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Antioxidant, Anti-urease Activities and Genotoxic Effects of Terfezia Claveryi Methanol Extracts on Human Lymphocytes/Terfezia Claveryi Metanol Ekstrelerinin Antioksidan, Anti-ureaz Aktiviteleri ve Insan Lenfosit Hucreleri Uzerindeki Genotoksik Etkileri.

INTRODUCTION

Edible mushrooms have been used for nourishment, as functional foods to prevent ailments, and as a source of medicine to treat diseases. Edible mushrooms are used for lowering cholesterol, lowering blood pressure, strengthening the immune system against diseases, fighting tumors and improving liver function (1).

Desert truffle (Ascomycetes) is a kind of edible mushroom that grow in arid and semiarid areas on every continent other than Antarctica. It has been shown that Desert truffle contain rich fiber, protein, amino acids, fatty acids, minerals, vitamins, carbohydrates, potassium, phosphate, ascorbic acid, anthocyanins, esterified phenols, free phenolics, flavonoids, and carotenoids (2).

Terfezia and Picoa are two of the most common edible desert truffles in the world. Terfezia claveryi Chatin (Family, Terfeziaceae) is the dark brown color truffle and the juice of these mushrooms have been used in the Middle East to treat eye and skin diseases. T. claveryi contains saturated fatty acid, linoleic acid, protein, and carbohydrate compounds (3).

Turkey is a rich country in mushroom diversity and medicinal plants. Turkish people have a tradition of using various types of mushrooms for food, instead of using them for the treatment of various ailments (2). In consideration of the extensive use of mushrooms in human diet, it is an important issue to determine their bio-potential and toxicity. Therefore, the aim of this study was to comparatively reveal the antioxidant and anti-urease effects of T. claveryi methanol extracts obtained by using two extraction method followed by to evalute the genotoxicity of each extract.

MATERIALS AND METHODS

Chemicals

2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate), ascorbic acid, 2,2-diphenyl-1-picryl-hydrazyl, Folin Ciocalteu's phenol reagent, gallic acid and 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4',4''-disulfonic acid sodium salt were obtained from Fluka (Sigma-Aldrich; Buchs, Switzerland). All other reagents were of analytical grade.

Collection and identification of material

Terfezia claveryi Chatin were collected during the edible season from Batman-Sason, Turkey. The taxonomic identity of the material was confirmed by Assist. Prof. Dr. Ismail Senkardes. The voucher specimens were deposited in the herbarium of the Faculty of Pharmacy, Marmara University; herbarium numbers: MARE-18813.

Preparation of extracts

The fresh mushroom materials were washed carefully with water. Then, this parts were cut into small pieces. Two different extraction methods were followed to prepare crude extracts from this mushroom materials

Maceration: 94 g materials was extracted with methanol for 7 days at 25 [degrees]C.

Ultrasonic bath extraction: Mushroom materials (94 g) was extracted with 300 mL methanol for 30 min. in ultrasonic bath set at 30 [degrees]C.

The maceration methanol (MM) and Ultrasonic bath methanol (UM) extracts were filtered through filter paper and concentrated by rotary vacuum evaporator (Heidolph Hei-Vap; Schwabach, Germany). All the extracts obtained were stored at 4 [degrees]C for future analysis.

After extraction, studied concentrations were determined by preliminary experiments accordingly results of activities.

Extract yield percentage

The extraction yield is a measure of the solvent's efficiency to extract specific components from the original material (4). The percentage yield was obtained using this formula [A.sub.2]-[A.sub.1]/[A.sub.0] x 100. Where [A.sub.2] is the weight of the extract and the container, [A.sub.1] is the weight of the container alone and [A.sub.0] the weight of the initial dried sample.

Quantification of total phenolic contents

Extracts prepared at different concentrations were taken in 0.1 mL tubes and 4.5 mL of water was added to them. Then Folin-Ciocalteu reagent (diluted 1/3 with distilled water) and 0.3 mL of 2 % sodium carbonate solution were added to the mixture. The mixture was allowed to stand at room conditions for 2 hours, and then absorbance was measured at 760 nm against the reference. The total phenolic compounds contained in the extracts were given as mg gallic acid equivalents / mg extract (5).

