Antioxidant and antimicrobial activities of four Astragalus species growing wild in Turkey Türkiye’de doğal yayılış gösteren dört Astragalus türünün antioksidan ve antimikrobiyal aktiviteleri

Objective: The objective of the present study was to investigate the phenolic compositions, antioxidant, antimicrobial and cytotoxic activities of four Astragalus species, two of which are endemic to Turkish flora. Methods: The total phenolic and flavonoid contents of methanol extracts obtained from four Astragalus species were detected using Folin-Ciocalteu and aluminum chloride colorimetric assays. Their phenolic compositions were identified by Liquid chromatography–mass spectro-metry (LC-MS). The antioxidant activity was assayed with phosphomolybdenum, 2,2-diphenyl-1-picrylhydrazyl radical scavenging (DPPH), hydrogen peroxide scavenging, β -carotene bleaching activity, ferric-ion reducing power (FRAP) and cupric ions (Cu 2 + ) reducing antioxidant capacity (CUPRAC) methods. Antimicrobial activities of the extracts were studied by agar well-diffusion assay. The cytotoxic effects of the extracts on MCF-7 (human breast cancer cell lines) were determined by MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazoliumbromide) method. Results: The extracts exerted moderate antioxidant and reducing activity with low phenolic contents. The main component in the extracts was determined as ferulic acid. The extracts demonstrated no antibacterial activity except P. aeruginosa . A. talasseus showed the highest cytotoxic activity on MCF-7 during 48 h. Conclusion: It is believed that the results of this study will contribute to research recently increasing for the use of natural antioxidant and antimicrobial compounds in many industrial fields such as food, pharmacy and medicine.


Introduction
Humankind has used herbal products to obtain nutrients, medicines, oils and many other useful products throughout history.It is stated that there is a relation between a diet with high consumption of herbal products such as fruit and vegetables and a decrease in risk for many chronic diseases [1,2].The majority of the antioxidant capacity of a fruit or vegetable may be from phenolic compounds [3].
The oils and extracts obtained from plants have been used as natural agents in many industry such as food, pharmacy, alternative medicine and natural therapy because of their antimicrobial activity.In order to prolong the storage stability of foods, synthetic antioxidants are mainly used in industrial processing [4].Recently, there has been a significant increase in the search for natural antioxidants to replace synthetic antioxidants in recent years [5].Because the use of synthetic antioxidants which have many side effect such as cancer have limited.The antioxidative status is associated with reduced incidence of many diseases [6].Thus, investigation and identification of new antioxidant compounds are very important.
The richest genus with 425 taxa is Astragalus L. (Fabaceae) in Turkish flora.Of these 425 taxa, 201 are endemic and the endemism rate is about 47% [7][8][9].Researches on Astragalus species in Turkey have resulted in the isolation of cycloartane and triterpenoid saponins [10][11][12][13][14].These compounds exert different biological activity such as immuno stimulant, antiviral, cardiovascular, anti-protozoal and cytotoxic [11,15,16].The roots of Astragalus species are well-known in folk medicine due to their biological activities such as hepatoprotective, antioxidative, antibacterial, antiperspirant, antihypertensive, antidiabetic, diuretic and tonic [17][18][19][20].It has also been used in the treatment of diabetes mellitus, nephritis, leukemia and uterine cancer.In the district of Anatolia, located in South Eastern Turkey, an aqueous extract of the roots of Astragalus is traditionally used against leukemia and for its wound-healing properties [20].
Noteworthiness of Astragalus species are great due to have a high nutritional values, obtain gum glue as a pharmaceutical emulsifying agent, used as firewood and animal feedstuffs, and in erosion prevention [21].
Although biological activities of many Astragalus species have been investigated, the antioxidant and antimicrobial activities of A. gummifer, A. microcephalus, A. talasseus and A. acmophyllus have not previously been published as far as our literature survey could ascertain.With present study, it is aimed to examine the phenolic compositions, total phenolic and flavonoid contents, antioxidant, antimicrobial and cytotoxic activities of the methanol extracts obtained from the underground parts of four different Astragalus taxa, two of which are endemic in Turkey.

