MS technique and their pharmacological properties: A comparative study

Industrial Crops & Products 131 (2019) 266–280

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Metabolomic profile of Salvia viridis L. root extracts using HPLC–MS/MS technique and their pharmacological properties: A comparative study

T

⁎

Gokhan Zengina, , Fawzi Mahomoodallyb, Carene Picot-Allainb, Alina Diuzhevac, József Jekőd, Zoltán Cziákyd, Aleksandra Cvetanoviće, Abdurrahman Aktumseka, Zoran Zekoviće, Kannan R.R. Rengasamyf a

Department of Biology, Science Faculty, Selcuk University, Campus, Konya, Turkey Department of Health Sciences, Faculty of Science, University of Mauritius, Réduit, Mauritius c Department of Analytical Chemistry, Pavol Jozef Šafárik University in Košice, Košice, Slovakia d Agricultural and Molecular Research and Service Institute, University of Nyíregyháza, Nyíregyháza, Hungary e Faculty of Technology, Bulevar Cara Lazara 1, 21000, Novi Sad, Serbia f REEF Environmental Consultancy, #2 Kamaraj Street, S.P. Nagar, Puducherry, 605 001, India b

A R T I C LE I N FO

A B S T R A C T

Keywords: Salvia viridis Diabetes Alzheimer’s disease Hyperpigmentation Extraction Phyto-pharmaceuticals

Several Salvia species have received due scientific attention regarding their therapeutic virtues, yet little is known about the pharmacological potential of Salvia viridis L. roots. This study, therefore, attempts to explore the phytochemical composition, enzyme inhibitory potential, and antioxidant activities of S. viridis ethanolic root extracts obtained by different extraction methods, namely microwave-assisted extraction, maceration, supercritical fluid extraction, Soxhlet extraction, and ultrasonic assisted extraction. The extract produced by ultrasonic assisted extraction possessed the highest phenolic and flavonoid contents (111.41 mg gallic acid equivalent/g extract and 23.46 mg rutin equivalent/g extract). S. viridis ethanolic root extract obtained by ultrasonic assisted extraction showed highest radical scavenging (240.00 and 302.85 mg Trolox equivalent TE/g for DPPH (1,1-diphenyl-2-picrylhydrazyl) and ABTS (2,2′-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid) assays, respectively) and reducing (970.74, 704.27 mg TE/g, and 2.84 mmol TE/g for CUPRAC (cupric reducing antioxidant capacity), FRAP (ferric reducing antioxidant power), and phosphomolybdenum assays, respectively) activities. Chemical profiles of these extracts were investigated by HPLC–MS/MS, and the profiles (23 components) of the supercritical fluid extract was different from other extraction techniques. The study reports for the first time, the inhibitory action of ethanolic root extract of S. viridis on key enzymes related to Alzheimer’s disease (acetylcholinesterase, butyrylcholinesterase), diabetes (α-amylase, α-glucosidase), and skin hyperpigmentation disorders (tyrosinase). Data generated from this study appraises the multiple biological activities of plants belonging to the Salvia genus. Scientific evidence gathered in this study support further investigations which might lead to the development of new pharmaceutical entities for the management of diabetes, Alzheimer’s disease, and skin hyperpigmentation conditions.

1. Introduction It is generally agreed that medicinal plants represent promising alternatives for the management of numerous human ailments. Multiple lines of evidence attest of the ethnopharmacological use of plants by several folk populations since ancient times (Zengin et al., 2019). Indeed, naturally occurring plant compounds underpin drug development. It is noteworthy highlighting that nearly one-quarter of new molecular entities were derived from naturally occurring compounds (Patridge et al., 2016; Mollica et al., 2017). Additionally, the rising ⁎

prevalence of besetting diseases, along with the ever-increasing demand for more effective and safer drugs, provide further impetus for research. The type of extraction method is one of the most critical steps in the phytochemical studies, and thus several techniques have been considered to extract phytochemicals from plants. In recent years, conventional methods (maceration, soxhlet, etc.) have been replaced by advanced techniques including ultra-sonication, microwave, and supercritical methods, which have several advantages such as ecofriendly, reduce use of solvents and extraction times. In this respect, many researchers have been focusing on the best extraction techniques

Corresponding author. E-mail address: [email protected] (G. Zengin).

https://doi.org/10.1016/j.indcrop.2019.01.060 Received 13 December 2018; Received in revised form 26 January 2019; Accepted 28 January 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.

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2.2.4. Soxhlet extraction (SE) Five grams of root samples were separately extracted with ethanol (96%, w/w) in a Soxhlet apparatus for 6 h.

with high efficiency of solvents (Ameer et al., 2017; Belwal et al., 2018). The Salvia genus is the most prominent genus of the Lamiaceae family and it contains approximately 900 species. Species of this genus are widely distributed in temperate, subtropical, and tropical regions (Sharifi-Rad et al., 2018). The most common applications of Salvia species include the food, cosmetics, and pharmaceutical industries. Several genera of this species have been used in traditional medicine, for instance, S. cavaleriei has been used to treat dysentery, boils, injuries, haemoptysis; S. desoleria has been used to treat central nervous system, menstrual, and digestive problems; S. bucharica has been used to manage liver disorders (Hao et al., 2015). Salvia species have been reported to possess multiple bio-pharmaceutical activities, namely, antioxidant, antimicrobial, antidiabetic, neuroprotective, anti-inflammatory, and cytotoxic properties (Zengin et al., 2018a). Salvia viridis L. (synonym Salvia horminum L.), commonly known as ‘Red topped sage’, naturally occurs in the Mediterranean region. It is a perennial, annual or biennial herb, having the erect stem of 50 cm and 4–8 axillary flowers (Rungsimakan and Rowan, 2014). S. viridis has been used in traditional medicine as gargle against sore gum (Grzegorczyk-Karolak and Kiss, 2018). In Turkey, an infusion of the shoot, flowers, and leaves of S. viridis have been used against a sore throat, throat inflammation, antitussive, ulcer, intestinal spasm, and gynaecological complications (Sharifi-Rad et al., 2018). According to our literature search, no study has reported the inhibitory activity of S. viridis roots on key enzymes related to skin hyperpigmentation problems, Alzheimer’s disease, and diabetes. Besides, this study evaluates the possible effect of different extraction techniques on the bioactivity of S. viridis roots. It is anticipated that data generated by this study will provide new horizons into the possible biological activity of S. viridis a poorly studied medicinal plant from the Salvia genus.

2.2.5. Ultrasonication-assisted extraction (UAE) The powdered roots (2 g) was extracted with 50 mL of ethanol (96%, w/w) for 60 min in a sonication bath at 30 °C. The obtained extracts were concentrated under vacuum at 40 °C by using a rotary vacuum evaporator. All samples were stored at 4 °C in the dark until use. 2.3. Profile of bioactive compounds Concerning our previous studies (Uysal et al., 2017), the total amount of phenolics (TPC) (by standard Folin-Ciocalteu method) and flavonoids (TFC) (by AlCl3 method) were determined. The final results were expressed as equivalents of standard compounds (gallic acid (mg GAE/g) for TPC and rutin (mg RE/g) for TFC), respectively). All HPLC–MS/MS experiments were carried out on Dionex Ultimate 3000RS UHPLC instrument from Thermo Scientific (USA). The chromatographic runs were conducted at the temperature 25 ± 1 °C using a Thermo Accucore C18 (100 mm x 2.1, mm i. d., 2.6 μm) column. The mobile phase was composed of water acidified with 0.1% formic acid “A” and acetonitrile with 0.1% formic acid “B”. Mass spectrometry was performed on Thermo Q-Exactive Orbitrap mass spectrometer (Thermo Scientific, USA) equipped with electrospray ionization probe interface in positive and negative-ion mode. For collection and analyzing data Thermo Scientific Xcalibur 3.1 and TraceFinder Clinical Research 3.3 softwares (Thermo Scientific, USA) were used. Compounds were identified by their exact molecular masses, isotopic patterns and fragmentations. In the cases of fragmentations an own database was used. All analytical details were given in supplementary material (Zengin et al., 2018c).

2. Materials and methods

2.4. Determination of antioxidant and enzyme inhibitory effects

2.1. Collection of plant material

The root extracts were tested as sources of enzyme inhibitors on some enzymes, including α-amylase, α-glucosidase, cholinesterases, and tyrosinase. The procedures of these assays are as reported in our earlier work (Uysal et al., 2017). The enzyme inhibitor effects were evaluated as equivalents of acarbose (for α-amylase and α-glucosidase), galantamine (for AChE and BChE), and kojic acid (for tyrosinase). Different experiments spectrophotometrically screened antioxidant capacity of the root extracts as phosphomolybdenum, quenching of radicals (DPPH and ABTS), reduction potentials (FRAP and CUPRAC), and ferrous ion chelating. The findings were expressed as standard compounds equivalents (mg TE/g and mg EDTAE/g). The procedures of assays were given reported in our earlier work (Uysal et al., 2017).

Salvia viridis roots were collected in Adana-Turkey, in June 2017. The taxonomic identification was performed by the botanist Dr. Murad Aydin Sanda (Muş Alpaslan University, Department of Molecular Biology, Muş, Turkey) and one voucher specimen was deposited at the herbarium of Selçuk University, Konya, Turkey. The roots were dried at the room temperature until a constant weight was recorded. The samples were then ground using a laboratory mill. 2.2. Extraction techniques 2.2.1. Microwave-assisted extraction (MAE) Five grams of root sample was extracted with 100 mL of 96% (w/w) ethanol (1:20 ratio). The extractions were performed in an open system at 600 W during 30 min.

2.5. Statistical analysis One-way ANOVA was done to determine any differences between the different extraction methods following by Tukey’s test. p < 0.05 were assigned to be statistically significant. The heat map and Pearson linear correlation were employed to recognize any relationship between phytochemical contents and the observed biological activities. Besides, principal component (PCA) and partial least squares discriminant analysis (PLS-DA) analysis were performed to classify the performed extraction methods. The statistical procedures were performed by R software v. 3.5.1.

