International Journal of Biological Macromolecules

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Shikimic acid from Artemisia absinthium inhibits protein glycation in diabetic rats

Abdulrahman L. Al-Malki

Department of Biochemistry, Faculty of Science, Experimental Biochemistry Unit, King Fahd Medical Research Center, Bioactive Natural Products Research Group, King Abdulaziz University, Jeddah, Saudi Arabia

a b s t r a c t a r t i c l e i n f o

Article history:

Received 11 May 2018

Received in revised form 7 September 2018 Accepted 12 September 2018

Available online 15 September 2018

This study investigated the impact of Shikimic Acid (SA) obtained from leaves ofArtemisia absinthiumon protein glycation in the retina of diabetic rats. The GC/MS analysis ofA. absinthiumshowed that the most abundant bioactive compound was SA (C7H10O5) with a measured retention Index (RI) of 1960 compared to that of the reference sample (1712). Male albino rats were divided into two main groups, Group I (control) and Group II (diabetic); Group II was further divided into four subgroups: Group IIa (diabetic control), Group IIb (diabetic rats were given SA orally [50 mg/kg, body weight (bw)/day], Group IIc diabetic rats were given SA orally [100 mg/kg, bw/day], and Group IId (diabetic rats were given metformin orally [100 mg/kg, bw/day] as positive control). The data obtained suggested that SA reduced glucose and glycated hemoglobin levels. In addition, SA also decreased the formation of glucose-derived advanced glycation end products. Interestingly, SA showed in- terference with the release of inﬂammatory mediators in retina and possess antioxidant potential. In conclusion, SA protected the tissues from detrimental effects of hyperglycemia and enhanced antioxidant activity. SA could be a potential lead in the process of drug development in the future to prevent retinopathy in diabetic subjects.

Keywords:

Artemisia absinthium Shikimic acid Antiglycation Hyperglycemia Retinopathy Diabetic rats

1. Introduction

In the recent few years, natural product research has attracted the attention of the researchers globally toﬁnd a suitable cure for the various diseases and disorders. Natural products have shown to demonstrate signiﬁcant therapeutic activities that control different cell signaling pathways and exert cytotoxic, and genotoxic effects.

The rationale for using these natural products is that they have increased bioavailability and enhanced efficacy [1,2]. In fact, some natural products have been considered as lead compounds to obtain biologically active treatment regimens with increased efficiency and efficacy for the therapeutic use [3–7]. Therefore, the development of novel natural products as therapeutic agents could be promising to treat various disease conditions and could alsofind suitable targets for drug discovery [5,8,9].

Artemisia absinthiumL. (Asteraceae), commonly known as wormwood, has pharmaceutical and medicinal effects such as antimicrobial, insecticidal, antiparasitic, hepatoprotective, and antioxidant activity.

The chemical composition ofA. absinthiumL. extracts includes sesquiterpene lactone, sesquiterpene lactone-pinene and β-thujone, α- thujone, sabinyl acetate, andβ-thujone [10]. Elevated blood glucose can bind with the amino group of tissue proteins non-enzymatically to

form a nonfunctional glycated protein which undergoes changes to form fructosamine and is converted to form stable advanced glycated end products (AGEs) [10]. Protein glycation is a sequential but highly complex process that begins with the non-catalytic binding of sugar as glucose, fructose or their derivatives to the amino group of a protein.

This is accompanied by molecular rearrangements of functional proteins prior to the production of glycated proteins (GP) and crosslinking of these proteins [11]. These modiﬁed proteins are not recycled in different organs such as collagen of connective tissue, nephron, retina, blood vessels that lead to dysfunction of these organs. In diabetic patients, the rate of formation of AGEs is increased leading to deleterious effect in vital tissues as retina, neurons, nephrons and heart [11].

Moreover, aldose reductase plays an important role in the conversion of glucose 6-phosphate to sorbitol that acts as a mediator towards the development of retinopathy [12]. Prolonged use of foods rich in polyphenols such as pomegranate can prevent some diseases due to its high phenolic content which acts as a natural defense against envi- ronmental stress worldwide. Numerous phenolic compounds have been shown to prevent non-enzymatic reaction of protein with mono- saccharides in vivo [13]. Phenolic compounds have been widely studied for their potent antioxidant activity against free radical production even in controlling the progression of cancers. Shikimic acid (SA) was considered one of the important phenolic isolated from different fruits and plants [14].

