Phytochemical Composition, Antioxidant, and Enzyme Inhibition Activities of Methanolic Extracts of Two Endemic Onosma Species

Onosma species have been used as a dye for hundreds of years due to their dark red pigments. These species have also been used by mankind in the treatment of various diseases since ancient times. This work analyzed the phytochemical composition in methanol extract of two endemic Onosma species (O. lycaonica and O. papillosa). Methanolic extract of these species varied in the content of flavonoids and phenolics. The flavonoids were found higher in O. papillosa [32.9 ± 0.3 mg QEs (quercetin equivalent)/g extracts] while the phenolics were higher in O. lycaonica [43.5 ± 1.5 mg GAEs (gallic acid equivalent)/g extracts]. ESI-MS/MS (electrospray ionization-mass spectrometry) revealed the presence of 25 compounds in O. lycaonica and 24 compounds in O. papillosa. The former was richer than the latter for apigenin, luteolin, eriodictyol, pinoresinol, apigenin 7-glucoside, rosmarinic acid, luteolin 7-glucoside, ferulic acid, vanillin, caffeic acid, 4-hydroxybenzoic acid, (+)-catechin3,4-dihydroxyphenylacetic acid. The O. papillosa exhibited low EC50 (1.90 ± 0.07 mg/mL) which indicated its strong phosphomolybdenum scavenging activity as compared to O. lycaonica. However, the O. lycaonica showed low IC50 or EC50 for 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+), cupric reducing antioxidant power (CUPRAC), ferric reducing antioxidant power (FRAP) and ferrous ion chelating activity, as compared to O. papillosa. The results proved the presence of potent antioxidant compounds in O. lycaonica. Further, the plant extracts significantly varied for enzyme inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), but the plant extracts did not significantly differ for inhibition of α-glucosidase, α-amylase, and tyrosinase. Onosma species deserve further research towards developing novel drugs to treat oxidative diseases.


Introduction
Onosma genus contains species rich in shikonins. Particularly the red dye of Onosma sp., has been used since ancient times both in fabric dyeing (especially in silk dyeing) and a dyestuff in foods [1]. Also, this pigment offers alternative solutions for cosmetics and medicine due to its bioactivity [2,3]. Hence, researchers have been prompted to analyse the bioactivities of Onosma sp. [4]. Oxidative stress stimulates various diseases. Free radicals and reactive oxygen species are oxidants formed as intermediates during various metabolic functions. Oxidants are simultaneously scavenged by elevated synthesis of suitable enzymes (superoxidase dismutase, hydroperoxides, catalase). Therefore, a balance between oxidants and antioxidants is essential to ensure a healthy metabolic activity defense system. However, if unbalancing of oxidants and antioxidants occurs, will lead to the oxidative stress [5,6], which is known to cause various diseases including atherosclerosis, coronary heart disease and aging diseases (cancer, arthritis, osteoporosis, cataracts, type 2 diabetes, Alzheimer's disease and hypertension) [7]. Several synthetic antioxidant molecules are consumed to prevent oxidative stress-related diseases but they cause adverse effects [8]. The consumption of antioxidant-rich foods such as green tea, fresh vegetables and fruits successfully prevents oxidative stress. Hence, it is essential to discover novel antioxidants from natural resources to erase the risk of oxidative stressmediated diseases.
Onosma species (family Boraginaceae) are globally distributed medicinal plants with a total of 180 species. In Turkey, alone there are 85 species of Onosma, including more than 40 endemic species [17,18], and they are traditionally used in the country as natural remedies to cure fever, bladder pain, kidney infections, blood diseases, burns, abdominal pain and wound healing [19]. The phytochemistry and ethnopharmacology of several Onosma spp., have been documented, but not for two endemic Onosma spp., (O. lycaonica and O. papillosa). Hence, the present work analyzed the phenolic and flavonoid compounds from these Onosma spp. by using LC-ESI-MS/MS, and also tested their bioactivities in terms of antioxidation and enzyme inhibition properties.
Onosma species are important medicinal plants due to their promising pharmacological properties, including antioxidant, anti-inflammatory, cytotoxicity, and enzyme inhibition activities [20], Moreover, these plants are traditionally recognized as folk medicines to cure several aging-related diseases [19]. Generally antioxidant, antimicrobial, enzyme inhibitory, anti-inflammatory, and anti-cardiovascular disease biological activities are related to the phenolics, tannins and flavonoids present in plants [21]. It is essential to study the phytochemistry of plants to establish a basis for the isolation of novel compounds as drug candidates to treat the aforesaid diseases. Therefore, the present study examined the phytochemical composition in the methanol extract of two endemic Onosma species (O. lycaonica and O. papillosa) by spectrophotometric and LC-ESI-MS/MS methods.

