Accepted Manuscript

Equisetum telmateia extracts: Chemical compositions, antioxidant activity andantimicrobial effect on the growth of some pathogenic strain causing poisoning andinfection

Marzie Yeganegi, Farideh Tabatabaei Yazdi, Seyed Ali Mortazavi, Javad Asili,Behrooz Alizadeh Behbahani, Adel Beig Babaei

PII: S0882-4010(17)31757-6

DOI: 10.1016/j.micpath.2018.01.014

Reference: YMPAT 2731

To appear in: Microbial Pathogenesis

Received Date: 22 December 2017

Revised Date: 8 January 2018

Accepted Date: 9 January 2018

Please cite this article as: Yeganegi M, Tabatabaei Yazdi F, Mortazavi SA, Asili J, Alizadeh BehbahaniB, Babaei AB, Equisetum telmateia extracts: Chemical compositions, antioxidant activity andantimicrobial effect on the growth of some pathogenic strain causing poisoning and infection, MicrobialPathogenesis (2018), doi: 10.1016/j.micpath.2018.01.014.

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Equisetum telmateia extracts: Chemical compositions, antioxidant activity and antimicrobial 1

effect on the growth of some pathogenic strain causing poisoning and infection 2

Marzie Yeganegia, Farideh Tabatabaei Yazdia*, Seyed Ali Mortazavia, Javad Asilib, Behrooz Alizadeh Behbahania, 3

Adel Beig Babaeic 4

a Department of Food Science and Technology, Ferdowsi University of Mashhad, Mashhad, Iran 5

b Department of Pharmacognosy School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran 6

c Department of Food Processing, Research Institute of Food Science and Technology, Mashhad, Iran 7

* Corresponding author, Email: tabatabai@um.ac.ir 8

Abstract 9

The aerial parts of Equisetum telmateia have been used as a source of biologically active compounds 10

to treat inflammatory, diarrhea, stomach-ache, eczema and mouth infections in traditional medicine. The 11

aim of this work is to evaluate the extraction yield, chemical compositions, antioxidant activity and 12

antimicrobial activity of E. telmateia extracts on Staphylococcus aureus, Bacillus cereus, Escherichia 13

coli, Salmonella typhi and Candida albicans. Chemical compositions E. telmateia was analyzed by high 14

performance liquid chromatography (HPLC) using a C18 column. Analysis of E. telmateia extract by 15

HPLC allowed the identification of Kaempferol 3-O-(6’’-O-acetylglucoside) as major compound. The 16

antioxidant activity of extracts was examined by measuring their ability to sequestrate 2,2-diphenyl-1-17

picrylhydrazyl (DPPH) radicals. The results showed that the DPPH (IC50 = 70.83 ± 0.2 µg/ml) were 18

obtained in the case of supercritical fluid extraction (SFE) extract. MIC microdilution assay were used to 19

determine the antimicrobial activities. Contrary to lower extraction yield (9.6 ± 0.5), the SFE extract 20

exhibited the highest antimicrobial potency with MIC and MBC values of 32 mg/ml against S.aureus 21

compared to the other extracts. The results suggest that SFE method is more appropriate for extraction of 22

E. telmateia biologically active substances with antimicrobial and antioxidant activity than conventional 23

solvent extraction methods. 24

25

Key words: Antimicrobial activity, Antioxidant activity, Biologically active compounds, Equisetum 26

telmateia, Microbial pathogens. 27

28

29

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1- Introduction 31

Equisetum telmateia Ehrh. with widespread distribution in Europe, western Asia, northwest Africa 32

and north America, is the largest member of the genus Equisetum (fam. Equisetaceae) [1]. The aerial parts 33

of this plant have been used for the treatment of prostatitis, stomach-ache, inflammatory and diarrhea, and 34

also for mouth infections, chronic eczema, strain and antifungal in traditional medicine [2, 3]. E. telmateia 35

contains a variety of phenolic compounds such as flavonoids as well as phenolic acid derivatives 36

including protocatechuic and p-hydroxybenzoic acids and various caffeic acid derivatives [4-6]. Recent 37

investigations support the efficiency of kaempferol and its derivatives in protection against several 38

pathological conditions such as viral diseases, inflammation, cancers and arteriosclerosis [7-9]. Similar 39

effects alongside antioxidant and antimicrobial activity have been reported for other classes of flavonoids 40

such as proanthocyanidins and catechins [10, 11]. Therefore, E. telmateia can be expected to serve as a 41

rich source of biologically active compounds. Many articles have been published highlighting the 42

biological activities of E. telmateia. For instance, Behnam Rassouli et al. [12] and Gurbuz and Yesilada 43