E-mail address:[email protected].

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International Journal of Biological Macromolecules

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A. absinthiumplays a vital role in catering to the essential-nutrient requirements of people in many countries [15]. Since SA is rich in most of the macro and micronutrients required for the vital biological processes in the body, this could solve the problem of malnutrition in many countries [16].

The aim of this study was to investigate the impact of SA extracted fromA. absinthiumleaves on the glycosylation of proteins in the retina of diabetic rats to prevent late complications such as retinopathy.

2. Materials and methods

2.1. Extraction of SA from Artemisia absinthium

Artemisia absinthiumleaves were collected from hills of ASIR city (Saudi Arabia) and identiﬁed by botanists at Botany department (KAU, Jeddah). Extraction of SA fromA. absinthiumleaves was done with 95%

hexane (Avantor PA, U.S.A) and identiﬁcation of the active compounds was done by GC/MS grade reagents from Fisher (NJ, U.S.A). Alkane standard solution from Sigma Aldrich (C8-C20, app. 40 mg L⁻¹for each compound in hexane) was used for calculation of modiﬁed retention index.

Working solution of alkane standard was prepared by dilution 1:10 of stock solution in hexane (ﬁnal concentration app. 4 mg L⁻¹for each compound) [17]. For hexane solvent, 1.0 mg of extracted plant samples were dissolved in 1 mL hexane. For methanol solvent, samples were prepared in the same manner by using methanol instead of hexane.

All extracted plant samples were dissolved in methanol. All samples were kept at−10 °C until analysis via gas chromatography–mass spectrometry (GC–MS).

2.1.1. Gas chromatography-mass spectrometry

Agilent GCMS 5975 (Agilent, CA, USA) system including Agilent 7890 A gas chromatography equipped with Agilent 5975C-VL MSD mass spectrometer with Agilent 7693 A automatic liquid sampler was used for analysis of plant extracts. 1μL of standard or sample solution was introduced into a GC–MS system using a hot injector with the pulsed splitless mode. Agilent HP-5MS (30 m in length × 0.25 mm id × 0.25μm thickness stationary phasefilm, Agilent, USA) was used for separation. Data acquisition was employed via Agilent ChemStation for GCMS software version. MassHunter version B.07.01 GC MSD Trans- lator and MassHunter Quantitative B.07.01 were used for conversion of ChemStation data and for processing. AMDIS (Automated Mass Spectral Deconvolution Identification System) software version 2.70 (2011) from NIST (National Institute of Standards and Technology, USA) was used for chromatographic deconvolution. NIST library (version 2.0g, 2011) was used for identification of compounds. The compound was identified via both electron impact ionization mass spectrum and modified retention indexes (programmed temperature n-alkane based retention index). According to Van Den Dool et al., modified retention index was calculated via the following equation [18]:

RI¼100nþ100 tx−tn

tnþ1−tn

RI is modiﬁed retention index, tx, tnandtn+1denotes retention time of target compound and alkane compounds in molecules which have‘n’ and‘n + 1’carbon atoms that eluted before and after target compound respectively.

2.2. Animal model

Male albino rats 150 ± 20 g (n = 75) were obtained from King Fahad Medical Research Center, KAU, Jeddah, Saudi Arabia and housed in clean sterile cages. The rats received rat chow and tap water ad libitum. Fifteen rats were considered as normal (Group I), received 0.25 mol/l citrate buffer. The remaining sixty rats were injected with a single dose of streptozocin (65 mg/kg bw)i.p. Blood sugar was checked

daily to conﬁrm the development of diabetes mellitus (DM). When blood glucoseN250 mg/dl, it was considered as diabetic and excluded if lesser than the above-stated value. Further, diabetic rats were divided into four groups as follows: Group IIa, diabetic but untreated; Group IIb, diabetic rats were given orally SA (50 mg/kg bw/day); Group IIc, diabetic were given orally SA (100 mg/kg bw/day); Group IId, diabetic rats were given metformin orally (100 mg/kg bw/day) as positive control. The doses of SA were given according to the previously published literature [18]. After 6 weeks of treatment, the animals were fasted overnight (12 h) and then administered with light anesthesia with 10% thiopental. Blood and retina were collected directly from animals.