Extraction
Dried underground parts of the plants at room temperature were ground to a fine powder with a grinder.Then the powdered plant material (10 g) was extracted using a Soxhlet type xtractor with 100 mL methanol at 60°C for 6 h.Thereafter, the extract was filtered and evaporated to dryness under vacuum at 40°C with a rotary evaporator.The extract yield was calculated and stored at 4°C [22].

Determination of total phenolic
Folin-Ciocalteau assay was performed to detect of the total phenolic amount in the extract [23].Forty microliter aliquot of the extract (1 mg/mL) was mixed with 200 μL of Folin-Ciocalteu reagent and 600 μL (20% Na 2 CO 3 ) of sodium carbonate.The mixture was vortexed and incubated at room temperature for 2 h.Absorbance was then read at 765 nm by the spectrophotometer.The standard curve was formed with gallic acid.Total phenolic was expressed in terms of milligrams of gallic acid equivalents (mg GAE)/g extract.

Total flavonoid
Total flavonoid was assessed using Aluminum chloride colorimetric assay according to Pourmorad et al. [24].The extract (0.5 mL) in methanol were separately mixed with 1.5 mL of methanol, 0.1 mL of 10% aluminum chloride, 0.1 mL of 1 M potassium acetate and 2.8 mL of distilled water.It remained at room temperature for 30 min; the absorbance of the reaction mixture was measured at 415 nm.The result is expressed as mg of quercetin equivalents (QE)/g extract.

Total antioxidant activity
The antioxidant activity of methanolic extracts was determined by the phosphomolybdenum method [25].0.4 mL of the methanolic extract (1 mg/mL) was mixed with 4 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate).The tubes were capped and incubated in water bath at 95°C for 90 min.After the samples had cooled to room temperature, the absorbance of the green phosphomolybdenum complex was measured at 695 nm.In the case of the blank, 0.4 mL of methanol was used in place of sample.The antioxidant activity was determined using a standard curve with ascorbic acid solutions as the standard.The antioxidant activity was expressed as mg of ascorbic acid equivalents (AAE)/g extract.

DPPH method
The ability of the extracts to scavenge DPPH radical was assessed spectrophotometrically [26].Fifty microliter aliquots of the proper extract dilution at a concentration range of 0.1-2 mg/mL was mixed with and 1 mL of the methanolic DPPH solution (0.1 mM).Methanol was used as a control instead of extract.The mixtures were left for 30 min at room temperature in the dark and the absorbance at 517 nm measured using methanol as blank.IC 50 (concentration causing 50% inhibition) value of the extract was determined graphically.The same procedure was repeated with BHT (butylated hydroxytoluene) as a positive control.The measurements were performed in triplicate and the results were averaged.Radical scavenging activity was expressed as percentage inhibition of DPPH radical and was calculated by following equation: Lipid peroxidation inhibitory effect in β-carotene bleaching system was assessed [27].β-Carotene (10 mg) was dissolved in 10 mL of chloroform (CHCl 3 ).An aliquot (0.2 mL) of this solution was added into a boiling flask containing 20 mg of linoleic acid and 200 mg of Tween 40.The chloroform was removed using a rotary evaporator at 40°C for 5 min.Distilled water (50 mL) was slowly added to the residue with vigorous agitation, to form an emulsion.The emulsion (5 mL) was added to a tube containing 0.2 mL of the extract solution (2 mg/mL).The test emulsion was incubated in a water bath at 50°C for 2 h, when the absorbance was measured at 470 nm.BHT was used as the positive control.

CUPRAC assay
The cupric ion reducing antioxidant activity of extract was detected [28].One milliliter each of 10 mM CuCl 2 , 7.5 mM neocuproine and NH 4 Ac buffer (1 M, pH 7.0) solutions were added into a test tube.Then, 0.5 mL of different concentrations of extracts (0.2-1 mg/mL) were mixed.The mixture absorbance was recorded against a blank at 450 nm after 30 min incubation at room temperature.Trolox was used as the positive control.