In vitro evaluation of antioxidant assays

2,2-diphenyl-1-picryl-hydrazyl (DPPH) free radical scavenging activity: DPPH solution (0.1 mM, 3.9 mL) was added to extracts (0.1 mL) prepared at different final concentrations (20 [micro]g/mL, 50 [micro]g/mL, 100 [micro]g/mL). Then the mixture was allowed to stand at room temperature for 30 min. The absorbance of the mixture was measured against the reference using a spectrophotometer (Shimadzu Corporation; Japan) at 517 nm. The experiment was repeated three times and the averages of the values and the standard deviation were calculated. The results obtained in the experiment were given as the concentration of the substance which provides the inhibition of 50 % of the radical (I[C.sub.50]) = mg / mL (6).

2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) radical cation scavenging assay: 40 [micro]L of extracts prepared at different final concentrations (20 [micro]g/mL, 50 [micro]g/mL, 100 [micro]g/mL), 3960 [micro]L of ABTS*+ working solution were combined. The absorbance of the mixture was measured against the reference at 734 nm for 6 min. The data obtained in this study were expressed as mM trolox / mg extract (7).

Ferric reducing/antioxidant power (FRAP) assay: The ferric reducing ability of different extracts were estimated by the method of Benzie and Strain (1996) (8). The FRAP reagent [300 mM acetate buffer (pH 3.6), TPTZ solution and 20 mM Fe[Cl.sub.3].6[H.sub.2]O] was kept at 37 [degrees]C for 30 min. in incubator device (Nuve; Ankara, Turkey). 3.8 mL of FRAP reagent was mixed with 0.2 mL of extract and after 4 min. the absorbance of the mixture was measured against the reference at 593 nm. FRAP values of the extracts were expressed as mM [Fe.sub.2]+/mg extract.

In vitro urease inhibitory activity

The indophenol method with some modifications was used to measure the urease inhibitory activity of the extracts (9). 5 mg /mL stock solutions from different extracts were prepared and working solutions were prepared at 2 mg/mL concentrations by diluting these solutions. 500 [micro]L of urease enzyme was added to 100 [micro]L of the working solutions and incubated at 37 [degrees]C for 30 min. in the incubator (Nuve; Ankara, Turkey). Then, 1100 [micro]L of urea was placed on this mixture and the mixture was incubated for 30 min. at 37 [degrees]C in the incubator. R1 (1 % phenol, 0.005 % sodium nitroprusside) and R2 (0.5 % NaOH, 0.1 % sodium hypochlorite) reagents were added respectively to the mixture. The mixture was incubated for 2 hours in the incubator (37 [degrees]C) (Nuve; Ankara, Turkey) and then the absorbance of mixture was read at 635 nm.

The % inhibition of urease enzyme was calculated by the formula:

% enzyme inhibition = [([A.sub.0] - [A.sub.1])] / [A.sub.0]] x 100].

[A.sub.0] : The absorbance of the control solution

[A.sub.1] :Absorbance of plant extracts and standard solutions.

Determination of genotoxic activity

Fresh venous blood sample (~30 mL) was obtained from a volunteer 30 years old non-smoking women and collected in lithium heparin tubes (Vacuette[R]). Lymphocytes were isolated from peripheral blood by centrifugation in a density gradient of Histopaque 1077 (Sigma Diagnostics, St. Louis, USA) (40 min, 400 g, 21[degrees]C). The viability of the cells was measured after isolation by the trypan blue exclusion assay and was found to be about 99 %. The cells were incubated for 30 minutes at 37[degrees]C with MM and UM extracts of Terfezia claveryi at 20, 50 and 100 [micro]g/mL final concentration. The viability of the cells was checked concurrently by trypan blue exclusion assay in all experiments at all tested concentrations of MM and UM extracts of T. claveryi. An equal volume of 0.4 % trypan blue reagent was added to the 40 [micro]l cell suspension and the percentage of viable cells was evaluated under a microscope.