2.2.2. Maceration (MAC) To produce macerated extracts, the root samples (5 g) were macerated with 100 mL of ethanol at room temperature for 24 h. 2.2.3. Supercritical fluid extraction (SFE) SFE extraction of the root samples was performed under the following conditions: 50 °C, 350 bar. The SFE process was carried out on laboratory scale high-pressure extraction plant (HPEP, NOVA, Swiss, Efferikon, Switzerland) described in detail by Pekić et al. (1995). The extraction process was carried out, and extraction yield was measured after 15, 30, 60, 90, 120, 180, 240, 300 and 360 min of extraction, in order to study the dynamics and kinetics of the process. The extraction was stopped after it went into the diffusion-limited process.

3. Results and discussion The last decades have witnessed a renewed interest in phytochemicals for the pharmaceutical and nutraceutical industries (Yahya et al., 2018). This shift has been fuelled by the alleged therapeutic potential of 267

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G. Zengin et al.

of plant material could also lead to degradation of polyphenols (ŠvarcGajić et al., 2013). This implies that MAE has the potential to be used for polyphenols extraction, but the extraction conditions, in the first place: the power of irradiation, and time should be optimized. As it was expected, the SFE, which is suitable for non-polar components, offers the lowers yield of polyphenolic compounds. Besides, previous studies have indicated that UAE optimised flavonoid extraction (Zhang et al., 2011; Zheng et al., 2016). HPLC–MS/MS screening was applied to S. viridis ethanolic root extracts obtained by different extraction methods. Compounds identification involved accurate molecular mass measurements (MS) and obtaining tandem mass spectra (MS/MS) followed by comparison with database, literature and retention time of the standards. A total of 76 compounds were identified in the extracts obtained by SE, UAE, and MAC, 73 compounds for MAE and 24 compounds for SFE (Tables 1–5; Figure S1-S5). The selective identification of compounds was provided by SFE extraction. The results of all extracts showed that the main components belong to salvianolic acids, polyphenols, flavonoids, and terpenoids. The presence of salvianolic acids was also reported in other Salvia species such as S. miltiorrhiza (Li et al., 2009), S. przewalski (salvianolic acid B and K) (Li et al., 2013; Ożarowski et al., 2017), S. chinensis (salvianolic acid B, D) (Li et al., 2013), S. officinalis (salvianolic acid L) (Lu and Foo, 2001), S. flava (salvianolic acid J) (Ai et al., 1994). Diterpenes namely, 1-oxomicrostegiol, viroxicin, and 3-oxomicrostegiol (m/z 313.18) were identified in all extracts. Viridoquinone (m/z 297.18) was found in all extracts except UAE extract and similar finding was reported in S. viridis roots (Rungsimakan and Rowan, 2014). Two unknown terpenoid isomers with m/z 485.36 were found in SFE extracts. Monoterpene glycoside lipedoside A (m/z 607.20), characterized by high antioxidant properties (Chen et al., 2002) and phenylpropanoid glycoside leucosceptoside A (m/z 637.21), identified in SE, UAE, MAE and MAC extracts, were also identified in S. viridis shoots (GrzegorczykKarolak and Kiss, 2018). Kynurenic acid (m/z 190.0), previously isolated from S. officinalis (Turski et al., 2015), was identified in MAE, SE, and UAE extracts of S. viridis ethanolic root extracts. Vanillin (m/z 153.44), present in all studied extracts, was also identified in S. digitaloides roots (Wu and Chan, 2014). Compounds such as caffeoylglucose (m/z 341.08), chlorogenic acid (m/z 355.10), apigenin-O-glucuronide (m/z 445.07), martynoside (m/z 651.22) were also isolated from S. viridis shoots (Grzegorczyk-Karolak and Kiss, 2018). Coniferyl aldehyde (m/z 179.07) was previously isolated from Salvia plebeia(Weng and Wang, 2000). The role of reactive oxygen species and free radicals in the pathogenesis of several human ailments is well established. A substantial body of evidence has demonstrated the ability of phytochemicals to influence important cellular and molecular mechanisms related to health problems, including oxidative-stress related ones (Zengin et al., 2019). In the present study, the antioxidant activity of S. viridis extracts was assessed using three different classes of antioxidant assays (radical scavenging, reducing power, and metal chelating). The results are present in Table 6. S. viridis ethanolic root extract obtained by UAE showed highest radical scavenging (240.00 and 302.85 mg TE/g for DPPH and ABTS assays, respectively) and reducing (970.74, 704.27 mg TE/g, and 2.84 mmol TE/g for CUPRAC, FRAP, and phosphomolybdenum assays, respectively) activities. UAE carry some advantages; namely use of low temperature preserving the integrity of phytochemicals, short extraction time, increased extraction yield, making it one of the most exciting techniques for the extraction of bioactive components from plant materials (Liu et al., 2018). The influence of the extraction technique on the antioxidant activity was already proven and detailed explained in the literature (Cvetanović et al., 2015). The antioxidant results were also correlated total bioactive components namely total phenolics and flavonoids content (Fig. 2A). In contradiction with the aforementioned antioxidant assays, the extract obtained by MAE (22.71 mg EDTAE/g) showed the most potent metal chelating activity. This finding was supported by correlation data

Fig. 1. Total bioactive compounds of the Salvia viridis extracts (Values expressed are means ± S.D. of three parallel measurements. MAE: Microwave assisted extraction; MAC: Maceration, SFE: Supercritical fluid extraction, SE: Soxhlet extraction; UAE: Ultrasonic assisted extraction. GAE: Gallic acid equivalent; RE: Rutin equivalent. Different letters indicate differences in the tested extracts (p < 0.05)).

phytochemicals for the management of a plethora of human ailments. Along with conventional extraction techniques such as maceration, percolation, and Soxhlet extraction, newer enhanced techniques have been developed to boost extraction of bioactive constituents from plants. Non-conventional methods having the particularity of producing higher extraction yield, being less destructive to extracted compounds, and being environmentally friendly, have gained increasing importance in drug discovery (Saha et al., 2018). In the present study, we have attempted to elucidate the possible effect of different extraction techniques on the biological activity of S. viridis roots. Phytochemicals are chemical compounds, ubiquitously occurring in plants, known to provide multiple health benefits such as anti-cancer, antibacterial, antiviral, anti-inflammatory, anti-diabetic, and antioxidant effects (Guldiken et al., 2018). Therefore, while determining the possible bioactivity of plants, assessing the phytochemical composition is a crucial step. The amounts of phenolics and flavonoids were determined using well-known, rapid spectrophotometric methods, namely Folin-Ciocalteu and Aluminum chloride tests, respectively. From Fig. 1, the phenolic and flavonoid contents of S. viridis ethanolic root extracts followed this order: UAE > MAC > SE > MAE > SFE. The total phenolics content of the hydro-ethanolic extract of S. viridis aerial part obtained by UAE (Grzegorczyk-Karolak and Kiss, 2018) was comparable to that of S. viridis ethanolic root extract obtained by the same extraction method (Fig. 1). S. viridis roots (102.03 mg GAE/g extract) possessed higher phenolics content compared to S. fruticosa roots (80 mg GAE/g extract) (Boukhary et al., 2016). As observed from Fig. 1, the extraction technique influenced extractability of flavonoids, and UAE obtained the highest flavonoid content. The differences among the extracts were influenced by the different phenomena which occur in different techniques. As it can be seen cavitation phenomenon has the most substantial impact on both phenols and flavonoids extraction. Conventional techniques (MAC and SE) were proven as a better choice than modern MAE technique. The reason could be the time of extractions. However, among the SE, MAC and MAE the differences in total phenols and flavonoids were not notable, but there was a remarkable difference in extraction times. Namely, the MAC and SE were performed 24 and 6 h, respectively, while MAE was performed only 30 min. Also, the reason for lower content could be possible degradation during the microwave irradiation. Data from the literature suggests that the content of polyphenolic compounds from different matrices increase up to certain levels and then decreases as a consequence of microwave irradiation (Ameer et al., 2017). Moreover, too high microwave irradiation 268

269

15,68 16,83 17,39 18,04 19,09 19,89 20,11 20,19 20,54 30,50 34,35 34,46 34,47 34,91 35,85 36,52 36,65 36,96 38,18 38,62 40,36 41,81 49,39 49,84

1* 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Vanillin Ethyl syringate Syringaldehyde (3,5-Dimethoxy-4-hydroxybenzaldehyde) Antiarol (3,4,5-Trimethoxyphenol) Indole-4-carbaldehyde Coumarin Coniferyl aldehyde (4-Hydroxy-3-methoxycinnamaldehyde) N-(2-Phenylethyl)acetamide Sinapyl aldehyde (3,5-Dimethoxy-4-hydroxycinnamaldehyde) Dimethoxy-trihydroxy(iso)flavone isomer 1 Dihydroxy-dimethoxy(iso)flavone Dimethoxy-trihydroxy(iso)flavone isomer 2 Genkwanin Dihydroxy-trimethoxy(iso)flavone Hydroxy-trimethoxy(iso)flavone Hydroxy-tetramethoxy(iso)flavone 1-Oxomicrostegiol Viroxocin Apigenin-4',7-dimethyl ether (4',7-Dimethoxy-5-hydroxyflavone) 3-Oxomicrostegiol Hexadecanedioic acid Viridoquinone Unknown terpenoid isomer 1 Unknown terpenoid isomer 2

Compound

* Confirmed by standard.

Rt, min

No.

Table 1 The chemical composition of SFE from Salvia viridis roots.

