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Industrial Crops & Products
journal homepage:www.elsevier.com/locate/indcropMultiple biological activities of two Onosma species (O. sericea and O.
stenoloba) and HPLC-MS/MS characterization of their phytochemical
composition
Jelena S. Katani
ć Stanković
a, Ramazan Ceylan
b,*
, Gokhan Zengin
b, Sanja Mati
ć
a, Tatjana Juri
ć
c,
Alina Diuzheva
d, József Jeko
e, Zoltán Cziáky
e, Abdurrahman Aktumsek
baDepartment of Science, Institute for Information Technologies Kragujevac, University of Kragujevac, Kragujevac, Serbia bSelçuk University, Faculty of Science, Department of Biology, Campus, Konya, Turkey
cUniversity of Novi Sad, Faculty of Agriculture, Novi Sad, Serbia
dDepartment of Forest Protection and Entomology, Faculty of Forestry and Wood Sciences, Czech University of Life Sciences, Prague, Czech Republic eAgricultural and Molecular Research and Service Institute, University of Nyíregyháza, Nyíregyháza, Hungary
A R T I C L E I N F O Keywords: Onosma Antigenotoxic Natural agents Flavonoids Enzyme inhibition A B S T R A C T
Members of the Onosma genus are widely used in folk medicine and they have great interest in the pharma-ceutical industry. In this study, phytochemical characterization, antioxidant activity (2,2-diphenyl-1-picrylhy-drazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS), ferric reducing antioxidant power (FRAP), cupric reducing antioxidant capacity (CUPRAC), metal chelation, and phosphomolybdenum assays), enzyme inhibition (acetylcholinesterase (AChE), butyrylcholinesterase (BChE),α-amylase, α-glucosi-dase, and tyrosinase), antimicrobial activity (microdilution method), genotoxic and antigenotoxic (by using Drosophila melanogaster larvae and Comet assay) potentials of Onosma sericea Willd. and Onosma stenoloba Hausskn. ex Riedl. were investigated. Additionally, the bioactive compounds were identified by high perfor-mance liquid chromatography-mass spectrometry/mass spectrometry (HPLC-MS/MS) analysis. Generally, O. sericea showed stronger antioxidant activity, while O. stenoloba extract exhibited stronger enzyme inhibitory abilities (cholinesterases andα-amylase). The protective effects of extracts, at the concentration range from 25 to 400μg/mL, against hydroxyl radical-induced DNA damage were dose-dependent, increasing with a higher dosage. The extracts at the highest concentration (80 mg/mL) showed the absence of genotoxicity in vivo. Antigenotoxic effects were evident after treatment with both extracts, with a percentage reduction of over 80 %. Overall antimicrobial activity of studied extracts was weak, with the lowest minimal inhibitory concentration values (MIC) of 2.5 mg/mL. Taken together, obtained results showed that tested Onosma species can be con-sidered as promising sources of bioactive phytochemicals for pharmacological purposes.
1. Introduction
A variety of plant species have been known as an important source of therapeutics. Since the antique ages, people have used traditional herbal medicine to treat many diseases (Cragg and Newman, 2013). The great chemical diversity of natural products from medicinal plants provides a basis for discovering the right targets which underlying various diseases. Within this context, plant species used in folk medi-cine are also largely studied in order to find new molecular entities derived from natural products and to struggle global health challenges such as cancer, Alzheimer disease, and diabetes mellitus (David et al., 2015;Mahomoodally et al., 2018;Zengin et al., 2018).
The genus Onosma belongs to the family Boraginaceae and contains more than 180 species distributed worldwide (Binzet and Eren, 2018; Cecchi et al., 2016). According toBinzet and Eren (2018), the genus includes 102 species, 59 of these are endemic to Turkey. The Onosma species are traditionally used in the treatment of blood disease, ab-dominal pain, bladder pain, burns, wounds, rheumatism, kidney irri-tation, strangury, fever, and as laxative, diuretic, anthelmintic, cooling, and astringent agents (Kumar et al., 2013; Mašković et al., 2015; Sarikurkcu et al., 2018). In Turkey, the plants from this genus are also used for wound healing, edema, and hemorrhoids (Altundag and Ozturk, 2011; Çakılcıoğlu and Türkoğlu, 2007; Tetik et al., 2013). However, to the best of our knowledge, the biopharmaceutical
https://doi.org/10.1016/j.indcrop.2019.112053
Received 24 August 2019; Received in revised form 30 October 2019; Accepted 15 December 2019 ⁎Corresponding author.
E-mail address:biyoram7@gmail.com(R. Ceylan).
Available online 21 December 2019
0926-6690/ © 2019 Elsevier B.V. All rights reserved.
2. Material and methods
2.1. Plant material and extraction procedure
The Onosma species were collected in Central Anatolia region of Turkey in June 2014 (O. serica: Yahyalı-Kayseri, 38° 01' 31" N, 35° 29' 33" E, 1416 m; O. stenoloba: Çamardı-Niğde, 37° 50' 05" N, 34° 58' 45" E, 1530 m). The taxonomical classification was performed by a local bo-tanist (Dr. Ahmet Savran from Omer Halis Demir University, Nigde-Turkey). The aerial parts were divided and dried for 10 days at room temperature. Thereafter, the samples were powdered with a laboratory mill.