[13] showed neuroprotective effects and antiulcerogenic activity of E. telmateia extracts respectively. 44

They represent that these protective effects and also anti-inflammatory and diuretic properties of E. 45

telmateia extracts could be due to the presence of flavonoids and compounds with antioxidant activity. 46

Correia et al. [5], Milovanovic et al. [6] and Stajner et al. [14] also reported the antimicrobial and potent 47

antioxidant effects of the polar extracts of E. telmateia. 48

The base of application of plant extracts in food preservation, pharmaceuticals and cosmetics is 49

their functional properties like antimicrobial and antioxidant activity, which is owing to a range of 50

biologically active compounds. Since these active compounds in herbal plants usually are in low 51

concentrations and also concerns about toxic organic solvent residues in plant extracts is increasing, a 52

great deal of study has been performed to develop more selective and effective extraction methods for 53

extraction of these compounds from the plant material [15, 16]. 54

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Supercritical fluid extraction (SFE), being an environmentally process, has appeared as an 55

alternative to conventional organic solvent extraction of plants for the recovery of high value natural 56

substances and phytochemicals with high purity [17, 18]. Using SFE, the active substances could be 57

extracted in its natural form under medium temperature conditions (40–60 °C) without degradation or 58

contamination. SFE provides a rapid extraction procedure due to the solvent’s low viscosity, high 59

diffusivity and solvation strength. Carbon dioxide (CO2) is the most appropriate solvent for supercritical 60

fluid extraction of thermolabile or sensitive to oxidation compounds due to its mild critical parameters 61

(31.1°C, 7.38 MPa). Besides that, carbon dioxide offers many other advantages more than other solvents, 62

since it is cheap, non-flammable, non-toxic and with high purity. Another advantage is that CO2 is 63

gaseous at room temperature and ordinary pressure, which makes the recovery of analytes very easy, so 64

the supercritical extracts does not contain organic solvents residual as in traditional solvent extraction 65

methods, which makes these products suitable for use in food, cosmetic and pharmaceutical industry [19, 66

20]. 67

The studies above mentioned used liquid-solvent-based methods to obtain the E. telmateia 68

extract. Taking into account the differences between the applied extraction methods, which may affect 69

primarily on bioactivity of the active substances and subsequently its preservation and therapeutic 70

properties, the aim of this study was to evaluate the overall extraction yield, antioxidant and antibacterial 71

activities of conventional and supercritical extracts of Equisetum telmateia Ehrh. as a posibly new source 72

of biologically active natural substanses. To the best of our knowledge, there are no previous reports on 73

antioxidant and antibacterial activities of E. telmateia supercritical extract. 74

75

2- Materials and methods 76

2-1- Collection of plant materials 77

The sterile stems of the E. telmateia were collected from Chenaran, Khorasan-Razavi province, Iran, 78

with the following geographical coordinates: 36°30'3.5"N 59°02'11.5"E. Collection was done in May 79

2015 and the plant identified by Mr. Joharchi. A voucher specimen, (no. 45394) was deposited at the 80

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Ferdowsi University of Mashhad Herbarium (FUMH), Mashhad, Iran. The stems were washed thoroughly 81

and then air-dried at room temperature for 3 days, finally milled to fine powder and stored in sterile dark 82

bottles at room temperature for further analysis. The average particle size of milled herbs was 0.40 mm 83

(used for all applied extractions). 84

85

2-2- Conventional organic solvent extraction (COSE) 86

The COSE method consists of a cold maceration (Mac) and fractionation (Frac) of the E. telmateia to 87

avoid thermal degradation, applied to obtain the E. telmateia extract. The extraction was done with 250 g 88

of air-dried and powdered aerial parts of E. telmateia placed in 2500 ml of ethanol (EtOH) for six days. 89