Serum was subjected to determination of malondialdehyde (MDA), glycated hemoglobin, fructosamine, total antioxidant activity. Retinas were rinsed in phosphate buffer saline to remove excess blood and stored at−80 °C till further analysis.

2.3. Determination of aldose reductase

Serum aldose reductase (AR) activity was determined spectrophoto- metrically by monitoring the decrease in nicotinamide adenine dinucle- otide phosphate hydrogen (NADPH) absorption at 340 nm at 37 °C usingDL-glyceraldehyde as a substrate. The AR activity represents the difference between the rate of NADPH oxidation with and without the substrate (μmol/mg protein) [19].

2.4. Biochemical analysis of retina tissue

Retinal tissues (0.1 g) were homogenized in 2 mL phosphate buffer (pH 7.3) containing a protease inhibitor. Samples were centrifuged at 12,000 rpm/10 min at 4 °C. Theﬁltrate was used for the determination of malondialdehyde (MDA), reduced glutathione (GSH), catalase, and superoxide dismutase by using commercial kits from BIORAD (En- gland), tumor necrosis factor, interleukin-1, and advanced glycated end products (AGEs) levels by using ELISA kits [20]. Protein levels were determined by Foline reagent using a standard curve [21].

2.5. Statistical analysis

All experiments were performed in duplicate, and the results were expressed as the Mean ± S.D. A one-way analysis of variance (ANOVA) was performed using GraphPad Prism 6 (Graph Pad Software, USA) to compare the differences. The individual comparisons were car- ried out by employing Tukey's test. Signiﬁcant differences are indicated as *pb0.05, **pb0.01 and ***pb0.001.

3. Results

The phytochemical analysis of the hexane extract ofA. absinthiumby GC–MS report has been depicted inFig. 1. The GC–MS analysis revealed the presence of seven compounds (Table 1). It was observed that SA formed the major constituent of the isolated compounds and proved to possess biological activity. It must be conferred that the further study of these phytoconstituents may be quite promising in designing a suitable therapeutic regimen.

3.1. Alteration of blood glucose levels

The body weight of the rats at the beginning of the study was similar in all the groups. At the end of the experiment, diabetic animals showed a signiﬁcant weight loss (pb0.001) compared to the untreated group.

The administration of STZ resulted in a significant increase in blood glucose levels in the diabetic group as compared to that of the control group (p= 0.01). Treatment of rats with 50 or 100 mg SA/kg bw or metformin resulted in a significant decrease in blood glucose compared with the untreated diabetic animals (pb0.05,Table 2). As a conse- quence of diabetes, HbA1c was significantly increased (pb0.05) in the

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untreated diabetic group. Treatment of animals with 50 or 100 mg SA/

kg bw or metformin improved two parameters in a dose-dependent manner (Table 2). The hypoglycemic action exerted by SA was lower than that of metformin (pb0.05).

3.2. Antioxidant activity

Additionally, the antioxidant activities of GSH, catalase, and SOD were signiﬁcantly reduced in the retinas of diabetic animals due to

STZ injection. Supplementation with various concentrations of SA or metformin resulted in a significant elevation of the GSH (pb0.05 for each) level and the activities of catalase and SOD (pb0.001,b0.01 and b0.05) respectively in a dose-dependent manner (Table 3). However, the lipid peroxidation in the retina was significantly elevated due to diabetes, while the administration of SA resulted in a dose-dependent decrease in MDA levels (Table 3). Moreover, serum aldolase reductase (Fig. 2A) was significantly reduced (pb0.01) in diabetic rats as compared to the control. Unexpectedly, the total antioxidant activity

Table 2

Body weight, glucose and HbA1c levels in all groups are represented as mean ± SD.

Parameters Group 1 Group IIa Group IIb Group IIc Group IId

Initial B·W(gm) 150 ± 9.50 155.60 ± 7 159.1 ± 6.50 152 ± 9.2 151 ± 9.5

Final B·W(gm) 189 ± 9.20 110.80 ± 8.20â 137 ± 12.50â,b 155 ± 8.9â,b 190 ± 12.3â,b

Glucose (mg/dL) 82 ± 0.35 285.90 ± 6.45â 175 ± 4.40â,b 155 ± 4.2^b 121 ± 0.97â,b

HbA1c (%) 5.2 ± 0.71 9.20 ± 0.34^a 7.8 ± 0.64^b 6.4 ± 0.48^b 6.1 ± 0.32^b

aComparison with control group.

b Comparison with untreated diabetes group.