Air-dried aerial parts of each plant species were extracted by ma-ceration with methanol to the ratio 1:20 (w/v) for one day at room temperature, to obtain the methanolic extracts of O. sericea and O. stenoloba. The methanolic solutions were evaporated under reduced pressure at 45 °C using a vacuum rotary evaporator (Heindolph, Germany) to dryness. The dried extracts were stored at 4 °C until use. 2.2. HPLC-MS/MS analysis
2.2.1. Sample preparation
One milligram of each extract was dissolved in 1 mL of metha-nol:water (ratio 70:30). Thereafter, the solutions were ultrasonicated for 10 min. The obtained solutions werefiltered through a 0.22 μm PTFEfilter membrane (Labex Ltd, Budapest, Hungary) before HPLC-MS/MS analysis.
2.2.2. Instruments and methods
A Dionex Ultimate 3000RS UHPLC instrument, equipped with Thermo Accucore C18 (100 mm x 2.1 mm i. d., 2.6 μm) analytical column thermostated at 25 °C ( ± 1 °C) was used for separation of compounds. A mobile phase consisting of water (A) and methanol (B) containing 0.1 % formic acid was pumped at aflow rate of 0.2 mL/min. A gradient elution method was applied as following: 0−3 min – 5 % B; 3−43 min – 100 % B; 43–61 – 100 % B; 61−62 min 5 % B, 62−70 min – 5 % B. The total run time was 70 min. Mass spectra were recorded using a Thermo Q Exactive Orbitrap mass spectrometer (Thermo Scientific, USA) equipped with an electrospray source. All samples were analyzed in different runs in positive and negative ion modes. Thermo Scientific Xcalibur 3.1 software (Thermo Scientific, USA) was used to collect and analyze data. TraceFinder 3.1 (Thermo Scientific, USA) was applied for targeted screening. The obtained chromatograms are given inFigs. 1 and 2.
2.3. Total bioactive components
The total bioactive components (phenolic andflavonoid) contents of the plant extracts were tested, as previously defined (Uysal et al., 2017) by using well-known methods such as Folin-Ciocalteu and AlCl3tests, respectively. The data were given as gallic acid and rutin equivalents, respectively.
chelating activity on ferrous ions, as previously defined (Uysal et al., 2017). The data were given as reference compounds (Trolox and EDTA) equivalents.
2.5. Enzyme inhibitory activity
Enzyme inhibitory potentials of two Onosma species were assessed using several enzyme inhibition assays. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition for anti-Alzheimer, α-amylase andα-glucosidase inhibition for anti-hyperglycemic, and tyr-osinase inhibition for anti-melanogenesis assays were performed ac-cording to the methods previously defined byBender et al. (2018). The data obtained by these assays were given as reference inhibitors equivalents: galantamine for AChE and BChE, kojic acid for tyrosinase, and acarbose forα-amylase and α-glucosidase.
2.6. In vitro DNA protective effect
The experiments were performed using Herring sperm DNA as previously defined byLin et al. (2008)andMatić et al. (2015). Several concentrations of extracts (25−400 μg/mL), the Herring sperm DNA as blank and quercetin (50μM) as a standard drug (Poorna et al., 2013) was loaded onto a 1 % agarose gel in 1xTAE buffer with ethidium bromide (10 mg/mL). The obtained DNA bands were recorded using ImageJ software (version 1.48 for Windows).
2.7. In vivo genotoxic and antigenotoxic activity
Theflies and larvae of the wild type strain of Drosophila melanogaster (Canton S, available from Bloomington Stock Centre, Indiana, USA) were used. These strains were cultured (conditions: a 12:12 h light/dark regime on standard corn medium containing agar, sugar and yeast, 60 % humidity and at 25 °C).
Third instar larvae of D. melanogaster (74 ± 2 h) were collected and transferred on standard corn medium. Then, negative and positive control groups (received standard Drosophila diet and 1 mM ethyl me-thanesulphonate, respectively) were designed. Regarding Onosma ex-tracts, six groups were prepared with O. sericea (range of 20−80 mg/ mL) as well as O. stenoloba extracts (range of 20−80 mg/mL), respec-tively. In addition, the extracts (80 mg/mL of O. sericea or O. stenoloba extracts) tested simultaneously with 1 mM EMS.
The comet assay was performed to evaluate genotoxicity and anti-genotoxicity, as described by Singh et al. (Singh et al., 1988) and Mukhopadhyay et al. (2004). The images were visualized and captured with afluorescence microscope (Nikon, Ti-Eclipse). One hundred ran-domly selected cells were analyzed per treatment. The total comet score and the percentage reduction (%R) in the comet score analyzed by qualitative method (Collins, 2004) were calculated byManoharan and Banerjee (1985)andWaters et al. (1990).