The alkaline Comet assay (10) with some modifications was performed to determine the genotoxic properties of T. claveryi extracts. MM and UM extracts of T. claveryi was added to the suspension of the cells to give final concentrations of 20, 50 and 100 [micro]g/mL. Lymphocytes were incubated with extracts for 30 minutes at 37[degrees]C. The experiment included 100 [micro]g /mL final concentration of DMSO as a solvent control since it was used to solved extracts and it was applied for 30 minutes at 37[degrees]C. Also, hydrogen peroxide at 100 [micro]M applied for 7 min at 4[degrees]C was included as a positive control. After treatment with extracts, the cells were washed and ready to spread onto microscope slide. Each experiment was carried out in triplicate.

Fully frosted microscope slides were covered with 1 % high melting agarose (HMA). Lymphocytes were mixed with 1% low melting point agarose (LMA) at the end of incubation and was spread onto covered microscope slides. After solidification of low-melting agarose, slides were immersed in cold lysing solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, pH 10 with 1% Triton X-100 and 10% DMSO) for at least 1h at +4[degrees]C. After the lysis, the slides were left in the cold electrophoresis buffer (0.3 M NaOH, 1mM EDTA, pH 13) for 30 min to allow the unwinding of the DNA. And then, slides were placed in a horizontal electrophoresis tank that was filled with the same cold buffer and electrophoresed for 30 min at 300 mA and 15 V. Slides were washed three times with neutralization solution (0.4 M Tris buffer, pH 7.5). At the end of all this steps; slides were treated with % 50, % 75 and % 100 cold Ethanol for 5 min, respectively for fixation of cells and then were left in room temperature to dry. The slides stained with 50 [micro]l ethidium bromide (EtBr - 20 [micro]g/mL) were visualized under a microscope with a fluorescence attachment (Olympus BX51; Tokyo, Japan) and were examined by eye. One hundred cells 50 per replicate were scored and total comet score was calculated according to following formula (11).

Arbitrary Unit: 0 x No Migration (NM) + 1 x Low Migration (LM) + 2 x Medium Migration (MM) + 3 x High Migration (HM) + 4 x Extensive Migration. Figure 1 presents microscope pattern of cells undamaged cells to damaged cells with appearance of comet.

Statistical Analysis

The normal distribution of continuous variables was tested by Shapiro--Wilk's test. The test statistic is defined by the two-independent sample t-test between two group. The statistical analysis was performed using a one-way analysis of variance (ANOVA), with a Tukey multiple comparison, post hoc test for comparisons of different treatments versus the respective controls. Results were expressed as mean [+ or -] standard deviation of the mean (SD), with n= 3. A difference at p<0.05 was considered statistically significant (SPSS, an IBM Company; USA).

RESULTS

Extract yield percentage

The yield percentages of T. claveryi was shown in Figure 2. According to in this study, MM extract (3.96 %) was found to have higher recovery than UM extract (2.57 %).

Antioxidant activity and total phenolic contents of T. claveryi extracts

Antioxidant activities of methanol extracts obtained from T. claveryi were given in Table 1. In the DPPH radical scavenging activity assay, it was found that the UM extract (I[C.sub.50]: 0.77 mg/mL) has stronger free radical scavenging activity than MM extract (p<0.001). When the activity results of both two the extracts were compared with the standard, it was found that these extracts had lower activity than standard compounds (BHA and ascorbic acid) (p<0.001).

In this study, It was found that the methanol extract (4.09 mM trolox/mg extract) obtained from maceration method exhibited stronger ABTS radical cation scavenging activity than ultrasonic bath methanol extract (p>0.05). According to results, it was found that both extracts had lower activity than the BHA compound used as standard (p<0.001).

According to the results obtained from the FRAP assay, UM extract (2.02 mM [Fe.sub.2]+/mg extract) has stronger ferric reducing antioxidant power activity than MM extract (p>0.05). The methanol extracts obtained from two different extraction methods showed lower ferric reducing antioxidant power activity than the BHA compound (p<0.001).