Table 1

Phytochemical analysis ofArtemisia absinthiumby GC/MS.

S. no Compound CAS number Measured RI Molecular formula

1 3,4-Dehydro-L-proline 4043-88-3 1014 C5H7NO2

2 2,4,5-Trimethyl-4H-pyrazol-3-one 17826-82-3 1039 C6H10N2O

4 3,5-Dihydroxy-6-methyl-2,3-dihydropyran-4-one 28564-83-2 1143 C6H8O4

5 3,6-Dimethyl-4,5,6,7-tetrahydro-1-benzofuran 494-90-6 1165 C10H14O

7 Shikimic acid 138-59-0 1690 C7H10O5

Fig. 1.GC/MS chromatogram separation of Shikimic acid (SA): GC/MS analysis ofArtemisia absinthiumshowed presence of SA (C7H10O5) with a measured Retention index (RI) of 1960 compared to that of reference RI (1712).

Table 3

Serum and retina malondialdehyde (MDA) levels and reduced glutathione (GSH), catalase, and superoxide dismutase (SOD) activity (p* value, all groups vs control;p** value, treated vs untreated) in retinas of different groups (mean ± S.D).

Groups Serum

MDA μmol/L

Retina MDA

μmol/mg protein

GSH μg/mg protein

Catalase U/mg protein

SOD U/mg protein Group I

Mean ± S.D.

55 ± 3.4 33 ± 2.8 512 ± 42 1359 ± 120 1835 ± 154

Group IIa Mean ± S.D.

118 ± 4.9â 182 ± 11â 213.6 ± 32â 412 ± 55â 1132 ± 68â

Group IIb Mean ± S.D.

83 ± 6.2â,b 162 ± 15â,b 289 ± 22â,b 741 ± 134â,b 1432 ± 106â,b

Group IIc Mean ± S.D.

73 ± 3.7â,b 163 ± 14â,b 340 ± 26 879 ± 66â,b 1543 ± 190â,b

Group IId Mean ± S.D.

68 ± 4.7 151 ± 17 337 ± 18 981 ± 76 1651 ± 143

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(Fig. 2B) was increased (pb0.001) in diabetic rats as compared to the control. This may be due to the triggered hyperactive antioxidant defense system following the initial dose. The SA supplementation (50 or 100 mg/kg bw) or metformin elevated these activities in a dose- dependent manner.

It was also established that both TNFα(Fig. 2C) and IL-1 (Fig. 2D) played a signiﬁcant role in the pathogenesis of diabetic retinopathy.

Upon administration of an STZ injection, the retinal levels of TNFαand IL-1 became significantly elevated (Fig. 2C) (pb0.001) for each group indicating a considerable rise in the levels of inflammation compared to normal control rats. The administration of SA (50 or 100 mg/kg bw) or metformin resulted in a significant dose-dependent reduction of TNFαand IL-1 levels (pb0.01). However, in the untreated diabetic group, enhanced formation of AGEs was observed as compared to the healthy control group. SA supplementation (50 or 100 mg/kg bw) or metformin resulted in a significant reduction of AGEs in a dose- dependent manner as indicated (Fig. 2E).

4. Discussion

Shikimic acid (SA) is a natural phenolic compound found in many plants and fruits as a bioproduct of tryptophan pathways. SA is an inter- mediate metabolite synthesized from tryptophan and exerts specific an- abolic effects on bone in in vitro conditions [22]. SA was found to reduce oxidative stress and exert its ability to attenuate the expression of the antioxidant genes in the diabetic retina of rats and to attenuate diabetic nephropathy [15]. The effect of SA on renal cell function is of particular importance since nephropathy develops in 5–10% of patients with both types of diabetes. Several cases of acute renal failure and renal insuffi- ciency in patients indicated that antioxidant analogs could potentially alter function retina. Moreover, previous studies demonstrated that SA exerts an anti-inflammatory effect on the diabetic retina of rats by

suppression of NF-βactivation [23]. SA may also reduce inﬂammation in renal cells, thereby delaying the progression of nephropathy [16].

