DOI: 10.17344/acsi.2018.4921 Acta Chim. Slov. 2019, 66, – 1 Scientific paper Two Acylated Isoscutellarein Glucosides with Anti-Inflammatory and Antioxidant Activities Isolated from Endemic Stachys Subnuda Montbret & Aucher ex Benth Ali Sen,1,* Fatih Goger, 2 Ahmet Dogan3 and Leyla Bitis1 1 Department of Pharmacognosy, Faculty of Pharmacy, Marmara University, Istanbul, Turkey, 2 Department of Pharmacognosy, Faculty of Pharmacy, Anadolu University, Eskisehir, Turkey 3 Department of Pharmaceutical Botany, Faculty of Pharmacy, Marmara University, Istanbul, Turkey * Corresponding author: E-mail: [email protected]; [email protected] Phone: +90 535 938 78 94; Fax: +90 216 345 29 52; e-mail Received: 12-27-2018 Abstract In this study, we report anti-inflammatory and antioxidant activities of two acylated isoscutellarein glucosides isolated from ethyl acetate extract of Stachys subnuda aerial part. 4ʹ-O-methylisoscutellarein-7-O-2ʹʹ-O-(6ʹʹʹ-O-acetyl-β-D-al- lopyranosyl)-β-D-glucopyranoside (SS1) and isoscutellarein-7-O-2ʹʹ-O-(6ʹʹʹ-O-acetyl-β-D-allopyranosyl)-β-D-glu- copyranoside (SS2) were isolated as major compounds from ethyl acetate extract (SSEA). Also, 2 hydroxycinnamic acid derivatives, and 5 isoscutellarein glucoside derivatives in the SSEA were identified using LC-MS/MS. SS1 with IC50 values of 2.35 and 1.98 μg/mL and SS2 with IC50 values 13.94 and 12.76 μg/mL showed fairly strong antioxidant activity against DPPH (1,1-diphenyl-2-picrylhydrazyl) and ABTS (2,2ʹ-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid) radicals, re- spectively. SS1 and SS2 inhibited 5-lipoxygenase (5-LOX) activity with IC50 values of 47.23 and 41.60 μg/mL, respectively. The results demonstrated that SS1 and SS2 have significant anti-inflammatory and antioxidant potential. Acylated flavo- noid glycosides have been first reported for Stachys subnuda. Also, the activities of SS1 and SS2 have been investigated for the first time in this study. Keywords: Stachys subnuda; acylated flavonoids; anti-inflammatory activity; antioxidant activity; LC-MS/MS subnuda in the literature.4 However, no study on isolation 1. Introduction of flavonoids from this species and their biological activi- The Stachys genus is one of the largest species of the ties have yet been reported. In the studies on other Stachys Lamiaceae family and is represented by about 300 species species, it has revealed that secondary metabolites are gen- worldwide.1 Also, Turkey is one of the richest countries in erally iridoids, flavone glycosides, diterpenes and essential terms of variety of Stachys species and this genus is repre- oils.2,5–7 Also, it has been reported that various extracts of sented by 91 species with an endemism rate of 48%. Stachys Stachys species have antioxidant, anti-proliferative, an- species known as “Deli sage” or “Mountain tea” in Anatolia ti-inflammatory, antiulcer, antinociceptive, antimicrobial are used in the skin diseases, ulcers, cancer, respiratory activities.8–10 There is no scientific information on chemi- diseases and kidney diseases by the people because of their cal composition of Stachys subnuda ethyl acetate extract antibacterial, anti-inflammatory, antipyretic, antioxidant and anti-inflammatory and antioxidant activities of its ac- and cytotoxic effects.2 In many countries, especially in the tive major compounds. Therefore, the aim of this study Mediterranean regions, Stachys species are consumed as was to test anti-inflammatory and antioxidant activities of herbal tea (Mountain Tea), food and herbal remedies.3 major compounds isolated from ethyl acetate extract of ae- There is only one report on the chemical composi- rial parts of endemic Stachys subnuda Montbret & Aucher tion and biological activity of the essential oil of Stachys Ex Benth. Sen et al.: Two Acylated Isoscutellarein Glucosides ... 2 Acta Chim. Slov. 2019, 66, – 2. Experimental ings were taken at 517 nm. The percent radical scavenging activity of extracts and standard against DPPH were calcu- 2. 1. Plant Material lated according to the following: Aerial parts of plant were collected in the flowering period from the Tunceli province of Turkey in 2015 and DPPH radical-scavenging activity (%) = (2) identified by Dr. Ahmet Dogan, a botanist of the Faculty of = [(A0–A1)/A0] × 100 Pharmacy, University of Marmara. Voucher specimens were deposited in the Herbarium of the Faculty of Phar- where A0 is the absorbance of the control (containing all macy, Marmara University (MARE No: 17720). reagents except the test extracts or compounds), and A1 is the absorbance of the extracts/standard. Extracts or com- 2. 