FRAP assay
Ferric ions reducing capacity of the extract was examined [29].FRAP is based on the reduction of ferric

H 2 O 2 scavenging activity
The extract (25-500 μg/mL) was mixed with 43 mM H 2 O 2 solution.After 10 min, the absorbance of the solution was read at 230 nm [30].Gallic acid, BHA and BHT were used as control.The percentages of scavenged hydrogen peroxide of extract and standards were calculated using the following equation:

Reducing power
The reducing power of the methanolic extract was detected [31].The extracts (0.5-10 mg/mL) were added to 2.5 mL potassium ferricyanide [K 3 Fe(CN) 6 ] and was stored for 20 min at 50°C.Then, the solution was centrifuged at 1000 g for 10 min after trichloroacetic acid (2.5 mL) was added.The upper layer solution was added to FeCl 3 (0.5 mL, 0.1%, w/v).The absorbance was read at 700 nm.
BHT was used as control.Higher absorbance of the reaction mixture indicated greater reducing power.

Chelating activity on ferrous ion
Ferrous ions (Fe 2+ ) chelating activity of the extract was examined [30].Aliquots of the extract (5 mg/mL) were added to FeCl 2 (2 mM, 0.1 mL).After incubation, ferrozine (5 mM and 0.2 mL) was added to the reaction solution.The absorbance was read at 562 nm.A lower absorbance indicates a higher chelating power.The chelating activity of the extract on Fe 2+ was compared with that of ethylenediamine tetraacetic acid (EDTA).Chelating activity was calculated by the following formula:

Determination of antimicrobial activity
The Agar-well diffusion assay was performed to detect of antimicrobial activity [22].Suspensions of each microorganism were regulated to 10 6 -10 7 colony-forming units (cfu)/mL and added to sterile growth medium.The mix was poured into Petri plates (9 cm).The wells (5 mm in diameter) were cut from the agar.The extracts were prepared at 1%, 2.5%, 5% and 10% concentrations in absolute methanol and 50 μL of extract solutions were applied to the wells.The growth inhibition zones were measured in millimeters.Inhibition zones of standard antibiotics namely Tetracycline TE-30 (10 mg/ mL) and Oxacillin OX-5 (10 mg/mL) were compared.

MTT assay
MCF-7 (Human breast cancer) cell line was incubated in Dulbecco's modified Eagle's medium (DMEM) supplemented with heat-inactivated 100 mL/L fetal bovine serum (Gibco), 100 U/mL penicillin, 100 mg/mL streptomycin.Fibroblast cell line was incubated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM glutamine.The cells were cultured in a humidified atmosphere of 5% CO 2 at 37°C, and the medium was changed every other day.
The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) assay was used to examine effects of four Astragalus extracts on the cell growth and survival [32]. 1 × 10 4 cells were dispersed to a 96 well plate.After 24 h incubation, the cells were treated with extracts (10, 25, 50, 100, 200 and 400 μg/mL) and incubated for 24 and 48 h.Then 10 mL of MTT in PBS (5 mg/mL) was added into each well and the cells were further incubated for 4 h.The MTT formazan crystals were solubilized by adding 200 mL of DMSO to each well.Absorbance was measured at 570 nm with Micro-plate Reader.The absorbance of the control cells was taken as 100% viability.The results are expressed as percentages of viable cells versus the respective controls.

Statistical analysis
Data from the experiments were subjected to analysis of variance (ANOVA) using SPSS [33] for Windows.Percentage data were transformed using arcsine √x before ANOVA.Means were separated at the 5% significance level by the least significant difference (LSD) test.Bivariate correlations were analyzed by Pearson's test using SPSS 10.0 on Windows.