The action of phenolic compounds on protein glycation (AGEs) is re- lated to their antioxidant potential, they prevent the oxidation and the subsequent formation of AGEs, sometimes referred to as autoxidation.

Moreover, previous studies have shown the role of SA derivatives as antifungal agents that can be used for plant protection. Since SA is present in cereals and many other plants, it is beneficial for human consumption in addition to being toxic to pathogens. Recent investigations have revealed that SA are the most bioactive components present in the rhi- zomes ofCimicifuga heracleifolia[24]. This medicinal plant is an anti- inflammatory source of a drug frequently used in Japanese traditional medicine and has an additional use as an AGEs inhibitor. All of these reported studies are in good agreement with our presentfindings. SA, which was used in this study, is a hydroxy-shikimic acid that is a hydroxy derivative of shikimic acid. Dorantes et al., reported the inhibition of growth of some foodborne pathogenic bacteria by SA that was extracted fromArtemisia absinthium[25].

The present study also demonstrated that inhibition of overproduction of ROS by SA (50 or 100 mg/kg bw) in the diabetic rats was indicated by the reduction of glucose and HbA1c levels as compared to the untreated group. Moreover, SA normalized the parameters of oxidative stress in diabetic retinas and prevented the activation of major pathways involved in hyperglycemia-induced vascular damage. SA also repressed downstream effectors of vascular response to injury. Additionally, the reduction of free radical overproduction suggests an indirect AGE-inhibiting effect of SA. The results obtained are very close to those for metformin, which was used as a positive control [26,27]. Interestingly, these results are also in accordance with those of a previous study which demonstrated that phenolic compounds reduce diabetic complications with the inhibition of free radical production.

Fig. 2.A) Serum aldose reductase activity. B) Serum total antioxidant activity. C) Retina tumor necrosis factor (TNF-α). D) Interleukin-1 and E) Advanced glycated end products (AGEs). The data is presented as Mean ± SD. * indicates comparison versus control (pb0.01); # denotes comparison versus diabetes (pb0.01).

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Interestingly, it was found that IL-1 plays a signiﬁcant role in the pathogenesis of proliferative diabetic retinopathy. Retinal IL-1 was found to be signiﬁcantly increased in diabetic rats as compared to normal control rats, SA treatment (50 or 100 mg/kg bw) attenuated the expression of IL-1 as compared to the untreated group. Various studies have shown that phenolic compounds such as SA inhibit IL-1- mediated diabetic retinopathy [28].

TNF-αcontributes to the development of diabetic retinopathy and significantly higher levels of TNF-αare found in patients with either type 1 or type 2 diabetes as compared to similarly aged healthy control subjects. TNF-αplays a significant role in the apoptotic pathway of retinal endothelial cells during early and late stages of diabetic retinopathy in a rat model. In the present study, TNF-αexpression was significantly increased in the diabetic rats as compared to normal ones and the groups treated with SA (50 or 100 mg/kg bw) showed a significant reduction in TNF-αlevels in relation to the untreated diabetic group.

The major constituents of plasma that contribute to total antioxidant capacity are albumin, urate, ascorbate, alpha tocopherol, and bilirubin.

Therefore, measurement of total plasma antioxidant capacity may give a more precise indication of the relationship between antioxidants and the pathogenesis.

Therefore, the measurement of total plasma antioxidant capacity may give a more precise indication of the relationship between antioxidants and the pathogenesis. In the present study, we demonstrated a signiﬁcant decrease in the total plasma antioxidant activity in the diabetic group as compared to the control group. After treatment with different doses of SA, the total antioxidant activity of diabetic rats was increased compared to that of the untreated diabetic group. Our results are in accordance with the results of some previous studies that suggest the possible role of free radicals in the pathogenesis of diabetic mellitus [29].

In conclusion, it can be suggested that SA may have potential bene- ﬁts in the prevention of the onset and progression of retinopathy in diabetic patients. SA may also suppress the formation of AGEs in diabetic subjects and will delay or prevent the late and irreversible complications of diabetes which require further investigations.

References

[1] A. Kuerban, S.S. Moselhy, Y.Q. Almulaiky, S.S. Razvi, Mohammed Nihal Hasan, Khalid Omar Abulnaja, Taha A. Kumosani, A. L-AL-Malki, Natural compounds that inhibit protein glycation: a review for recentﬁndings, Indo Am. J. Pharm. Sci. 4 (2017) 4027–4042,https://doi.org/10.5281/ZENODO.1045213.