C8H8O3 C11H14O5 C9H10O4 C9H12O4 C9H7NO C9H6O2 C10H10O3 C10H13NO C11H12O4 C17H14O7 C17H14O6 C17H14O7 C16H12O5 C18H16O7 C18H16O6 C19H18O7 C20H24O3 C20H24O3 C17H14O5 C20H24O3 C16H30O4 C20H24O2 C31H48O4 C31H48O4

Formula

297,18546 485,36309 485,36309

329,10252 359,11308 313,18037 313,18037 299,09195 313,18037

153,05517 227,09195 183,06574 185,08139 146,06059 147,04461 179,07082 164,10754 209,08139

[M + H]+

285,20659

329,06613 313,07122 329,06613 283,06120 343,08178

[M - H]− 125,0601 181,0500 155,0705 170,0575 118,0656 119,0493 161,0600 122,0968 191,0708 314,1530 298,0487 314,1533 268,0380 328,0590 314,0789 344,0895 295,1697 295,1696 284,0683 295,1696 267,1970 279,1747 453,3677 453,3720

Fragment 1 111,0445 155,0706 140,0471 154,0626 117,0575 103,0548 147,0443 105,0705 177,0551 271,0245 283,0254 271,0254 239,0357 313,0359 313,0711 343,0816 280,1465 227,1069 256,0735 280,1457 223,2067 269,1912 425,3792 425,3787

Fragment 2

239,1433 407,3676 407,3680

271,2418 271,2428

(Rungsimakan and Rowan, 2014)

(Rungsimakan and Rowan, 2014)

243,1019

Literature

(Rungsimakan and Rowan, 2014) (Rungsimakan and Rowan, 2014)

259,2427 259,2424

105,0705 79,0550 55,0187

65,0393 119,0496 90,9484 145,0288

285,0764 313,0703

125,0600

95,0498

Fragment 5

65,0394 123,0444 95,0498 139,0392

Fragment 4

117,0337 298,0122 300,0634 329,0660 243,1019 199,0757

255,0299

93,0341 140,0471 123,0444 153,0548 91,0547 91,0548 133,0651 103,0549 149,0601

Fragment 3

G. Zengin et al.

Industrial Crops & Products 131 (2019) 266–280

Rt

1,24 2,34 12,69 13,90 14,24 14,27 14,32 15,19 15,54 16,58 16,75 16,76 16,90 17,26 17,30 17,75 17,88 17,96 17,97 18,09 18,99 19,05 19,12 19,32 19,45 20,02 20,15 20,22 20,37 20,47 20,83 21,72 21,98 22,11 22,20 22,35 22,40 22,90 23,13 23,14 23,23 23,31 23,59 23,64 23,89 23,95 24,09 24,34 24,40 24,41 24,65 25,85 25,88 26,28

No.

1 2* 3 4 5 6* 7 8 9* 10 11 12 13 14 15 16* 17 18 19 20 21 22 23 24 25 26 27 28 29 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

Quinic acid Gallic acid (3,4,5-Trihydroxybenzoic acid) Caffeoylglucose isomer 1 Kynurenic acid Caffeoylglucose isomer 2 Chlorogenic acid (3-O-Caffeoylquinic acid) Caffeic acid Coumaroylhexose isomer 1 Vanillin Coumaroylhexose isomer 2 Feruloylhexose isomer 1 Ethyl syringate Coumaroylquinic acid Ethyl gallate Syringaldehyde (3,5-Dimethoxy-4-hydroxybenzaldehyde) 4-Coumaric acid Feruloylhexose isomer 2 Antiarol (3,4,5-Trimethoxyphenol) Caffeoylshikimic acid Sinapoylhexose Indole-4-carbaldehyde Luteolin-C-hexoside-C-pentoside isomer 1 Dimethoxy-hydroxybenzoic acid Ferulic acid Luteolin-C-hexoside-C-pentoside isomer 2 Coniferyl aldehyde (4-Hydroxy-3-methoxycinnamaldehyde) N-(2-Phenylethyl)acetamide Luteolin-C-hexoside-O-pentoside Coumaroylshikimate Sinapyl aldehyde (3,5-Dimethoxy-4-hydroxycinnamaldehyde) Myricetin-O-hexoside Apigenin-C-hexoside-O-pentoside Verbascoside Luteolin-O-(pentosyl)hexoside Luteolin-O-glucuronide Luteolin-7-O-glucoside (Cynaroside) Luteolin-O-(deoxyhexosyl)hexoside Isoquercitrin (Hirsutrin, Quercetin-3-O-glucoside) 3-[(1-Carboxyvinyl)oxy]benzoic acid Rosmarinic acid-O-hexoside Salvianolic acid isomer Lipedoside A isomer 1 Lipedoside A isomer 2 Leucosceptoside A Apigenin-O-(deoxyhexosyl)hexoside Cosmosiin (Apigetrin, Apigenin-7-O-glucoside) Apigenin-O-glucuronide Methoxy-trihydroxyflavone-O-glucuronide Methyl caffeate Rosmarinic acid (Labiatenic acid) N-trans-Feruloyltyramine Martynoside Ethyl caffeate 3-O-Methylrosmarinic acid

Name

Table 2 The chemical composition of MAE from Salvia viridis roots.

C7H12O6 C7H6O5 C15H18O9 C10H7NO3 C15H18O9 C16H18O9 C9H8O4 C15H18O8 C8H8O3 C15H18O8 C16H20O9 C11H14O5 C16H18O8 C9H10O5 C9H10O4 C9H8O3 C16H20O9 C9H12O4 C16H16O8 C17H22O10 C9H7NO C26H28O15 C9H10O5 C10H10O4 C26H28O15 C10H10O3 C10H13NO C26H28O15 C16H16O7 C11H12O4 C21H20O13 C26H28O14 C29H36O15 C26H28O15 C21H18O12 C21H20O11 C27H30O15 C21H20O12 C10H8O5 C24H26O13 C36H30O16 C29H36O14 C29H36O14 C30H38O15 C27H30O14 C21H20O10 C21H18O11 C22H20O12 C10H10O4 C18H16O8 C18H19NO4 C31H40O15 C11H12O4 C19H18O8

Formula

270 209,08139

314,13924

477,10331 195,06574

577,15574 433,11347

595,16630

581,15065

565,15574

209,08139

179,07082 164,10754 581,15065

146,06059

185,08139

183,06574

227,09195

153,05517

355,10291

190,05042

[M + H]+

373,09235

651,22890

359,07670

445,07709

463,08765 207,02935 521,12952 717,14557 607,20269 607,20269 637,21325

461,07201 447,09274

623,19760

479,08257

319,08178

579,13500 197,04500 193,05009 579,13500

335,07670 385,11348

163,03952 355,10291

337,09235 197,04500

325,09235 355,10291

179,03444 325,09235

341,08726

191,05557 169,01370 341,08726

[M - H]− 173,0449 125,0232 281,0675 162,0552 281,0672 163,0391 135,0440 265,0721 125,0600 265,0722 295,0831 181,0499 191,0557 169,0133 155,0704 119,0490 295,0828 170,0576 179,0343 267,0725 118,0655 489,1022 182,0215 178,0264 489,1036 161,0598 122,0967 449,1086 163,0392 191,0706 317,0314 433,1133 461,1680 449,1086 285,0411 327,0504 449,1083 301,0360 137,0233 359,0774 519,0942 461,1669 461,1667 461,1677 433,1135 271,0603 269,0460 301,0710 163,0391 197,0451 177,0549 475,1833 163,0392 197,0451

Fragment 1 171,0288 97,0283 251,0563 144,0449 251,0561 145,0286 107,0487 235,0611 111,0444 235,0612 235,0610 155,0705 173,0446 125,0231 140,0471 93,0331 235,0612 154,0626 161,0234 249,0619 117,0578 459,0893 166,9977 149,0598 459,0953 147,0442 105,0704 431,0975 155,0338 177,0548 316,0232 415,1028 315,1084 287,0553 217,0502 285,0411 287,0552 300,0280 135,0441 341,0885 339,0513 443,0985 443,0992 315,1095 271,0603 153,0181 175,0241 286,0476 145,0286 179,0343 145,0286 193,0501 145,0286 179,0342

Fragment 2

119,0499 113,0231 258,0520 135,0443 161,0234 121,0651 175,0391 135,0444 175,0391

271,0254 93,0331 323,0777 321,0415 297,0403 297,0400 193,0503

199,0400 284,0333

193,0502 153,0549 135,0441 223,0610 91,0548 399,0728 153,0547 137,0232 399,0730 133,0651 103,0549 413,0873 119,0490 149,0599 287,0200 337,0708 161,0234

123,0444

205,0503 93,0340 205,0505 193,0503 140,0470 163,0389

127,0385 81,0331 221,0453 116,0498 221,0454 135,0443

Fragment 3

160,0156 117,0339 160,0156

117,0339 135,0440

255,0302 87,0073 197,0450 295,0615 161,0234 161,0234 175,0392

151,0027 256,0376

145,0286 271,0246 313,0707 133,0284

369,0631 138,0311 134,0362 369,0621 119,0496 90,9483 329,0657

208,0373

175,0391 139,0392

95,0497

163,0391 65,0393 163,0392 175,0391 123,0444 119,0490

111,0440 69,0331 179,0343 89,0393 179,0343 117,0338

Fragment 4

107,0860 135,0440

107,0496 123,0439

161,0234 277,0507 135,0441 135,0440 160,0156

133,0282 151,0027

283,0602

55,0186

339,0520 105,0705 79,0553 299,0553

339,0513 121,0282

164,0469

160,0157 125,0600

145,0285 160,0155 95,0497 93,0332

145,0285

161,0235 107,0496

161,0235

85,0280

Fragment 5

(continued on next page)

(Grzegorczyk-Karolak and Kiss, 2018)

(Grzegorczyk-Karolak and Kiss, 2018)

(Grzegorczyk-Karolak and Kiss, 2018)

(Grzegorczyk-Karolak and Kiss, 2018) (Grzegorczyk-Karolak and Kiss, 2018) (Grzegorczyk-Karolak and Kiss, 2018)

(Grzegorczyk-Karolak and Kiss, 2018)

(Grzegorczyk-Karolak and Kiss, 2018) (Grzegorczyk-Karolak and Kiss, 2018)

(Grzegorczyk-Karolak and Kiss, 2018)

Literature

G. Zengin et al.

Industrial Crops & Products 131 (2019) 266–280

26,97 27,17 27,85 27,88 28,54 29,28 29,54 29,69 29,90 30,23 30,58 32,55 34,54 35,83 36,63 36,95 38,62 40,36 41,81

55 56* 57* 58 59 60 61 62* 63 64 65 66 67 68 69 70 71 72 73

Pentahydroxyflavone Naringenin Luteolin (3',4',5,7-Tetrahydroxyflavone) Di-O-methylellagic acid Dihydroxycinnamoyl-dihydroxyphenyl-ethenol isomer 1 Luteolin-O-(coumaroyl)hexoside Dihydroxycinnamoyl-dihydroxyphenyl-ethenol isomer 2 Apigenin (4',5,7-Trihydroxyflavone) Chrysoeriol (Scoparol, 3'-Methoxy-4',5,7-trihydroxyflavone) Tri-O-methylellagic acid Dimethoxy-trihydroxy(iso)flavone Methoxy-trihydroxyflavone Genkwanin Hydroxy-trimethoxy(iso)flavone 1-Oxomicrostegiol Viroxocin 3-Oxomicrostegiol Hexadecanedioic acid Viridoquinone

Name

* Confirmed by standard.