The resulting extract was evaporated to dryness at reduced pressure to obtain the crude ethanolic extract (90

CEE). Then, the CEE was re-dissolved in distilled water and partitioned with 300 ml of each solvent 91

petroleum ether, dichloromethane (DCM), ethyl acetate (EtAc) and n-Butanol (n-BuOH) (Kitzberger et 92

al. [21] with brief modification). The organic solvents were 99% pure and used in sequence according to 93

their increasing polarity values of 0.0, 3.1, 4.4 and 4, respectively [22]. In addition, a crude aqueous 94

extract (CAE) was obtained according to the foresaid procedure for obtaining CEE. The extraction yield 95

results were obtained by the mean value of the triplicated experiments and calculated as the dry weight of 96

the extract relative to the raw material weight as: 97

yield (%) = m/M × 100 (1) 98

where m is the dry weight of the extract (g) and M is the raw material weight (g) [23]. 99

100

2-3- Supercritical fluid extraction (SFE) 101

Supercritical CO2 extraction was performed with a laboratory-scale supercritical fluid extraction 102

apparatus (fabricated in Research Institute of Food Science and Technology (RIFST), Mashhad, Iran), 103

equipped with a 2.5 L stainless extraction vessel with manual control of temperature, pressure and flow 104

rate of the fluid. The CO2 was first liquefied before passing through a high-pressure pump. The CO2 105

liquid was then heated until it reached to supercritical state. Temperature in the extraction vessel was 106

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maintained with the aid of jacket for circulating water from thermostated bath. The working condition has 107

been selected taking into account the optimum condition for extraction of bioactive phenolics (according 108

to liza et al. [24]) and was carried out at pressure of 200 bar, temperature of 50°C and dynamic extraction 109

time of 60 min. 100 gr of ground plant material were placed into the extractor vessel. The sample was 110

extracted with pure CO2 (99.99%purity), obtained from Toos Oxygen Co. (Mashhad, Iran), and EtOH as a 111

co-solvent. The amount of EtOH, 1 L, was almost twice the sample volume and the flow rate of CO2 was 112

maintained at 0.03 L/min, which were shown to be appropriate in preliminary experiments. The EtOH 113

was added directly to the extractor together with the ground stems before extraction. A glass vial with a 114

rubber plug at the top used to collect the extracted analyte for further analysis. 115

116

2-4- Determination of Minimum inhibitory concentration (MIC) and Minimum 117

bactericidal/fungicidal concentration (MBC/MFC) 118

The obtained extracts and fractions from E. telmateia with SFE and COSE methods, were tested 119

separately against 2 Gram-positive bacteria (Staphylococcus aureus PTCC 1337 (Persian Type Culture 120

Collection) and Bacillus cereus PTCC 1247), 2 Gram-negative bacteria (Escherichia coli PTCC 1330 and 121

Salmonella typhi PTCC 1609), and one fungi strain (Candida albicans PTCC 5027). Mueller hinton agar 122

and culture broth were used for growing and diluting of the bacteria suspensions. The microbial 123

suspensions turbidity was made based on 0.5 McFarland [25]. In order to investigate MIC and 124

MBC/MFC values of different extracts, the modified microdilution method in culture broth was used 125

[26]. The extracts were dissolved in 0.5 ml of DMSO and the solution added to 9.5 ml of muller hinton 126

broth for the bacteria growth and sabouraud dextrose broth for fungi. Then, serial two-fold dilutions 127

were prepared by mixing 5 ml of the previous concentration with the same volume of culture broth, in a 128

concentration range from 1 to 512 mg/ml. Then, 200 µl of each diluted solution was inoculated in a sterile 129

96-well microplate with 20 µl of the bacterial or fungi suspensions. Besides, negative and positive 130

controls were used for each tested strain. After an overnight incubation at 37°C, 20 µl of ,2,3,5-131

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triphenyltetrazolium chloride (Sigma) (5 mg/ml) was added to each well as a colorimetric indicator of 132

bacterial and fungi growth and incubated for 3 h at 37°C. The MIC was determined as the lowest 133

concentration of the extracts that inhibited the microbial growth and showed no red dye production. 134