Results and discussion
The yields of methanolic extracts ranged from 9.78 to 16.38%.While the extract obtained from A. acmophyllus had the highest yield, A. talasseus had the least extract yield (Table 1).Identification of the phenolic compositions of the extracts was carried out using Liquid chromatography-mass spectrometry (LC-MS)/MS.The amounts of phenolic compounds in the extracts were calculated as ppm and the results are given in Table 2.
The total amounts of phenolic in the extracts evaluated by the Folin-Ciocalteu assay were indicated as gallic acid equivalent (GAE) and the results are given in Table 1. A. microcephalus (13.49± 0.0 mg GAE/g extract) had the highest phenolic content, while A. acmophyllus (5.49 ± 0.3 mg GAE/g extract) had the least amount of phenolic.
The amount of total flavonoid in the extracts measured by the aluminum chloride colorimetric assay was expressed as the quercetin equivalent (mg QE/g extract) and the results  The phenolic compositions of the Astragalus extracts tested were examined by LC-MS (Table 2).The amount of each compound is demonstrated as ppm.Phenolic compounds couldn't be determined in the extracts not shown in the table.Chlorogenic acid, epicatechin, catechin hidrate, rutin, quercetin, kaempferol, syringic acid, sinnamic acid and ferulic acid were identified.The main component in the extracts was determined as ferulic acid (1123.9ppm) followed by syringic acid (735.18ppm) and sinnamic acid (558 ppm).The least abundant compound was quercetin (293.5 ppm).
The total antioxidant activity of the extracts measured by the phosphomolybdenum method is indicated by the ascorbic acid equivalent (mg AAE/g extract) and the results are shown in Table 1. A. gumnifer (131.37 ± 0.2 mg AAE/g extract) had the lowest total antioxidant activity while A. acmophyllus (178.25 ± 0.3 mg AAE/g extract) had the highest total antioxidant activity among the extracts.
The percent inhibition values of the extracts caused on the oxidation of the β-carotene-linoleic acid emulsion are given in Table 1.As shown in Table 1, A. talasseus had the highest activity (80.60%) and A. acmophyllus (54.23%) had the lowest activity among the extracts.% Inhibition values of the extracts were lower than the inhibition values of BHA (107.96 ± 1.72%) and BHT (105.47 ± 2.28%).
The antiradical activities were tested by the DPPH assay, and the results were expressed as % inhibition.The results are shown in Figure 1.The obtained values were compared with the standard antioxidant BHT.The concentration causing the 50% inhibition (IC 50 ) was detected by plotting the graph including percent inhibition values versus concentrations for each extract (Table 1). A. acmophyllus had the lowest DPPH scavenging effect (IC 50 = 253.88μg/mL), A. microcephalus had the highest DPPH scavenging effect (IC 50 = 86.67 μg/mL).
The reduction activity of the copper (II)-neopurin complex of the extracts to the copper (I)-neucuproin complex was measured by the CUPRAC method [28].The resulting complex gives maximum absorbance at 450 nm.The results are shown in Figure 2 as absorbance values at 450 nm and compared with the values of Trolox.As shown in Figure 2, A. microcephalus (0.83) exerted the highest absorbance value among the extracts at 1 mg/mL, whereas A. acmophyllus (0.32) showed the lowest absorbance value.Depending on the concentration, the absorbance and activity were increased.The absorbance values of the extracts were much lower than that of the trolox (2.85) used as control.
FRAP is commonly used to measure of reduction of ferric iron (Fe 3+ ) to ferrous iron (Fe 2+ ) in the presence of antioxidants [34].The results are expressed as mM/L (Table 1).The obtained values were compared with the value of a standard L-ascorbic acid.A. microcephalus (0.97 mM/L) had the highest reducing power while A. acmophyllus had the least activity (0.60 mM/L) among the extracts.The iron ion-reducing powers of extracts were very lower than that of L-ascorbic acid (4.25 mM/L).
The hydrogen peroxide scavenging activity of the extracts was tested; the results are expressed as % inhibition and compared with the % inhibition values of BHT, BHA and Gallic acid.The % inhibition values of the extracts are shown in Figure 3.It was observed that hydrogen peroxide scavenging activity increased with increasing concentration.The concentrations (IC 50 ) causing 50% inhibition were detected by plotting the graph including percent inhibition values versus concentrations for each extract and values were given in Table 1. A. acmophyllus (IC 50 = 220.03μg/mL) had the least hydrogen peroxide scavenging The reducing powers of the extracts have been measured.In this bioanalytical method used in antioxidant studies, the color of test solution transforms into green color due to the reducing activities of the antioxidant substances in the medium [35].The results are shown in Figure 4 as absorbance values at 700 nm.The obtained values were compared with the values of standard antioxidant, BHT. A. microcephalus (2.172) had the highest absorbance value while A. gummifer had the least absorbance value (1.118) among the extracts at 10 mg/mL.Increased absorbance value indicates increased antioxidant activity.The reducing power of A. microcephalus was quite close to that of BHT (2.249) at this concentration.