2. Extraction pounds concentration providing 50% inhibition (IC50) was calculated from the graph plotting inhibition percent- Dried and ground aerial parts of Stachys subnuda (10 age against extracts concentration. Tests were carried out g) for activities were extracted with CH3OH (3 × 100 mL), in triplicate. BHA, Ascorbic acid and Trolox were used as using an ultrasonic bath. After filtration and evaporation, positive control. the residue (SSM) was dissolved in 50 mL 50% aqueous methanol, and subjected to solvent-solvent partition be- 2. 5. ABTS Radical-Scavenging Activity tween n-hexane (3 × 50 mL), chloroform (3 × 50 mL) and ethyl acetate (3 × 50 mL). The n-hexane, chloroform, ethyl Free radical scavenging capacity of isolated com- acetate and aqueous methanol extracts were coded as SSH, pounds and extracts was evaluated according to the previ- SSC, SSEA and SSAM, respectively. Also, about 140 g of ously reported procedure.12 ABTS radical cations were the plant was weighed for isolation and similar extraction prepared by mixing equal volume of ABTS (7 mM in H2O) procedures described above were carried out. All extracts and potassium persulfate (4,9 mM in H2O), allowing them were stored under refrigeration for further analysis. to react for 12–16 h at room temperature in the dark. Then, ABTS radical solution was diluted with 96% ethanol to an 2. 3. In vitro Anti-Inflammatory Activity absorbance of about 0.7 at 734 nm. 10 µL of sample in DMSO at different concentrations (125–0.24 µg/mL) were The anti-inflammatory activity was evaluated accord- added to 190 µL of ABTS radical solution in a well of 96- ing to the method described by Phosrithong et al.11 An ali- well plate. The mixture was shaken vigorously and allowed quot of 500 μL at different concentrations of isolated com- to stand in the dark at room temperature for 30 min. Ab- pounds was added to 250 μL of 0.1 M borate buffer pH 9.0, sorbance readings were taken at 734 nm. The percent rad- followed by addition of 250 μL of type V soybean lipoxy- ical scavenging activity of extracts and standards against genase solution in buffer (20.000 U/mL). After the mixture ABTS were calculated according to the following: was incubated at 25 °C for 5 min, 1000 μL of 0.6 mM linole- ic acid solution was added, mixed well and the change in ABTS radical-scavenging activity (%) = (3) absorbance at 234 nm was recorded for 6 min. Indometha- = [(A0–A1)/A0] × 100 cin was used as a reference standard. The percent inhibition was calculated from the following equation: where A0 is the absorbance of the control (containing all reagents except the test extracts or compounds), and A1 is % inhibition= [(Acontrol–Asample) / Acontrol] × 100 (1) the absorbance of the extracts/standards. Extracts or com- pounds concentration providing 50% inhibition (IC50) A dose-response curve was plotted to determine the was calculated from the graph plotting inhibition percent- IC50 values. IC50 is defined as the concentration sufficient age against extracts concentration. Tests were carried out to obtain 50% of a maximum anti-inflammatory activity. in triplicate. BHA, ascorbic acid and trolox were used as All tests and analyses were performed in triplicates. positive control. 2. 4. DPPH Radical Scavenging Activity 2. 6. Quantitative Determination of the Total Free radical scavenging capacity of isolated com- Phenolic Contents of Stachys Subnuda pounds and extracts were evaluated according to the pre- Extracts viously reported procedure using the stable DPPH.12 Brief- Total phenolic compound content of extracts was de- ly, 10 µL of sample in DMSO at different concentrations termined according to Gao et al.13 The assay was adapted to (125–0.24 µg/mL) were added to 190 µL methanol solu- the 96 well microplate format. 10 µL of extracts in various tion of DPPH (0.1 mM) in a well of 96-well plate. The mix- concentrations were mixed with 20 µL Folin-Ciocalteu re- ture was shaken vigorously and allowed to stand in the agent (Sigma), 200 µL of H2O, and 100 µL of 15% Na2CO3 dark at room temperature for 30 min. Absorbance read- and the absorbance was measured at 765 nm after 2 h incu- Sen et al.: Two Acylated Isoscutellarein Glucosides ... Acta Chim. Slov. 2019, 66, – 3 bation at room temperature. Gallic acid was used as a stan- 2. 9. LC-MS/MS Analysis dard and the total phenolic contents of extracts were ex- pressed as mg/g gallic acid equivalents (GAE) LC-MS/MS analysis was carried out using an Ab- sciex 3200 Q trap MS/MS dedector. Experiments were 2. 7. Quantitative Determination of the Total performed with a Shimadzu 20A HPLC system coupled to an Applied Biosystems 3200 Q-Trap LC- MS/MS instru- Flavonoid Contents of Stachys Subnuda ment equipped with an ESI source operating in negative Extracts ion mode. For the chromatographic separation, ODS 150 Total flavonoid compound content of extracts was × 4.6 mm, i.d., 3 µm particle size, octadecyl silica gel ana- determined according to Zhang et al.14 The assay was lytical column operating at 40 °C has been used. The sol- adapted to the 96 well microplate format. 25 µL of extracts vent flow rate was maintained at 0.5 mL/min.