2.8. Antimicrobial activity evaluation
For the comparative analysis of the potential antimicrobial activity of O. sericea and O. stenoloba extracts were used eight bacterial species,
two Gram-positive (Enterococcus faecalis ATCC 92912 and Micrococcus lysodeikticus ATCC 4698), and six Gram-negative strains (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 10145, Pseudomonas fluorescens FSB 28, Klebsiella pneumoniae ATCC 70063, Agrobacterium tumefaciens FSB 11, and Azotobacter chroococcum FSB 14). Antifungal activity of Onosma extracts was monitored on a yeast (Candida albicans ATCC 10259) and ten fungal strains (Aspergillus niger FSB 31, Aspergillus glaucus FSB 32, Trichoderma longibrachiatum FSB 13, Trichoderma har-zianum FSB 12, Penicillium canescens FSB 24, Penicillium cyclopium FSB 23, Doratomyces stemonitis FSB 41, Phialophora fastigiata FSB 81, Alternaria alternata FSB 51 and Fusarium oxysporum FSB 91). The above microbial strains were obtained from the Faculty of Chemistry, University of Belgrade (Belgrade, Serbia) and Laboratory for Microbiology, Department of Biology, Faculty of Science, University of Kragujevac (Kragujevac, Serbia). These cultures were stored and cul-tured as previously reported (Katanić et al., 2015).
The microdilution method was applied for testing of antibacterial and antifungal activities of O. sericea and O. stenoloba methanolic ex-tracts (CLSI, 2012;Sarker et al., 2007), using the procedure described in Katanić et al. (2015). All tests for evaluation of antibacterial activity were performed in Mueller–Hinton broth (MHB) and antifungal tests were done using Sabouraud dextrose broth (SDB). The tests were per-formed by a serial dilution technique using 96-well microtiter plates. The starting concentration of tested extracts was 10 mg/mL, and after added into thefirst row of the plate the double dilutions were made in 50μL of MHB or SDB. The indicator solution (10 μL of resazurin; 270 mg resazurin in 40 mL of sterile distilled water) and 30μL of MHB were added to each well. For tests with fungal strains, 40 μL of SDB was added since no indicator was used. At the end, 10μL of bacterial cell suspension (1.0 × 106CFU/mL) or fungal spore suspension (1.0 × 104 CFU /mL) were added. After incubation for 24 h at 37 °C for the bac-teria, or for 48 h at 28 °C for fungi, respectively, the indicator color change (from purple to pink or colorless) was monitored as positive. The results were expressed as minimum inhibitory concentrations (MICs). In antibacterial tests, the lowest concentration of the extract without color change was defined as the MIC and for antifungal
activity, the lowest concentrations without visible growth of fungi were taken as MICs. All tests were repeated in triplicate. Erythromycin was used as a standard antibiotic, ketoconazole was used as the reference antimycotic against the tested fungi and nystatin was applied as the control for C. albicans (Zengin et al., 2017).
2.9. Statistical analysis
The differences between the extracts for antioxidant and enzyme inhibition tests were analyzed using the Student t-test (α = 0.05). This treatment was carried out using SPSS v. 14.0 program. Regarding other assay, the observations were given as mean ± SEM and statistical evaluation of data was done using one-way analysis (ANOVA) followed by post hoc analysis using Bonferroni or the T3 Dunnett tests using SPSS statistical software package, version 13.0 for Windows. The significance level was set at p < 0.05.
3. Results and discussion
3.1. Total phenolics and HPLC-MS/MS analysis
Polyphenols, also named as polyhydroxyphenols, are a large group of chemical compounds that are synthesized by plant metabolism and can be provided through nutrition. Polyphenols, including phenolic acids,flavonoids, tannins, their derivatives, and metabolites, are known to have various bioactivities such as antioxidant, anti-inflammatory, antimicrobial, and cardioprotective and many other activities (Shahidi and Ambigaipalan, 2015;Sies, 2010). The determination of phenolic compounds in plant materials can be used for a better understanding of the potential health-beneficial effects.
The results of the spectrophotometric evaluation of total phenolics andflavonoid contents in tested Onosma extracts are shown inTable 1. The results showed that the methanolic extract of O. sericea was richer more than twice in total phenolic content compared with O. stenoloba extract (69.79 and 32.46 mg GAE/g, respectively). Also, the O. sericea extract had much higherflavonoid content (53.62 mg RE/g) compared Fig. 1. Total ion chromatogram of the methanolic extract of Onosma sericea in the negative (A) and positive ion mode (B).
with O. stenoloba. Both plants O. sericea and O. stenoloba were not studied so far regarding their phenolic content. OnlySivaci et al. (2015) showed that O. sericea was quite rich in total phenolics in vegetative and particularly in the reproductive period. On the other hand, many recent studies were focused on the determination of phenolic com-pounds in different Onosma species growing in Turkey. Hence, the study of Sarikurkcu et al. (2018) showed a high content of phenolic com-pounds in O. gigantea methanolic extract (9μmol GAEs), but with low quantity of totalflavonoids (2 μmol REs/g). The methanolic extract of another plant from genus Onosma which grows in Turkey, O. tauricum var. tauricum, contained a high amount of phenolic compounds (16 μmol GAEs/g), with a slightly higher content of total flavonoids (3 μmol REs/g) (Kirkan et al., 2018). Moreover, the methanolic extract of O. heterophyllum also contained a significant amount of phenolic com-pounds (7.5μmol GAEs/g), again with a lower flavonoid presence (0.6 μmol REs/g) (Ozer et al., 2018). In the cited studies, ethyl acetate and water extracts of respected Onosma species were also tested for total phenolic content and it was clearly showed that water extracts had a much higher content of both total phenolics andflavonoids compared with the methanolic extracts. In a recent study on O. isauricum extracts (ethyl acetate, methanolic, and water extracts) it was shown that me-thanolic extract had the highest content of total phenolics (63 mg GAE/ g) with highflavonoid quantity (43 mg RE/g) (Zengin et al., 2019). These results are comparable to those reported in this study, particu-larly for O. sericea extract, where it can be seen that O. sericea was slightly richer in phenolic compounds content than O. isauricum.