The metal chelating activity of the extracts was measured at 5 mg/mL and the results are indicated as % inhibition (Figure 5 Asgarpanah et al. [36] showed that the methanolic extract obtained from aerial parts of A. squarrosus contained low phenolic (23.3 mg/g) and flavonoid (26.0 mg/g) contents thus had weak antiradical activity by DPPH assay.Thirty phenolic compounds in A. membranaceus were determined by Jun et al. [15].Similarly, rutin, chlorogenic acid, kaempferol, ferulic acid and syringic acid were detected in Astragalus taxa tested in our study.Adıgüzel et al. [37] reported that the A. microcephalus, A. erinaceus, A. psoraloides, A. macrocephalus, and A. argyroides were strong DPPH scavenger (IC 50 = 20.3-38.0 μg/ mL), strong inhibitor on β-carotene-linoleic acid oxidation (23.8-88.1%)and extracts had inhibitory effect on the microorganisms tested.In our study, % inhibition on βcarotene-linoleic acid oxidation and IC 50 values on DPPH assay of Astragalus taxa tested varied 54.23-80.60%and 86.67-253.88μg/mL, respectively.
A. sinicus acetone extract exerted antiradical activity (95.1% at 10.0 mg/mL) and chelating effect (93.2%, at 10.0 mg/mL) with 279.9 and 175.2 mg/g total phenolic and flavonoid contents.Also, the reducing powers of ethanol, hexane, and water extract of A. sinicus were increased from 0.06 to 0.79, 0.08 to 0.69, and 0.09 to 0.39 OD 700 nm, when the concentrations were increased from 0.5 to 5 mg/ mL, respectively [17].According to our literature survey, ferric reducing power by FRAP and cupric reducing power by CUPRAC assay of Astragalus taxa studied were determined with this study at first time.
The antimicrobial activities of the extracts were studied by agar diffusion assay against 15 microorganisms at 1, 2.5, 5 and 10% concentrations (Table 3).The tested Astragalus extracts showed low antibacterial activity against only P. aeruginosa among the tested  cytotoxic activity against HL-60 and SMMC-7721 cells (IC 50 = 5 and 10 μg/mL) [18].These differences may be attributed to the genotypic variation, climatic conditions, using different bacteria, different activity assay and different extract concentrations [40].
Ionkova et al. [41], showed the cytotoxic activities of saponins isolated from A. membranaceus against five different cancer cell lines.It has been detected that dichloromethane extracts of A. gombiformis showed strong cytotoxic effect against the human A549 lung epithelial carcinoma cell line (IC 50 = 85 ± 21.7 μg/mL) for 48 h and antibacterial activity [19].
In conclusion, from the results it is clear that Astragalus taxa tested in this study showed moderate antioxidant activity.The extracts demonstrated no antibacterial activity except P. aureginosa.To our knowledge, this is the first report on biological and chemical study of four Astragalus taxa tested.Astragalus extracts tested may be use food or medicinal applications that need natural antioxidant agents due to their antioxidant activities.Further researches on identify, isolate and characterize of the specific compounds from the extracts may be carry out to understand the action mechanisms.
concentration causing 50% inhibition) values of the extract and standards were determined graphically.

Figure 1 :Figure 2 :
Figure 1: % Inhibition values of Astragalus extracts and BHT as positive control by DPPH assay.

Figure 3 :Figure 4 :
Figure 3: % Inhibition values of the Astragalus extracts and standards by H 2 O 2 assay.

Table 1 :
The yields, total phenolic contents, total flavonoid contents and antioxidant activities of Astragalus species.
*In each column, means of three independent experiments (±standard deviation) with different superscript letters are significantly different (p < 0.05).Total phenolic content expressed as gallic acid equivalent (GAE), total flavonoid content expressed as quercetin equivalent (QE), total antioxidant activity expressed as ascorbic acid equivalent (AAE).

Table 2 :
The quantity of some phenolic compounds (ppm) determined in Astragalus extracts by LC-MS.

Table 1 .
As shown in Table1, A. microcephalus had the highest flavonoid content (2.19 ± 0.0 mg QE/g extract), while A. acmophyllus (0.76 ± 0.0 mg QE/g extract) had the least amount of flavanoid among the extracts.