humulene1.1923.399C15H24
15. Phytol0.2432.921C20H40O
16. Isoledene1.9823.910C15H24
17. Platambin3.2828.660C15H26O2
18. Thunbergol0.4932.189C20H34O
19. Longifolenaldehyde1.8523.987C15H24O
20. (-)-Spathulenol 17.8726.736C15H24O
21. Isobornyl propionate0.3516.796C13H22O
22. Cedran-diol, 8S,140.3516.796C15H26O
23. Isomyocorene1.5930.280C10H16
24. 3,4-Decadiene0.3014.676C11H20
25. Cyclononene0.0913.389C9H16
26. Isoaromadendrene epoxide7.3924.050C15H24O
27. dl-Thioctic acid0.1434.952C8H14O2S2
28. Isobornyl propionate0.3516.796C13H22O
29. cis-Linaloloxide0.1611.835C10H18O
30. 1-Eicosene0.4633.299C20H40
31. 5-Caranol, (1S,3R,5S,6R)-(-)-0.4320.714C10H18O
32. α-Santalol 0.3014.676C15H24O
33. trans-2-Caren-4-ol0.8321.091C10H16O
34. 17-(Acetyloxy)-kauran-18-al0.2921.335C23H36O
35. Coprostan-3 β16β-diol0.4122.068C27H48O2
36. cis-Linaloloxide0.2214.477C10H18O
37. 2,6-Dimethyl-3,7-octadiene-2,6-diol0.3213.888C10H18O2
38. 8,14-Cedranoxide, 2.8228.793C15H24O
39. dl-Thioctic acid0.1434.952C8H14O2S2
40. Artemisia alcohol0.0913.289C10H18O
41. Alloaromadendrene oxide-(1)1.6227.113C15H24O
42. Lavandulol, trifluoroace0.1114.621C12H17F3O2
43. (6Z)-Nonen-1-ol0.7718.473C9H18O
44. cis-13-Octadecenoic acid0.4633.299C18H34O2
45. 1,6,9-Tetradecatriene0.2015.975C14H24
46. 1,3,5-Heptatriene0.2213.578C7H10
47. Aristolene epoxide1.9625.552C15H24O
48. γ-Gurjunene0.8823.044C15H24
49. Epi-β-Santalol0.2213.578C15H24O
50. Oxiranecarboxaldehyde, 3-methyl-3-(4-methyl-3-pentenyl)-5.4429.670C10H16O2
51. 5-Caranol, (1S,3R,5S,6R)-(-)0.4320.714C10H18O
52. (+)-δ3-Carene3.8528.948C10H16
53. Caryophyllene oxide 5.3125.230C15H24O
54. Morphinan-6-one, 4,5-epoxy-3,14-dihydroxy-17-(2-propenyl)-, (5 α)-0.2536.550C19H21NO4
55. Pentadecyl pentafluoropropionate0.9824.465C18H31F5O2
56. Murolan-3,9(11)-diene-10-peroxy2.5625.297C15H24O2
57. β-Cedren-9-α-ol1.9628.793C15H24O
58. (-)-Neoclovene-(I), dihydro9.9829.866C15H26
59. Neogammacer-22(29)-ene1.5732.489C30H50
60. 7,8-Epoxy- α -ionone1.5930.280C13H20O2
Total identified compounds98.18
Table 3.

