Chemical composition and bio-functional perspectives of Erica arborea L. extracts obtained by different extraction techniques: Innovative insights

Industrial Crops & Products 142 (2019) 111843

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Chemical composition and bio-functional perspectives of Erica arborea L. extracts obtained by different extraction techniques: Innovative insights

T

Gokhan Zengina,⁎,1, Aleksandra Cvetanovićb,1, Uroš Gašićc, Alena Stupard, Gizem Bulute, Ismail Senkardese, Ahmet Dogane, Roumita Seebaluck-Sandoramf, Kannan R.R. Rengasamyg, Kouadio Ibrahime Sinana, Mohamad Fawzi Mahomoodallyf a

Department of Biology, Science Faculty, Selcuk University, Campus, Konya, Turkey Faculty of Technology, University of Novi Sad, Bulevar cara Lazara 1, 21000, Novi Sad, Serbia c University of Belgrade–Faculty of Chemistry, P.O. Box 51, 11158, Belgrade, Serbia d Institute of Food Technology, University of Novi Sad, Bulevar cara Lazara 1, 21000, Novi Sad, Serbia e Department of Pharmaceutical Botany, Pharmacy Faculty, Marmara University, Istanbul, Turkey f Department of Health Sciences, Faculty of Science, University of Mauritius, Réduit, Mauritius g Department of Bio-resources and Food Science, Konkuk University, Seoul, South Korea b

A R T I C LE I N FO

A B S T R A C T

Keywords: Green extraction Phenolics Antioxidant Enzyme inhibition Multivariate analysis

Erica arborea L., also known as Estrella Gold, is traditionally used for several purposes. In this research, five different extraction techniques: accelerated solvent extraction (ASE), microwave-assisted extraction (MAE), maceration (MAC), soxhlet (SOE) and ultrasound-assisted extraction (UAE) were used to compare the total phenolic, flavonoids, total antioxidant activity and enzymatic activities of E. arborea extracts obtained different extraction techniques. The total phenolic and flavonoid contents were in the order of ASE > MAE > SOE > MAC > UAE. All extracts showed antioxidant, anticholinesterase, anti-tyrosinase and anti-diabetic activities. A highly sensitive method using ultra-high-pressure liquid chromatography coupled with linear ion trap-Orbitrap tandem mass spectrometry (UHPLC–LTQ–Orbitrap–MS) has been used for the qualitative analysis of obtained extracts. Seventy-two polyphenolic compounds were identified in all extracts. However, 20 components were quantified among the extracts. ASE was found to be a better extraction technique as compared to the other extraction techniques. E. arborea can be exploited in the discovery of bioactive natural products for the treatment of Alzheimer’s disease, diabetes and pigmentation problems.

1. Introduction

and activities such as: antiulcer, antibacterial, cytotoxic, anti-oedema, antidiarrheal and healing agent (Guendouze-Bouchefa et al., 2015). Turkey’s traditional medicine uses different parts of E.arborea (mostly leaves and flowers) as a natural diuretic and urinary antiseptic as well as for the treatment of constipation. The phytochemicals present in the plant include flavonoids, and phenolic, and the newly isolated constituents are (-)-epicatechin and quercitrin (Ay et al., 2007). In earlier reports, the presence of several phenolics including quercitrin (51.42 mg/g dry extract) (Pavlović et al., 2013), ferulic acid (25.6 mg/g dry extract) (Luís et al., 2011) and myricetin (11.45 μg/mg dry extract) (Marquez-Garcia et al., 2009) were reported as main components in E. arborea leaves. Applied extraction technique possess entirely affects the quality of obtained extracts, in the first place, their composition and

The Ericaceae is a species-rich family consisting of 4100 species divided into 124 genera, among which Erica is one of the three most widespread genera in the Mediterranean. Plants from the Ericaceae family are well-known for their medicinal properties, and many of their therapeutical benefits are closely related to polyphenols in their composition. Erica arborea is also known as tree heath which belongs to the Erica genus. It is a shrub which can reach 4 m in height. The plants of these species widely occur throughout an area of Mediterranean together with the western part of Portugal, as well as in the Canary Islands, in Northern Africa, Morocco, Tunisia and Algeria. E. arborea is classified as an astringent plant, whose aerial parts have been used for ages in the treatment of many diseases because of their health benefits

⁎

Corresponding author. E-mail address: [email protected] (G. Zengin). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.indcrop.2019.111843 Received 29 May 2019; Received in revised form 3 September 2019; Accepted 3 October 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.

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2015). Soxhlet extraction is the reference extraction technique in which plant material is extracted until its exhaustion with a solvent at the boiling point (Saini and Keum, 2018). In the frame of this work, five different techniques were applied for the extraction of bioactives from E. arborea. Taking into account that the applied techniques (ASE; MAE, UAE; MAC; SOW) rely on a different mechanism of action, different composition of the obtained extracts is expected to affect their bioactivity. To have a deeper insight into changes of chemical composition, which are primarily the results of application of different extraction approaches, the obtained extracts were analysed by LC–MS analysis. Extracts were further tested for their biological activities by in vitro antioxidant and enzyme-inhibitory assays.

concentration of desired components. The choice of extraction technique and the parameters during the process may have a crucial role in the successful isolation of valuable bioactive (Tomšik et al., 2017). In the process of choosing an appropriate technique, some of the criteria that must be fulfilled are the following: environmentally concern, maximization of target compounds recovery, and minimal degradation of beneficial molecules (Cai et al., 2016). Taking all these requirements into consideration, different modern techniques have been developed. One of the technique that has gained huge attention is the accelerated solvent extraction (ASE). It implies the use of liquid solvents under conditions of increased temperature (100 to 180 °C) and pressure (100 to 150 bar), influencing the changes in performances of the extraction processes making them more successful than the conventional one (Švarc-Gajić, 2012). Changes in extraction temperature and pressure make series of effects which have direct impact on solvent properties as well as on the interactions with plant matrix. By increasing the temperature, the solubility of the analytes becomes better as well as the high diffusivity influences better the mass transfer. The strength of the interactions between the analyte and the matrix decreases (Van der Wals, hydrogen, dipole-dipole bonds) which affects the kinetics of the desorption process by increasing its rate. Simultaneously, the density, the surface tension and the viscosity of the solvent are decreased improving the penetration of the solvent in the pores of matrix (Cvetanović et al., 2019). Generally, this technique has become very popular since it usually relies on green solvents and at the same time offers a faster procedure with increased reproducibility (Kang et al., 2016). Strongly controlled conditions, together with high extraction yield in ASE, make this technique one of the most commonly used in the last few years (Gomes et al., 2017). Apart from ASE, microwave-assisted extraction (MAE) is one of the most promising extraction technique. This approach combines the traditional solvent extraction and microwaves. Microwave energy is efficiently transmitted to the matter through the molecular interactions with the electromagnetic field, whereby rapid energy transfer to the solvent and the material is achieved (Criado et al., 2004). Rapid warming is caused by the direct interaction of microwaves with solvent molecules causes rapid warming (Ng and Hupé, 2003). Some of the advantages of this technique over traditional techniques are the following: requires a lower amount of plant material and solvents, it is not a time-consuming technique and offers high extraction yield. On the other hand, this modern technology includes expensive equipment and require high operator skill (Shang et al., 2018). Another modern extraction technique which offers higher reproducibility in a shorter period in comparison with the traditional ones is the ultrasound-assisted extraction (UAE). This technique does not require high temperatures, and it often requires lower amount of solvents (Fu et al., 2006). The cavitation phenomenon dominates the process of ultrasound extraction, which is caused by the propagation of ultrasound through a medium. When a cavitation bubble collapses near the cell wall, an ultrasound micro-jet is formed that directs the solvent toward the cell wall. Due to this ultrasound effect, the solvent penetrates into the plants’ cell, dissolving the components and transporting them out of the matrix (Švarc-Gajić, 2012). In the last 10 years all of the modern, techniques mentioned above have been successfully applied for the isolation of polyphenolic compounds and they are considered as potential alternatives to the traditional solid-liquid extraction method for obtaining secondary metabolites from plants. Numerous papers have dealed with polyphenols extraction from different matrices (medicinal/ aromatic plants, plant waste, side-stram from agro-industry, atc.) by ASE, MAE or UAE (Mašković et al., 2017; Radojković et al., 2018; Veličković et al., 2017). No matter the fact that numerous modern techniques have been developed, traditional maceration process still represents one of the most used technique. The procedure of maceration is simple and does not require expensive and complicated equipment, but it is a timeconsuming technique (usually performed for 1–3 days) (Azwanida,

