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A Comparative Study on the in vitro Antioxidant and Antimicrobial Potentials of Three Endemic Ononis L. Species from Turkey/Turkiye'den Uc Endemik Ononis L. Turunun in vitro Antioksidan ve Antimikrobiyal Potansiyelleri Uzerine Karsilastirmali Bir Calisma.

INTRODUCTION

Antioxidants have been recognized as potential therapeutics for preventing different human diseases (e.g., cancer, aging, cardiovascular diseases, asthma, acute central nervous system injury, neurodegenerative disease, and malaria). (1,2) Medicinal plants and herbs have played an important role in the health care of ancient and modern cultures. They are promising as natural antioxidant sources. (3,4) The number of studies on new natural antioxidants with plant origin is increasing. (5) Ononis L. genus (Leguminosae) is represented by 18 species, 4 of which are endemic to Turkey. (6) The Ononis species has various pharmacologic properties, such as antioxidant, aperient, diuretic, antimicrobial, analgesic, antiviral, cytotoxic, anti-inflammatory and anti-diarrheal activities. In Turkish folk medicine, O. spinosa L. has been used for the urinary tract, kidney stones, inflammatory diseases, wounds healing, and skin disorders. (1,7-9) Suntar et al. (10) reported that water and ethanolic extracts of the herb O. macrosperma demonstrated the highest activity in both wound models and anti-inflammatory activity. In previous studies, some Ononis species were found to contain different component groups such as isoflavone, triterpene, sterol, pterocarpane, and resorcinol derivatives, flavonoids, isocoumarins, and hydroxycinnamic acids. (1,11-13) A DPPH* (1,1-diphenyl-2-picrylhdrazyl radical) radical is a stable radical with maximum absorbance at 517 nm, and when reduced to hydrazine derivatives by electron and hydrogen atom transfer from substances with antioxidant properties, its absorbance is decreased. (14) Lipid peroxidation is a chain reaction that causes the deterioration of biologic systems, and is the accumulative effect of reactive oxygen species. Reactive free radicals start the reaction by the deletion of allylic hydrogen atoms from the methylene group of unsaturated fatty acids. (15) Thiobarbituric acid (TBA) and DPPH methods have been used to evaluate the antioxidant capacities of the plant extracts/component. (16) Medicinal plants represent potential sources of natural antioxidant and antimicrobial agents for food and medicinal purposes. The purpose of this investigation was to study antioxidant activities using TBA and DPPH [thin layer chromatography (TLC) screening], determine phenolic contents using spectrophotometry, and the antimicrobial activity with disc diffusion assays of herb and root extracts of three Ononis species. This is the first study on the antioxidant capacity and antimicrobial activities of the three Ononis species.

MATERIALS AND METHODS

Plant materials

Ononis species were gathered from different provinces of Turkey in their natural habitats. Voucher specimens were stored in the Herbarium of the Faculty of Pharmacy at the University of Ankara, Turkey (AEF). O. sessilifolia was collected from the Camardi county of Nigde in June 2007 (AEF 23979); O. basiadnata was collected from the Gulnar county of Icel in June 2007 (AEF 23968); and O. macrosperma was collected from the Elmali county of Antalya in May 2008 (AEF 24698).

Extraction of plants

The herb and root of the O. basiadnata and O. sessilifolia and the herb of O. macrosperma were powdered and then 50 g of the herbs and roots were macerated separately with 500 mL of water for 5 h at 60[degrees]C. Afterwards, the water extracts were filtered, frozen, and lyophilized. One hundred grams of plant material was macerated with ethanol (EtOH) for 5 h at 50[degrees]C. The extracts were then filtered and evaporated until dry. Ethanolic extracts were dispersed in methanol: water (1:9), partitioned with dichloromethane (DCM), ethyl acetate (EtOAc) and n-butanol (BuOH). Afterward, the fractions were evaporated until dry.

Determination of total phenolic content

The total phenolic contents of extracts were evaluated using the Folin-Ciocalteu assay as gallic acid equivalents (GAE). (17)

Antioxidant capacities of extracts

DPPH test

The antioxidant capacity of extracts (EtOH and water extracts; DCM, EtOAc and BuOH fractions) were evaluated with rapid TLC screening. (18)

TBA test (measurement of malondialdehyde value)

The amount of malondialdehyde formed in the reaction mixture was determined using the TBA reagent spectrophotometrically. (18)

Antimicrobial activity

Test microorganisms

The test microorganisms used in the experiment were Gram-positive bacteria (Staphylococcus aureus ATCC 25923, Staphylococcus aureus (MRSA) (clinical isolate), Bacillus subtilis ATCC 25923); Gram-negative bacteria (Escherichia coli ATCC 25922) and fungus: (Candida albicans ATCC 10231).