Rt

No.

Table 2 (continued)

C15H10O7 C15H12O5 C15H10O6 C16H10O8 C17H14O6 C30H26O13 C17H14O6 C15H10O5 C16H12O6 C17H12O8 C17H14O7 C16H12O6 C16H12O5 C18H16O6 C20H24O3 C20H24O3 C20H24O3 C16H30O4 C20H24O2

Formula

297,18546

329,10252 313,18037 313,18037 313,18037

315,08687

315,08687

[M + H]+

285,20659

269,04500 299,05556 343,04540 329,06613 299,05556 283,06120

593,12952

301,03483 271,06065 285,03991 329,02975

[M - H]− 273,0393 177,0183 217,0503 314,0075 205,0498 285,0410 205,0500 227,0353 284,0331 328,0226 314,1529 284,0331 268,0382 314,0793 295,1692 295,1694 295,1698 267,1970 279,1748

Fragment 1 178,9984 165,0185 199,0400 298,9842 163,0391 284,0329 163,0391 225,0559 256,0378 312,9996 271,0252 256,0372 239,0355 313,0710 280,1461 227,1068 280,1465 223,2067 269,1916

Fragment 2

239,1432

133,0284 117,0328 300,0632 243,1018 199,0756 243,1019

151,0027 151,0026 151,0027 270,9896 153,0545 145,0282 153,0549 201,0554 227,0344 297,9760

Fragment 3

285,0759

145,0285 151,0026 151,0024

145,0286

107,0123 119,0489 133,0283

Fragment 4

135,0443 117,0332 107,0119

135,0444

107,0126 107,0127

Fragment 5

(Rungsimakan and Rowan, 2014)

(Rungsimakan and Rowan, 2014) (Rungsimakan and Rowan, 2014) (Rungsimakan and Rowan, 2014)

Literature

G. Zengin et al.

Industrial Crops & Products 131 (2019) 266–280

271

Rt

1,24 2,38 12,82 13,92 14,31 14,33 14,43 15,29 15,60 16,66 16,78 16,82 16,96 17,34 17,37 17,85 17,94 18,00 18,05 18,12 19,05 19,10 19,12 19,40 19,50 20,06 20,19 20,28 20,45 20,51 20,91 21,77 22,03 22,11 22,35 22,39 22,44 22,97 23,15 23,18 23,27 23,36 23,64 23,69 23,95 23,99 24,15 24,36 24,42 24,43 24,70 25,76 25,90 25,93

No.

1 2* 3 4 5 6* 7 8 9* 10 11 12 13 14 15 16* 17 18 19 20 21 22 23 24 25 26 27 28 29 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

Quinic acid Gallic acid (3,4,5-Trihydroxybenzoic acid) Caffeoylglucose isomer 1 Kynurenic acid Caffeoylglucose isomer 2 Chlorogenic acid (3-O-Caffeoylquinic acid) Caffeic acid Coumaroylhexose isomer 1 Vanillin Coumaroylhexose isomer 2 Ethyl syringate Feruloylhexose isomer 1 Coumaroylquinic acid Ethyl gallate Syringaldehyde (3,5-Dimethoxy-4-hydroxybenzaldehyde) 4-Coumaric acid Feruloylhexose isomer 2 Antiarol (3,4,5-Trimethoxyphenol) Caffeoylshikimic acid Sinapoylhexose Indole-4-carbaldehyde Luteolin-C-hexoside-C-pentoside isomer 1 Dimethoxy-hydroxybenzoic acid Ferulic acid Luteolin-C-hexoside-C-pentoside isomer 2 Coniferyl aldehyde (4-Hydroxy-3-methoxycinnamaldehyde) N-(2-Phenylethyl)acetamide Luteolin-C-hexoside-O-pentoside Coumaroylshikimate Sinapyl aldehyde (3,5-Dimethoxy-4-hydroxycinnamaldehyde) Myricetin-O-hexoside Apigenin-C-hexoside-O-pentoside Verbascoside Luteolin-O-(pentosyl)hexoside Luteolin-7-O-glucoside (Cynaroside) Luteolin-O-glucuronide Luteolin-O-(deoxyhexosyl)hexoside Isoquercitrin (Hirsutrin, Quercetin-3-O-glucoside) 3-[(1-Carboxyvinyl)oxy]benzoic acid Rosmarinic acid-O-hexoside Salvianolic acid isomer Lipedoside A isomer 1 Lipedoside A isomer 2 Leucosceptoside A Apigenin-O-(deoxyhexosyl)hexoside Cosmosiin (Apigetrin, Apigenin-7-O-glucoside) Apigenin-O-glucuronide Methoxy-trihydroxyflavone-O-glucuronide Methyl caffeate Rosmarinic acid (Labiatenic acid) N-trans-Feruloyltyramine 3-O-Methylellagic acid Martynoside Ethyl caffeate

Name

Table 3 The chemical composition of SE from Salvia viridis roots.

C7H12O6 C7H6O5 C15H18O9 C10H7NO3 C15H18O9 C16H18O9 C9H8O4 C15H18O8 C8H8O3 C15H18O8 C11H14O5 C16H20O9 C16H18O8 C9H10O5 C9H10O4 C9H8O3 C16H20O9 C9H12O4 C16H16O8 C17H22O10 C9H7NO C26H28O15 C9H10O5 C10H10O4 C26H28O15 C10H10O3 C10H13NO C26H28O15 C16H16O7 C11H12O4 C21H20O13 C26H28O14 C29H36O15 C26H28O15 C21H20O11 C21H18O12 C27H30O15 C21H20O12 C10H8O5 C24H26O13 C36H30O16 C29H36O14 C29H36O14 C30H38O15 C27H30O14 C21H20O10 C21H18O11 C22H20O12 C10H10O4 C18H16O8 C18H19NO4 C15H8O8 C31H40O15 C11H12O4

Formula

272 209,08139

314,13924

477,10331 195,06574

579,17139 433,11347

595,16630

581,15065

565,15574

209,08139

179,07082 164,10754 581,15065

146,06059

185,08139

183,06574

227,09195

153,05517

355,10291

190,05042

[M + H]+

315,01410 651,22890

359,07670

445,07709

463,08765 207,02935 521,12952 717,14557 607,20269 607,20269 637,21325

447,09274 461,07201

623,19760

479,08257

319,08178

579,13500 197,04500 193,05009 579,13500

335,07670 385,11348

163,03952 355,10291

355,10291 337,09235 197,04500

325,09235

179,03444 325,09235

341,08726

191,05557 169,01370 341,08726

[M - H]− 173,0444 125,0232 281,0671 162,0552 281,0672 163,0392 135,0441 265,0721 125,0601 265,0721 181,0499 295,0829 191,0557 169,0134 155,0705 119,0489 295,0832 170,0575 179,0342 267,0725 118,0655 489,1051 182,0215 178,0264 489,1029 161,0599 122,0969 449,1087 163,0391 191,0708 317,0306 433,1137 461,1679 449,1087 327,0504 285,0411 449,1084 301,0360 137,0234 359,0776 519,0959 461,1671 461,1679 461,1675 433,1135 271,0604 269,0460 301,0710 163,0392 197,0452 177,0549 299,9917 475,1844 163,0392

Fragment 1

193,0502 145,0287

171,0286 97,0282 251,0564 144,0444 251,0563 145,0287 107,0488 235,0611 111,0445 235,0611 155,0705 235,0609 173,0447 125,0231 140,0471 93,0332 235,0612 154,0626 161,0234 249,0619 117,0578 459,0941 166,9977 149,0597 459,0933 147,0442 105,0705 431,0979 155,0340 177,0550 316,0225 415,1030 315,1077 287,0553 285,0411 217,0496 287,0552 300,0280 135,0441 341,0942 339,0506 443,0986 443,1014 315,1102 271,0604 153,0181 175,0239 286,0476 145,0287 179,0344 145,0287

Fragment 2

175,0392 135,0445

119,0496 113,0231 258,0520 135,0444 161,0235 121,0652

271,0253 93,0332 323,0779 321,0411 297,0404 297,0414 193,0502

284,0333 199,0397

193,0502 153,0549 135,0441 223,0608 91,0548 399,0740 153,0547 137,0233 399,0727 133,0652 103,0549 413,0873 119,0490 149,0600 287,0193 337,0712 161,0234

123,0444

205,0503 93,0341 205,0504 140,0471 193,0503 163,0394

127,0388 81,0331 221,0456 116,0500 221,0453 135,0445

Fragment 3

160,0156 117,0339

117,0340 135,0441

255,0303 87,0073 197,0451 295,0616 161,0234 161,0235 175,0392

256,0376 151,0026

145,0287 271,0253 313,0711 133,0284

369,0625 138,0310 134,0362 369,0619 119,0495 90,9483 329,0660

208,0371

175,0392 139,0392

95,0498

163,0391 65,0394 163,0391 123,0445 175,0391 119,0492

111,0439 69,0331 179,0343 89,0390 179,0343 117,0339

Fragment 4

107,0858

107,0496 123,0439

161,0234 277,0492 135,0443 135,0442 160,0156

151,0027 133,0281

283,0605

55,0187

299,0554

339,0503 105,0704

339,0499 121,0282

164,0469

160,0155 125,0601

145,0285 95,0496 160,0155 93,0332

145,0285

161,0234 107,0496

161,0234

85,0280

Fragment 5

(continued on next page)

(Grzegorczyk-Karolak and Kiss, 2018)

(Grzegorczyk-Karolak and Kiss, 2018)

(Grzegorczyk-Karolak and Kiss, 2018)

(Grzegorczyk-Karolak and Kiss, 2018) (Grzegorczyk-Karolak and Kiss, 2018) (Grzegorczyk-Karolak and Kiss, 2018)

(Grzegorczyk-Karolak and Kiss, 2018)

(Grzegorczyk-Karolak and Kiss, 2018) (Grzegorczyk-Karolak and Kiss, 2018)

(Grzegorczyk-Karolak and Kiss, 2018)

Literature

G. Zengin et al.

Industrial Crops & Products 131 (2019) 266–280

26,31 27,02 27,23 27,91 27,95 28,59 29,33 29,59 29,73 29,97 30,27 30,63 32,58 34,46 34,59 35,84 36,52 36,63 36,95 38,61 40,38 41,81

55 56 57* 58* 59 60 61 62 63* 64 65 66 67 68 69 70 71 72 73 74 75 76

3-O-Methylrosmarinic acid Pentahydroxyflavone Naringenin Luteolin (3',4',5,7-Tetrahydroxyflavone) Di-O-methylellagic acid Dihydroxycinnamoyl-dihydroxyphenyl-ethenol isomer 1 Luteolin-O-(coumaroyl)hexoside Dihydroxycinnamoyl-dihydroxyphenyl-ethenol isomer 2 Apigenin (4',5,7-Trihydroxyflavone) Chrysoeriol (Scoparol, 3'-Methoxy-4',5,7-trihydroxyflavone) Tri-O-methylellagic acid Dimethoxy-trihydroxy(iso)flavone Methoxy-trihydroxyflavone Dihydroxy-dimethoxy(iso)flavone Genkwanin Hydroxy-trimethoxy(iso)flavone Hydroxy-tetramethoxy(iso)flavone 1-Oxomicrostegiol Viroxocin 3-Oxomicrostegiol Hexadecanedioic acid Viridoquinone

Name

* Confirmed by standard.