2. Materials and methods 2.1. Collection of plant material Erica arborea samples were collected at Omerli catchment area (Turkey) identified by Dr. Ismail Senkardes from Marmara University (Istanbul, Turkey), (Voucher Number: MARE-19367). The leaves of the plant previously were dried naturally (in the shade at room temperature for ten days). The dried plant materials were grounded by a laboratory mill (particle size about 1 mm), and then the powdered plant materials were stored in darkness at room temperature. 2.2. Preparation of plant extracts The powdered leaf samples of Erica arborea were extracted using five different techniques, and the selection of the techniques was performed similarly to our previous paper (Sut et al., 2019). Accelerated solvent extraction (ASE) was performed by the Dionex extraction system (ASE 350, Sunnyvale, CA, USA). The extraction cells were filled with diatomic earth (250 mg) and dry plant material (1 g). Extraction was performed using 96% ethanol at 120 °C under the 1500 psi. Heating was performed for 6 min, and rinsing the cells was done by the fresh solvent. Nitrogen was used as purged gas for 30 s. Collection of the obtained extracts was done in 50 mL tubes. Microwave-assisted extraction (MAE) was carried out using 96% ethanol and a sample-to-solvent ration of 1:20. Extraction was accomplished after 30 min at 600 W. Ultrasound-assisted extraction (UAE) was carried out for one hour in a sonication bath keeping the same solvent (96% ethanol) and sample-to-solvent ratio (1:20) as in the case of MAE. Traditional extraction – maceration (MAC) was conducted by mixing 5 g of dry plant material with 100 mL of 96% ethanol. The mixture was macerated for 24 h. Soxhlet extraction (SOE) was performed according to the standardized Soxhlet procedure, with 96% ethanol for 6 h. The obtained extracts were then filtered and evaporated under reduced pressure at 40 °C until dryness. The dried extracts were stored at 4 °C protected from the light until analysis. 2.3. Profile of bioactive compounds The content of two important groups of secondary metabolites: phenols and flavonoids were measured spectrophotometrically by using well-known methods with Folin-Ciocalteu and AlCl3, respectively (Uysal et al., 2017). Gallic acid equivalent (mg GAE/g extract), as well as rutin equivalent (mg RE/g extract), were used as a measure of their content. 2.4. UHPLC-LTQ OrbiTrap MS qualitative analysis of phenolic compounds Analysis of phenolic compounds was done by UHPLC system (ThermoFisher Scientific Accela) coupled with LTQ OrbiTrap MS 2

Industrial Crops & Products 142 (2019) 111843

G. Zengin, et al.

according to the method previously described in detail by (Vasić et al., 2019). The phenolic compounds were quantified using the appropriate standards (Sigma-Aldrich, Steinheim, Germany). The basic solution of the mixture of all the phenolics with concentrations of 1000 mg/L was prepared in methanol (HPLC grade), while the working solutions were prepared by diluting the basic solution in the mobile phase. Extracts in methanol (10 mg/mL) were used for the analysis. According to the peak area, quantification was done, and the results were expressed as mg/kg dry extract. Tentative identification of some compounds in the absence of standards was achieved by high-resolution mass spectrometry (HRMS) and MSn fragmentation. 2.5. Determination of antioxidant and enzyme inhibitory effects

Fig. 1. Total bioactive compounds of the studied extracts. Values expressed are means ± S.D. of three parallel measurements. GAE: Gallic acid equivalent; RE: Rutin equivalent. Different letters indicate significant differences in the extracts (p < 0.05).

The ability of the extracts to inhibit different biologically important enzymes was monitored by in vitro assays using α-amylase, α-glucosidase, cholinesterases, and tyrosinase (Uysal et al., 2017). Obtained results were expressed in the way prescribed by official methods by using appropriate standards (Uysal et al., 2017). Antioxidant capacity of the tested extracts was measured FRAP, CUPRAC, DPPH and ABTS assays. All details about the used tests as well as the way of results expression are given in our earlier work (Uysal et al., 2017).

preferred over the UAE in order to obtain extract with high antioxidant potential. Phenolics are the primary sources of natural antioxidants with strong action. Carbonated tea infusion made by E. arborea leaves was characterized by Suna et al. (2018). According to that investigation TPC in dried leaves was 749.48 ± 34.46 mg GAE/100 g chemical extract, and 249.50 ± 18.10 mg GAE/100 g physiological extract (bioaccessible phenols). Guendouze-Bouchefa et al. (2015) studied the methanol extract for total phenolic content of E. arborea and obtained 70.8 ± 2.5 mg GAE/g extract. Jiménez-Zamora et al. (2016) investigated the total phenolic content and antioxidant activity of several herbs (during for six months storage process at 50 °C and room temperature). In their study, total phenolic content was found to be 44 ± 7–119 ± 5 mg GAE/L in E. multiflora at different storage conditions The variations in these results may link several factors such as the time of taking samples, choice of parts tested, environmental differences, and determination methods (Suna et al., 2018).

2.6. Statistical analysis One-way ANOVA, followed by Tukey’s multiple ranges, was done to investigate significant differences (p < 0.05) between the tested samples. Correlation map was generated to pinpoint the link between the studied biological activities and total bioactive compounds (TPC and TFC). Unsupervised principal component analysis, supervised Partial Least Squares Discriminant Analysis (PLS-DA) and Hierarchical cluster analysis for both biological activities and samples, using “ward” as linkage rule and the Euclidean similarity measure, were conducted. The statistical procedures were achieved by R software v. 3.5.1. 3. Results and discussion

3.2. UHPLC-Orbitrap MS profiles

3.1. Phytochemical compounds

Using LCeMS analysis, 72 different phenolic compounds were identified in tested E. arborea samples. All of these components could be divided into four different groups: 1) phenolic acids and derivatives (21 compounds); 2) flavan-3-ols and proanthocyanidins (6 compounds); 3) flavonoid glycosides (33 compounds); and 4) flavonoid aglycones (9 compounds). Among all identified compounds, the presence of twenty of them was certified using available analytical standards, while the others were identified by HRMS and multi-stage mass spectrometry (MSn). Table 1 summarized all obtained chromatographic and MS data, while the content of some compounds quantified using available standards are presented in Table 2. All compounds from Table 1 were detected in all E. arborea extracts obtained by different extraction techniques. Chromatograms of the extracts obtained by different extraction techniques are given in Fig. 2.