Ampicillin sulbactam (20 [micro]g), ciprofloxacin (5 [micro]g), fluconazole (25 [micro]g) and cefotaxime (30 [micro]g) were used as control drugs. ATCC strains were obtained from the culture collection of the Refik Saydam Health Institution of Health Ministry, Ankara.

Media

Mueller-Hinton agar (Difco, Detroit, MI, USA) was used for bacteria, and MHA supplemented with 2% glucose and 0.5 [micro]g/mL methylene blue was used for C. albicans.

Disc diffusion assay

Antimicrobial activities of the extracts and fractions were evaluated by using the disc diffusion technique. (19,20)

RESULTS

Phenolic compounds are known as a main class of active compounds determined by Folin-Ciocalteu assay. The highest total phenolic contents of all extracts and fractions of the three species were determined in the EtOAc fractions. The results of total phenolic contents of extracts and fractions are shown in Table 1.

When all DCM fractions and EtOAc fractions, excluding O. basiadnata root, were compared using propyl gallate, they were shown to have a high radical scavenging effect with qualitative DPPH. Yellow zones on a purple ground were marked for the DCM and EtOAc fractions of all species. In DPPH test, EtOH extracts were generally active from the water extract, especially the root and herb extracts of O. sessilifolia (Figure 1).

The most significant results in the TBA method were obtained in EtOH extracts of O. macrosperma herb (OMH) (I[C.sub.50]=0.13[+ or -]0.17 [micro]g/mL), O. sessilifolia herb (I[C.sub.50]=1.41[+ or -]0.58 [micro]g/mL) and root (I[C.sub.50]=1.96[+ or -]0.39 [micro]g/mL) (Table 2). A survey of the published literature shows that the antioxidant activity of O. sessilifolia, O. basiadnata and O. macrosperma has not been subjected to research so far.

In our antimicrobial activity studies, the extracts and fractions of three endemic Ononis species were examined against various bacteria and fungi. First, EtOH extracts were prepared, and then extracted with DCM, EtOAc and BuOH. The antimicrobial activity of the water extract was also examined. Water extracts of herb and root parts had no antimicrobial activity against Gram (-), Gram (+) bacteria and yeast. All of the BuOH extracts showed moderate activity compared with the standards. Some EtOAc fractions also demonstrated less activity against E. coli, S. aureus, MRSA and C. albicans. Apart from OMH, other DCM extracts showed no activity against Gram (+), Gram (-) bacteria and fungi. All EtOH extracts showed less activity against some bacteria. In addition to this, they showed moderate activity against C. albicans according to fluconazole (Table 3, 4, 5 ,6).

DISCUSSION

In the literature, there are some studies on the antioxidant activities and phenolic contents of other Ononis species. Leaf methanolic extract of O. natrix has significant total phenolic content (51 mg GAE/g DW) and flavonoid content (14.76 CE/g DW). (21) The antioxidant activity and total phenolic contents of O. natrix used in folk medicine in Jordanian were identified as follows: according to antioxidant capacity results, the aqueous extract has 82.0[+ or -]1.5 [micro]mol TE/g, methanolic extract has 76.7[+ or -]2.0 [micro]mol TE/g dry weight; in total phenolic content, the aqueous extract has 16.9[+ or -]0.4 mg GAE/g, methanolic extract has 21.1[+ or -]0.7 mg GAE/g dry weight. (9) In another study, O. spinosa root infusion was evaluated in both DPPH inhibition (20.5[+ or -]0.8%) and total phenolic content 3.09[+ or -]0.01 mg GAE/g extract. (22) Although the ethanolic extract of O. spinosa indicated concentration-dependent superoxide anion radical scavenging capacity (I[C.sub.50]=1.35 mg/mL), the extract showed no concentration-dependent inhibitory effect on lipid peroxidation. (23) Unlike the present study, in our lipid peroxidation experiment, significant results were obtained (Table 2).

The present study shows that the three Ononis species contained phenolic compounds, which inhibit the oxidation of lipids by donating hydrogen atoms to scavenge free radicals. (24) Phenolic compounds have been shown to be more effective antioxidants than vitamin A and C. (25) Our results showed that there seemed to be good compatibility between the phenolic content and antioxidant capacity of the extracts because the EtOAc fractions with a higher phenolic content showed higher DPPH radical scavenging capacity.