Rt

No.

Table 3 (continued)

C19H18O8 C15H10O7 C15H12O5 C15H10O6 C16H10O8 C17H14O6 C30H26O13 C17H14O6 C15H10O5 C16H12O6 C17H12O8 C17H14O7 C16H12O6 C17H14O6 C16H12O5 C18H16O6 C19H18O7 C20H24O3 C20H24O3 C20H24O3 C16H30O4 C20H24O2

Formula

297,18546

329,10252 359,11308 313,18037 313,18037 313,18037

315,08687

315,08687

[M + H]+

285,20659

269,04500 299,05556 343,04540 329,06613 299,05556 313,07122 283,06120

593,12952

373,09235 301,03483 271,06065 285,03991 329,02975

[M - H]− 197,0451 273,0424 177,0186 217,0501 314,0073 205,0498 285,0411 205,0498 227,0342 284,0332 328,0227 314,1530 284,0333 298,0488 268,0382 314,0787 344,0893 295,1694 295,1693 295,1696 267,1975 279,1749

Fragment 1 179,0342 178,9978 165,0179 199,0398 298,9839 163,0392 284,0337 163,0392 225,0555 256,0380 312,9999 271,0252 256,0376 283,0253 239,0347 313,0710 343,0814 280,1461 227,1069 280,1462 223,2063 269,1901

Fragment 2

239,1433

133,0277 255,0301 117,0334 300,0631 329,0661 243,1017 199,0757 243,1018

175,0392 151,0028 151,0027 151,0027 270,9883 153,0549 145,0286 153,0549 201,0557 227,0345 297,9759

Fragment 3

285,0760 313,0736

145,0287 151,0027 151,0027

145,0286

160,0156 107,0124 119,0490 133,0283

Fragment 4

135,0444 117,0332 107,0125

135,0444

107,0126 107,0125

135,0441

Fragment 5

(Rungsimakan and Rowan, 2014)

(Rungsimakan and Rowan, 2014) (Rungsimakan and Rowan, 2014) (Rungsimakan and Rowan, 2014)

Literature

G. Zengin et al.

Industrial Crops & Products 131 (2019) 266–280

273

Rt

1,26 2,42 12,89 13,84 14,41 14,49 14,54 15,35 15,72 16,73 16,86 16,87 16,99 17,41 17,45 17,93 17,98 18,08 18,11 18,13 19,09 19,15 19,16 19,44 19,56 20,14 20,25 20,35 20,48 20,58 20,97 21,82 22,06 22,17 22,41 22,45 22,51 22,72 23,02 23,11 23,19 23,29 23,39 23,67 23,72 24,01 24,07 24,15 24,41 24,45 24,46 24,75 25,93 25,98

No.

1 2* 3 4 5* 6 7 8 9* 10 11 12 13 14 15 16* 17 18 19 20 21 22 23 24 25 26 27 28 29 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

Quinic acid Gallic acid (3,4,5-Trihydroxybenzoic acid) Caffeoylglucose isomer 1 Kynurenic acid Chlorogenic acid (3-O-Caffeoylquinic acid) Caffeoylglucose isomer 2 Caffeic acid Coumaroylhexose isomer 1 Vanillin Coumaroylhexose isomer 2 Ethyl syringate Feruloylhexose isomer 1 Coumaroylquinic acid Ethyl gallate Syringaldehyde (3,5-Dimethoxy-4-hydroxybenzaldehyde) 4-Coumaric acid Feruloylhexose isomer 2 Antiarol (3,4,5-Trimethoxyphenol) Caffeoylshikimic acid Sinapoylhexose Dimethoxy-hydroxybenzoic acid Indole-4-carbaldehyde Luteolin-C-hexoside-C-pentoside isomer 1 Ferulic acid Luteolin-C-hexoside-C-pentoside isomer 2 Coniferyl aldehyde (4-Hydroxy-3-methoxycinnamaldehyde) N-(2-Phenylethyl)acetamide Luteolin-C-hexoside-O-pentoside Coumaroylshikimate Sinapyl aldehyde (3,5-Dimethoxy-4-hydroxycinnamaldehyde) Myricetin-O-hexoside Apigenin-C-hexoside-O-pentoside Verbascoside Luteolin-O-(pentosyl)hexoside Luteolin-O-glucuronide Luteolin-7-O-glucoside (Cynaroside) Luteolin-O-(deoxyhexosyl)hexoside Apigenin-C-hexoside-O-deoxyhexoside Isoquercitrin (Hirsutrin, Quercetin-3-O-glucoside) 3-[(1-Carboxyvinyl)oxy]benzoic acid Rosmarinic acid-O-hexoside Salvianolic acid isomer Lipedoside A isomer 1 Lipedoside A isomer 2 Leucosceptoside A Apigenin-O-(deoxyhexosyl)hexoside Cosmosiin (Apigetrin, Apigenin-7-O-glucoside) Apigenin-O-glucuronide Methoxy-trihydroxyflavone-O-glucuronide Methyl caffeate Rosmarinic acid (Labiatenic acid) N-trans-Feruloyltyramine Martynoside Ethyl caffeate

Name

Table 4 The chemical composition of UAE from Salvia viridis roots.

C7H12O6 C7H6O5 C15H18O9 C10H7NO3 C16H18O9 C15H18O9 C9H8O4 C15H18O8 C8H8O3 C15H18O8 C11H14O5 C16H20O9 C16H18O8 C9H10O5 C9H10O4 C9H8O3 C16H20O9 C9H12O4 C16H16O8 C17H22O10 C9H10O5 C9H7NO C26H28O15 C10H10O4 C26H28O15 C10H10O3 C10H13NO C26H28O15 C16H16O7 C11H12O4 C21H20O13 C26H28O14 C29H36O15 C26H28O15 C21H18O12 C21H20O11 C27H30O15 C27H30O14 C21H20O12 C10H8O5 C24H26O13 C36H30O16 C29H36O14 C29H36O14 C30H38O15 C27H30O14 C21H20O10 C21H18O11 C22H20O12 C10H10O4 C18H16O8 C18H19NO4 C31H40O15 C11H12O4

Formula

274 209,08139

314,13924

477,10331 195,06574

579,17139 433,11347

595,16630 579,17139

581,15065

565,15574

209,08139

179,07082 164,10754 581,15065

146,06059

185,08139

183,06574

227,09195

153,05517

190,05042 355,10291

[M + H]+

651,22890

359,07670

445,07709

463,08765 207,02935 521,12952 717,14557 607,20269 607,20269 637,21325

461,07201 447,09274

623,19760

479,08257

319,08178

579,13500 193,05009 579,13500

335,07670 385,11348 197,04500

163,03952 355,10291

355,10291 337,09235 197,04500

325,09235

341,08726 179,03444 325,09235

191,05557 169,01370 341,08726

[M - H]− 173,0446 125,0232 281,0674 162,0552 163,0392 281,0674 135,0441 265,0722 125,0601 265,0722 181,0500 295,0828 191,0556 169,0134 155,0706 119,0490 295,0828 170,0576 179,0343 267,0725 182,0215 118,0656 489,1054 178,0264 489,1046 161,0600 122,0969 449,1086 163,0392 191,0707 317,0299 433,1137 461,1672 449,1086 285,0410 327,0512 449,1085 433,1136 301,0359 137,0234 359,0776 519,0934 461,1674 461,1673 461,1674 433,1136 271,0603 269,0460 301,0709 163,0392 197,0452 177,0550 475,1821 163,0392

Fragment 1 171,0286 97,0281 251,0564 144,0438 145,0287 251,0564 107,0491 235,0611 111,0445 235,0610 155,0705 235,0609 173,0448 125,0232 140,0472 93,0332 235,0609 154,0627 161,0235 249,0617 166,9979 117,0577 459,0939 149,0596 459,0942 147,0443 105,0704 431,0985 155,0337 177,0549 316,0234 415,1032 315,1111 287,0554 217,0499 285,0411 287,0553 415,1037 300,0281 135,0441 341,0901 339,0526 443,0991 443,1001 315,1088 271,0603 153,0183 175,0240 286,0476 145,0287 179,0342 145,0287 193,0501 145,0287

Fragment 2

119,0495 113,0231 258,0517 135,0444 161,0234 121,0652 175,0392 135,0444

337,0709 271,0253 93,0332 323,0778 321,0408 297,0414 297,0411 193,0504

199,0399 284,0332

193,0503 153,0549 135,0441 223,0611 153,0547 91,0548 399,0729 137,0231 399,0729 133,0652 103,0547 413,0877 119,0489 149,0600 287,0194 337,0709 161,0235