When the amounts of the phenolic compounds in the extracts were compared, it was found that the MM extract (0.0097[+ or -]0.0002 mgGAE/mg extract) contained more phenolic contents than the UM extract (p<0.05) (Table 2).

According to the three antioxidant activity tests, UM extract exhibited stronger free radical scavenging and ferric reducing antioxidant power activity than MM extract but the results were not found statistically significant except DPPH method. In addition, MM extract had stronger ABTS radical cation scavenging activity than UM extract however statistically not significant. It was also found that MM extract had higher total phenolic contents than other extract. Therefore, a linear relationship was found between ABTS radical cation scavenging activity and phenolic compounds in this study. However, a linear relationship was not observed between total phenolic contents and antioxidant activity (DPPH, FRAP assay) of maceration methanol extract.

Urease inhibitory activity

The results for the assessment of urease inhibitory activity of T. claveryi methanol extracts (25 [micro]g/mL) obtained through different extraction methods were shown in Table 2. The UM extract (31.012 %) exhibited stronger anti-urease activity than MM extract (27.125 %) (p>0.05). In the present study, the results of two different extraction methods showed that ultrasonic bath method were the most suitable method to get the strongest anti-urease activity. According to this study, both extracts from two extraction methods exhibited lower anti-urease activity than thiourea (96.85 %) used as standard (p<0.001). A linear relationship wasn't found between anti-urease activity and total phenolic contents in this study.

Genotoxic activities of T. claveryi extracts

Lymphocytes were exposed two methanolic extracts that extracted by different method at three concentration (20 [micro]g/mL, 50 [micro]g/mL and 100 [micro]g/mL) for 30 min at 37[degrees]C. The comet assay was performed to determine the DNA damaging activity of the extracts as it is a sensitive method. Our results revealed that extracts of T. claveryi induced DNA damage in lymphocytes (compared with DMSO solvent control, p<0.001) and total comet scores was higher in cells which had been incubated with MM extracts (p<0.05). Table 3 shows total comet scores in lymphocytes after exposures of different methanolic extracts of T. claveryi and the mean total comet score distributions of different methanol extracts at different concentrations are shown in Figure 3.

DISCUSSION

According to the literature review, there are some studies on the antioxidant activity of different extracts from T. claveryi. The antioxidant activity of methanol extract from T. claveryi powder have been reported before. That study have reported that methanol extract (100 [micro]g/[micro]L) had stronger DPPH radical scavenging activity (7.31 %) (2). In another study, antioxidant activity of hexane, ethyl acetate, ethanol, methanol and water extracts obtained from hot maceration method were examined. According to results, ethanol extract exhibited stronger DPPH (I[C.sub.50]:52.10 [micro]g/mL) and ABTS (I[C.sub.50]:64.76 [micro]g/mL) radical scavenging activity than other extracts. In addition, hexane extract (105.8 [micro]mol Fe 2+/mg) showed the highest ferric reducing antioxidant power activity (3). According to the work of Neggaz and co-worker, methanol extract (I[C.sub.50] 8.56 mg/mL) obtained from Soxhlet method showed stronger DPPH radical scavenging activity than chloroform and ethyl acetate extracts (12).

Unlike above discussed studies, in current study, the antioxidant activities of methanol extracts obtained from two different extraction methods were examined using by the three different methods (DPPH, ABTS, FRAP). In addition, the amounts of phenolic contents contain in the extracts and the effects of extraction methods on antioxidant activity were examined. Our study showed that UM extract had higher free radical scavenging and ferric reducing antioxidant power activity than MM extract. In addition, MM extract exhibited higher total phenolic contents and ABTS radical scavenging activity than UM extract. Each extracts exhibited lower antioxidant activity than standards.

Although the bio-potential of T. claveryi were investigated in many studies (2, 3), the toxicity of this species are still poorly explored. To the best of our knowledge, this research is the first study that investigates the genotoxic activity of T. claveryi. According to the our results, each methanolic extracts have a potential genotoxic activity in lymphocytes and extract of maceration exhibited higher DNA damage than extract of ultrasonic bath.