This study evaluated the extraction efficiency of bioactive compounds using ASE, microwave (MAE), ultrasound (UAE), maceration (MAC) and Soxhlet (SOE) extraction techniques. The results of the total bioactive compounds and the total antioxidant capacity obtained using different extraction techniques are illustrated in Fig. 1. The total phenolic contents obtained were in the order of ASE > MAE > SOE > MAC > UAE. A similar trend was also observed for total flavonoids, i.e. ASE sample was the richest with these compounds. The highest content was obtained from ASE extract (56.57 ± 0.58 mg RE/g). Extraction techniques exert significant influences on the characteristics of the obtained extracts. In this study, ASE was found to be the best extraction technique compared to the four different techniques used. In ASE, the efficiency rates (solubility or diffusion capacity) of the extracting solvents increase as the temperature increases. However, the viscosities of extraction solvents and the interactions between solute and matrix decrease. Additionally, because ASE offers better recovering of target analytes in comparison to conventional techniques, but also because of the possibility of full automatization, priority is given to this technique (Kang et al., 2016). According to Mutalib (2015), MAE is preferred as compared to the UAE They could be explained with some points: (i) the MAE increase the efficiency through solid matrix of the dried plant material, (ii) faster mixing of the liquid (solvent of extraction) occurs, (iii) provide the highest amount, standard and purity of active components in the extract. Additionally, in our research MAE showed to be

3.2.1. Phenolic acids and derivatives Analysis of available MS data revealed in the determination of 21 various phenolic acids and their related derivatives. Phenolic acid was found in free form, as to form of glycosides and esters (mostly with quinic acid). For example, compounds 8 and 9 at 4.31 and 5.22 min, respectively, showing the same molecular ion at 341 m/z, were identified as caffeic acid hexoside isomers. The MS2 base peak of these compounds was found at 179 m/z (loss of 162 Da), and the MS3 base peak at 135 m/z, which was obtained by further loss of 44 Da (CO2 group). Compound 19 (7.38 min and 693 m/z) with MS2 base peak at 517 m/z (loss of one feruloyl group – 176 Da) was tentatively identified 3

179,03498

C9H7O4–

5,85

15

477,06746

C21H17O13–

5,73

14

137,02442 153,01933

C7H5O3– C7H5O4–

5,40 5,55

12 13

353,08781

C16H17O9–

5-O-Caffeoylquinic acida

5,30

11

341,08781 341,08781 337,09289

C15H17O9– C15H17O9– C16H17O8–

Caffeic acid hexoside isomer 1 Caffeic acid hexoside isomer 2 3-O-p-Coumaroylquinic acid

4,73 5,22 5,27

8 9 10

353,08781

C16H17O9–

4,66

7

331,06707 329,08781 315,07216 299,07724 153,01933

4

8,16 Dihydroxybenzoic acid ethyl ester Flavan-ols and proanthocyanidis 4,78 A type prodelphinidin trimer

5,08

5,43

5,48

5,57

5,88

5,98

6,59

21

23

24

25

26

27

28

29

22

7,94

20

289,07176 863,18289

C15H13O6– C45H35O18−

Epicatechina

B type procyanidin dimer isomer 2

A type procyanidin trimer

577,13515

577,13515

C30H25O12–

B type procyanidin dimer isomer 1

C30H25O12

607,10933

C30H23O14−

−

289,07176

C15H13O6–

Catechina

A type prodelphinidin dimer

305,06668

911,16763

(Epi)gallocatechin

C45H35O21

181,05063

C9H9O4– −

499,12404

693,20362

C25H23O11–

C32H37O17

C15H13O7−

Caffeoylcoumaroylquinic acid

Ferulic acid feruoyl dihexoside

7,38

19

167,03498 −

C8H7O4–

Vanillic acida

6,80

18

163,04007

C9H7O3–

p-Coumaric acida

6,77

17

337,09289

C16H17O8–

6,36

16

5-O-p-Coumaroylquinic acid

Caffeic acid

a

Methyl-ellagic acid hexoside

p-Hydroxybenzoic acid 2,5-Dihydroxybenzoic acida

a

3-O-Caffeoylquinic acid

Gallic acid hexoside Vanillic acid hexoside Dihydroxybenzoic acid hexoside isomer 2 Hydroxybenzoic acid hexoside 3,4-Dihydroxybenzoic acida

C13H15O10– C14H17O9– C13H15O9– C13H15O8– C7H5O4–

4,16 4,21 4,41 4,43 4,46

2 3 4 5 6

Calculated mass, [M–H]–

315.07216

Molecular formula, [M–H]–

C13H15O9–

Phenolic acids and derivatives 4.07 Dihydroxybenzoic acid hexoside isomer 1

1

Compound name

tR, min

No

577,13342

863,18181

289,07040

577,13312

607,10852

289,07123

305,06527

911,16687

181,05011

499,12234

693,20264

167,03421

163,03975

337,09125

179,03438

477,06516

137,02408 153,01923

353,08670

341,08737 341,08685 337,09192

353,08755

331,06622 329,08691 315,07197 299,07703 153,01901

315.07138

Exact mass, [M–H]–

Table 1 High resolution MS data and negative ion mode MS4 fragmentation of phenolics found in E. arborea extracts.

3,00

1,25

4,70

3,52

1,33

1,83

4,62

0,83

2,87

3,41

1,41

4,61

1,96

4,87

3,35

4,82

2,48 0,65

3,14

1,29 2,81 2,88

0,74

2,57 2,73 0,60 0,70 2,09

2.48

Δ ppm

179(30), 135(10)

95(75), 79(20),

109(10)

125(5)

152(50), 109(15),

753(100), 725(20), 575(15), 483(10), 437(25), 305(10) 261(50), 221(70), 219(70), 179(100), 165(35) 271(5), 245(100), 205(40), 179(15), 125(5) 589(20), 579(30), 439(100), 305(40), 301(15) 559(10), 451(30), 425(100), 407(50), 289(25), 287(10) 271(5), 245(100), 205(40), 179(15), 125(5) 711(100), 693(20), 573(25), 451(25), 411(40), 289(15) 559(10), 451(30), 425(100), 407(50), 289(25), 287(10)

153(100), 109(10)

153(10), 152(80), 124(10), 123(100), 108(20) 517(100), 499(80), 485(20), 401(10), 355(10), 323(30) 361(5), 337(100), 163(10)

119(100)

191(100), 179(5), 163(10)

135(100)

109(10), 93(100) 136(5), 125(10), 109(100), 95(20), 79(10) 315(100)

191(10), 179(100), 135(10) 179(100), 135(10) 191(10), 173(10), 163(100), 119(10) 191(100), 179(5)

153(100), 108(10) 169(100), 167(100) 153(100), 137(100) 109(100), 59(10) 191(100),

MS2 Fragments, (% Base Peak)

227(35), 203(100), 187(30), 175(15), 161(25) 693(100), 559(80), 541(30), 425(10), 407(25), 285(5) 407(100), 381(5), 273(10)

227(30), 203(100), 187(25), 175(10), 161(20) 421(60), 313(70), 301(100), 261(20), 243(30) 407(100), 381(5), 273(10)

725(100), 617(10), 559(25), 575(70), 441(10), 423(30) 164(100), 151(40), 135(30)

355(15), 337(45), 295(15), 265(40), 235(50), 193(100) 191(10), 173(60), 163(100), 119(10) 109(100)

287(100), 271(15), 259(10), 243(10), 199(5) 135(60), 117(15), 107(100), 91(55), 79(15) 173(75), 127(100), 111(40), 93(60), 85(90) 119(60), 101(20), 93(25), 91(100), 72(10) 108(100)