MBC/MFC was measured by subculturing 100 µl from each well (without any color change) on the 135

mueller hinton agar plates and incubation at 37°C for 24 h for bacteria. MBC/MFC was considered as 136

the lowest concentration of the extracts that had bactericidal/fungicidal effect without any visible growth 137

[26]. All experiments were performed in triplicate. 138

139

2-5- Antioxidant Activity 140

The antioxidant activity of extracts and fractions from E. telmateia obtained in different 141

conditions was investigated using the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) method. 142

The DPPH radical-scavenging activity was measured by the method described by Radojevic et al. 143

[27]. Briefly, the stock solution of the plant extract was prepared in methanol to achieve the concentration 144

of 1 mg/ml. Dilutions were made in a concentration range of 500 to 7.81 µg/ml. 1mL of each diluted 145

solution were mixed with 1 mL of methanolic solution containing DPPH radicals (0.2 mM). After 30 min 146

at room temperature (23 °C) in darkness the absorbance was measured at 517 nm. The blank samples 147

contained all the reagents except the extract. The radical scavenging ability was calculated using the 148

following formula: 149

I% = (A blank – A sample/A blank)×100 150

Antioxidant activity was also represented as the IC50 (inhibition concentration at 50%), i.e., the effective 151

concentration of the extract to scavenge 50% of DPPH radicals. Vitamin C and BHT was used as positive 152

controls. The scavenging ability and IC50 of all extracts were determined considering the mean value of 153

triplicated experiments. 154

155

2-6- High performance liquid chromatography (HPLC) Analysis 156

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Polyphenol composition of the supercritical fluid extract of E. telmateia was analyzed by HPLC 157

(Knauer, Germany) using a C18 column (4.6 mm ID × 150 mm (5 µm), with a UV detector (at 350 nm), 158

as previously described by Milovanović et al. [6]. Elution profile: A=0.15% phosphoric acid in H2O–159

MeOH 77:23 (v/v, pH=2); B= MeOH. Isocratic: 0–3.6 min 100% A; gradient: 3.6 min 100% A-linear-160

24.0 min; 80.5% A-isocratic-30 min linear- 60 min; 51.8% A-linear-67.2 min; 100% B; flow rate: 1.0 161

ml/min. The injected volume was 20 µl. Due to the poor reproducibility of retention times in HPLC 162

chromatograms, we computed the relative retention indices (as the ratio between retention time of the 163

component in question and naringenin as the internal standard). The peak identity was checked by 164

comparison of their relative retention indices with the previously published ones [4] and by co-injection 165

with naringenin, quercetin, kaempferol, apigenin and rutin (Sigma Chemical Co. St. Louis, USA). The 166

percentage composition of the extracts (Table 3) was calculated from the HPLC peak areas. 167

168

2-7- Statistical analysis 169

The results of the extraction yield and antioxidant activity were statistically assessed by a one-170

way analysis of variance (ANOVA) using the software Statistical for Windows 7.0 (Statsoft Inc., USA) in 171

order to detect significant differences among the values. The significant differences (p < 0.05) were 172

analyzed by Tukey test. 173

174

3- Results and discussion 175

3-1- Antimicrobial activity 176

Table 1 shows the MIC and MBC/MFC results of E.telmateia extracts obtained by the different 177

methods (SFE, Mac and Frac) and solvents against four bacteria and one fungi. All the MIC and MBC 178

values against Gram-negative bacteria were over 128 mg/mL, excepting the supercritical extract and EtAc 179

fraction, that showed MIC and MBC of 128 mg/mL for E.coli and MIC of 128 mg/mL for S.typhi. the 180

results of E.telmateia extracts obtained by COSE and SFE showed that the extracts were generally more 181

effective on Gram-positive bacteria, mostly S.aureus, comparing to Gram-negative ones. Extracts 182