160,0156 117,0339

117,0338 135,0441

313,0709 255,0301 87,0073 197,0452 295,0616 161,0235 161,0235 175,0392

151,0028 256,0381

145,0286 271,0245 313,0711 133,0285

369,0623 134,0363 369,0623 119,0496 90,9483 329,0660

208,0375 138,0313

175,0392 139,0394

95,0498

163,0392 65,0394 163,0392 123,0445 175,0392 119,0488

205,0504 93,0341 205,0504 140,0471 193,0503 163,0391 123,0445

111,0441 69,0331 179,0342 89,0393 117,0340 179,0343

Fragment 4

127,0388 81,0333 221,0455 116,0502 135,0444 221,0454

Fragment 3

107,0861

107,0500 123,0439

161,0234 277,0507 135,0440 135,0441 160,0156

283,0602

133,0281 151,0022

283,0605

55,0187

299,0554

339,0518 105,0704

339,0517

164,0469 121,0283

160,0158 125,0601

145,0285 95,0495 160,0158 93,0331

145,0285

107,0496 161,0234

161,0235

85,0281

Fragment 5

(continued on next page)

(Grzegorczyk-Karolak and Kiss, 2018)

(Grzegorczyk-Karolak and Kiss, 2018)

(Grzegorczyk-Karolak and Kiss, 2018)

(Grzegorczyk-Karolak and Kiss, 2018) (Grzegorczyk-Karolak and Kiss, 2018) (Grzegorczyk-Karolak and Kiss, 2018)

(Grzegorczyk-Karolak and Kiss, 2018)

(Grzegorczyk-Karolak and Kiss, 2018) (Grzegorczyk-Karolak and Kiss, 2018)

(Grzegorczyk-Karolak and Kiss, 2018)

Literature

G. Zengin et al.

Industrial Crops & Products 131 (2019) 266–280

26,34 27,09 27,28 27,95 27,99 28,64 29,38 29,64 29,78 30,00 30,33 30,67 32,61 34,49 34,61 35,90 36,57 36,67 36,99 38,24 38,64 40,40

55 56 57* 58* 59 60 61 62 63* 64 65 66 67 68 69 70 71 72 73 74 75 76

3-O-Methylrosmarinic acid Pentahydroxyflavone Naringenin Luteolin (3',4',5,7-Tetrahydroxyflavone) Di-O-methylellagic acid Dihydroxycinnamoyl-dihydroxyphenyl-ethenol isomer 1 Luteolin-O-(coumaroyl)hexoside Dihydroxycinnamoyl-dihydroxyphenyl-ethenol isomer 2 Apigenin (4',5,7-Trihydroxyflavone) Chrysoeriol (Scoparol, 3'-Methoxy-4',5,7-trihydroxyflavone) Tri-O-methylellagic acid Dimethoxy-trihydroxy(iso)flavone Methoxy-trihydroxyflavone Dihydroxy-dimethoxy(iso)flavone Genkwanin Hydroxy-trimethoxy(iso)flavone Hydroxy-tetramethoxy(iso)flavone 1-Oxomicrostegiol Viroxocin Apigenin-4',7-dimethyl ether (4',7-Dimethoxy-5-hydroxyflavone) 3-Oxomicrostegiol Hexadecanedioic acid

Name

* Confirmed by standard.

Rt

No.

Table 4 (continued)

C19H18O8 C15H10O7 C15H12O5 C15H10O6 C16H10O8 C17H14O6 C30H26O13 C17H14O6 C15H10O5 C16H12O6 C17H12O8 C17H14O7 C16H12O6 C17H14O6 C16H12O5 C18H16O6 C19H18O7 C20H24O3 C20H24O3 C17H14O5 C20H24O3 C16H30O4

Formula

329,10252 359,11308 313,18037 313,18037 299,09195 313,18037

315,08687

315,08687

[M + H]+

285,20659

269,04500 299,05556 343,04540 329,06613 299,05556 313,07122 283,06120

593,12952

373,09235 301,03483 271,06065 285,03991 329,02975

[M - H]− 197,0452 273,0427 177,0178 217,0505 314,0078 205,0502 285,0411 205,0501 227,0342 284,0331 328,0227 314,1531 284,0331 298,0488 268,0382 314,0789 344,0895 295,1696 295,1697 284,0685 295,1697 267,1972

Fragment 1 179,0342 178,9980 165,0179 199,0399 298,9837 163,0392 284,0331 163,0392 225,0558 256,0378 312,9999 271,0254 256,0377 283,0255 239,0352 313,0710 343,0818 280,1466 227,1069 256,0734 280,1462 223,2065

Fragment 2

(Rungsimakan and Rowan, 2014)

Literature

243,1019

135,0444 117,0331 107,0124

135,0444

107,0122 107,0126

135,0440

Fragment 5

(Rungsimakan and Rowan, 2014) (Rungsimakan and Rowan, 2014)

285,0760 313,0708

145,0287 151,0026 151,0025

145,0286

160,0156 107,0126 119,0489 133,0283

Fragment 4

133,0281 255,0301 117,0328 300,0633 329,0662 243,1019 199,0757

175,0392 151,0028 151,0028 151,0027 270,9889 153,0550 145,0286 153,0549 201,0551 227,0340 297,9757

Fragment 3

G. Zengin et al.

Industrial Crops & Products 131 (2019) 266–280

275

Rt

1,24 2,36 12,78 13,69 14,37 14,37 14,43 15,24 15,72 16,62 16,76 16,85 16,87 17,27 17,46 17,78 17,88 17,95 18,08 18,13 18,86 19,07 19,15 19,30 19,46 20,16 20,24 20,37 20,39 20,58 20,87 21,85 21,98 22,21 22,30 22,36 22,53 22,92 22,94 23,04 23,15 23,32 23,58 23,63 24,02 24,05 24,08 24,30 24,32 24,41 24,76 25,84 25,97 26,20

No.

1 2* 3 4 5 6 7* 8 9* 10 11 12 13 14 15 16* 17 18 19 20 21 22 23 24 25 26 27 28 29 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

Quinic acid Gallic acid (3,4,5-Trihydroxybenzoic acid) Caffeoylglucose isomer 1 Kynurenic acid Caffeic acid Caffeoylglucose isomer 2 Chlorogenic acid (3-O-Caffeoylquinic acid) Coumaroylhexose isomer 1 Vanillin Coumaroylhexose isomer 2 Feruloylhexose isomer 1 Ethyl syringate Coumaroylquinic acid Ethyl gallate Syringaldehyde (3,5-Dimethoxy-4-hydroxybenzaldehyde) 4-Coumaric acid Feruloylhexose isomer 2 Caffeoylshikimic acid Antiarol (3,4,5-Trimethoxyphenol) Sinapoylhexose Dimethoxy-hydroxybenzoic acid Luteolin-C-hexoside-C-pentoside isomer 1 Indole-4-carbaldehyde Ferulic acid Luteolin-C-hexoside-C-pentoside isomer 2 Coniferyl aldehyde (4-Hydroxy-3-methoxycinnamaldehyde) N-(2-Phenylethyl)acetamide Luteolin-C-hexoside-O-pentoside Coumaroylshikimate Sinapyl aldehyde (3,5-Dimethoxy-4-hydroxycinnamaldehyde) Myricetin-O-hexoside Apigenin-C-hexoside-O-pentoside Verbascoside Luteolin-O-(pentosyl)hexoside Luteolin-O-glucuronide Luteolin-7-O-glucoside (Cynaroside) Luteolin-O-(deoxyhexosyl)hexoside Isoquercitrin (Hirsutrin, Quercetin-3-O-glucoside) 3-[(1-Carboxyvinyl)oxy]benzoic acid Rosmarinic acid-O-hexoside Salvianolic acid isomer Lipedoside A isomer 1 Lipedoside A isomer 2 Leucosceptoside A Apigenin-O-(deoxyhexosyl)hexoside Cosmosiin (Apigetrin, Apigenin-7-O-glucoside) Apigenin-O-glucuronide Rosmarinic acid (Labiatenic acid) Methyl caffeate Methoxy-trihydroxyflavone-O-glucuronide N-trans-Feruloyltyramine Martynoside Ethyl caffeate 3-O-Methylrosmarinic acid

Name

Table 5 The chemical composition of MAC extract from Salvia viridis roots.

C7H12O6 C7H6O5 C15H18O9 C10H7NO3 C9H8O4 C15H18O9 C16H18O9 C15H18O8 C8H8O3 C15H18O8 C16H20O9 C11H14O5 C16H18O8 C9H10O5 C9H10O4 C9H8O3 C16H20O9 C16H16O8 C9H12O4 C17H22O10 C9H10O5 C26H28O15 C9H7NO C10H10O4 C26H28O15 C10H10O3 C10H13NO C26H28O15 C16H16O7 C11H12O4 C21H20O13 C26H28O14 C29H36O15 C26H28O15 C21H18O12 C21H20O11 C27H30O15 C21H20O12 C10H8O5 C24H26O13 C36H30O16 C29H36O14 C29H36O14 C30H38O15 C27H30O14 C21H20O10 C21H18O11 C18H16O8 C10H10O4 C22H20O12 C18H19NO4 C31H40O15 C11H12O4 C19H18O8

Formula

276 209,08139

195,06574 477,10331 314,13924

579,17139 433,11347

595,16630

581,15065

565,15574

209,08139

179,07082 164,10754 581,15065

146,06059

185,08139

183,06574

227,09195

153,05517

355,10291

190,05042

[M + H]+

373,09235

651,22890

445,07709 359,07670

463,08765 207,02935 521,12952 717,14557 607,20269 607,20269 637,21325

461,07201 447,09274

623,19760

479,08257

319,08178

193,05009 579,13500

385,11348 197,04500 579,13500

163,03952 355,10291 335,07670

337,09235 197,04500

325,09235 355,10291

325,09235

179,03444 341,08726

191,05557 169,01370 341,08726

[M - H]− 173,0446 125,0232 281,0674 162,0552 135,0441 281,0673 163,0392 265,0723 125,0601 265,0721 295,0831 181,0499 191,0557 169,0134 155,0706 119,0490 295,0829 179,0343 170,0579 267,0727 182,0215 489,1049 118,0656 178,0266 489,1044 161,0600 122,0968 449,1086 163,0392 191,0706 317,0311 433,1134 461,1671 449,1088 285,0412 327,0530 449,1086 301,0360 137,0234 359,0778 519,0930 461,1674 461,1674 461,1673 433,1135 271,0605 269,0461 197,0452 163,0392 301,0712 177,0550 475,1838 163,0392 197,0452