173(75), 127(100), 111(40), 93(60), 85(90) 93(100) 81(85), 67(100), 63(60)

173(75), 127(100), 111(40), 93(60), 85(90) 135(100) 135(100) 119(100)

125(100) 152(100), 123(70), 108(20) 109(100) 93(100) 81(100), 68(25), 65(15)

109(100)

MS3 Fragments, (% Base Peak)

185(20), 157(15) 657(40), 525(45), 297(30),

567(100), 407(5) 285(100),

175(100),

185(20), 175(100), 157(10) 273(15), 257(100), 175(70) 297(30), 285(100),

(continued on next page)

188(70), 161(40), 283(15), 215(20), 389(30), 243(70) 188(60), 161(35), 675(10), 541(10), 389(30), 243(70)

707(10), 689(20), 599(100), 581(20), 541(20), 423(30) 120(100), 108(20)

–

119(100)

178(30), 149(70), 134(100)

80(35), 78(100)

–

109(30), 99(40), 85(100)

259(100), 243(50), 231(10), 215(30) –

– –

109(40), 99(50), 85(100)

135(100), 107(50) 107(100), 79(20) 119(100)

109(30), 99(40), 85(100)

123(25), 109(10), 85(10), 81(100) 107(100), 81(10) 124(5), 108(100) – – –

MS4 Fragments, (% Base Peak)

G. Zengin, et al.

Industrial Crops & Products 142 (2019) 111843

Flavonoid glycosides 5,75 Myricetin 7-O-hexoside

6,03

6,20

6,23

6,46

6,54

6,57

6,61

6,68 6,80

6,86

6,87

7,06

7,17

7,21

7,36

7,42 7,49

7,54

7,62

7,63

7,97

8,05

8,14

8,17

8,30

31

32

33

34

35

36

37

38

39 40

41

42

43

44

45

46

47 48

49

50

51

52

53

54

55

56

463,08820 609,14611 449,07255

C21H19O12– C27H29O16– C20H17O12–

5 433,07763 477,10385 447,09329 445,07763 449,10894 447,09329 583,10933 625,11989

C20H17O11– C22H21O12– C21H19O11– C21H17O11– C21H21O11– C21H19O11– C28H23O14− C30H25O15–

Isorhamnetin 3-O-glucoside

Kaempferol 3-O-glucoside (Astragalin)a

Quercetin 3-O-(6"-p-coumaroyl)hexoside isomer 2

Isorhamnetin 3-O-(6"-caffeoyl)hexoside

Quercetin 3-O-(6"-p-coumaroyl)hexoside isomer 1 Myricetin 3-O-(6"-benzoyl)hexoside

Kaempferol 7-O-(6"-benzoyl)hexoside

Kaempferol 3-O-rhamnoside

Quercetin 3-O-(6"-p-hydroxybenzoyl) hexoside Myricetin 3-O-(6"-p-coumaroyl)hexoside

Eriodictyol 7-O-hexoside Quercetin 3-O-rhamnoside (Quercitrin)a

Apigenin 7-O-hexuronide

Quercetin 3-O-pentoside

567,11441 609,12498 583,10933 639,13554 609,12498

C28H23O13– C30H25O14– C28H23O14− C31H27O15– C30H25O14–

431,09837

593,15119

C27H29O15–

Kaempferol 7-O-(6"-hexosyl)hexoside

C21H19O10

449,10894

C21H21O11–

Taxifolin 3-O-rhamnoside

−

463,08820 463,08820

C21H19O12– C21H19O12–

Myricetin 3-O-rhamnoside Quercetin 3-O-glucoside (Isoquercitrin)a

8-Methoxy-myricetin 3-O-rhamnoside

493,09876

465,10385

479,08311

C21H19O13–

C21H21O12

449,07255

C20H17O12–

−

479,08311

575,11950

C30H23O12–

C21H19O13–

Calculated mass, [M–H]–

Molecular formula, [M–H]–

C22H21O13−

Taxifolin 3-O-hexoside

Quercetin 3-O-(6"-rhamnosyl)glucoside (Rutin)a Myricetin 3-O-pentoside

Myricetin 7-O-rhamnoside

Myricetin 3-O-hexoside

Myricetin 7-O-pentoside

A type procyanidin dimer

6,90

30

Compound name

tR, min

No

Table 1 (continued)

609,12408

639,13531

583,10803

609,12396

567,11440

431,09645

625,11920

583,10730

449,10676 447,09148

445,07559

447,09113

477,10171

433,07558

593,14990

449,10693

463,08794 463,08590

493,09644

465,10193

449,07076

609,14453

463,08789

479,08176

449,07082

479,08176

575,11749

Exact mass, [M–H]–

1,48

0,36

2,23

1,67

0,02

4,45

1,10

3,48

4,85 4,05

4,58

4,83

4,49

4,73

2,17

4,48

0,56 4,97

4,70

4,13

3,99

2,59

0,67

2,82

3,85

2,82

3,49

Δ ppm

317(30), 316(100), 287(5), 271(10), 179(5) 477(15), 463(100), 323(5), 315(10), 301(20) 463(100), 301(20)

463(100), 301(20)

479(100), 461(5), 317(15), 316(15) 327(5), 285(90), 284(100), 255(10) 286(15), 285(100), 257(5)

463(100), 301(40), 300(2)

287(100), 151(10) 301(100), 300(35), 284(20)

357(20), 315(50), 314(100), 300(5), 285(10), 271(10) 327(20), 285(80), 284(100), 255(10) 269(100), 175(15)

343(5), 301(80), 300(100)

431(10), 323(20), 303(100), 285(90), 177(5), 151(30) 286(15), 285(100), 257(5)

478(10), 347(50), 346(100), 332(15), 331(15), 209(5) 317(50), 316(100) 301(100), 300(30)

313(30), 303(100), 151(20)

343(10), 301(100), 300(40), 271(10), 255(5), 179(5) 317(40), 316(100)

317(100), 316(40)

317(40), 316(100)

317(100), 316(40)

317(100), 316(40)

539(10), 449(30), 423(100), 411(50), 289(25), 285(10)

MS2 Fragments, (% Base Peak)

267(50), 257(100), 241(40), 229(50), 213(30) 421(5), 343(5), 301(100), 300(50) 287(40), 271(100), 270(40), 242(10), 179(10), 151(5) 343(5), 301(100), 300(50), 179(5), 151(5) 421(50), 341(30), 327(30), 313(50), 301(100), 300(30)

461(5), 359(5), 317(50), 316(100), 179(5) 255(100), 227(10)

269(10), 225(100), 201(40), 183(30), 151(40) 151(100) 273(25), 257(20), 179(100), 151(75) 343(5), 301(100), 300(40)

267(50), 257(100), 241(40), 229(50), 213(30) 271(100), 255(60), 179(10), 151(10) 300(30), 285(100), 271(75), 257(10), 243(25) 255(100), 227(10)

287(30), 271(100), 179(40) 273(25), 257(20), 179(100), 151(75) 285(100), 177(10), 125(10)

299(100), 287(30), 271(60), 255(20), 195(30), 167(15) 299(100), 287(30), 271(60), 255(20), 195(30), 167(15) 287(40), 271(100), 270(40), 242(10), 179(10), 151(5) 299(100), 287(30), 271(60), 255(20), 195(30), 167(15) 273(25), 257(20), 179(100), 151(75) 287(40), 271(100), 270(40), 242(10), 179(10), 151(5) 285(10), 177(5), 151(100), 125(5), 107(5) 331(100)

405(10), 313(15), 297(15), 285(100)

MS3 Fragments, (% Base Peak)