The Chemical Composition of Chloroform Extract of Teucrium persicum

Compound NameEthyl Acetat Extract (%)Retention TimeChemical Formula
1. Ledol2.3439.378C15H26O
2. Myrcenylacetat0.1523.590C12H20O2
3. Ethisterone3.6442.229C21H28O2
4. Culmorin6.4235.153C15H26O2
5. Phytol0.5223.686C20H40O
6. Ocimene0.1522.695C10H16
7. γ-Elemene0.5228.672C15H24
8. Isophytol0.2222.543C20H40O
9. Camphene0.2224.594C10H16
10. 7-Nonenamide0.5928.199C9H17NO
11. α-Farnesene5.0227.937C15H24
12. α-Irone0.4929.085C14H22O
13. γ-Santalol0.5228.672C15H24O
14. (-)-Spathulenol14.832.569C15H24O
15. Falcarinol0.8632.521C17H24O
16. γ-himachalene0.1127.000C15H24
17. Longifolenaldehyde11.531.589C15H24O
18. Cholesterol0.5934.438C27H46O
19. Tetratetracontane1.6137.164C44H90
20. 1-Heptatriacotanol4.3835.197C37H76O
21. 7-Nonenamide0.3126.359C9H17NO
22. γ-Gurjunenepoxide-(2)3.2035.704C15H24O
23. Tetracosane1.0443.099C24H50
24. Cadala-1(10),3,8-triene0.4030.234C15H22
25. 5(10)-Estrene-3β,17α-diol4.0841.250C18H28O2
26. 17-(acetyloxy)-Kauran-18-al0.6833.619C23H36O3
27. Tricosane0.5239.841C23H48
28. 8S,14-Cedrandiol0.4940.161C15H26O2
29. Diazoprogesterone0.8632.531C21H30N4
30. beta.-Cedren-9-α-ol2.6135.586C15H24O
31. Pyridine0.068.695C5H5N
32. Eucalyptol0.1116.237C10H18O
33. cis-Linalol oxide0.2217.469C10H18O2
34. Trans-Linalool oxide 2.5621.428C10H18O2
35. trans-3-Penten-2-ol0.1118.347C5H10O
36. 1,5,8-Menthatriene0.1319.276C10H14
37. (Z)6-Pentadecen-1-ol0.0619.656C15H30O
38. E-12-Tetradecenal0.7223.085C14H26O
39. Dodecane0.1321.564C12H26
40. 2,4-Hexadiene, (E,Z)0.2223.252C6H10
41. 1-Chloro-2-phenylazetidine0.0615.393C4H8ClN
42. Propanedioic acid, 2-propenyl0.0623.995C10H16O4
43. 17-Octadecenal0.1324.079C18H34O
44. 13-Tetradece-11-yn-1-ol0.1324.763C14H24O
45. 3-Octadecyne0.1124.814C18H34
46. 5-Octen-1-ol1.0925.692C8H16O
47. Guanidine, N-[3-[(2-bromophenyl)0.5929.592C10H14BrN3
48. cis-Z-α-Bisabolene epoxide0.4029.592C15H24O
49. Ledene oxide-(II)0.2730.538C15H24O
50. Alloaromadendrene oxide-(1)4.6531.767C15H24O
51. Epianastrephin0.2231.027C12H18O2
52. α-Bulnesene0.7431.171C15H24
53. (E)-3(10)-Caren-4-ol0.9932.821C10H16O
54. 2-Tetradecene1.0932.158C14H28
55. Calarene epoxide3.2035.704C15H24O
56. Ledene oxide-(I)4.2435.831C15H24O
57. Diepicedrene-1-oxide1.4535.957C15H24O
58. (-)-Neoclovene-(I), dihydro-1.4537.722C15H26
59. [1,2,4]Triazolo[1,5-a]pyrimidine0.0445.210C5H4N4H28O
60. (-)-Isolongifolol, methyl ether1.2939.933C16H28OO
61. Ergost-22-en-3-ol, (3β, 5α, 22E,24R)0.3628.376C28H48O
Total identified compounds96.83

4.6. The Cytotoxic Effects of Chloroform and Ethyl Acetate Fractions on Different Cell Lines

In this study, two human cancer cell lines including PC-3 (human prostate adenocarcinoma), SW-480 (human colorectal adenocarcinoma), and normal HEK-293 (human embryonic kidney) cells were treated with different concentrations of chloroform and ethyl acetate extracts of T. persicum for 48 hours followed by measuring the viability of cells by MTT. As shown in Figure 1, the chloroform extract of T. persicum showed a higher toxicity effect on PC-3 cells compared to the ethyl acetate extract. However, the ethyl acetate extract showed greater growth inhibition on SW-480 cells compared to the chloroform extract. As shown in Figure 1, the chloroform extract exerted higher inhibitory effects on SW-480 cells compared to PC-3 and HEK-293 cells. The IC50 values of chloroform extract for PC-3, SW-480, and HEK-293 cells were calculated as 39, 4.453, and 6.574 μg/mL, respectively. Our MTT results demonstrated that although 10 and 25 μg/mL of ethyl acetate extract caused a small increase in the number of viable PC-3 cells (up to 15%), the treatment of PC-3 cells with higher concentrations of the extract led to a considerable reduction in cell viability (Figure 1). The ethyl acetate extract exerted stronger inhibitory effect on the viability of SW-480 cells followed by HEK-293 cells (Figure 1). The IC50 values of ethyl acetate extract for PC-3, SW-480, and HEK-293 cells were calculated as 65.9, 1.65, and 5.181 μg/mL, respectively (Table 4).

Chloroform and ethyl acetate extracts of Teucrium persicum reduces the viability of PC-3, SW-480, and HEK-293T cell lines. The cells were seeded in 96-well plates and allowed to grow to 70% confluency. The cells were then treated with different concentrations of the extract for 48 hours, and the cell viability was calculated using the MTT assay. The shown data are mean ± SD of three separate experiments repeated in 10 wells (*: P < 0.05; **: P < 0.01; ***: P < 0.001 vs. control).
Chloroform and ethyl acetate extracts of Teucrium persicum reduces the viability of PC-3, SW-480, and HEK-293T cell lines. The cells were seeded in 96-well plates and allowed to grow to 70% confluency. The cells were then treated with different concentrations of the extract for 48 hours, and the cell viability was calculated using the MTT assay. The shown data are mean ± SD of three separate experiments repeated in 10 wells (*: P < 0.05; **: P < 0.01; ***: P < 0.001 vs. control).
Figure 1.