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obtained from SFE by SC-CO2 with MIC and MBC values of 32 mg/mL and Frac by the solvent EtAc 183

with MIC and MBC values of 64 mg/mL showed the lowest MIC and MBC values against S.aureus. The 184

higher resistance of the Gram-negative bacteria is most likely due to the presence of an outer-membrane 185

permeability barrier and to its two-layer cell membrane, compared to the single membrane of the Gram-186

positive bacteria, which restricts access of the antimicrobial substances to their targets in the bacterial 187

cells [28]. These results are in accordance with those from Oliveira et al. [16], Smith-Palmer et al. [28], 188

and Michielin et al. [29] showing that Gram-negative bacteria were usually more resistant to the 189

antimicrobials 190

Table 1. Determination of minimal inhibitory concentration (MIC, mg/ml), minimal bactericidal/ fungicidal concentration (MBC/MFC, mg/ml) of the different extracts from E. telmateia obtained by supercritical and

conventional (maceration and fractionation) extraction methods.

191

originating from plants and even showed no effect, comparing to Gram-positive bacteria. 192

We observed that, all investigated extracts and fractions had antimicrobial activity against all 193

tested microorganism, but this effect depends on the solvent and method of the extraction. The control 194

sample did not show any effect on the growth of tested microbial species (data not shown). All the MIC 195

and MBC/MFC results of DCM and n-BuOH fractions against tested microorganisms were over 128 196

mg/mL. Comparing the usage of various solvents in COSE, table 1 shows that Frac with EtAc presented 197

lower MIC and MBC/MFC values when compared with other fractions and Mac extracts. 198

When we compared the extraction methods, MIC and MBC/MFC values of SFE and COSE 199

extracts showed that supercritical extract was the most effective E.telmateia extract, with the lowest MIC 200

= 32-128 mg/mL and MBC/MFC = 32-128 mg/mL value for all tested microorganisms, while the COSE 201

Extraction Method

Solvent Microorganism

S. aureus B. cereus E. coli S. typhi C. albicans MIC MBC MIC MBC MIC MBC MIC MBC MIC MFC

SFE SC-CO2 + EtOH

32 32 64 64 128 128 128 ˃128 32 32

Maceration EtOH 128 ˃128 ˃128 ˃128 ˃128 ˃128 ˃128 ˃128 128 128 Water 128 128 128 128 ˃128 ˃128 ˃128 ˃128 128 128

Fractionation DCM ˃128 ˃128 ˃128 ˃128 ˃128 ˃128 ˃128 ˃128 ˃128 ˃128 EtAc 64 64 64 128 128 128 128 ˃128 64 64 n-BuOH ˃128 ˃128 ˃128 ˃128 ˃128 ˃128 ˃128 ˃128 ˃128 ˃128

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extracts except EtAc fraction presented lower antimicrobial effectiveness against all tested 202

microorganisms (MIC and MBC/MFC ≥128 mg/mL). EtAc fraction presented the slightly close MIC 203

values to supercritical extract (MIC = 64 mg/mL, MBC/MFC = 64˃ 128 mg/mL). A similar behavior was 204

also reported by Oliveira et al. [16], Vagi et al. [30], Benelli et al. [31] and Petrović et al. [32] for extracts 205

from other raw materials, indicating low MIC results and higher effectiveness for the extracts obtained by 206

SFE, when compared to COSE for the tested microorganism. Mazzutti et al. [17] described that although 207

different extraction methods applied (soxhlet, maceration and SFE) produced extracts with similar 208

antimicrobial potential, however,supercritical technigue allowed the extraction of antimicrobial 209

substances using lower amount of solvent in shorter time. The results of this study point toward the use of 210

SFE to obtain E. telmateia extract with antimicrobial activity against different microorganisms according 211

to the extracting conditions used. 212

213

3-2- Antioxidant activity 214

The model of sequestrate the stable DPPH radicals has been commonly used as a tool to assess 215

the free radical-scavenging abilities of materials due to the sensitivity, simplicity and speed of this method 216

[33]. Fig. 1 describes the scavenging ability of different extracts and fractions from E. telmateia on DPPH 217

radicals. 218

In this work, a dose-dependent DPPH radical-scavenging ability was observed for all tested 219

extracts and fractions. The scavenging abilities of samples extracted by SC-CO2 extraction and COSE on 220