CONCLUSION

In this study, DPPH free radical scavenging activity of UM extract was found to be stronger than MM extract however, activity of both of extracts found lower than standards. It was also found that UM extract had a lower genotoxic effect than the MM extract. This findings may be related to the recovery of the extraction methods as well as to the different composition of the extracts. This study is a preliminary study and it is aimed to analyze the composition of these different extracts in further study. In addition, the genotoxic activity of this species should be confirmed with further studies and in different cell lines.

REFERENCES

[1] Gadallah MGE, Ashoush IS. Value addition on nutritional and sensory properties of biscuit using desert truffle (Terfezia claveryi) Powder. Food Nutr Sci. 2016; 7: 1171-1181.

[2] Akyuz M, Kirbag S, Bircan B. Medical characteristics of arid-semi arid truffle (Terfezia and Picoa) in the Elazig-Malatya region of Turkey. Hacettepe J. Biol. & Chem. 2015; 43 (4): 301-308.

[3] Dahham SS, Al-Rawi SS, Ibrahim AH, Abdul Majid AS, Abdul Majid AMS. Antioxidant, anticancer, apoptosis properties and chemical composition of black truffle Terfezia claveryi. Saudi J Biol Sci. 2016; http://dx.doi.org/10.1016/j.sjbs.2016.01.03 1-7.

[4] Murugan R, Parimelazhagan T. Comparative evaluation of different extraction methods for antioxidant and anti-inflammatory properties from Osbeckia parvifolia Arn. An in vitro approach. J.King Saud Univ. 2014; 26: 267--275.

[5] Taskin T, Ozakpinar OB, Gurbuz B, Uras F, Gurer US, Bitis L. Identification of phenolic compounds and evaluation of antioxidant, antimicrobial and cytotoxic effects of the endemic Achillea multifida. IJTK. 2016; 15(4): 594-603.

[6] Wei F, Jinglou C, Yaling C, Yongfang L, Liming C, Lei P. Antioxidant, free radical scavenging, anti-inflammatoryand hepatoprotective potential of the extract from Parathelypteris nipponica (Franch.et Sav.) Ching. J Ethnopharmacol. 2010; 130: 521-528.

[7] Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. FRBM. 1999; 26: 1231--1237.

[8] Benzie IF, Strain JJ. The ferric reducing ability of plasma (FRAP) as a measure of "antioxidant power": the FRAP assay. Anal Biochem. 1996; 239(1): 70-76.

[9] Jaffary SRA. Ahmed SW, Shakeel S, Asif HM, Usmanghani K. Evaluation of in vitro urease and lipoxygenase inhibition activity of weight reducing tablets. Pak. J. Pharm. Sci. 2016; 29(4):1397-1400.

[10] Singh NR, McCoy MT, Tice RR, Schneider EL. A simple technique for quantification of low levels of DNA damage in indiviuals cells. Exp Cell Res. 1988; 175: 184-191.

[11] Collins AR, Oscoz AA, Brunborg G, Isabel Gaiva o I, Giovannelli L, Marcin Kruszewski M, Smith CC, Stetina R. The Comet assay: topical issues. Mutagenesis 2008, 23: 143--151.

[12] Neggaz S, Fortas Z, Chenni M, Abed DE, Ramli B, Kambouche N. In vitro evaluation of antioxidant, antibacterial and antifungal activities of Terfezia claveryi Chatin. Phytotherapie. doi 10.1007/s10298-015-0993-4. 1-7.

Turgut Taskin (1), Tugce Yesil Devecioglu (2), Ismail Senkardes (3)

(1) Marmara University, Faculty of Pharmacy, Department of Pharmacognosy, Turkey

(2) Marmara University, Faculty of Pharmacy, Department of Pharmaceutical Toxicology, Turkey

(3) Marmara University, Faculty of Pharmacy, Department of Pharmaceutical Botany, Turkey

ORCID IDw of the authors:

Cite this article as: Taskin T., Yesil Devecioglu T., Senkardes I. Antioxidant, Anti-urease Activities and Genotoxic Effects of Terfezia claveryi Methanol Extracts on Human Lymphocytes Clin Exp Health Sci 2018; DOI: 10.5152/clinexphealthsci.2017.651