Phytochemical Composition
Although ESI-MS/MS is a frequently used method for the quantification the phyto compounds, it is essential to standardize the operating conditions for sensitive target compounds based on their MRM ionization modes. Therefore, the present study standardized the analytical parameters of LC-ESI-MS/MS in response to negative and positive ionization using the standard molecules (Table S1). After the establishment of LC-MS operating conditions, a total of 31 standard flavonoids/phenolic compounds were used at different concentrations to prepare a standard curve. The results of standard compounds were fitted to the calibration curve, and their linear equations and R 2 values are presented in the Supplementary Information (Table S2). The LC-MS/MS mediated quantification of phytocompounds in two Onosma species are presented in Table 2, which shows that out of 31 compounds studied, a total of 25 compounds were present in O. lycaonica while 24 compounds were observed in O. papillosa ( Figure 1 [11,13,20,[24][25][26]. Moreover, (+)-catechin and eriodictyol were present in O. lycaonica but absent in O. papillosa. Similarly, the earlier research on the phytochemical analysis of Onosma species has indicated that the compounds such as pyrocatechol, (−)-epicatechin, taxifolin and 2-hydroxycinnamic acid are not observed in Onosma species while (+)-catechin and eriodictyol are not commonly observed in Onosma species [11,13,20,[24][25][26]. Levels of a total of 15 compounds (apigenin, luteolin, eriodictyol, pinoresinol, apigenin 7-glucoside, rosmarinic acid, luteolin 7-glucoside, ferulic acid, vanillin, caffeic acid, 4-hydroxybenzoic acid, (+)-catechin3,4-dihydroxyphenylacetic acid) were found to be high by their concentration in the methanolic extract of O. lycaonica compared to O. papillosa. These variations in quantity and occurrence of phytochemicals of Onosma species is probably due to difference in the extraction methods and ecological conditions of the plant species (climate, soil properties and altitude) [24].

