*Contributed equally
For thousands of years, medicinal herbs have been an integral part of traditional medicine, since a number of them exhibit potent antioxidant properties, mainly associated with their rich content in bioactive compounds. Based on these attributes, nowadays, medicinal herbs are used for industrial purposes (e.g., as natural food additives) and are also evaluated as chemopreventive strategies for diseases associated with the disruption of redox homeostasis. In that frame, the aim of the present study was to appraise the redox properties of various medicinal or edible herbs originating from the region of Epirus in Greece. The antioxidant, reducing and antigenotoxic effects of herb decoction extracts were evaluated using a series of
Medicinal plants have attained a commanding role in the global health care system as sources of various phytochemicals, several of which possess potent antioxidant properties. Such products cover a large part of the global market, exceeding $100 billion annually and this coverage is expected to reach $550 billion by 2030 with a compound annual growth rate of 18.9% (
Natural products offer a plethora of advantages to the drug development process compared to conventional synthetic compounds. To begin with, natural products can be found in high abundance in nature, allowing scientists to yield almost endless quantities of these. Notably, natural products have a higher structural complexity and scaffold diversity than typical synthetic small-molecule libraries (
Synthetic antioxidants were the food industries first candidates used to counteract the potential adverse effects of various food products on the health of consumers (
Several methodologies have been developed for the purpose of evaluating the antioxidant capacity of crude natural extracts or pure isolated chemical compounds that are derived from natural sources (
All the herbs were derived from local producers in the Epirus region of Greece. To determine the total phenolic content, Folin-Ciocalteu reagent and gallic acid were purchased from Sigma-Aldrich; Merck KGaA. For the appraisal of the antiradical and reducing activities of the herb decoction extracts, 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), horseradish peroxidase (HRP), hydrogen peroxide (H2O2) solution 30%, methanol (MeOH), 1,1-diphenyl-2-picrylhydrazyl (DPPH•), ferric chloride, 2-deoxyribose, nicotinamide adenine dinucleotide (NADH), nitroblue tetrazolium (NBT) and phenazine methosulfate (PMS) were obtained from Sigma-Aldrich; Merck KGaA. Furthermore, trichloroacetic acid (TCA) and 2-thiobarbituric acid (TBA) were obtained from Merck KGaA. To estimate the potential antigenotoxic properties, pBluescript (SK+) plasmid DNA was purchased from Stratagene; Agilent Technologies, Inc. and 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) from Sigma-Aldrich; Merck KGaA. With respect to the tested cell line, EA.hy926 endothelial cells were donated by Professor George Koukoulis (University of Thessaly, Larissa, Greece). For cell cultures, Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline (PBS) and trypsin-EDTA solution 0.25% were purchased from Gibco; Thermo Fischer Scientific, Inc. The TACS XTT Cell Proliferation assay kit was purchased from R&D Systems, Inc. Finally, to determine the intracellular GSH and ROS levels, mercury orange and 2,7-dichlorofluorescein diacetate (DCF-DA) were purchased from Sigma-Aldrich; Merck KGaA. All solvents were of analytical grade.
To prepare herb decoction extracts, 2 g of dry herb leaves were added to 200 ml tap water, followed by boiling for 3 min. Subsequently, the boiled samples were allowed to stand for 5 min. The resulting decoction was filtered, followed by lyophilization of the total filtrate. The yield of the products following extraction is presented
The TPC of the samples was determined using Folin-Ciocalteu reagent. Briefly, 1 ml dH2O, 100 µl Folin-Ciocalteu reagent and 20 µl of each sample were added to test tubes and the mixture was incubated for 3 min at 25˚C under dark conditions. Subsequently, 280 µl of a sodium carbonate solution (25% w/v) and 600 µl dH2O were added, followed by incubation for 1 h at 25˚C under ambient conditions in the dark, and the absorbance was then determined at 765 nm using a spectrophotometer (Hitachi, U-1900 UV/VIS, Hitachi High-Technologies Corporation). A test tube containing Folin-Ciocalteu reagent and dH2O was used as a blank. The phenolic content was determined using a standard curve of gallic acid (0, 50, 150, 250 and 500 µg/ml), and the results are expressed as mg of gallic acid per g of dry sample.