Chloroform and ethyl acetate extracts of Teucrium persicum reduces the viability of PC-3, SW-480, and HEK-293T cell lines. The cells were seeded in 96-well plates and allowed to grow to 70% confluency. The cells were then treated with different concentrations of the extract for 48 hours, and the cell viability was calculated using the MTT assay. The shown data are mean ± SD of three separate experiments repeated in 10 wells (*: P < 0.05; **: P < 0.01; ***: P < 0.001 vs. control).

Table 4.

The IC50 Values of Chloroform and Ethyl Acetate Extracts of Teucrium persicum

Cell LineChloroform Extract (μg/mL)Ethyl Acetate Extract (μg/mL)
PC-33965.9
SW-4804.4531.65
HEK-2936.5745.181

5. Discussion

There is a body of evidence suggesting that plants and their phytochemicals may have therapeutic properties; some of them are currently used in the clinics to treat various diseases, including different types of cancer (17-19).

Teucrium persicum is an Iranian endemic plant with potentially unknown properties, which has been used in Iranian traditional medicine to treat various diseases as well as hyperlipidemia, diabetes, and obesity (6).

This study aimed to investigate the antioxidant and cytotoxic potential of chloroform and ethyl acetate extracts of T. persicum. According to the results from our previous studies, the methanolic extract of T. persicum had significant antioxidant and cytotoxic properties (13). Our biochemical analyses indicated that chloroform and ethyl acetate extracts of T. persicum contained noticeable total phenolic and flavonoid contents. Moreover, it was found that 2.5 μg/mL of chloroform extract was required to scavenge 50% of DPPH radicals (compared to 8 ± 0.005 μg/mL of ascorbic acid) (P < 0.05). This value for ethyl acetate was detected to be 3 μg/mL.

According to the results from MTT experiments, PC-3 cells were discovered to be less sensitive than SW-480 and even HEK-293 cells. It was also found that the chloroform extract was capable of exerting stronger inhibitory effect on PC-3 cells, but the opposite was the case as for SW-480 and HEK-293 cells (Table 4). Taking the laboratory results into consideration, these different inhibitory effects may have been attributed to the synergistic effects of two or more compounds in the extracts (19, 20). Previous reports have indicated that Teucrium species contain considerable amounts of phenolic and flavonoid compounds that are the main cause of their biological activities (11-13). Therefore, it is suggested that the cytotoxic potential of the ethyl acetate and chloroform extracts of T. persicum may be due to the presence of phenolic and flavonoid compounds. Compared to our previous study (16), the present study showed that the chloroform and ethyl acetate extracts of T. persicum had more phenolic contents than the methanolic extract.

Due to the difference in polarity of solvents used in this study, the chemical profile of these three extracts may have been different and, therefore, the variability in antioxidant and cytotoxicity properties of these extracts may have been reasonable. GC and GC/MS analysis facilitated the identification of 60 and 61 compounds representing 98.18% and 96.52% of the chloroform and ethyl acetate extracts, respectively. The relative concentrations of the identified components of chloroform and ethyl acetate extracts are presented in Tables 2 and 3, respectively.

Previous studies conducted to analyze the essential oils of Teucrium species had already documented the general dominance of the sesquiterpenes group (21-23). Our results were in agreement with the findings from the given studies.

Reviewing the literature demonstrated that some of these compounds including α-pinene, β-caryophyllene, and β-caryophyllene-oxide had significant anti-inflammatory, antioxidant, anti-hypercholesterolemic, anticancer, and antibacterial effects (24-29). Laboratory findings had already suggested that the presence of various compounds in the essential oils or extracts of plants may have been the underlying cause of their biological properties (30-32). Therefore, it was hypothesized that one or more compounds may have been responsible for the inhibitory effects of chloroform and ethyl acetate extracts of T. persicum.

5.1. Conclusions

The results from our previous studies and the current study suggested that the antioxidant and cytotoxic effects of T. persicum extracts may have been attributed to the presence of various compounds, including phenolic and flavonoid compounds. However, it was recommended that further studies should be conducted in order to investigate phytochemical profiles of T. persicum extract, facilitate the process of studying its anti-cancer potential, and investigate the underlying molecular mechanism(s) by isolating and identifying the effective compounds.

Acknowledgements

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