Antioxidant Properties
Most oxidative stress-related human diseases occur due to impairment of the balance between oxidant and antioxidant molecules. The screening of the antioxidant activity of plant extracts provides a basis for discovering novel phytocompounds with bio-health-promoting efficiency. The free radicals such as DPPH, ABTS and phosphomolybdenum are scientifically accepted for the screening of antioxidant molecules from medicinal plants [24,27]. Therefore, the current study screened the free radicals for scavenging activity of two Onosma species in comparison with standard antioxidants such as Trolox, ethylenediaminetetraacetic acid, butylated hydroxyanisole, and butylated hydroxytoluene, and the results are shown in Table 3. Phosphomolybdenum scavenging occurs through reduction of Mo (V) to Mo (V) by the interaction of antioxidant molecules from plant extracts or other antioxidant molecules with phosphomolybdenum [28]. In the present work, the methanol extract of the two plants showed a considerable level of phosphomolybdenum scavenging activity, and the EC 50 value was found to be low for O. papillosa (1.90 ± 0.07 mg/mL) as compared to O. lycaonica (2.05 ± 0.07 mg/mL  DPPH is a stable free radical and its scavenging reaction occurs through replacement of the nitrogen atom a by hydrogen atom of an oxidant molecules [29]. In the current study, O. lycaonica exhibited a stronger DPPH savaging activity than O. papillosa, as indicated by a lower IC 50 value of 2.69 ± 0.10 mg/mL and a higher Trolox equivalent value (92.6 ± 3.6 mg TEs/g extract) of O. lycaonica as compared to O. papillosa (IC 50 −3.41 ± 0.05 mg/mL, 73.1 ± 1.0 mg TEs/g extracts). A similar trend of IC 50 and Trolox equivalent was also observed for ABTS scavenging activity. The IC 50 and Trolox equivalents of the ABTS+ scavenging significantly varied between the plants studied, with a low IC 50 (2.18 ± 0.01 mg/mL) and high Trolox equivalent value (130.6 ± 0.4 mg TEs/g extract) for O. lycaonica. Thus, O. lycaonica was a stronger ABTS+ scavenger as compared to O. papillosa. The sample that exhibits less IC 50 , EC 50 with high Trolox equivalent is considered to be stronger in antioxidant activity than the sample showing high IC 50 , EC 50 with low Trolox equivalent [11] and this is in accordance with our results on free radical scavenging activity of the Onosma species.
Antioxidant molecules scavenge free radicals such as DPPH and ABTS+ through an electron or hydrogen atom transfer mechanism whereas FRAP reduction occurs through an electron reaction related to pH [30]. The methanol extract of O. lycaonica showed a low EC 50 for CUPRAC reduction (1.10 ± 0.01 mg/mL) and FRAP reduction (0.69 ± 0.01 mg/mL) as compared to O. papillosa. The EC 50 of CUPRAC and FRAP reduction along with the Trolox equivalents indicated that O. papillosa was potent in reducing the CUPRAC and FRAP. O. lycaonica showed higher ferrous ion chelating activity than O. papillosa, which was evidenced by the low IC 50 (2.32 ± 0.16 mg/mL) and high EDTAEs (21.60 ± 1.45 mg EDTAEs/g extract) of O. lycaonica (Table 3). Moreover, both plants exhibited low EC 50 or IC 50 values compared to standard antioxidants tested such as Trolox, ethylenediaminetetraacetic acid, butylated hydroxyanisole, and butylated hydroxytoluene (Table 3). Overall, the methanol extract of O. lycaonica exhibited better antioxidant activity than the O. papillosa one, but the phosphomolybdenum scavenging activity difference between the plants was not significant (p < 0.05). The higher antioxidant activity of O. lycaonica might be attributed to the presence of high levels of antioxidant molecules as evidenced by LC-ESI-MS/MS. The LC-ESI-MS/MS analysis revealed that the total amounts of 15 compounds were found to be higher in O. lycaonica than O. papillosa (Table 2). Particularly, the antioxidant substances such as apigenin [14], luteolin [31], pinoresinol [32], apigenin 7-glucoside [14], rosmarinic acid [33], ferulic acid [34], vanillin [35], and caffeic acid [36] were found to be higher in the methanol extract of O. lycaonica.

Enzyme Inhibition Assay
The enzyme inhibitory effect of methanol extract of two Onosma species (O. lycaonica and O. papillosa) was assessed for Alzheimer's disease (AChE, BChE), diabetes (α-amylase and α-glucosidase) and photocarcinogenesis (tyrosinase)-related enzymes by spectrophotometric assays and the results are shown in Table 4. Moreover, the enzyme inhibitory activity was compared to standard enzyme inhibitors such as galantamine for AChE, and BChE, acarbose for α-amylase and α-glucosidase, kojic acid for tyrosinase.
The AChE and BChE inhibition activities varied significantly between the plants, but did not vary for α-glucosidase, α-amylase, and tyrosinase inhibition activities (p < 0.05). O. lycaonica exhibited high AChE inhibition activity (IC 50 −1.32 ± 0.02 mg/mL) while O. papillosa showed high BChE inhibition (IC 50 −4.96 ± 0.07 mg/mL). Thus, both plants showed potent inhibition of the targeted enzymes. Although the IC 50 and standard galantamine equivalent, acarbose equivalent and kojic acid equivalent did not vary between the two Onosma species analyzed in the present study, an earlier work has reported significant variations between the enzyme inhibitory activity of O. sericea and O. stenoloba [12]. Thus the methanol extract of Onosma species is a promising source for the isolation of enzyme inhibitors, as disclosed in earlier reports [12,22,24].