According to previous studies, the BuOH extracts of O. spinosa (4 mg/disc) had moderate antifungal activity against Aspergillus flavus, Fusarium moniliforme and C. albicans in comparison with miconazole nitrate at 40 [micro]g/disc. Petroleum benzene, EtOH, and water extracts showed high activity against Gram (+) and Gram (-) bacteria. (26) In another study, EtOH extract of O. spinosa demonstrated significant activity against Gram-positive (E. coli and P. aeruginosa), Gram-negative (S. aureus) bacteria and fungi (C. albicans). (27) In our study, similar results were obtained, EtOH extracts and BuOH fractions of all species showed generally high activity against Gram (+), Gram negative (-) bacteria and C. albicans.

CONCLUSION

Further studies are being conducted to determine the characterization and identification of active components responsible for the antioxidant and antimicrobial activities. Natural products are commonly a source for active compounds that have important potential for developing new therapeutic agents. Ononis species can be introduced as new plant source for antioxidant and antimicrobial agents.

Conflict of Interest: No conflict of interest was declared by the authors.

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[iD] Ayse BALDEMIR (1*), [iD] Aysegul KOROGLU (2), [iD] Nurten ALTANLAR (3), [iD] Maksut COSKUN (2)

(1) Erciyes University, Faculty of Pharmacy, Department of Pharmaceutical Botany, Kayseri, Turkey

(2) Ankara University, Faculty of Pharmacy, Department of Pharmaceutical Botany, Ankara, Turkey

(3) Ankara University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology, Ankara, Turkey

(*) Correspondence: E-mail: aysebaldemir@gmail.com, Phone: +90 536 835 07 57 ORCID-ID: orcid.org/0000-0003-2473-4837

Received: 31.03.2017, Accepted: 22.06.2017

DOI: 10.4274/tjps.62533
Table 1. Total phenolic contents of the extracts and fractions of
Ononis species

Species                             Total phenolic contents
                               ([mg.sub.gallicacid]/[g.sub.extr])
                                        [+ or -] SD (*)
                              Water extracts       EtOH extracts

O. sessilifolia herb (OSH)   37.19[+ or -]1.58   132.01[+ or -]4.16
O. sessilifolia root (OSR)   14.78[+ or -]0.79   145.33[+ or -]1.57
O. basiadnata herb (OBH)     80.33[+ or -]0.79   111.44[+ or -]0.79
O. basiadnata root (OBR)     20.15[+ or -]0.64    91.17[+ or -]5.55
O. macrosperma herb (OMH)    46.17[+ or -]3.78    67.19[+ or -]0.64

Species                              Total phenolic contents
                               ([mg.sub.gallicacid]/[g.sub.extr])
                                         [+ or -] SD (*)
                                DCM fraction         EtOAc fraction

O. sessilifolia herb (OSH)   208.11[+ or -]3.93   413.67[+ or -]5.50
O. sessilifolia root (OSR)   221.44[+ or -]2.36   327.08[+ or -]0.79
O. basiadnata herb (OBH)     105.33[+ or -]4.16   620.89[+ or -]12.57
O. basiadnata root (OBR)     119.22[+ or -]2.36   242.56[+ or -]4.84
O. macrosperma herb (OMH)     63.67[+ or -]4.21   467.03[+ or -]3.93

Species                           Total phenolic contents
                             ([mg.sub.gallicacid]/[g.sub.extr])
                                      [+ or -] SD (*)
                                       BuOH fraction

O. sessilifolia herb (OSH)          124.77[+ or -]0.79
O. sessilifolia root (OSR)          131.44[+ or -]2.36
O. basiadnata herb (OBH)             89.77[+ or -]4.71
O. basiadnata root (OBR)             88.11[+ or -]0.79
O. macrosperma herb (OMH)           156.44[+ or -]4.71

(*) SD: Standard deviation, EtOH: Ethanol, DCM: Dichloromethane, EtOAc:
Ethyl acetate, BuOH: n-butanol

Table 2. Antioxidant capacities of extracts and fractions of Ononis
species in TBA test

Species           I[C.sub.50] value ([micro]g/mL) [+ or -] SD (*)
             Water ext.             EtOH ext.              DCM frac.

OSH       NE (*)                 1.41[+ or -]0.58      14.38[+ or -]1.32
OSR       >1000[+ or -]8.55      1.96[+ or -]0.39      26.03[+ or -]0.24
OBH       >1000[+ or -]3.11     24.19[+ or -]2.21      49.73[+ or -]1.41
OBR       >1000[+ or -]7.99     15.57[+ or -]0.95      26.06[+ or -]1.19
OMH       NE                     0.13[+ or -]0.17   >1000[+ or -]2.85
Propyl        3.72[+ or -]1.6
gallate

Species   I[C.sub.50] value ([micro]g/mL) [+ or -] SD (*)
                   EtOAc frac.          BuOH frac.