Fragment 1 171,0287 97,0282 251,0566 144,0440 107,0489 251,0563 145,0287 235,0612 111,0445 235,0612 235,0612 155,0705 173,0447 125,0232 140,0472 93,0331 235,0612 161,0234 154,0627 249,0616 166,9977 459,0924 117,0580 149,0598 459,0945 147,0444 105,0705 431,0980 155,0343 177,0550 316,0229 415,1028 315,1102 287,0554 217,0502 285,0412 287,0553 300,0282 135,0442 341,0916 339,0522 443,0990 443,0990 315,1097 271,0605 153,0182 175,0240 179,0343 145,0287 286,0477 145,0287 193,0502 145,0286 179,0344

Fragment 2

119,0494 113,0232 161,0235 135,0445 258,0528 121,0652 175,0393 135,0444 175,0393

271,0256 93,0332 323,0780 321,0408 297,0414 297,0430 193,0503

199,0402 284,0333

193,0503 135,0441 153,0550 223,0609 153,0548 399,0727 91,0549 137,0233 399,0730 133,0652 103,0546 413,0877 119,0494 149,0600 287,0201 337,0710 161,0235

123,0445

221,0453 135,0444 205,0504 93,0341 205,0504 193,0502 140,0471 163,0394

127,0391 81,0334 221,0453 116,0499

Fragment 3

160,0157 117,0339 160,0156

135,0441 117,0339

255,0301 87,0073 197,0452 295,0617 161,0235 161,0235 175,0393

151,0026 256,0382

145,0287 271,0250 313,0710 133,0285

134,0363 369,0623 119,0496 90,9484 329,0661

139,0393 208,0376 138,0314 369,0621

175,0393

95,0498

179,0343 117,0339 163,0392 65,0394 163,0392 175,0393 123,0443 119,0489

111,0438 69,0331 179,0344 89,0391

Fragment 4

107,0858 135,0441

123,0439 107,0498

161,0235 277,0506 135,0442 135,0442 160,0156

133,0280 151,0028

283,0604

55,0187

299,0554

339,0517 105,0704

125,0601 164,0469 121,0283 339,0534

160,0156

145,0285 160,0157 95,0497 93,0332

161,0234 107,0495 145,0285

161,0235

85,0280

Fragment 5

(continued on next page)

(Grzegorczyk-Karolak and Kiss, 2018)

(Grzegorczyk-Karolak and Kiss, 2018) (Grzegorczyk-Karolak and Kiss, 2018)

(Grzegorczyk-Karolak and Kiss, 2018) (Grzegorczyk-Karolak and Kiss, 2018) (Grzegorczyk-Karolak and Kiss, 2018)

(Grzegorczyk-Karolak and Kiss, 2018)

(Grzegorczyk-Karolak and Kiss, 2018) (Grzegorczyk-Karolak and Kiss, 2018)

(Grzegorczyk-Karolak and Kiss, 2018)

Literature

G. Zengin et al.

Industrial Crops & Products 131 (2019) 266–280

26,94 27,18 27,83 27,87 28,65 29,24 29,64 29,66 29,88 30,20 30,56 32,49 34,38 34,49 35,90 36,56 36,66 36,99 38,23 38,64 40,33 41,82

55 56* 57* 58 59 60 61 62* 63 64 65 66 67 68 69 70 71 72 73 74 75 76

Pentahydroxyflavone Naringenin Luteolin (3',4',5,7-Tetrahydroxyflavone) Di-O-methylellagic acid Dihydroxycinnamoyl-dihydroxyphenyl-ethenol isomer 1 Luteolin-O-(coumaroyl)hexoside Dihydroxycinnamoyl-dihydroxyphenyl-ethenol isomer 2 Apigenin (4',5,7-Trihydroxyflavone) Chrysoeriol (Scoparol, 3'-Methoxy-4',5,7-trihydroxyflavone) Tri-O-methylellagic acid Dimethoxy-trihydroxy(iso)flavone Methoxy-trihydroxyflavone Dihydroxy-dimethoxy(iso)flavone Genkwanin Hydroxy-trimethoxy(iso)flavone Hydroxy-tetramethoxy(iso)flavone 1-Oxomicrostegiol Viroxocin Apigenin-4',7-dimethyl ether (4',7-Dimethoxy-5-hydroxyflavone) 3-Oxomicrostegiol Hexadecanedioic acid Viridoquinone

Name

* Confirmed by standard.

Rt

No.

Table 5 (continued)

C15H10O7 C15H12O5 C15H10O6 C16H10O8 C17H14O6 C30H26O13 C17H14O6 C15H10O5 C16H12O6 C17H12O8 C17H14O7 C16H12O6 C17H14O6 C16H12O5 C18H16O6 C19H18O7 C20H24O3 C20H24O3 C17H14O5 C20H24O3 C16H30O4 C20H24O2

Formula

297,18546

329,10252 359,11308 313,18037 313,18037 299,09195 313,18037

315,08687

315,08687

[M + H]+

285,20659

269,04500 299,05556 343,04540 329,06613 299,05556 313,07122 283,06120

593,12952

301,03483 271,06065 285,03991 329,02975

[M - H]− 273,0427 177,0179 217,0505 314,0078 205,0501 285,0412 205,0502 227,0353 284,0333 328,0230 314,1532 284,0333 298,0489 268,0383 314,0788 344,0896 295,1697 295,1697 284,0684 295,1697 267,1973 279,1748

Fragment 1 178,9974 165,0179 199,0399 298,9843 163,0393 284,0334 163,0392 225,0556 256,0381 312,9998 271,0254 256,0379 283,0255 239,0354 313,0712 343,0819 280,1465 227,1070 256,0732 280,1462 223,2066 269,1904

Fragment 2

239,1432

(Rungsimakan and Rowan, 2014)

(Rungsimakan and Rowan, 2014)

Literature

243,1020

135,0445 117,0333 107,0125

135,0446

107,0128 107,0127

Fragment 5

(Rungsimakan and Rowan, 2014) (Rungsimakan and Rowan, 2014)

285,0761 313,0713

145,0287 151,0028 151,0024

145,0288

107,0125 119,0492 133,0284

Fragment 4

133,0284 255,0302 117,0331 300,0634 329,0660 243,1019 199,0758

151,0030 151,0026 151,0027 270,9891 153,0552 145,0285 153,0545 201,0551 227,0345 297,9758

Fragment 3

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277

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G. Zengin et al.

Table 6 Antioxidant properties of Salvia viridis extracts*. Extraction method MAE MAC SFE SE UAE

DPPH (mg TE/g) c

199.95 ± 4.83 218.59 ± 5.47b 4.89 ± 0.93e 177.46 ± 2.13d 240.00 ± 4.57a

ABTS (mg TE/g) b

280.50 ± 6.52 293.28 ± 8.78a 32.59 ± 2.62c 276.35 ± 6.83b 302.85 ± 7.12a

CUPRAC (mg TE/g) 858.75 922.64 138.61 910.06 970.74

± ± ± ± ±

c

7.35 17.21b 3.89d 7.39b 6.58a

FRAP (mg TE/g) c

653.26 ± 13.00 705.89 ± 12.12a 99.94 ± 1.71d 674.37 ± 5.48b 704.27 ± 8.09a

Phosphomolybdenum (mmol TE/g) 2.37 2.68 1.34 2.40 2.84

± ± ± ± ±

b

0.14 0.07a 0.06c 0.04b 0.01a

Metal chelating (mg EDTAE/g) 22.71 15.60 14.19 20.33 19.53

± ± ± ± ±

1.11a 0.57b 3.82b 1.17a 0.90a

* Values expressed are means ± S.D. of three parallel measurements. MAE: Microwave assisted extraction; MAC: Maceration, SFE: Supercritical fluid extraction, SE: Soxhlet extraction; UAE: Ultrasonic assisted extraction. TE: Trolox equivalent; EDTAE: EDTA equivalent. Different letters indicate differences in the tested extracts (p < 0.05).

potent inhibitor of tyrosinase, is a widely used therapeutic agent for the management of skin hyperpigmentation conditions. However, the utilization of kojic acid by the pharmaceutical and cosmetic industry to thwart excessive melanin production has been associated with many side effects (Zengin et al., 2015). Interestingly, phytochemicals have proved to be natural enzyme inhibitors, thereby opening new avenues for research and development of novel therapeutic solutions. In this study, S. viridis extracts obtained by different extraction techniques were tested for possible enzyme inhibitory activity against α-amylase, α-glucosidase, acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and tyrosinase. Additionally, this study was aimed at identifying the most suitable extraction technique for the isolation of potent enzyme inhibitors from S. viridis roots. It has been previously reported that S. viridis, as part of the herbal mixture, was traditionally used to manage diabetes type 1 (Bayat et al., 2017). Additionally, several Salvia species, namely S. chloroleuca (Asghari et al., 2015), S. mirzayanii (Rouzbehan et al., 2017) and S. sclarea (Zengin et al., 2018b) have been documented to possess α-amylase and α-glucosidase inhibitory properties. From data summarized in Table 7, S. viridis showed stronger

shown in Fig. 2A. The antioxidant potential of S. viridis aerial parts has been reported previously (Erdemoglu et al., 2006; Grzegorczyk-Karolak and Kiss, 2018), but this study is, as far as we know, the first one to report the antioxidant potent of S. viridis roots extract. Evidence gathered from the literature indicates the presence of diterpenoids in the roots of S. viridis (Rungsimakan and Rowan, 2014). Diterpenoids isolated from an S. barrelieri have been reported to exhibit potent antioxidant activity (Kolak et al., 2009). Besides, rosmarinic acid, known for its potent antioxidant properties (Adomako-Bonsu et al., 2017), has also been identified from S. viridis roots (Rungsimakan and Rowan, 2014). The synergistic effect of these phytochemicals along with other bioactive constituents might justify the observed antioxidant activity. To control global health problems, enzymes involved are considered as key targets (Vujanović et al., 2019). For instance, the modulation of α-amylase and α-glucosidase, key enzymes responsible for the digestion of starch, is considered as an important therapeutic strategy for the management of postprandial glucose peaks (Picot and Mahomoodally, 2017). The need for novel enzyme inhibitors is primarily fueled by the side effects of currently existing inhibitors. For example, kojic acid, a