Standard Phytochemicals and Chemicals
2,5-Dihydroxybenzoic acid, pyrocatechol, chlorogenic acid, 4-hydroxybenzoic acid, (−)-epicatechin, caffeic acid, gallic acid, (+)-catechin, vanillin, syringic acid, taxifolin, p-coumaric acid, sinapic acid, ferulic acid, 2-hydroxycinnamic acid, rosmarinic acid, pinoresinol, luteolin, quercetin, apigenin and HPLC grade of methanol and formic acid were obtained from Sigma-Aldrich (St. Louis, MO, USA). 3-Hydroxybenzoic acid, vanillic acid, apigenin 7-glucoside, 3,4-dihydroxyphenylacetic acid, luteolin 7-glucoside, eriodictyol, hesperidin, and kaempferol were obtained from Fluka (St. Louis, MO, USA). The hyperoside protocatechuic acid and verbascoside were purchased from HWI Analytik (Ruelzheim, Germany). Ultra-pure water (18.2 mΩ/cm) was prepared by using a Milli-Q water purification system (Milli-Q Millipore Merck KGaA Darmstadt, Germany). All the chemicals and reagents used in the biological assay were obtained from Sigma-Aldrich. . The aerial parts of the samples were separated, and shadow air-dried for several weeks without direct exposure of sunlight. Afterwards, these samples were cut into small pieces. The plant sample weighed at 5 g was immersed in 100 mL of methanol at ambient room condition for 24 h and filtered using the Whatman No.1. filter paper. The extraction was repeated two times and the extracts were pooled together for each plant species, and concentrated using a rotary evaporator [11] and the extracts were preserved at 4 • C for further experiments.

Analysis of Total Phenolics and Flavonoids
The content of total phenolics in the plant extracts was determined by spectrophotometric assay using Folin-Ciocalteu reagent according to the methods described earlier [37,38]. In brief, 0.25 mL of plant extract was mixed with Folin-Ciocalteu reagent (1 mL, 1:9) and vigorously vortexed for 3 min followed by 0.75 mL of 1% Na 2 CO 3 were added and incubated for 2 h in room temperature. After incubation, the sample was measured for optical density (OD) at 760 nm, and the content of total phenolics is presented as gallic acid equivalents. The total flavonoids in the extracts were measured according to the methods described earlier [39]. In brief, the 2% of aluminum chloride solution was prepared in methanol. Then the plant extract was mixed with AlCl 3 solution at 1:1 ratio. A blank, prepared by mixing methanol with AlCl 3 at the same ratio. The plant and blanks were incubated at room temperature for 10 min then the OD was measured at 415 nm, and the content of flavonoids is presented as quercetin equivalents.

Analysis of Phytochemical Composition by LC-ESI-MS/MS
The selected phytochemical constituents were analysed in the methanolic extracts of Onosma spp., by using LC-ESI-MS/MS (1260 Infinity liquid chromatography system hyphenated to a 6420 Triple Quad mass spectrometer, Agilent Technologies, Santa Clara, CA, USA) equipped with Poroshell 120 EC-C18 (100 mm × 4.6 mm I.D., 2.7 µm) column. In order to analyze the compounds, the mobile phases were prepared using different combinations of formic acid, ammonium acetate, methanol and acetic acid according to target compounds isomeric resolution as described in our earlier study [40]. The LC-ESI-MS/MS analysis was operated according to the methods described earlier [40].

Statistical Analysis
Biological assays and phytochemical composition analysis were performed for three times. The statistical analysis such as descriptive statistics, and one-way ANOVA and post hoc test (Tukey's) and student t-test were performed to observe the significance (p < 0.05) between extracts (O. lycaonica and O. papillosa) by using statistical software package SPSS v. 22.0 (PSS Inc, Chicago, IL, USA).

Conclusions
This work reports the phytochemical composition, antioxidant and enzyme inhibitory activity of two Onosma species endemic to Turkey. The methanol extract of O. lycaonica exhibited high levels of~15 antioxidant-related compounds and antioxidant activities as compared to O. papillosa. Both plant species showed considerable enzyme inhibitory activity. The present results evidenced that these endemic Onosma species (O. lycaonica and O. papillosa) represent promising sources for the isolation of pharmacologically important drug candidate molecules to treat various oxidative diseases, including diabetes, cancer, Alzheimer's disease and photocarcinogenesis.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/plants10071373/s1, Table S1: ESI-MS/MS Parameters and analytical characteristics for the Analysis of target analytes by MRM Negative and Positive Ionization Mode, Table S2: Calibration curves and sensitivity properties of the method.

Data Availability Statement:
The data presented in this study are available in the article.