OSH           51.18[+ or -]3.31   NE
OSR          146.35[+ or -]2.73   NE
OBH          532.01[+ or -]5.58   NE
OBR            3.10[+ or -]1.14   138.93[+ or -]2.26
OMH           61.56[+ or -]3.61   570.41[+ or -]7.14
Propyl
gallate

(*) Non-effective; SD: Standard deviation, OSH: O. sessilifolia herb,
OSR: O. sessilifolia root, OBH: O. basiadnata herb, OBR: O. basiadnata
root, OMH: O. macrosperma herb, EtOH: Ethanol, DCM: Dichloromethane,
EtOAc: Ethyl acetate, BuOH: n-butanol

Table 3. Results of the antimicrobial activity of O. sessilifolia herb
(OSH) and root (OSR) extracts (inhibition zones in mm)

Extracts/Drugs   S. aureus    MRSA   B. subtilis   E. coli   C. albicans
OSA                 ATCC    isolate   ATCC 25923    ATCC        ATCC
                   25923                            25922       10231

OSH EtOH ext.         10       12         -            7          13
OSR EtOH ext.         -         5         -           -           11
OSH DCM               -        -          -           -           -
OSR DCM               -        -          -           -           -
OSH EtOAc             -        -          -           15          -
OSR EtOAc             -        -          -           -           -
OSH BuOH              11       15         15          17          12
OSR BuOH              14       11         10          16          17
OSH water ext.        -        -          -           -           -
OSR water ext.        -        -          -           -           -

OSH: O. sessilifolia herb, OSR: O. sessilifolia root, EtOH: Ethanol,
DCM: Dichloromethane, EtOAc: Ethyl acetate, BuOH: n-butanol

Table 4. Results of the antimicrobial activity of O. basiadnata herb
(OBH) and root (OBR) extracts (inhibition zones in mm)

Extracts/Drugs   S. aureus    MRSA   B. subtilis   E. coli   C. albicans
OBA                 ATCC    isolate   ATCC 25923    ATCC        ATCC
                   25923                            25922       10231

OBH EtOH ext.       11          -         12          10         11
OBR EtOH ext.       10           7        -           14         10
OBH DCM             -           -         -           -          -
OBR DCM             -           -         -           -          -
OBH EtOAc           10          11                    -           7
OBR EtOAc           -           -                     14         -
OBH BuOH            12          12        14          14         -
OBR BuOH            11          14        17          17         14
OBH water ext.      -           -         -           -          -
OBR water ext.      -           -         -           -          -

OSR: O. sessilifolia root, OBH: O. basiadnata herb, OBR: O. basiadnata
root, OMH: O. macrosperma herb, EtOH: Ethanol, DCM: Dichloromethane,
EtOAc: Ethyl acetate, BuOH: n-butanol

Table 5. Results of the antimicrobial activity of O. macrosperma herb
(OMH) extracts (inhibition zones in mm)

Extracts/      S. aureus    MRSA     B. subtilis   E. coli   C. albicans
Drugs            ATCC      Isolate      ATCC        ATCC        ATCC
OMH              25923                 25923        25922       10231

EtOH ext.         10          9          -            -           16
DCM frac.         12         -           -             7          -
EtOAc fract.      -          10          -            -           -
BuOH frac.        12         15          -            16          13
Water ext.        -          -           -            -           -

OMH: O. macrosperma herb, EtOH: Ethanol, DCM: Dichloromethane, EtOAc:
Ethyl acetate, BuOH: n-butanol

Table 6. Inhibition zones of standard antibiotics (inhibition zones in
mm)

Reference       S. aureus    MRSA    B. subtilis  E. coli   C. albicans
Substances        ATCC      isolate     ATCC        ATCC     ATCC 10231
                  25923                25923       25922

Ampicillin          30         -         21          -            -
sulbactam
Ciprofloxacin       -          27        -           -            -
Fluconazole         -          -         -           -            32
Cefotaxime          -          -         -           25           -
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Title Annotation:ORIGINAL ARTICLE
Author:Baldemir, Ayse; Koroglu, Aysegul; Altanlar, Nurten; Coskun, Maksut
Publication:Turkish Journal of Pharmaceutical Sciences
Article Type:Report
Date:Aug 1, 2018
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