(continued on next page)

255(20), 239(30), 229(100), 213(30), 163(60) 273(60), 255(40), 229(10), 179(100), 151(80), 107(10) 243(100), 227(30), 215(10), 199(10) 271(60), 255(40), 179(100), 151(70) 273(20), 255(10), 229(12), 179(100), 151(90), 107(20)

271(90), 255(50), 179(100), 151(70), 107(10) 287(40), 271(100), 270(40), 242(10), 179(10), 151(5) 227(100), 211(60)

107(100) 151(100)

225(5), 197(50), 181(100)

227(100), 211(60)

255(20), 239(30), 229(100), 213(30), 163(60) 243(100), 227(80), 215(20), 199(20) 270(100)

257(10), 241(100), 175(60)

303(100), 287(15), 275(15), 259(20), 205(10), 191(20) 271(15), 243(100), 227(30) 151(100)

243(100), 227(30), 215(10), 199(10) 107(100), 83(5), 65(10)

271(100), 255(20), 243(10), 231(40), 227(20), 199(10) 271(100), 255(20), 243(10), 231(40), 227(20), 199(10) 243(100), 227(30), 215(10), 199(10) 271(100), 255(20), 243(10), 231(40), 227(20), 199(10) 151(100)

267(10), 257(80), 241(100), 217(50), 163(40), 125(40)

MS4 Fragments, (% Base Peak)

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8,78

8,88

9,02

9,43

Quercetin 3-O-(6"-cinnamoyl)hexoside isomer 1 9,63 Quercetin 3-O-(6"-cinnamoyl)hexoside isomer 2 Flavonoid aglycones 7,07 Taxifolina

7,76

7,95

8,69 8,72

8,79

9,21

9,57

9,73

59

60

61

62

65

66

67 68

69

70

71

72

64

63

8,70

58

6

Kaempferola

Apigenin

a

Limocitrin

Quercetina

Eriodictyol Luteolina

a

Aromodedrin

Myricetin

Kaempferol 3-O-(6"-benzoyl)hexoside

Myricetin 3-O-(6"-cinnamoyl)hexoside

Quercetin 3-O-(6"-benzoyl)hexoside isomer 1 Quercetin 3-O-(6"-benzoyl)hexoside isomer 2 Quercetin 3-O-(6"-(iso)valeryl)hexoside

8,50

57

Compound name

tR, min

No

Table 1 (continued) Calculated mass, [M–H]– 567,11441 567,11441 547,14571 609,12498 551,11950 593,13006 593,13006

303,05103 317,03029 287,05611 287,05611 285,04046 301,03537 345,06159 269,04554 285,04046

Molecular formula, [M–H]– C28H23O13– C28H23O13– C26H27O13– C30H25O14– C28H23O12– C30H25O13– C30H25O13–

C15H11O7– C15H9O8− C15O11O6− C15O11O6− C15H9O6− C15H9O7− C17H13O8– C15H9O5− C15H9O6− 285,04004

269,04523

345,06134

301,03395

287,05597 285,03985

287,05536

317,02917

303,05017

593,12970

593,12958

551,11881

609,12451

547,14496

567,11285

567,11346

Exact mass, [M–H]–

1,47

1,15

0,72

4,72

0,49 2,14

2,61

3,53

2,84

0,61

0,81

1,25

0,77

1,37

2,75

1,68

Δ ppm

225(100), 201(30), 183(20), 159(10), 151(25), 149(50) 255(100), 227(10)

285(100), 179(50), 177(10), 151(30), 125(10) 287(40), 277(20), 193(20), 179(100), 151(40), 107(5) 269(10), 259(100), 243(15), 201(10) 151(100), 107(10) 257(40), 241(100), 217(50), 199(70), 175(70) 283(15), 271(60), 257(25), 179(100), 151(80) 330(100), 281(5)

343(5), 301(100), 300(50), 271(15), 255(5), 179(5) 343(5), 301(95), 300(100), 271(15), 255(5), 179(5) 463(30), 445(5), 343(5), 301(100), 300(90), 271(10) 479(10), 317(30), 316(100), 287(5), 271(15) 327(20), 285(80), 284(100), 255(60), 227(20) 447(100), 301(60), 300(50), 271(10), 255(5), 179(5) 447(100), 301(60), 300(50), 271(10), 255(5), 179(5)

MS2 Fragments, (% Base Peak)

197(30), 196(20), 183(50), 181(100), 169(20), 157(10) 211(100), 195(5), 167(15)

315(100)

241(25), 215(100), 173(30), 151(20), 125(65) 107(100) 255(50), 227(100), 211(75), 197(35), 183(85) 151(100)

257(10), 241(100), 217(20), 199(15), 175(60) 151(100)

301(100), 300(40)

301(100), 300(40)

283(10), 271(100), 255(40), 229(10), 179(70), 151(60) 287(40), 271(100), 270(40), 242(10), 179(10), 151(5) 255(100), 227(10)

283(10), 271(100), 255(40), 229(10), 179(70), 151(60) 271(100), 255(40)

MS3 Fragments, (% Base Peak)

271(35), 255(30), 179(100), 151(80) 271(35), 255(30), 179(100), 151(80)

211(60)

227(30), 215(10),

227(70), 215(10),

227(60), 215(20),

227(70), 215(10),

211(40), 137(100)

287(100), 271(30), 259(10), 243(20), 175(20) 141(80), 117(100)

107(100), 83(10)

200(25), 187(10), 173(100), 158(15) 65(100) –

223(10), 213(100), 199(60), 197(50), 173(20) 107(100), 83(10), 65(5)

283(25), 229(10), 283(25), 229(10),

243(100), 199(10) 243(100), 199(20) 243(100), 199(10) 243(100), 199(10) 227(100),

MS4 Fragments, (% Base Peak)

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as ferulic acid feruloyl dihexoside. In this case, MS2 secondary peaks were found at 499 m/z (loss of ferulic acid – 194 Da) and 323 m/z (mass of deprotonated hexosylhexoside). This compound also showed the MS3 base peak at 193 m/z, which confirmed the presence of another ferulic acid. Dihydroxybenzoic acid ethyl ester (compound 21) at 8.16 min and 181 m/z generated MS2 base peak at 153 m/z (mass of deprotonated dihydroxybenzoic acid) and MS3 base 109 m/z (further loss of CO2 group).

Table 2 Concentration (mg/kg) of individual phenolics found in Erica arborea extracts. Compound names Phenolic acids and derivatives 3,4-Dihydroxybenzoic acid 5-O-Caffeoylquinic acid p-Hydroxybenzoic acid 2,5-Dihydroxybenzoic acid Caffeic acid p-Coumaric acid Vanillic acid Flavan-3-ols and proanthocyanidins Catechin Epicatechin Flavonoid glycosides Quercetin 3-O-(6"-rhamnosyl) glucoside (Rutin) Quercetin 3-O-glucoside (Isoquercitrin) Isorhamnetin 3-O-glucoside Kaempferol 3-O-glucoside (Astragalin) Quercetin 3-O-rhamnoside (Quercitrin) Flavonoid aglycones Taxifolin Eriodictyol Luteolin Quercetin Apigenin Kaempferol Σ