DPPH radicals were 9.25% to 96.11% and from 0.3% to 73.4%, respectively, with concentration 221

increasing from 7.81 to 500 µg/ml. 222

The DPPH radical-scavenging ability of extracts was compared together using IC50 value of each 223

extract (table 2). The extract with lower IC50 value represented higher antioxidant ability. The DPPH 224

results showed antioxidant ability of the investigated extracts and fractions orderly SFE (70.83 ± 0.2 225

µg/mL), EtOH (96.14 ± 0.3 µg/ml), EtAc (164.32 ± 0.4 µg/ml), Water (425.48 ± 0.1 µg/ml), DCM 226

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(404.88 ± 0.2 µg/ml) and n-BuOH (455.75 ± 0.1 µg/ml) indicating that SFE extract had a higher 227

scavenging activity on DPPH radicals than extracts and fractions extracted by COSE. 228

Fig 1. Determination of scavenging ability of the different extracts from E. telmateia obtained by supercritical and conventional (Maceration and Fractionation) extraction methods on DPPH radicals.

SFE extract also showed a highly comparable capacity in sequestrating of radicals with synthetic 229

product BHT (IC50 = 41.82 ± 0.2 µg/mL). Milovanović et al. [6] measured the antioxidant activity of 230

ethanol 231

(80%) extracts of five Serbian Equisetum species and showed that E. telmateia extract presented the 232

highest antioxidant activity among other species. They attributed the higher antioxidant activity of E. 233

telmateia to the high content of caffeic acid and Kaempferol acetylated derivatives. 234

235

Table 2. IC50 values obtained for the E. telmateia extracts in DPPH assay

Extraction Method Solvent IC50 (µg/ml)

SFE SC-CO2 + EtOH

70.83 ± 0.2

Maceration EtOH Water

96.14 ± 0.3 425.48 ± 0.1

Fractionation DCM EtAc

n-BuOH

404.88 ± 0.2 164.32 ± 0.4 455.75 ± 0.1

BHT - 41.82 ± 0.2

0

20

40

60

80

100

120

7 .81 15 .62 31 .25 62.5 125 250 500

SFE water

EtOH EtAC

DCM n-BuOH

Sca

ven

gin

g ab

ility

(%

)

Concentration (µg/ml)

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Vitamin C - 36.39 ± 0.3 236

237

238

3-3- HPLC Analysis 239

The best results for antimicrobial and antioxidant activity of E. telmateia extracts obtained for 240

supercritical extract. For that reason, the supercritical extract was analyzed by HPLC in order to 241

determine the contents of bioactive polyphenol compounds. 242

Table 3 shows the identified components and the respective relative peak area of E. telmateia 243

extract obtained by SFE. Few components were identified in extract, possibly because non-polar and 244

volatile substances are not detectable by the HPLC analysis used in this work, and because the extracts 245

are complex mixtures of polar and non-polar compounds, in agreement to the solvents (CO2 + EtOH) 246

used for the extraction. Among the substances identified, flavonoids of kaempferol glycosides and its 247

acetylated derivatives comprise the highest amount (59.1%) of chemical composition of SFE extract and 248

kaempferol 3-O-(6’’-O-acetylglucoside) with % area peak of 25.3 is the major identified component. 249

Contrary to this work, Milovanović et al. [6] report Kaempferol 3,7-O-diglucoside as main component of 250

hydroalcoholic extract of E. telmateia and in Veit’s et al. [4] E. telmateia samples, kaempferol 3-O-(6’’-251

O-acetylglucoside) was not detected. The differences in the result of biological activity and chemical 252

composition of different extracts of the same species can be explained by different locations from which 253

the plant came, different methods of extraction and the solvents used. 254

Other important flavonoid detected in the SFE extract was catechin (11.1%) that was only 255

detected in Correia’ et al. [5] study alongside with proanthocyanidins .The HPLC analysis of SFE extract 256

of E. telmateia also allowed the identification of phenolic acid derivatives including: protocatechuic acid 257

(2.4%), p-OH-benzoic acid (4.5%), 5-O-caffeoyl shikimic acid (15.2%) and monocaffeoyl meso-tartaric 258

acid (1.3%). Caffeic acid conjugates in total 16.5% comprise the most abundant phenolic acids in extract. 259

5-O-Caffeoyl shikimic acid (18.4%) and monocaffeoyl meso-tartaric acid (2.2%) were detected by 260