The radical scavenging capacity (RSC) of the tested herb decoctions was evaluated using a slightly modified method of the DPPH• assay (
The ABTS•+ RSC of the tested samples was determined as previously described by Cano (
The superoxide anion radical scavenging ability of the herb decoctions was assessed using the method of Gülçin
The reducing power capacity was determined according to the method described in the study by Yen and Duh (
The assay was performed using a procedure previously described (
Following the cell-free based assays and the characterization of the antioxidant properties of the tested herb decoctions, the four most potent herb decoction extracts were assessed for their cytotoxic and intracellular antioxidant properties in the EA.hy926 cell line. EA.hy926 is a stable human endothelial cell line derived by hybridizing human umbilical vein endothelial cells, namely human umbilical vein endothelial cells (HUVECs), with the A549 human lung carcinoma cells. The endothelial cells were cultured in 25 cm2 tissue culture flasks and incubated for 24 h at 37˚C in 5% CO2 and 80-95% humidity to reach ~70-80% confluency. The cell culture medium used was DMEM containing 1 g/l D-glucose, 4 mM L-glutamine and supplemented with 10% (v/v) FBS, 100 units/ml penicillin and 100 units/ml streptomycin. A morphology examination at high and low culture densities was conducted using a microscope (Kern, OCL251, KERN & SOHN GmbH; data not shown) to authenticate the state of cells, through their phenotypic characteristics. According to the international guidelines on good cell culture practice (
Cell viability was assessed using the XTT assay kit (R&D Systems, Inc.). Briefly, 104 cells were seeded into a 96-well plate with their respective complete medium. Following a 24-h incubation, the cells were treated with increasing concentrations of the Epirus herb decoctions in serum-free medium for an additional 24 h. Subsequently, 50 µl of the XTT test solution were prepared by mixing 50 µl XTT-labeling reagent with 1 µl XTT activator, and 50 µl of the XTT test solution were added to each well. Following a 4-h incubation, the optical density was measured at 450 and 630 nm (reference wavelength) using a microplate reader (Bio-Tek ELx800; Bio-Tek Instruments, Inc.). Cell cultures in serum-free medium were used as a negative control. Moreover, the absorbance of every tested sample concentration alone in serum-free medium and XTT test solution was also measured at 450 nm using a plate reader (EL808; BioTek Instruments, Inc.). The absorbance values that were obtained in wells that contained only herb decoctions extracts were subtracted from the ones that acquired from wells that contained the respective extract concentration and seeded cells. Data were calculated as follows: Cell viability (% of control)=(ODsample/ODcontrol) x100, where ODcontrol and ODsample indicate the optical density of the negative control and the test compounds, respectively. All experiments were carried out in duplicate and at least on two separate occasions.
The endothelial cells were seeded in 25 cm2 culture flasks for GSH and ROS determination and incubated for 24 h at 37˚C in 5% CO2 and 80-95% humidity to reach about 70-80% confluency. The culture medium was then removed and replaced with serum-free medium containing the herb decoction extracts tested at different concentrations. Following a 24-h incubation, the cells were trypsinized, collected and washed twice following consecutive centrifugations at 300 x g for 10 min at 5˚C. After each centrifugation the supernatant was discarded, and the cellular pellet was resuspended in PBS. After the second wash the cellular pellet (106 cells/ml) was ready for intracellular staining for determinations of GSH and ROS levels using flow cytometry with mercury orange and DCF-DA, respectively. The fluorescent mercury orange binds directly to GSH, whereas DCF-DA is deacetylated by esterases within the cells, and is further converted to fluorescent DCF by the oxidative action of ROS. A 400 µM stock solution of mercury orange was created in acetone and stored at 4˚C, while a fresh 400 µM stock solution of DCF-DA was prepared in methanol. To assess the GSH and ROS levels, the cells (106 cells/ml) were resuspended in PBS and incubated in the presence of mercury orange (40 µΜ) or DCF-DA (10 µΜ) in the dark at 37˚C for 30 min. Following incubation, the cells were washed with PBS to remove the excess dye, centrifuged (300 x g, 10 min, 4˚C) and resuspended in PBS. The cells were then submitted to flow cytometric analysis using a FACSCalibur flow cytometer (BD Biosciences) with excitation and emission length at 488 and 530 nm for ROS, and at 488 and 580 nm for GSH. Forward angle and right-angle light scattering representable of the cells size and cell internal complexity, respectively, were measured. Analyses were performed on 10,000 cells per sample, at a flow rate of 1,000 events/sec, and fluorescence intensities were measured on a logarithmic scale. Data were analyzed using BD Cell Quest software 6.0 (BD Biosciences). Each experiment was repeated at least three times.