ASE

MAE

MAC

SOE

UAE

51.21 583.28 80.21 8.80 20.33 7.85 3.74

21.26 319.92 31.32 5.88 8.30 5.92 1.54

12.51 457.66 38.71 4.34 9.19 3.58 5.23

14.09 461.07 29.81 6.85 9.87 6.04 5.72

10.91 353.36 41.74 3.93 8.86 4.15 8.67

27.43 588.00

17.75 337.90

26.28 536.91

23.05 432.32

27.17 562.69

18.00

9.47

24.69

17.98

14.11

633.41

335.45

524.03

470.69

507.01

8.62 475.95

7.25 273.28

13.26 437.22

8.11 347.25

3.14 402.06

32.76

23.65

31.08

21.42

14.18

28.08 10.43 2.92 598.72 9.42 19.21 3208.37

17.07 7.15 1.87 365.83 0.79 11.38 1802.98

20.67 4.57 0.45 391.91 0.21 11.00 2553.5

19.55 8.79 0.89 546.16 0.18 21.04 2450.88

3.47 5.37 0.50 314.53 0.35 8.98 2295.18

3.2.2. Flavan-3-ols and proanthocyanidins The LC/MS analysis of E. arborea extracts allowed tentative identification of some flavan-3-ols and proanthocyanidins (A and B types) based on their exact molecular masses in the negative ionization mode and MS4 fragmentation. From the group A-type proanthocyanidins, we were able to identify procyanidin and prodelphinidin dimers and trimers, while from the group of B type derivatives, two isomers of procyanidin dimers were identified (Table 2). Fragmentation patterns of all these derivatives were following previously reported literature data (Jaiswal et al., 2012; Lin et al., 2014; Lv et al., 2015). 3.2.3. Flavonoid glycosides Among thirty-three flavonoid glycosides, presence of five of them was achieved using available standards (quercetin 3-O-(6″-rhamnosyl) glucoside – 35, quercetin 3-O-glucoside – 40, isorhamnetin 3-O-glucoside – 44, kaempferol 3-O-glucoside – 45, and quercetin 3-O-rhamnoside – 48). Numerous other glycoside derivatives were identified, and their identification was confirmed by neutral loss of 132 Da (pentosides), 146 Da (rhamnosides), 162 Da (hexosides), 176 Da (hexuronides), 308 Da (rhamnosylhexosides), and 324 Da (dihexosides). Also, many different acylhexose derivatives with various hydroxycinnamic and hydroxybenzoic acid were found. According to the mass spectrometry data proposed by Cuyckens and Claeys (2005), it was easy to determine the glycosylation site of flavonoid glycoside derivatives.

Fig. 2. Proposed fragmentation pathway of compound 55 (isorhamnetin 3-O-(6″-caffeoyl)hexoside). 7

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Fig. 3. Chromatograms of Erica arborea extracts.

Identification of 8-methoxy-myricetin 3-O-rhamnoside (compound 38), at 6.61 min and 493 m/z, was proposed by examination of its MS fragmentation. MS2 base peak of this compound was found at 347/346 m/z, and it was obtained by loss of rhamnose unit (146 Da). This compound is found to be specific to Erica sp. because its presence was previously reported in the aerial parts of Erica verticillata (Gournelis, 1995). Compound 55 (8.17 min and 639 m/z) showed MS2 base peak at 463 m/z (obtained by loss of caffeic acid residue (162 Da) and methyl group (15 Da)) was tentatively identified as isorhamnetin 3-O-(6″-caffeoyl) hexoside. Secondary MS2 peak found at 323 m/z corresponding to deprotonated caffeoyl hexoside. Fragmentation pattern of this compound is proposed in Fig. 3. Compound 60 at 8.88 min and 609 m/z gave MS2 base peak at 316/317 m/z (loss of cinnamoyl hexose – 292 Da) and MS3 spectrum which corresponds to the fragmentation of myricetin. High intensity of MS2 fragment at 316 m/z showed that glycosylation site was in 3-O-position and this compound was tentatively identified as myricetin 3-O-(6″-cinnamoyl) hexoside.

that non-optimized condition in MAE could lead to degradation of flavonoids and especially magnetron power higher 400 W may allow to unwanted effects towards to flavonoids. According to the literature, optimal extraction conditions of flavonoids by ethanol in MAE are magnetron power of 320 W and time of 5 min (Švarc-Gajić et al., 2013) which are drastically different conditions than those used in our experiment. Both used traditional techniques MAC and SOX approved as a better choice for the polyphenols extraction from E. arborea than both modern approaches UAE and MAE, which can be explained with mild conditions in MAC and SOX processes. Extraction of other Erica species was performed in the literature where a similar situation was recorded. Namely, in the case of E. carnea L. literature data suggest MAC and SOE extraction as techniques which offers a high concentration of polyphenolic compounds (Veličković et al., 2017). Comparing the TPC values obtained using the Folin-Ciocalteu (F-C) method and by the results UPLC-MS/MS analysis some differences were noticed, which are probably the consequences in accuracy and specificity of the quantification methods. The F-C assay relies on electron transfer reaction which measures the reductive capacity of antioxidants, and the used Folin reagent reacts with phenolic components and non-phenolic reducing substances to form chromogens, which are easy to be detected spectrophotometrically. Therefore, the accuracy of F-C assay is usually affected by the structures of phenolic components and easily disturbed by other substances, such as amino acids with reducing properties (Folin and Ciocalteu, 1927). Extraction of E. arborea by ethanol in subcritical conditions was performed for the first time in our study. The obtained subcritical ethanolic extract was opulent with all monitored components among which quercetin 3-O-glucoside was with the highest concentration. Apart from its bound form, this compound is also presented in its free form (a form of aglycone). Content of its aglycone (quercetin) was much higher than the content of all other aglycones. For example, this compound was presented in ASE extracts in the yield of 18.66% of total aglycones, while luteolin was presented in the yield of 0.09%. Together with luteolin, apigenin was presented in deficient concentration in ASE sample, which implies that flavons had the lowest tendency to be extracted with ethanol under the subcritical conditions. On the other hand, the subcritical-ethanolic medium was proven an excellent choice for the extraction of flavanols, flavanones and flavan-3ols, but also glucosides of flavonoids and phenolic acids. The

3.2.4. Flavonoid aglycones Nine flavonoid aglycones were identified in the E. arborea extract, and the presence of six of them was confirmed in comparison to available standards (Table 1). Limocitrin (compound 70), known to be present in the flowers of Erica cinerea (Kaouadji et al., 1992), was identified at retention time 9.21 min. It produced an MS2 base peak at 330 m/z, generated by the elimination of methyl group (15 Da) and MS3 base peak at 315 m/z, obtained by further loss of methyl group. 3.2.5. Quantitative data Quantitative analysis was performed for 20 detected components where the differences among the extracts were noticed regarding the applied extraction technique (Table 2). Dominant components from the classes of phenolic acids, flavan-3-ols, flavonoid glucosides and aglycones were 5-O-caffeoylquinic acid, epicatechin, isoquercitrin and quercetin, respectively. The highest yield was observed for the glycosides which are presented in ASE, MAE, MAC, SOE and UAE extracts in the content of 35.37%, 34.68%, 39.13%, 34.43% and 40.35%, respectively. Generally, the highest concentration of all quantified polyphenols was achieved in ASE sample, while the lowest concentration was recorded in MAE sample. The lowest concentration of polyphenols, especially flavonoids, in MAE sample could be explained by the fact 8

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Table 3 Antioxidant properties of tested extracts. Extraction techniques ASE MAE MAC SOE UAE