Fig. 2. Statistical evaluations (A: Correlation coefficients between total bioactive compounds and biological activities (Pearson Correlation Coefficient (R), p < 0.05); B: Distribution of biological activities on the correlation circle based on PCA; C: Heatmap of extracts in according to bioactive compounds and biological activities; D: Distribution of the extraction methods on the correlation circle based on PCA, individual sample replications (n = 4) are given in the class prediction model score plot; PPBD: Phosphomolybdenum; TPC: Total phenolics content; TFC: Total flavonoids content.). 278

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Table 7 Enzyme inhibitory properties of Salvia viridis extracts*. Extraction method

AChE inhibition (mg GALAE/g)

MAE MAC SFE SE UAE

4.08 3.60 5.21 4.23 3.62

± ± ± ± ±

0.05b 0.15c 0.01a 0.07b 0.20c

BChE inhibition (mg GALAE/g) 5.63 5.45 6.33 5.60 5.44

± ± ± ± ±

0.02b 0.05c 0.02a 0.04b 0.01c

Tyrosinase inhibition (mg KAE/g) 159.75 162.56 151.54 159.58 158.38

± ± ± ± ±

1.80a 0.78a 2.14c 0.35a 0.14b

α-Amylase inhibition (mmol ACAE/g) 0.56 0.73 0.73 0.66 0.72

± ± ± ± ±

0.03c 0.01a 0.01a 0.02b 0.03a

α-Glucosidase inhibition (mmol ACAE/g) 1.66 1.63 1.64 1.61 1.65

± ± ± ± ±

0.01a 0.01b 0.01ab 0.02b 0.01a

* Values expressed are means ± S.D. of three parallel measurements. GALAE: Galatamine equivalent; KAE: Kojic acid equivalent; ACAE: Acarbose equivalent. MAE: Microwave assisted extraction; MAC: Maceration, SFE: Supercritical fluid extraction, SE: Soxhlet extraction; UAE: Ultrasonic assisted extraction. TE: Trolox equivalent; EDTAE: EDTA equivalent. Different letters indicate differences in the tested extracts (p < 0.05).

inhibitory action against α-glucosidase compared to α-amylase. It has been proposed that low α-amylase versus strong α-glucosidase inhibition could address the major drawbacks of currently used hypoglycemic agents. Indeed, excessive inhibition of α-amylase has been found to result in bacterial fermentation of undigested carbohydrate in the colon, eventually causing gastrointestinal problems (Picot and Mahomoodally, 2017). The root extracts of S. viridis showed inhibitory activity against both AChE and BChE (Table 7). The observed inhibitory activity against AChE might be related to the presence of β-sitosterol (Bahadori et al., 2016) which has been previously identified from S. viridis roots (Rungsimakan and Rowan, 2014). It is noteworthy mentioning that S. viridis ethanolic root extract obtained by SFE, showed higher inhibitory action against both cholinesterases (5.21 and 6.33 mg GALAE/g). A group of authors (Lee et al., 2011) previously reported the tyrosinase inhibitory activity of S. viridis aerial parts. Data gathered from this study demonstrated that S. viridis roots also exhibited tyrosinase inhibitory activity (Table 7). Tyrosinase, responsible for melanogenesis, is the main target for the management of epidermal hyperpigmentation problems (Zengin et al., 2018c). Other species of the Salvia genus have also been reported to possess tyrosinase inhibitory activity. For instance, S. sclarea water extract, ethanolic extract of S. cryptantha and S. cyanescens aerial parts (Süntar et al., 2011; Zengin et al., 2018b). Furthermore, rosmarinic acid, present in several species of the Salvia genus, identified in S. viridis roots has been reported to exhibit inhibitory action against tyrosinase (Oliveira et al., 2013). To provide new insights into the extraction methods in this study performed, we used several statistical analysis. In Principal Component Analysis (PCA), two principal components were obtained as 89.5% of total variance (Fig. 2B). The PCA and correlation analysis were similar and strong correlation was observed between total bioactive components and DPPH, ABTS, phosphomolybdenum, CUPRAC and FRAP assays. To provide a certain classification for the extraction methods, a heat map (Fig. 2C) was carried out, as well as PCA (Fig. 2D). Based on these results, the extraction methods were divided into three groups. SE, MAC, and UAE were classified in the same group. However, other groups contained SFE and MAE.

Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.indcrop.2019.01.060. References Adomako-Bonsu, A.G., Chan, S.L.F., Pratten, M., Fry, J.R., 2017. Antioxidant activity of rosmarinic acid and its principal metabolites in chemical and cellular systems: importance of physico-chemical characteristics. Toxicol. In Vitro 40, 248–255. Ai, C.-B., Deng, Q.-H., Song, W.-Z., Li, L.-N., 1994. Salvianolic acid J, a depside from Salvia flava. Phytochemistry 37, 907–908. Ameer, K., Shahbaz, H.B., Kwon, J., 2017. Green extraction methods for polyphenols from plant matrices and their byproducts: a review. Compr. Rev. Food Sci. Food Saf. 16, 295–315. Asghari, B., Salehi, P., Sonboli, A., Nejad Ebrahimi, S., 2015. Flavonoids from Salvia chloroleuca with α-Amylsae and α-glucosidase inhibitory effect. Iran. J. Pharm. Res. 14, 609–615. Bahadori, M.B., Dinparast, L., Valizadeh, H., Farimani, M.M., Ebrahimi, S.N., 2016. Bioactive constituents from roots of Salvia syriaca L.: acetylcholinesterase inhibitory activity and molecular docking studies. S. Afr. J. Bot. 106, 1–4. Bayat, M., Uslu, N., Erdem, E., Efe, Y.S., Variyenli, N., Arican, F., Kurtoglu, S., 2017. Complementary and alternative medicine used for children with type 1 diabetes mellitus. Iran. J. Pediatr. 27 (4) e11210. Belwal, T., Ezzat, S.M., Rastrelli, L., Bhatt, I.D., Daglia, M., Baldi, A., Devkota, H.P., Orhan, I.E., Patra, J.K., Das, G., Anandharamakrishnan, C., Gomez-Gomez, L., Nabavi, S.F., Nabavi, S.M., Atanasov, A., 2018. A critical analysis of extraction techniques used for botanicals: trends, priorities, industrial uses and optimization strategies. Trends Analyt. Chem. 100, 82–102. Boukhary, R., Raafat, K., Ghoneim, A.I., Aboul-Ela, M., El-Lakany, A., 2016. Anti-inflammatory and antioxidant activities of Salvia fruticosa: an HPLC determination of phenolic contents. Evid. Based Complement. Altern. Med. 2016, 1–6. Chen, Z.-Y., Wong, I.Y.F., Leung, M.W.S., He, Z.-D., Huang, Y., 2002. Characterization of antioxidants present in bitter tea (Ligustrum pedunculare). J. Agric. Food Chem. 50, 7530–7535. Cvetanović, A., Švarc-Gajić, J., Mašković, P., Savić, S., Nikolić, L., 2015. Antioxidant and biological activity of chamomile extracts obtained by different techniques: perspective of using superheated water for isolation of biologically active compounds. Ind. Crop. Prod. 65, 582–591. Erdemoglu, N., Turan, N.N., Cakõcõ, I., Sener, B., Aydõn, A., 2006. Antioxidant activities of some Lamiaceae plant extracts. Phytother. Res. 20, 9–13. Grzegorczyk-Karolak, I., Kiss, A., 2018. Determination of the phenolic profile and antioxidant properties of Salvia viridis L. shoots: a comparison of aqueous and hydroethanolic extracts. Molecules 23, 1468. Guldiken, B., Ozkan, G., Catalkaya, G., Ceylan, F.D., Ekin Yalcinkaya, I., Capanoglu, E., 2018. Phytochemicals of herbs and spices: health versus toxicological effects. Food Chem. Toxicol. 119, 37–49. Hao, D.C., Gu, X.-J., Xiao, P.G., 2015. 14 - phytochemical and biological research of Salvia medicinal resources. In: Hao, D.C., Gu, X.-J., Xiao, P.G. (Eds.), Medicinal Plants. Woodhead Publishing, pp. 587–639. Kolak, U., Kabouche, A., Ozturk, M., Kabouche, Z., Topcu, G., Ulubelen, A., 2009. Antioxidant diterpenoids from the roots of Salvia barrelieri. Phytochem. Anal. 20, 320–327. Lee, C.-J., Chen, L.-G., Chang, T.-L., Ke, W.-M., Lo, Y.-F., Wang, C.-C., 2011. The correlation between skin-care effects and phytochemical contents in Lamiaceae plants. Food Chem. 124, 833–841. Li, Y.-G., Song, L., Liu, M., Wang, Z.-T., 2009. Advancement in analysis of Salviae miltiorrhizae Radix et Rhizoma (Danshen). J. Chrom. A 1216, 1941–1953. Li, M.-h., Li, Q.-q., Liu, Y.-z., Cui, Z.-h., Zhang, N., Huang, L.-q., Xiao, P.-g., 2013. Pharmacophylogenetic study on plants of genus Salvia L. from China. Chin. Herb. Med. 5, 164–181. Liu, Y., Luo, X., Lan, Z., Tang, J., Zhao, P., Kan, H., 2018. Ultrasonic-assisted extraction and antioxidant capacities of flavonoids from Camellia fascicularis leaves. CyTA - J. Food 16, 105–112. Lu, Y., Foo, L.Y., 2001. Salvianolic acid L, a potent phenolic antioxidant from Salvia officinalis. Tetrahedron Lett. 42, 8223–8225.

4. Conclusion This study has revealed the antioxidant and enzyme inhibitory properties of S. viridis ethanolic root extracts, for the first time. It was noted that the choice of extraction technique influenced the antioxidant properties of S. viridis ethanolic root extracts. This study appraises the possible therapeutic application of S. viridis ethanolic root extracts for the management of chronic complications such as Alzheimer’s disease, diabetes, and skin hyperpigmentation disorders. Additionally, scientific evidence gathered in this investigation support the need for a further study which might lead to the development of new therapeutic entities for the management of the ailments as mentioned above.

279

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