For
Initially, the TPC of all the herb decoction extracts, that were supplied to us by Epirus local producers, was determined. According to the results, the highest polyphenolic content was observed in the sage extract (
According to the DPPH• assay, the decoction extract that derived from oregano (
In the ABTS•+ assay, the extract derived from oregano (
In the superoxide assay, the extract that was derived from sage (
The extract that exhibited the highest reducing power capacity was the one derived from lemon beebrush (
The plasmid relaxation assay revealed that rosemary (
Four of the extracts that displayed the highest cell-free antioxidant capacity in the methods tested were screened using the EA.hy926 cells for cytotoxicity and antioxidant-related parameters. More specifically, oregano (
Initially, the authors wished to examine whether these four extracts exerted any cytotoxic effects. For this purpose, XTT cell proliferation assay was performed using EA.hy926 cells. The
Subsequently, the present study examined whether sublethal concentrations of the herb decoction extracts were able to alter the intracellular levels of GSH and ROS, since they both play crucial roles in physiology, particularly in cells with a cancerous profile. The sublethal concentrations of all four herbs decoction extracts were unable to affect the ROS levels as compared with the control group (
The present study aimed to determine the redox-related properties of well-known and routinely used herb decoctions derived from Epirus region, Greece, predominantly for their extensive use in everyday life, their integral part in human diet, and eventually for their potential exploitation as chemopreventive agents. The results suggest the potent antioxidant activity of Epirus medicinal and aromatic herbs. Moreover, the lack of cytotoxicity and the alterations induced in the GSH/ROS equilibrium represent promising paraphernalia for activity, strengthening the logic of using herbal decoctions as a prevention strategy against oxidative stress related diseases which currently stand out as the dominant threats of human health (
The range of the TPC in the tested decoctions was from 0.44 mg gallic acid/ml for the
The potent antioxidant potential that the polyphenolic compounds of the herb decoctions possess is a manifestation that has been already reported (
The experiments performed in the present study clearly indicated that the extracts of
The results obtained herein correspond with those of other studies examining the antioxidant properties of the medicinal and aromatic herbs
In the global literature, there is a constant debate as to the plant herb biologically active substances that can affect the activity and metabolism of cells. Cell-free methodologies are able to provide valuable preamble data concerning their efficacy; however, cell-based
The high energy demand of cancer cells, and concomitantly, their intense metabolic rates lead to abundant ROS production in the cellular environment, derived primarily from the mitochondria and the endoplasmic reticulum. Albeit the continuous and elevated ROS levels can result in the death of normal cells, through the induction of oxidative stress, the high rate of ROS generation in cancer cells is compensated by the equally high activation of the respective antioxidant mechanisms (
To address the above, the present study evaluated the cytotoxicity exerted by the four most potent decoction extracts in order to determine the effects of non-cytotoxic concentrations of these on the intracellular GSH and ROS levels. The results from XTT assay revealed that
The assessment of the effects of the extracts on the antioxidant capacity of endothelial cells was based on the measurement of the GSH and ROS levels using flow cytometry. The regulation of intracellular GSH levels following extract treatment is crucial, since GSH is considered a significant endogenous antioxidant molecule in cells (
In the present study, the results revealed that
In the present study,
In the present study, the
Even though
The increase of the ‘spare capacity’ between the GSH and ROS levels in the cancer endothelial cell line, as proposed by the results of the present study may be part of a further disruption of their redox status compared to normal cells. This phenomenon may compose a critical step for the selection of appropriate chemopreventive strategies based on appropriate configurations of the redox potential of cancer cells. Chinese herbs have been associated with the metabolic reprogramming of cancer cells, enabling their experimental use as therapeutic compounds against metabolism-related diseases (
Αn uncertainty that the present study generates lies in the obvious discrepancy between results in decoctions examined in the authors' laboratory and originating from the same plant type, but have been provided by different producers. This could relate to the fact that herb biological properties are dependent on differences in the exact geographical location and cultivation micro-environment conditions. Previously, Karydas
In conclusion, the results of the present study support the promising role of the tested decoctions as a source of antioxidant active compounds in follow-up
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
ZS, IDK, PV and FT analyzed and interpreted the data regarding the antioxidant activity of the herbs. ZS, IDK and PV were major contributors to the writing of the manuscript. KA, NG and DK, were involved in the design and conception of the study, and also confirm the authenticity of all the raw data. All authors read and approved the final manuscript.