DPPH (mg TE/g) a

209.59 ± 4.37 179.17 ± 2.93b 92.19 ± 1.46c 76.43 ± 0.90d 66.61 ± 3.41e

ABTS (mg TE/g) 359.45 325.87 176.27 185.15 148.06

± ± ± ± ±

CUPRAC (mg TE/g) a

18.22 9.48b 9.25c 6.28c 1.96d

872.24 782.27 449.96 442.15 340.90

± ± ± ± ±

a

9.87 10.12b 11.72c 7.75c 6.89d

FRAP (mg /TE) 590.53 532.11 328.78 324.48 227.36

± ± ± ± ±

a

4.19 9.52b 8.42c 5.74c 1.44d

Metal chelating (mg EDTAE/g) 4.74 3.49 3.80 1.43 5.13

± ± ± ± ±

b

0.30 0.40c 0.16c 0.01d 0.42a

Phosphomolybdenum (mmol TE/g) 1.84 1.66 1.50 1.66 1.26

± ± ± ± ±

0.03a 0.04b 0.05c 0.05b 0.03d

*Values expressed are means ± S.D. of three parallel measurements. TE: Trolox equivalent; EDTAE: EDTA equivalent. Different letters indicate significant differences in the extracts (p < 0.05).

differences among the compound in the same group of phenols could be explained by the fact that solubility of each molecule in subcritical medium is caused by their structure: number and position of functional group, presence of conjugated double bonds, polar groups (hydroxyl, carboxyl, etc.) and glycoside moieties (Cvetanović et al., 2019). Advantages of ASE for the extraction of polyphenols from E. arborea have also reflected from the fact that this technique was performed only a few minutes which is drastically lower time than for MAC (24 h) or SOE (6 h) but also for UAE (60 min) and MAE (30 min). Also, this technique requires a much lower amount of plant material and solvent volume.

investigated the antioxidant capacity of E. arborea extracts and fraction and found the ethyl acetate extract was active against DPPH radical. Isolated compounds from E. arborea ethyl acetate namely (-)-epicatechin and quercitrin showed antioxidant activity. Phenolic compounds are main antioxidants in the plant extracts, and they exhibit several antioxidant ways including reducing agents, singlet oxygen quenchers, radical scavengers, hydrogen donors, and metal chelators (Khadri et al., 2010). Isolation of new flavonoids and phenylethanoid glycosides from E. arborea extracts have demonstrated antioxidant activities (Nazemiyeh et al., 2008).

3.3. Antioxidant activity

3.4. Enzymatic activity

In this research, antioxidant assays namely DPPH, ABTS, CUPRAC, FRAP and metal chelating were used to measure the antioxidant capacities of E. arborea extracts obtained from the five different extraction techniques (Table 3). The DPPH scavenging activities of the extracts were in the order of ASE > MAE > SOE > MAC > UAE. The ASE extract showed the highest antioxidant activity against all tested assays. All extracts were found to be good reducing agents and ABTS cation scavenger. CUPRAC assay uses the copper(II)-neocuproine [Cu(II)-Nc] reagent as the chromogenic oxidizing agent and is based on the cupric reducing ability to reduce compounds to cuprous (Al-Rimawi et al., 2016). The results showed that CUPRAC antioxidant activity of E. arborea followed a similar trend as FRAP antioxidant activity. The total antioxidant capacity of the different extracts was evaluated by the phosphomolybdenum method and was expressed as Trolox equivalents (TE). The total antioxidant of the extracts was found to follow the order as radical scavenging and reducing power assays. ROS generation can be avoided by redox-active metal catalysis, which involves chelating of the metal ions (Ebrahimzadeh et al., 2008). In contrast to the other antioxidant assays the chelating activity of the extracts were in the order of UAE > ASE > MAC > MAE > SOE. The different results could be explained with the presence of non-phenolic chelators (peptides, polysaccharides, etc.) in the extracts and this fact was also reported in earlier studies (Wang et al., 2009; Kalogeropoulos et al., 2013). ASE proved to be an excellent extraction technique as compared to the other four different techniques. A study carried out by Amezouar et al. (2013) demonstrated that ethanolic extract of the plant showed antioxidant and anti-inflammatory activity. Ay et al. (2007)

Treatment of neurodegenerative diseases such as Alzheimer’s disease (AD) is limited, and new medicines that alleviate symptomology and restrict disease progression are required Cholinesterase inhibitors (ChEIs), and high amount of antioxidant are used against AD (Nwidu et al., 2017). Currently, plant-derived alkaloids such as rivastigmine and galantamine are used in the management of AD (Machado et al., 2015). However, some adverse effects have been reported with the use of ChEIs such as hepatotoxicity, gastrointestinal disturbances, nausea, vomiting, diarrhoea and dizziness. Therefore, there is a need to discover and develop natural ChEIs that are safe, affordable and effective worldwide. The extracts of E. arborea exhibited inhibitory effects of the activity of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes in the range of 3.71–4.91 mg GALAE/g and 5.52–6.18 mg GALAE/g respectively (Table 4). According to Table 4, MAE and UAE extracts showed better activity on cholinesterases compared to other extracts. Studies have showed that various potent AChE inhibitors had been obtained from natural sources such as terpenoids, flavonoids and phenolic compounds (Murray et al., 2013). These findings are different from the total bioactive component results, in which ASE was found to be the best method. This fact may be attributed to the non-phenolic cholinesterase inhibitors, especially alkaloids and terpenoids and this approach was also supported by several researchers, who reported a weak correlation between total bioactive components and cholinesterase inhibition (Ozer et al., 2018). Kuş et al. (2017) studied the anticholinesterase activities of aqueous extract of various plant parts of E. manipulifora. The aerial parts extract of E. manipuliflora showed the potent inhibitory activity against AChE enzyme at 200 μg/mL concentrations. Tyrosinase is a well-known

Table 4 Enzyme inhibitory properties of tested extracts. Extraction techniques

AChE inhibition (mg GALAE/g)

ASE MAE MAC SOE UAE

3.71 4.33 4.91 4.67 4.91

± ± ± ± ±

0.03d 0.03c 0.14a 0.18b 0.11a

BChE inhibition (mg GALAE/g) 5.52 5.69 5.99 5.97 6.18

± ± ± ± ±

0.05d 0.06c 0.06b 0.13b 0.01a

Tyrosinase inhibition (mg KAE/g) 177.43 180.29 171.18 172.20 171.05

± ± ± ± ±

2.30a 1.87a 3.45b 1.82b 1.14b

Amylase inhibition (mmol ACAE/g) 0.52 0.39 0.48 0.38 0.39

± ± ± ± ±

0.04a 0.02bc 0.06ab 0.03c 0.06bc

Glucosidase inhibition (mmol ACAE/g) 1.63 1.64 1.60 1.57 1.62

± ± ± ± ±

0.02a 0.01a 0.01b 0.01c 0.01ab

*Values expressed are means ± S.D. of three parallel measurements. GALAE: Galatamine equivalent; KAE: Kojic acid equivalent; ACAE: Acarbose equivalent. Different letters indicate significant differences in the extracts (p < 0.05). 9