[2] K.V. Ramana, S.S. Singhal, A.B. Reddy, Therapeutic potential of natural pharmacological agents in the treatment of human diseases, Biomed. Res. Int. (2014) 1–4,https://

doi.org/10.1155/2014/573452.

[3] G.M. Cragg, D.J. Newman, Natural product drug discovery in the next millennium, Pharm. Biol. 39 (Suppl. 1) (2001) 8–17,https://doi.org/10.1076/phbi.39.s1.8.0009.

[4] L. Katz, R.H. Baltz, Natural product discovery: past, present, and future, J. Ind.

Microbiol. Biotechnol. 43 (2016) 155–176,https://doi.org/10.1007/s10295-015- 1723-5.

[5] M. Lahlou, The success of natural products in drug discovery, Pharmacol. Pharm.

(2013) 17–31,https://doi.org/10.4236/pp.2013.43A003.

[6] L.-X. Liao, X.-M. Song, L.-C. Wang, H.-N. Lv, J.-F. Chen, D. Liu, G. Fu, M.-B. Zhao, Y.

Jiang, K.-W. Zeng, P.-F. Tu, Highly selective inhibition of IMPDH2 provides the basis of antineuroinﬂammation therapy, Proc. Natl. Acad. Sci. U. S. A. 114 (2017) E5986–E5994,https://doi.org/10.1073/pnas.1706778114.

[7] L.-C. Wang, L.-X. Liao, H.-N. Lv, D. Liu, W. Dong, J. Zhu, J.-F. Chen, M.-L. Shi, G. Fu, X.- M. Song, Y. Jiang, K.-W. Zeng, P.-F. Tu, Highly selective activation of heat shock protein 70 by allosteric regulation provides an insight into efﬁcient neuroinﬂammation

inhibition, EBioMedicine 23 (2017) 160–172,https://doi.org/10.1016/j.ebiom.2017.

08.011.

[8] S. Mathur, C. Hoskins, Drug development: lessons from nature (review), Biomed.

Rep. (2017) 612–614,https://doi.org/10.3892/br.2017.909.

[9] A.A. Siddiqui, F. Iram, S. Siddiqui, K. Sahu, Role of natural products in drug discovery process, Int. J. Drug Dev. Res. 6 (2014) 172–204.

[10] K. Msaada, N. Salem, O. Bachrouch, S. Bousselmi, S. Tammar, A. Alfaify, K. Al Sane, W.

Ben Ammar, S. Azeiz, A. Haj Brahim, M. Hammami, S. Selmi, F. Limam, B. Marzouk, Chemical composition and antioxidant and antimicrobial activities of wormwood (Artemisia absinthiumL.) essential oils and phenolics, J. Chem. (2015) 1–12, https://doi.org/10.1155/2015/804658.

[11] K.S. Bora, A. Sharma, The genusArtemisia: a comprehensive review, Pharm. Biol. 49 (2011) 101–109,https://doi.org/10.3109/13880209.2010.497815.

[12]K.S. Bora, A. Sharma, Phytochemical and pharmacological potential ofArtemisia absinthiumLinn. andArtemisia asiaticaNakai: a review, J. Pharm. Res. 2 (2010) 325–328.

[13] S. Kordali, R. Kotan, A. Mavi, A. Cakir, A. Ala, A. Yildirim, Determination of the chemical composition and antioxidant activity of the essential oil ofArtemisia dracunculus and of the antifungal and antibacterial activities of TurkishArtemisia absinthium, A. dracunculus,Artemisia santonicum, andArtemisia spicig, J. Agric. Food Chem. 53 (2005) 9452–9458,https://doi.org/10.1021/jf0516538.

[14] H. Chiasson, A. Bélanger, N. Bostanian, C. Vincent, A. Poliquin, Acaricidal properties ofArtemisia absinthiumandTanacetum vulgare(Asteraceae) essential oils obtained by three methods of extraction, J. Econ. Entomol. 94 (2001) 167–171,https://doi.

org/10.1603/0022-0493-94.1.167.

[15] K.A. Tariq, M.Z. Chishti, F. Ahmad, A.S. Shawl, Anthelmintic activity of extracts ofAr- temisia absinthiumagainst ovine nematodes, Vet. Parasitol. 160 (2009) 83–88, https://doi.org/10.1016/j.vetpar.2008.10.084.