Correspondence Author/Sorumlu Yazar: Turgut Taskin E-mail/E-posta: turguttaskin@marmara.edu.tr
Table 1. Effects of extracting methods on the antioxidant activity of
T. claveryi extracts

Samples        DPPH (I[C.sub.50]: mg/mL)
               Ultrasonic bath         Maceration

Methanol       0.77[+ or -]0.01 (a,b)  1.17[+ or -]0.02 (a,b)
BHA            0.006[+ or -]0.6
Ascorbic acid  0.004[+ or -]0.9

Samples        ABTS (mM trolox/mg extract)
               Ultrasonic bath        Maceration

Methanol        3.71[+ or -]0.03 (b)  4.09[+ or -]0.03 (b)
BHA            52.63[+ or -]0.08
Ascorbic acid

Samples        FRAP assay (mM [Fe.sub.2]+/mg extract)
               Ultrasonic bath        Maceration

Methanol        2.02[+ or -]0.05 (b)  1.92[+ or -]0.06 (b)
BHA            16.91[+ or -]0.02
Ascorbic acid

Values are mean of triplicate determination (n = 3) [+ or -] standard
deviation
(a) Significancy of T. claveryi extracts doses compared with ascorbic
acid at p<0.001
(b): Significancy of T. claveryi extracts doses compared with BHA at
p<0.001
DPPH: 2,2-diphenyl-1-picryl-hydrazyl
ABTS: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate)
FRAP:ferric reducing/antioxidant power
BHA:Butylated hyroxyanisole

Table 2. Total phenolic contents and urease inhibitory activity of
methanol extracts from T. claveryi.

Samples   Total phenolics (mgGAE/mg extract) (n=3)
          Ultrasonic bath       Maceration

Methanol  0.0084[+ or -]0.0003  0.0097[+ or -]0.0002
          p=0.005 (*)
Thiourea

Samples   Urease inhibition (%) (25 [micro]g/mL) (n=2)
          Ultrasonic bath        Maceration

Methanol  31.012[+ or -]2.6 (b)  27.125[+ or -]1.9 (b)

Thiourea                         96.85[+ or -]0.9

GAE-Gallic acid equivalents
(b) Significancy of T. claveryi extracts doses compared with Thiourea
at p<0.001
(*) The test statistic is defined by the two independent sample t-test
(p<0.05)

Table 3. Total comet scores in lymphocytes after exposures of
methanol-maceration and methanol-ultrasonic bath extracts of T.
claveryi at 20, 50 and 100 [micro]/mL final concentration for 30 min at
37[degrees]C.

Sample                  Concentration    Total Comet Score (a)
                                         (mean[+ or -]SD)

Negative control          0               31.67[+ or -]10.50
Methanol-maceration      20 [micro]g/mL  343[+ or -]20.66 (***)
extract of T. claveryi   50 [micro]g/mL  349[+ or -]5.2 (***)
                        100 [micro]g/mL  344.66[+ or -]13.32 (***)
Methanol-ultrasonic      20 [micro]g/mL  303.66[+ or -]13.43 (***)
bath extract of          50 [micro]g/mL  311[+ or -] (***)
T. claveryi             100 [micro]g/mL  325.33[+ or -]10.79 (***)
Solvent control DMSO    100 [micro]g/mL   45.33[+ or -]10.02
Positive Control
[H.sub.2][O.sub.2]      100 [micro]M     355[+ or -]5.03

Total comet score: 0 x No Migration (NM) + 1 x Low Migration (LM) + 2 x
Medium Migration (MM) + 3 x High Migration (HM) + 4 x Extensive
Migration (EM). a Significancy of T. claveryi extracts doses compared
with DMSO solvent control at (*) p< 0.05; (**) p<0.005; (***) p<0.001
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Title Annotation:Original Article / Ozgun Arastirma
Author:Taskin, Turgut; Devecioglu, Tugce Yesil; Senkardes, Ismail
Publication:Clinical and Experimental Health Sciences
Article Type:Report
Date:Sep 1, 2018
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