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Milovanović et al. [6] and protocatechuic acid and p-OH-benzoic acid detected by Correia et al. [5] with 261

caffeic acid derivatives. 262

The better antimicrobial results of SFE extract can be attributed to the phenolic substances 263

identified such as catechin, kaempferol derivatives and p-OH-benzoic acid, well known as antimicrobial 264

Table 3. Phenolic profile of the supercritical fluide extract of E. telmateia determined by HPLC 265

Compounds relative peak area % Kaempferol 3-O-glucoside 5.9 Kaempferol 3-O-(6"-O-acetylglucoside)-7-O-rhamnoside 9.5 Kaempferol 3-O-(6’’-O-acetylglucoside) 25.3 Kaempferol 3-O-glucoside-7-O-rhamnoside 2.1 Kaempferol 3,7-O-diglucoside 8.6 Kaempferol 3-O-(6’’-O-acetylglucoside)-7-O-glucoside 7.7 Catechin 11.1 Protocatechuic acid 2.4 p-OH-benzoic acid 4.5 5-O-Caffeoyl shikimic acid 15.2 Monocaffeoyl meso-tartaric acid 1.3

266

compounds [11, 34] and the increase in the solubility of E.telmateia kaempferol derivatives and catechin 267

in the solvent mixture (EtOH/CO2) because of the interaction between solvent mixture and E.telmateia 268

bioactive flavonoids at applied SFE condition. Conventional techniques to obtain flavonoids cause loss of 269

flavonoids due to hydrolysis, ionisation and oxidation during extraction [24]. According to Garcia-Salas 270

et al. [35], phenolic compounds are very sensitive to light and air and will degrade by these two factors 271

easily. In addition, the short extraction time that SFE technique presents (less than 1 h) reduce the adverse 272

effects of enzymatic activity. Therefore, the higher antimicrobial effect of supercritical extract also can be 273

attributed to the short procedure time and possibility to perform extraction in the absence of light and air, 274

which protect bioactivity of the phenolic compounds in the extract compared with COSE procedure [35, 275

36]. Several studies documented the higher efficiency of SFE in extraction of biologically active 276

components over conventional extraction methods. According to Kotnik et al. [37], a maximum content 277

of 28 mg/g for matricine as an active compound in the extract of chamomile flower obtained by SFE 278

compared with trace amounts in conventional extracts (soxhlet= 1.08 mg/g, maceration= 1.01 mg/g and 279

distillation= 0.14 mg/g). They also showed that chalikulene, the degradation product of matricine that is 280

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formed thermally was detected in the extract obtained by steam distillation, while it was found only in 281

traces in SFE extracts. Finally, the results obtained by this work and above-mentioned studies determine 282

the SFE as a powerful technology to obtain antimicrobial components, improving the use of natural 283

products, which produced extracts with more efficient antimicrobial potential. 284

Former studies have demonstrated that the number of DPPH molecules that are reduced seems to 285

be correlated with the number of electron-donating hydroxyl groups in the antioxidant molecule. This 286

structural requirement could be linked to the presence of phenolic compounds, which are known to be 287

widely distributed in natural herb and spice extracts. Phenolic compounds have a high reducing ability to 288

eliminate free radicals because of both their alcoholic hydroxyl group and conjugated π electrons of the 289

benzene ring [38]. For sample extracted by SC-CO2 extraction, its high antioxidant activity could be 290

attributed to the high presence of phenolic hydroxyl group flavonoids and phenolic acid derivatives. In a 291

similar study He et al. [33] compared the extraction yield of flavonoids and their antioxidant activity from 292

Citrus grandis (L.) Osbeck extracts obtained by supercritical carbon dioxide and conventional solvent 293

extraction. Their results indicated that SFE extract have a significantly higher antioxidant activity than 294

EtOH extract due to high presence of phenolic hydroxyl group flavonoids. 295

296

3-4- Comparison of SFE Extraction yield and COSE 297

Different methods for extraction of natural compounds have different extraction efficiency and 298

yield. The extraction yield results of E. telmateia, comparing different extraction methods and solvents 299

(Mac, Frac and SFE), are presented in Fig. 2 with their standard deviation data. For the COSE techniques 300