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highlighted PC1 and PC2 as the best separating pair of PCs, accounting for 86.08% of total’ variance (Fig. 4). PCA biplot (Fig. 4B) showed clustering of different extraction methods along the first axis, in substance, ASE and MAE, two green technologies for the extraction of bioactive compounds clustered together to the positive side of the first axis, were distinguishable from the three traditional methods namely MAC, UAE and SOE. Moreover, SOE stood out from MAC and UAE. When compared to MAC and UAE, SOE is known to be a method for high-temperature extraction. This serious disadvantage could give rise to bioactive compounds degradation, responsible for excellent biological activities observed in the other extracts. The PCA exploration of dataset gave clear indications about biological activities differences among experimental used methods. To confirm PCA results, we performed supervised sPLS-DA analysis (sPLS-DA). sPLS-DA is a very robust technique that can provide the advantage of additional information regarding methods classification. As referred to sPLS-DA score plot and heatmap a clear separation between the methods was observed. This result confirmed the biological activities variations of extraction methods previously highlighted by PCA with a similar pattern (Fig. 4D). ASE and MAE exhibited similar biological activities. As opposed to conventional extraction (SOE, MAC and UAE), they are known to be a technique with a significant reduction in solvent consumption and extraction time. Further, MAC and UAE seemed to have some similarities The biological activities accountable for the discrimination between the studied methods are reported in Fig. 4E. Among them, antioxidant activities, with high VIP scores recorded accounting for most of the discrimination potential. Lastly, it should be noted that by observing the balance error rate plot, the best performance of our supervised model was achieved for ncomp = 2, ensuring us that the plot based on the first two axes were good quality projection. Multivariate statistical analysis is commonly used to increase the interpretability of large datasets in many different disciplines including phytochemical studies. Earlier studies on the use of multivariate statistical analysis to discriminate different techniques of plant extraction based on numerous biological activities datasets were reported. For instance, in our

crucial enzyme in melanin biosynthesis, and its inhibitors are essential due to their potential use as hypopigmenting agents. Since plants consist of a rich source of bioactive chemicals which are generally free from harmful side effects, there is an increase in interest for finding tyrosinase inhibitors from natural sources (Pintus et al., 2015). MAE extract showed strong inhibitory activity against tyrosinase enzyme at 180.29 ± 1.87 mg KAE/g. Diabetes mellitus is one major metabolic disease, and it is characterized by lack of insulin or no insulin respond in the cells. (Kumaresan et al., 2014). α-Glucosidase and α-amylase inhibitors (also known as starch blockers) are considered one effective way to control postprandial hyperglycemia (Thilagam et al., 2013; Wickramaratne et al., 2016). The examined extracts showed glucosidase inhibitory activity at the range of 1.7-1.60 mmol ACAE/g. The extracts exhibited activity against amylase enzyme at the range of 0.380.52 mmol ACAE/g. The enzymatic activity of the different extracts could be attributed to the presence of phenolic compounds and flavonoids. The chemical constituents of plants might be changing with several factors, including plant parts, growth conditions or the age of the plant (Arya et al., 2010). The phytochemical properties also vary according to the geographical regions, season and time of collection, different climatic conditions as well as extraction conditions, which could explain the difference in activities of extracts using different extraction techniques. The extracts showed various enzymatic activities such as antioxidant, anticholinesterase, anti-tyrosinase and anti-diabetic. However, it is crucial to test and validate the safety of these extracts for traditional use and also serve as a guide in the quest for novel active compounds (Latif et al., 2014). Acute toxicity evaluated using mice revealed that the extract was non-toxic at a dose of 5 g/kg body weight (Amezouar et al., 2013). 3.5. Discriminant analysis The unsupervised multivariate exploratory method principal component analysis was applied to the pre-processed data to provide a general overview of the dataset. Exploration of the scree plot

Fig. 4. Unsupervised and supervised multivariate statistical analyses presented as PCA and sPLS-DA outcomes. A&B PCA percentage of explained variance and representation of methods in the PCA space (PC1 vs PC2); C: sPLS-DA balance error rate plot reflecting the performance of the supervised model; D&E: loading plot of sPLS-DA and Heatmap showing clear separation between studied methods; F: discriminant biological activities assessment by VIP approach; G: relationship between bioactive compounds and biological activities. 10

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previous study (Sut et al., 2019), Anthemis cotula extracts were separated by using PLS-DA, and the extracts were classified by three groups. The first group was characterized by maceration and UAE, the second group contained MAE and SE and the last group included only ASE. Regarding the relationships between total bioactive compounds and biological activities, a high positive correlation was found between phytochemical contents (TPC and TFC) and PPBD (r = 0.93; r = 0.93), DPPH (r = 0.91; r = 0.94), ABTS (r = 0.93; r = 0.95), CUPRAC (r = 0.94; r = 0.96), FRAP (r = 0.96; r = 0.98) and inhibition of tyrosinase enzyme (r = 0.77; r = 0.76). These findings were consistent with previous reports that showed that phenolic compounds, especially flavonoids were contributed the most in antioxidant assays (Zengin et al., 2019). In addition, several studies reported remarkable antioxidant abilities of epicatechin, isoquercitrin, astragalin and quercetin, which were major flavonoid in E. arborea extracts (Choi et al., 2013; Li et al., 2016; Yilmaz and Toledo, 2004). No correlation was found between TPC, TFC and metal chelating ability. As previously mentioned, this implies that the metal chelating effect observed in Erica arborea extracts is dependent on other phytochemicals, including polysaccharides and peptides.

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4. Conclusion Erica arborea has been used traditionally to manage various diseases. Therefore, this research was conducted to examine the potential of Erica arborea in contemporary medicine using different extraction techniques to isolate bioactive compounds. The present study revealed that the different extracts exhibited antioxidant activities against various assays. The presence of 72 different polyphenol compounds from the different subclasses (phenolic acids and derivatives; flavan-3-ols and proanthocyanidins; flavonoid glycosides; flavonoid aglycones) was confirmed using UHPLC-LTQ OrbiTrap MS qualitative analysis. Quantification was done for the 20 polyphenols among which quercetin and isoquercitrin were the most dominant. Content of all observed polyphenols was much higher in the extract obtained with ethanol under the elevated temperature and pressure, and among the applied extraction techniques, ASE shows to be the most suitable extraction to obtain extract rich in phenolic compounds and high antioxidant activity. The result suggests that extraction of E. arborea can be one of the steps in order to exploited and discover bioactive natural products for the treatment of Alzheimer’s disease, diabetes and pigmentation problems making this potential plant candidate for the future development of various functional products and medicaments. Declaration of Competing Interest We wish to confirm that there are no known conflicts of interest associated with this publication, and there has been no significant financial support for this work that could have influenced its outcome. References Al-Rimawi, F., Rishmawi, S., Ariqat, S.H., Khalid, M.F., Warad, I., Salah, Z., 2016. Anticancer activity, antioxidant activity, and phenolic and flavonoids content of wild Tragopogon porrifolius plant extracts. Evid. Complement. Alternat. Med. 2016, 1–7. Amezouar, F., Badri, W., Hsaine, M., Bourhim, N., Fougrach, H., 2013. Antioxidant and anti-inflammatory activities of Moroccan Erica arborea L. Pathologiebiologie 61, 254–258. Arya, V., Yadav, S., Kumar, S., Yadav, J., 2010. Antimicrobial activity of Cassia occidentalis L (leaf) against various human pathogenic microbes. Life Sci. Med. Res. 9, e12. Ay, M., Bahadori, F., Öztürk, M., Kolak, U., Topçu, G., 2007. Antioxidant activity of Erica arborea. Fitoterapia 78, 571–573. Azwanida, N., 2015. A review on the extraction methods use in medicinal plants, principle, strength and limitation. Med. Aromat. Plants 4, 3–8. Cai, Z., Qu, Z., Lan, Y., Zhao, S., Ma, X., Wan, Q., Jing, P., Li, P., 2016. Conventional, ultrasound-assisted, and accelerated-solvent extractions of anthocyanins from purple sweet potatoes. Food Chem. 197, 266–272. Choi, J., Kang, H.J., Kim, S.Z., Kwon, T.O., Jeong, S.-I., Jang, S.I., 2013. Antioxidant effect of astragalin isolated from the leaves of Morus alba L. against free radical-induced

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