By chromatographic screening, twenty-two and twenty-seven com-pounds were identified in O. sericea and O. stenoloba samples, respec-tively, wherein ten compounds were similar for both extracts. The most important compounds presented in high amounts in both extracts are given in Fig. 3. The results are represented in Tables 2 and 3. The identification of compounds was performed by comparison of retention times, extracted mass and fragments with a literature survey, database data and data obtained for standards. Most of the compounds belong to phenolic andflavonoid groups, among them are apigenin, kaempferol, and their derivatives, as well as derivatives of rosmarinic acid, and salvianic acid A were the most represented. These compounds were also identified in O. tauricum (Kirkan et al., 2018). Earlier reports showed the presence of apigenin and its derivatives in O. hespidum (Kumar et al., 2013). In O. sericea, a fatty acid, namely stearic acid (m/z 283.26) was identified, the presence of it was confirmed before in O. irrigans species (Yuldasheva et al., 2013). O. stenoloba was characterized by the presence of pyrrolizidine alkaloids such as echimidine and heliosupine (m/z 398.22), intermedine and lycopsamine (m/z 300.18). Previously, echimidine was identified in O. alboroseum and O. stellulatum species; heliosupine and lycopsamine were found in O. alboroseum and O. are-naria (El-Shazly and Wink, 2014). In addition to the presentedfindings, the same studies regarding the genus Onosma or the family Bor-aginaceae were added inTables 2 and 3.
3.2. Antioxidant properties
Two radicals namely, DPPH·and ABTS·+, are used most frequently
complex to (Fe2+) complex (Antolovich et al., 2002). The CUPRAC assay represents a cupric reduction assay improved as a different ver-sion of the FRAP assay. This way measures the power of antioxidants to reduce cupric ion (Cu2+) to cuprous (Cu+) ion (Apak et al., 2004). Herein, FRAP and CUPRAC assays were used to determine the reducing power of Onosma extracts and the results are shown in Table 4. O. sericea extract exhibited much higher reducing activities (215.65 and 359.63 mg TEs per g extract in FRAP and CUPRAC assays, respectively) compared with O. stenoloba extract.
The total antioxidant capacity and chelating effects of Onosma ex-tracts were also tested. Again, the results followed a similar trend (Table 4) where O. sericea extract showed greater total antioxidant potential and metal chelating activity (2.46 mmol TE/g and 24.56 mg EDTAE/g, respectively) compared with the O. stenoloba. In the available literature, only antioxidant effects of O. sericea aerial parts extracts (in vegetative and reproductive periods) were tested on DPPH radical and showed significantly higher DPPH-scavenging potential in the re-productive period compared with the vegetative stage and also com-pared with O. rascheyana extracts (Sivaci et al., 2015). It can be linked with the high content of total phenolic compounds and pigments (chlorophyll b, carotenoids) in O. sericea extracts. In other research studies, several Onosma species showed noticeable antioxidant activity via different mechanisms.Özgen et al. (2003)reported that O. argen-tatum root extract exerted a high level of antioxidant activity in the TBA method. The study performed byMašković et al. (2015)showed that aerial part extract of O. aucheriana from Serbia had the prominent total antioxidant capacity, scavenging activity, and inhibitory effects against lipid peroxidation, mainly because of the high content of phenolic compounds, particularly rosmarinic acid as proven natural antioxidant (Alagawany et al., 2017). Also, recent studies of Onosma species from Turkey: O. heterophyllum (Ozer et al., 2018), O. gigantea (Sarikurkcu et al., 2018), O. tauricum var. tauricum extracts (Kirkan et al., 2018), and O. isauricum (Zengin et al., 2019), demonstrated high total anti-oxidant potential of selected species, along with good radical scaven-ging, reducing, and chelating properties. In all listed species, rosmarinic acid was the most dominant phenolic compound, which means that its antioxidant properties significantly contribute to the general anti-oxidant activity of Onosma species. In O. sericea and O. stenoloba ex-tracts tested in this study, rosmarinic acid was identified only in O. sericea, but its derivatives were presented in both extracts. Besides this, many other phenolics with proven antioxidant potential were founded in O. sericea and O. stenoloba extracts. For example, ferulic acid, pre-sented in both species, is well-known for its antioxidant potential, scavenging free radicals and enhancing the cell stress (Mancuso and Santangelo, 2014). Salvianic acid A, also detected in both extracts, exhibited many benefits among which antioxidant potential plays a crucial role (Zhang et al., 2016). Flavonoids (quercetin, kaempferol, apigenin) and their derivatives, which presence in extracts was verified in this study, possess quite diverse pharmacological activities, mostly based on their antioxidative effects (Procházková et al., 2011). Also, the presence of diosmin, diosmetin, cirsiliol, and verbascoside in O. sericea extract may significantly contribute, not only to its antioxidant poten-tial, but also to its overall biological activity (Alipieva et al., 2014; Arroo et al., 2014;Patel et al., 2013;Prasad et al., 2019).