[16]A.H. Gilani, K.H. Janbaz, Preventive and curative effects ofArtemisia absinthiumon acetaminophen and CCl4-induced hepatotoxicity, Gen. Pharmacol. 26 (1995) 309–315.

[17] J.M. Canadanovic-Brunet, S.M. Djilas, G.S. Cetkovic, V.T. Tumbas, Free-radical scav- enging activity of wormwood (Artemisia absinthiumL) extracts, J. Sci. Food Agric.

85 (2004) 265–272,https://doi.org/10.1002/jsfa.1950.

[18] H. van Den Dool, P. Dec. Kratz, A generalization of the retention index system including linear temperature programmed gas—liquid partition chromatography, J.

Chromatogr. A 11 (1963) 463–471,https://doi.org/10.1016/S0021-9673(01) 80947-X.

[19] T. Jiratanan, R.H. Liu, Antioxidant activity of processed table beets (Beta vulgarisvar, conditiva) and green beans (Phaseolus vulgarisL.), J. Agric. Food Chem. 52 (2004) 2659–2670,https://doi.org/10.1021/jf034861d.

[20] J. Safaei-Ghomi, A.H. Ebrahimabadi, Z. Djafari-Bidgoli, H. Batooli, GC/MS analysis and in vitro antioxidant activity of essential oil and methanol extracts ofThymus caramanicusJalas and its main constituent carvacrol, Food Chem. 115 (2009) 1524–1528,https://doi.org/10.1016/j.foodchem.2009.01.051.

[21] B.N. Singh, B.R. Singh, R.L. Singh, D. Prakash, R. Dhakarey, G. Upadhyay, H.B. Singh, Oxidative DNA damage protective activity, antioxidant and anti-quorum sensing potentials ofMoringa oleifera, Food Chem. Toxicol. 47 (2009) 1109–1116,https://

doi.org/10.1016/j.fct.2009.01.034.

[22] A. Basile, S. Sorbo, B. Conte, B. Golia, S. Montanari, R. Castaldo Cobianchi, S. Esposito, Antioxidant activity in extracts fromLeptodictyum riparium(Bryophyta), stressed by heavy metals, heat shock, and salinity, Plant Biosyst. 145 (2011) 77–80,https://doi.

org/10.1080/11263504.2010.509935.

[23] S. Irshad, M. Butt, H. Younus, A. Professor, In-vitro antibacterial activity of two medicinal plants neem (Azadirachta indica) and peppermint, J. Pharm. 01 (2011) 9–14.

[24] P.G. Pietta, Flavonoids as antioxidants, J. Nat. Prod. 63 (2000) 1035–1042.

[25] M. Friedman, P.R. Henika, R.E. Mandrell, Bactericidal activities of plant essential oils and some of their isolated constituents againstCampylobacter jejuni,Escherichia coli, Listeria monocytogenes, andSalmonella enterica, J. Food Prot. 65 (2002) 1545–1560.

[26]K.M. Schuenzel, M.A. Harrison, Microbial antagonists of foodborne pathogens on fresh, minimally processed vegetables, J. Food Prot. 65 (2002) 1909–1915.

[27] M. Sokmen, J. Serkedjieva, D. Daferera, M. Gulluce, M. Polissiou, B. Tepe, H.A.

Akpulat, F. Sahin, A. Sokmen, In vitro antioxidant, antimicrobial, and antiviral activities of the essential oil and various extracts from herbal parts and callus cultures of Origanum acutidens, J. Agric. Food Chem. 52 (2004) 3309–3312,https://doi.org/10.

1021/jf049859g.

[28] A. Rezaeinodehi, S. Khangholi, Chemical composition of the essential oil ofArtemisia absinthiumgrowing wild in Iran, Pak. J. Biol. Sci. 11 (2008) 946–949.

[29] Y. Jiang, N. Wu, Y.-J. Fu, W. Wang, M. Luo, C.-J. Zhao, Y.-G. Zu, X.-L. Liu, Chemical composition and antimicrobial activity of the essential oil of Rosemary, Environ.

Toxicol. Pharmacol. 32 (2011) 63–68,https://doi.org/10.1016/j.etap.2011.03.011.