Not applicable.
Not applicable.
DK is an Editor of the journal, but had no personal involvement in the reviewing process, or any influence in terms of adjudicating on the final decision, for this article. The other authors declare that they have no competing interests.
Cytotoxic concentration threshold (µg/ml) of
Effects of
Effects of
Total phenolic content, IC50 and AU0.5 values of Epirus herb decoction extracts evaluated using
Code | Herb | Common name | TPC (mg gallic acid/ml) | DPPH• IC50 (µg/ml) | ABTS•+ IC50 (µg/ml) | Superoxide IC50 (µg/ml) | Reducing power AU0.5 (µg/ml) | Plasmid relaxation assay IC50 (µg/ml) |
---|---|---|---|---|---|---|---|---|
1 | Chamomile | 0.51 | 97.10±2.97 | 45.09±5.73 | 43±1.42 | 46±5.49 | 154±6.78 | |
2 | Ironwort | 0.85 | 33.23±0.67 | 25.16±0.57 | 26±2.45 | 14±1.69 | 112±9.24 | |
3 | Peppermint | 0.53 | 50.29±3.28 | 37.24±0.90 | 45±3.78 | 32±1.42 | 125±8.75 | |
4 | Perforate St. John's wort | 1 | 16.11±3.49 | 12.47±0.70 | 32±5.21 | 11±0.78 | 138±4.59 | |
5 | Basil | 0.69 | 26.31±1.31 | 22.92±0.17 | 11.5±1.01 | 22±1.57 | 163±9.36 | |
6 | Chamomile | 0.29 | 94.29±9.63 | 64.97±1.15 | 45.5±2.47 | 36±2.59 | 158±4.97 | |
7 | Peppermint | 0.69 | 16.30±1.27 | 11.29±0.26 | 28±2.35 | 7±0.24 | 157±8.52 | |
8 | Lemon balm | 0.72 | 20.64±1.78 | 8.33±0.51 | 28±1.89 | 8.3±0.36 | 79±5.47 | |
9 | Bay laurel | 0.51 | 72.85±8.22 | 27.21±0.47 | 45±1.93 | 7±0.58 | 174±9.63 | |
10 | Linden | 0.72 | 23.07±0.26 | 14.23±0.64 | 28±2.41 | 18±0.49 | 140±2.89 | |
11 | Peppermint | 0.67 | 28.11±0.63 | 23.63±0.08 | 37.5±1.58 | 15±2.14 | 87±3.56 | |
12 | Ironwort | 0.79 | 36.55±0.11 | 33.56±0.67 | 37±2.63 | 18±2.39 | 156±8.54 | |
13 | Sage | 0.44 | 26.68±1.22 | 19.07±0.09 | 6.5±0.25 | 8±0.35 | 54±4.51 | |
14 | Pennyroyal | 0.82 | 27.53±1.24 | 14.32±0.53 | 29±1.28 | 12.5±1.54 | 65±5.62 | |
15 | Basil | 0.52 | 42.88±2.36 | 27.53±8.82 | 23.5±2.54 | 125±7.98 | >200 | |
16 | Lemon beebrush | 0.64 | 23.68±1.12 | 28.3±0.16 | 27±3.56 | 9±0.86 | 50±2.78 | |
17 | Yarrow | 0.28 | 50.50±3.50 | 35.81±7.34 | 26.5±3.41 | 45±3.97 | 135±5.41 | |
18 | Lemon Beebrush | 0.83 | 10.25±0.25 | 8.29±1.13 | 49±2.39 | 3.5±0.13 | 26±2.14 | |
19 | Perforate St. John's wort | 0.83 | 27.89±0.43 | 13.78±0.67 | 63±5.78 | 13.5±0.89 | 36±1.36 | |