3.3. Enzyme inhibitory properties
Alzheimer's disease (AD) is a progressive degenerative disease as-sociated with impaired neurological function. AD is asas-sociated with loss of cholinergic system by decreasing levels of acetylcholine in brain regions responsible for learning, memory, and behavioral functions. The cholinesterase inhibition increases the levels of acetyl choline in the synaptic region and enables the regulation of functions in these regions (dos Santos et al., 2018). The cholinesterase inhibition potential of Onosma extracts is shown inTable 5. In this study, O. stenoloba ex-tract showed higher cholinesterase inhibition activity (4.34 mg GALAE/ g for AChE, and 3.44 mg GALAE/g for BChE) compared with O. sericea extract (3.74 mg GALAE/g for AChE, and 0.51 mg GALAE/g for BChE). Diabetes mellitus is a metabolic disease characterized by chronically elevated plasma glucose levels. Inhibition of carbohydrate-hydrolyzing enzymes such asα-amylase and α-glucosidase is a significant strategy to
prevent hyperglycemia by controlling the breakdown of carbohydrates (Tundis et al., 2010). O. sericea and O. stenoloba extracts were also examined for their possibleα-amylase and α-glucosidase inhibition. As shown inTable 5, O. stenoloba extract exhibited higher inhibitory ac-tivity (43.74 mmol ACAE/g) than O. sericea (33.38 mmol ACAE/g) for the α-glucosidase inhibition, while for the α-amylase inhibition O. stenoloba showed lower inhibitory activity (0.89 mmol ACAE/g) than O. sericea (1.26 mmol ACAE/g).
Tyrosinase is an copper-containing enzyme which plays an im-portant role in the biosynthesis of melanin. Especially, the inhibition of tyrosinase activity is very important against photocarcinogenesis (Ullah et al., 2016). According to obtained results for tyrosinase inhibition shown inTable 5, O. sericea and O. stenoloba exhibited modest activity (136.35 and 135.68 mg KAE/g, respectively).
Many recentfindings showed the significant inhibitory activity of different Onosma species towards those five tested enzymes connected Fig. 3. The most important compounds in Onosma sericea and O. stenoloba extracts.
with chronic diseases (Kirkan et al., 2018;Mašković et al., 2015;Ozer et al., 2018;Sarikurkcu et al., 2018).
Observed enzyme inhibitory activity can be linked with the che-mical composition of the tested extracts. For example, echimidine,
heliosupine, inertmedine, and lycopsomine (from pyrrolizidine alka-loids) found in the chemical composition of the O. stenoloba have been reported as potential cholinesterase inhibitors (Benamar et al., 2017; Moreira et al., 2018). Also, rosmarinic acid and its derivatives have
10 10 4 10 21.95 Verbascoside C29H36O15 [M-H]− 623.19946 461.1677 161.0233 11 22.35 Kaempferol-O-hexoside C21H20O11 [M-H]− 447.09390 285.0409 199.0397 175.0028 151.0028 133.0281 107.0123 12 22.91 Quercetin-O-hexoside C21H20O12 [M-H]− 463.08856 301.0365 300.0284 271.0249 255.0288 178.9988 151.0021 13 23.11 Rosmarinic acid-O-hexoside C24H26O13 [M-H]− 521.13092 359.0735 341.0862 323.0775 197.0447 179.0339 161.0233 14 24.23 Apigenin-O-hexoside C21H20O10 [M+H]+ 433.11347 271.0600 153.0182 145.0286 119.0492 15 24.28 Methyl caffeate C10H10O4 [M+H]+ 195.06662 163.0389 145.0284 135.0442 117.0337 89.0386 16 24.60 Apigenin-O-rhamnosylhexoside C27H30O14 [M+H]+ 579.17130 433.1114 271.0601 153.0183 145.0284 119.0493 91.0542 17 24.91 Diosmin C28H32O15 [M+H]+ 609.18195 463.1255 301.0706 286.0471 129.0545 85.0290 71.0497 18 26.42 O-Methylrosmarinic acid isomer C19H18O8 [M-H]− 373.09241 197.0431 179.0341 174.9552 159.8630 135.0440 123.0438 19 29.92 Tricin C17H14O7 [M+H]+ 331.08178 315.0498 316.0577 287.0549 286.0468 270.0524 153.0184 20 29.95 Cirsiliol C17H14O7 [M-H]− 329.06680 314.0436 313.0363 299.0199 285.0407 271.0249 21 29.97 Diosmetin C16H12O6 [M+H]+ 301.07074 286.0474 258.0523 229.0484 153.0185 22 48.30 Stearic acid C18H36O2 [M-H]− 283.26462 265.2538
Rt:retention time, min.; Fr.: fragment.m/z.
Table 3
HPLC/MS/MS screening of methanol extract of Onosma stenoloba.