(Mac and Frac) the results from Fig. 2 show an enhancement in the extraction yield with the solvent 301

polarity. 302

The best extraction yields were obtained by maceration assay using water 17.20 ± 0.4% and 303

ethanol15.26 ± 0.5% as solvent. The lowest yield value was provided by the Frac with DCM (0.2 ± 0.33) 304

a non-polar solvent, indicating that the E. telmateia contains many intermediate to high polarity 305

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compounds, extracted ideally by increasing solvent polarity. SFE with CO2 plus ethanol as co-solvent 306

could produce an 307

Fig 2. Yield results for E. telmateia extraction using different technigues: SFE with CO2 plus EtOH as co-solvent; Mac with EtOH and H2O; Frac with DCM, EtAc and n-butanol.

0.56% which was lower than Mac extraction, but above the values obtained by DCM, EtAc and n-308

Butanol. This result confirms the increase in the polarity of CO2 by adding ethanol. The results reported 309

in terms of the extraction yield evaluate quantitatively the effectiveness of the technique and show SFE 310

method was less efficient than Mac as COSE method in order to recovery of E. telmateia substances; 311

nonetheless assessment of the qualitative effectiveness of the extraction methods showed that qualitative 312

effectiveness is not directly related to their yield values, so that SFE extract exhibit the highest biological 313

activity among all tested extracts. Therefore, SFE technique can be suggested as an appropriate extraction 314

method to recovery biological substances from E. telmateia stems. Vagi et al. [30] in comparison of 315

composition and antimicrobial activity of Origanum majorana L. reported that despite lower extraction 316

yield results (SFE with 3.8 g/100 g dry material and ethanolic with 9.1 g/100 g dry material), supercritical 317

fluid extracts presented considerably stronger antimicrobial efficiency in comparison to the insignificant 318

inhibitory effects of the ethanolic extract because of the existence of higher concentration of the 319

biologically active compounds (the SFE product contained approximately 21% of biologically active 320

compounds; meanwhile the ethanolic extract contained only approximately 9% of). 321

322

9.60

17.2015.26

0.60 0.32 0.20

-2.000.002.004.006.008.00

10.0012.0014.0016.0018.0020.00

yiel

d (

%)

Fractionation Maceration SFE

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323

324

4- Conclusion 325

This is the first effort to examine the overall extraction yield, chemical composition and 326

antimicrobial and antioxidant activity of E. telmateia supercritical extract. The results of this study show 327

that supercritical fluid extraction method is effective to obtain E. telmateia extract with good recovery of 328

antimicrobial and antioxidant active compounds as an alternative extraction method. Mac presented 329

highest overall extraction yield, particularly when using water as solvent, although this extract shows very 330

low antimicrobial (MIC and MBC ≥ 128 mg/mL) and antioxidant (IC50 = 425.48 ± 0.1 µg/mL) activity. 331

Despite lower overall yield result of SFE extract, this extract was the most effective E.telmateia extract, 332

with the lowest MIC and MBC (32-128 mg/mL) values and inhibited the growth of all bacteria and fungi 333

tested. In general, all investigated extracts were more effective against Gram-positive bacteria (S. aureus 334

and B. cereus) comparing to Gram-negative (E. coli and S. typhi). In DPPH assays also SFE extract of 335

E.telmateia showed the most potent antioxidant ability and showed a highly comparable ability in 336

sequestrating of radicals with BHT. Further studies are necessary to investigate the cytotoxicity of 337

extracts from E. telmateia, to evaluate its safety. Results presented here may recommend that the SFE 338

technique is appropriate to obtain functional substances from a natural source for the pharmaceutical, 339

cosmetic and food industry. 340

341

5- Acknowledgement 342

We gratefully acknowledge the Department of Food Science and Technology, Ferdowsi 343

University of Mashhad for financial support of this work. 344

345

346

347

348

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349

350

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Highlights

� Equisetum telmateia extracts a strong antimicrobial activity on the growth of some

pathogenic strain causing poisoning and infection.

� Chemical compositions Equisetum telmateia was analyzed by HPLC.

� Equisetum telmateia extracts showed greater inhibitory effect on Gram-positive bacteria.