20 | Ironwort | 0.48 | 90.75±0.26 | 36.96±0.52 | 32.5±5.02 | 30±4.07 | 83±3.65 | |
21 | Lemon Balm | 0.62 | 17.75±3.7 | 8.95±0.30 | 9±0.98 | 7±0.69 | 40±2.89 | |
22 | Lemon balm | 0.69 | 18.21±0.05 | 9.06±0.62 | 23±1.25 | 7.2±0.78 | 32±3.45 | |
23 | Hawthorn | 0.89 | 20.72±1.83 | 14.05±0.23 | 59±4.13 | 25±2.19 | 42±2.56 | |
24 | Sage | 0.76 | 23.26±1.89 | 20.96±2.63 | 37.5±4.52 | 7.5±0.67 | 35±3.74 | |
25 | Ironwort | 0.44 | 80.99±0.16 | 49.58±0.04 | 48±6.02 | 10±0.92 | 156±8.59 | |
26 | Ironwort | 0.64 | 39.36±0.15 | 32.23±0.39 | 59±5.49 | 26±1.28 | 145±9.63 | |
27 | Sage | 0.51 | 27.37±1.05 | 16.73±0.12 | 20.5±1.43 | 30±4.59 | 50±1.49 | |
28 | Spearmint | 0.92 | 29.35±0.52 | 20.71±1.83 | 24±0.97 | 10.5±2.30 | 53±2.15 | |
29 | Ironwort | 0.52 | 88.22±10.73 | 38.05±2.05 | 25±1.49 | 59±5.62 | 145±7.46 | |
30 | Sage | 1.04 | 20.44±0.01 | 14.73±0.46 | 20.5±1.34 | 6.1±0.34 | 56±3.54 | |
31 | Linden | 0.41 | 33.46±2.26 | 23.74±1.16 | 20.5±2.96 | 12.5±0.57 | 45±2.57 | |
32 | Rosemary | 0.66 | 11.63±4.32 | 12.27±0.38 | 14.5±1.09 | 7.5±0.48 | 25±1.27 | |
33 | Rosemary | 0.45 | 17.14±1.10 | 14.17±0.19 | 27±2.56 | 9.7±0.92 | 36±2.37 | |
34 | Oregano | 0.68 | 14.11±0.93 | 8.88 ±0.40 | 20.5±1.74 | 9.5±0.81 | 45±2.18 | |
35 | Oregano | 0.78 | 16.77±0.66 | 11.53±0.20 | 20.5±1.85 | 11±0.74 | 49±4.37 | |
36 | Oregano | 0.52 | 21.86±1.76 | 11.63±0.14 | 43±2.94 | 12±0.93 | 55±5.04 | |
37 | Oregano | 0.39 | 24.09±1.36 | 14.72±0.88 | 23.5±2.36 | 9±0.53 | 93±2.05 | |
38 | Winter savory | 0.61 | 25.49±4.66 | 20.03±2.29 | 30±1.86 | 24±2.19 | 84±6.54 | |
39 | Oregano | 0.62 | 20.99±1.10 | 11.77±0.22 | 15±2.45 | 12±1.24 | 65±4.83 | |
40 | Garden thyme | 0.87 | 21.52±0.89 | 23.68±0.84 | 15.5±1.75 | 4±0.02 | 67±4.09 | |
41 | Oregano | 0.62 | 22.25±0.01 | 13.72±0.11 | 18±1.27 | 9.5±0.35 | 54±2.51 | |
42 | Garden thyme | 0.59 | 41.68±0.07 | 30.76±5.04 | 34±1.93 | 6.5±0.56 | 56±3.64 | |
43 | Oregano | 0.73 | 17.43±3.39 | 13.19±0.21 | 38±2.54 | 10±0.84 | 70±5.06 | |
44 | Oregano | 0.77 | 16.81±0.30 | 12.22±0.06 | 16.5±1.41 | 5.8±0.86 | 78±4.23 | |
45 | Rosemary | 0.93 | 18.96±0.59 | 15.36±1.15 | 19±1.23 | 10.5±1.07 | 82±5.82 | |
46 | Oregano | 0.51 | 6.60±1.50 | 7.85±0.56 | 12±1.07 | 7.5±0.41 | 35±3.06 | |
47 | Oregano | 0.67 | 18.74±4.96 | 14.22±0.24 | 12±1.17 | 9.4±0.45 | 46±4.09 |