№ Rt Compound Formula MS1Exact mass Fr. 1 Fr. 2 Fr. 3 Fr. 4 Fr. 5 Fr. 6 References
1 1.10 C6 sugar alcohol C6H14O6 [M+H]+ 183.08687 165.0761 147.0651 129.0549 111.0444 99.0445 83.0497 2 1.13 Carnitine C7H15NO3 [M+H]+ 162.11250 103.0394 102.0919 85.0289 60.0815 57.0343 3 1.25 Galactosamine or Glucosamine C6H13NO5 [M+H]+ 180.08701 162.0759 144.0656 126.0551 98.0605 96.0449 84.0449 4 1.28 Betaine C5H11NO2 [M+H]+ 118.08654 59.0737 58.0658
5 1.30 Gluconic or Galactonic acid C6H12O7 [M-H]− 195.05048 177.0393 159.0287 129.0181 111.0069 99.0075 75.0072
6 1.34 Quinic acid C7H12O6 [M-H]− 191.05544 173.0077 171.0285 127.0390 111.0074 109.0280 93.0330
7 1.80 Adenosine C10H13N5O4 [M+H]+ 268.10358 136.0618 119.0346 69.0341 57.0343
8 4.43 Salvianic acid A C9H10O5 [M-H]− 197.04501 179.0341 135.0440 123.0438 72.9915
9 8.78 Intermedine C15H25NO5 [M+H]+ 300.18060 156.1020 138.0915 120.0810 94.0656 82.0657 54.7251 (Roeder et al., 1990)
10 14.85 Lycopsamine C15H25NO5 [M+H]+ 300.18110 156.1007 138.0912 120.0792 94.0645 82.0653 55.0547 (Ahmad et al., 2018)
11 18.00 Caffeoylshikimic acid isomer C16H16O8 [M-H]− 335.07602 179.0341 161.0232 135.0439
12 18.76 Heliosupine C20H31NO7 [M+H]+ 398.21732 380.2083 220.1331 120.0810 83.0497
13 19.06 Vicenin-2 C27H30O15 [M-H]− 593.15210 473.1090 383.0777 353.0672 503.1213
14 19.14 Echimidine C20H31NO7 [M+H]+ 398.21732 380.2083 220.1331 120.0810 83.0497 (Ahmad et al., 2018)
15 19.40 Ferulic acid C10H10O4 [M-H]− 193.04987 178.0263 149.0596 137.0227 134.0361 121.0275 (Sarikurkcu et al.,
2018) 16 20.48 Isoferulic acid C10H10O4 [M-H]− 193.04987 178.0263 149.0596 137.0227 134.0361 121.0275 17 21.90 Rosmarinic acid-di-O-hexoside C30H36O18 [M-H]− 683.19189 521.1281 359.0997 323.0779 197.0449 179.0339 161.0232 18 23.05 Quercetin-O-hexoside C21H20O12 [M-H]− 463.08826 301.7912 300.0276 271.0260 255.0298 151.0016 19 23.10 Rosmarinic acid-O-hexoside C24H26O13 [M-H]− 521.13037 359.0704 341.0892 323.0785 197.0450 179.0339 161.0233 20 24.06 Apigenin-O-hexoside C21H20O10 [M+H]+ 433.11380 271.0600 153.0182 145.0289 119.0491 21 24.34 Methyl caffeate C10H10O4 [M+H]+ 195.08774 163.0389 153.0548 145.0284 135.0442 117.0337 107.0496 22 24.60 Apigenin-O-rhamnosylhexoside C27H30O14 [M+H]+ 579.17125 433.1114 271.0601 153.0183 145.0284 119.0493 23 24.62 Kaempferol-O-hexoside C21H20O11 [M-H]− 447.09375 285.0409 284.0330 227.0335 256.0373 255.0297 24 25.16 Isorhamnetin-O-rhamnosylhexoside C28H32O16 [M-H]− 623.16528 315.0490 314.0439 300.0272 299.0213 255.0287 243.0295
25 28.13 Luteolin C15H10O6 [M-H]− 285.04074 217.0508 199.0396 175.0391 151.0025 149.0233 133.0283 (Sarikurkcu et al.,
2018) 26 29.79 Trihydroxyisoflavone C15H10O5 [M+H]+ 271.06000 243.0649 215.0232 153.0181 149.0233 91.0545
27 45.04 Ursolic acid C30H48O3 [M-H]− 455.35330 407.3308
shown promising effects in the treatment of AD and cardiovascular disease-induced dementia (Habtemariam, 2018).Zhang et al. (2016) showed that salvianic acid A can be used as a multifunctional com-pound for the treatment of AD. Generally, polyphenolic comcom-pounds can influence the synthesis of acetylcholine by inhibiting AChE and/or BChE activity, but they also can have cognitive benefits (Del Rio et al., 2013). Ferulic acid, with its antioxidant and anti-inflammatory prop-erties, could prove beneficial effects in AD, but also it can act as a natural α-glucosidase inhibitor and has beneficial effects on the re-duction of Diabetes mellitus in experimental models (Mancuso and Santangelo, 2014). Flavonoids and their derivatives have been reported to lower the risk for diabetes and obesity through different mechanisms, including inhibition of the key enzymes (Fraga et al., 2019). Rosmarinic and some other hydroxycinnamic acids and their derivatives showed significant anti-tyrosinase and photoprotection potential which candi-dates this group of polyphenolics for their use in cosmetic formulations (Taofiq et al., 2017). Flavonoids can act also as tyrosinase inhibitors, mostly by competitive inhibition for the oxidation of L-dopa by tyr-osinase. Some of the most activeflavonoids are quercetin, myricetin, and kaempferol, but their derivatives were found to be less effective than the corresponding aglycones (Chang, 2009).
3.4. In vitro and in vivo antigenotoxicity
The in vitro DNA protective ability of the O. sericea and O. stenoloba methanolic extracts at different concentrations (25, 50, 100, 200, and 400μg/mL) on hydroxyl radical is shown inFig. 4. In concentration range from 25 to 400 μg/mL, the DNA-protective effects of Onosma extracts against OH radical-induced DNA damage were dose-depen-dent, increasing with a higher dosage, indicating the protective effect of the extracts.
In the present study, the in vivo genotoxic effect and antigenotoxic potential of O. sericea and O. stenoloba extracts were studied against the EMS-induced DNA damage in the third instar larvae of D. melanogaster (Table 6). The analysis of the cells from the anterior midgut of D. melanogaster treated with the O. sericea and O. stenoloba extracts re-vealed that lower concentrations of both extracts (20 and 40 mg/mL) induced significant increases in the total score, which ranged from 47.3–37, when compared with the negative control (21.1). Larvae treated for 24 h with EMS alone induce significant increases in DNA damage with a total score of 150.1. No significant differences were observed between the group treated with 80 mg/mL and the negative control. Therefore, the same concentration was established along with EMS, and the third instar larvae were allowed to feed on it for 24 h. The
simultaneous treatment with 80 mg/mL plus EMS showed a significant reduction in DNA damage when compared with the treatment with EMS only. The antigenotoxic effect was evident after the treatment with both extracts with a percentage reduction of over 80 %.
To the best of our knowledge, there are no reports in the literature regarding the genotoxic and/or antigenotoxic potential of these two species. Therefore, the data presented here could be assumed as thefirst report on the antigenotoxic potential of O. sericea and O. stenoloba Table 4
The antioxidant activity of Onosma sericea and O. stenoloba extracts.
Samples DPPH scavenging (mg TE/ g extract)
ABTS scavenging (mg TE/ g extract)
FRAP (mg TE/g extract)
CUPRAC (mg TE/g extract)
Total antioxidant capacity (mmol TE/g extract)
Metal chelating activity (mg EDTAE/g extract)
O. sericea 130.23 ± 5.31*,a 235.53 ± 4.62a 215.65 ± 2.51a 359.63 ± 14.83a 2.46 ± 0.35a 24.65 ± 2.21a
O. stenoloba 53.96 ± 0.78b 95.60 ± 2.30b 76.48 ± 3.26b 142.88 ± 1.49b 1.16 ± 0.05b 5.51 ± 0.81b
Different superscript letters (a and b) in the same column indicate significant difference (p < 0.05).
* Values expressed are means ± S.D. of three parallel measurements. TE, trolox equivalents; EDTAE, EDTA equivalents.
Table 5
Enzyme inhibitory effects of Onosma sericea and O. stenoloba methanolic extracts.
Extracts AChE activity inhibition (mg GALAE/g extract)
BChE activity inhibition (mg GALAE/g extract)
Tyrosinase activity inhibition (mg KAE/g extract)
α-amylase activity inhibition (mmol ACAE/g extract)
α-glucosidase activity inhibition (mmol ACAE/g extract)
O. sericea 3.74 ± 0.18*,b 0.51 ± 0.06b 136.35 ± 1.43a 1.26 ± 0.07a 33.38 ± 6.44b
O. stenoloba 4.34 ± 0.11a 3.44 ± 0.70a 135.68 ± 1.44a 0.89 ± 0.02b 43.74 ± 0.16a
Different superscript letters (a and b) in the same column indicate significant difference (p < 0.05).
* Values expressed are means ± S.D. of three parallel measurements. AChE, acetylcholinesterase; BChE, butyrylcholinesterase; GALAE, galanthamine equivalents; ACAE, acarbose equivalents; KAE, kojic acid equivalents.
Fig. 4. Protective effect of O. sericea (A) and O. stenoloba (B) against hydroxyl radical-induced DNA damage. 1: DNA control; 2: DNA damage control; 3: standard drug quercetin (50 μM); 4-8: 25, 50, 100, 200, and 400 μg/mL. *p < 0.05 when compared with the negative control group; **p < 0.05 when compared with the EMS control group.
extracts. Polyphenols have a wide range of biological activities and have been increasingly studied due to its preventive and therapeutic potential (Pandey and Rizvi, 2009). The protective effect of extracts on DNA may be attributed to the phenolics andflavonoids presented in extracts or the synergies between them.
3.5. In vitro antimicrobial potential
The results of the antibacterial activity of O. sericea and O. stenoloba methanolic extracts evaluated by the microdilution method are re-ported inTable 7. Minimal inhibitory concentration (MIC) values for both extracts were in the range of 2.5−10 mg/mL. Both Onosma ex-tracts showed moderate antibacterial activity only on a few strains, namely A. chroococcum and E. coli with MIC values 2.5 and 5 mg/mL, respectively. O. sericea extract showed certain activity on Gram-positive strain M. lysodeikticus with MIC 10 mg/mL, while O. stenoloba extract exerted antibacterial properties on E. faecalis and A. tumefaciens with MIC values 5 and 10 mg/mL, respectively. The extracts applied at the concentration of 10 mg/mL had no influence on the growth and de-velopment of all other bacterial strains (MIC > 10 mg/mL). The same results were obtained for the yeast C. albicans which suggest the ab-sence of extracts’ activity. Opposed to our results, Khaledi et al. (2018) have recently demonstrated antimicrobial effect O. sericeum on E. fae-calis with MIC of 0.25 mg/mL.
Antifungal activity results of tested Onosma extracts (Table 7) were
in a similar range as for the bacterial strains. Both extracts were the most active on the growth of P. fastigiata and F. oxysporum, with MIC values 2.5 and 5 mg/mL, respectively. O. sericea extract also showed moderate activity on both applied Penicillium species in the MIC range of 2.5−5 mg/mL, while O. stenoloba was active only on P. cyclopium (MIC 10 mg/mL). Also, O. sericea showed moderate antifungal potential on both Trichoderma spp. with MIC 10 mg/mL. All other fungal strains were resistant to the impact of both tested extracts at the highest ap-plied concentration of 10 mg/mL. The reference standards, ery-thromycin, ketoconazole, and nystatin, showed their high antimicrobial activity with significantly lower MIC values (from 0.078 to 10 μg/mL) than tested extracts.
According to available literature, there are no significant reported data regarding the antimicrobial activity of aerial parts of the Onosma spp. tested in this study. Most of the scientific investigations were fo-cused on root extracts of other plant species from genus Onosma L. and isolated natural products, e.g.O. visianii, O. argentatum, O. hispidum (Naz et al., 2006;Özgen et al., 2003;Vukic et al., 2017).
4. Conclusion
To our knowledge, this study is thefirst report showing the phy-tochemical characterization and biological activity of O. sericea and O. stenoloba from Turkey. O. sericea extract exhibited higher antioxidant activity in all antioxidant assays, compared with O. stenoloba. O.
20 mg/mL 65.8 ± 0.81 21.1 ± 0.74 13.1 ± 0.5 0.00 ± 0.00 0.00 ± 0.00 47.3 ± 0.81*,‡ 40 mg/mL 61.4 ± 0.73 34.1 ± 0.82 4.5 ± 0.44 0.00 ± 0.00 0.00 ± 0.00 43.1 ± 0.24*,‡ 80 mg/mL 79.6 ± 0.74 16.1 ± 0.28 4.3 ± 0.94 0.00 ± 0.00 0.00 ± 0.00 24.7 ± 0.41‡
80 mg/mL + EMS 68.6 ± 0.54 30.3 ± 0.54 1.1 ± 0.21 0.00 ± 0.00 0.00 ± 0.00 32.5 ± 0.45*,‡ 91.2
a Values represented mean ± SEM from three independent experiments. b %R—percentage reduction.
c NC—negative control group. dEMS—ethyl methanesulfonate, 1 mM.
* p < 0.05 when compared with the negative control group. ‡ p < 0.05 when compared with the EMS control group.
Table 7
Antimicrobial activity of O. sericea and O. stenoloba methanol extracts and referent compounds.
MIC values*
Bacterial species O. sericea O. stenoloba Erythromycin Fungal species O. sericea O. stenoloba Ketoconazole
E. coli 5 5 0.156 A. niger > 10 > 10 0.625 M. lysodeikticus 10 > 10 0.078 A. glaucus > 10 > 10 2.5 E. faecalis > 10 5 10 T. longibrachiatum 10 > 10 1.25 P. aeruginosa > 10 > 10 > 10 T. harzianum 10 > 10 5 P.fluorescens > 10 > 10 0.3125 P. canescens 5 > 10 1.25 K. pneumoniae > 10 > 10 > 10 P. cyclopium 2.5 10 0.156 A. tumefaciens > 10 10 0.3125 D. stemonitis > 10 > 10 5 A. chroococum 2.5 2.5 5 P. fastigiata 2.5 2.5 10
Yeast Nystatin A. alternata > 10 > 10 5
C. albicans > 10 > 10 1.25 F. oxysporum 5 5 0.3125
MIC values of antibiotic and antimycotics are presented according to theZengin et al. (2017), as the part of the same study. * MIC - minimum inhibitory concentration values given as mg/mL for plant extracts and asμg/mL for referent compounds.
stenoloba extract showed a high level of inhibitory activity against acetylcholinesterase, butyrylcholinesterase, and α-glucosidase en-zymes. Both O. sericea and O. stenoloba showed moderated anti-microbial activity, but they were able to protect DNA from damage both in vitro and in vivo. The presence of different classes of phenolic com-pounds in both tested extracts may be the main reason for the exibited bioactivity. All experimental data showed that the tested extracts can be used for various purposes and focus on new studies in the direction of evaluating the most active metabolites and their potential benefits for human health.
Declaration of Competing Interest None.
Acknowledgments
This study was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Grant No.III43004).
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