Determination of the Chemical Composition, Antioxidant, and Enzyme Inhibitory Activity of Onosma mollis DC

Onosma species have long been used traditionally for respiratory tract infections, abdominal pain, wound treatment, burns, and constipation. This study aims to investigate the chemical composition and in vitro antioxidant and enzyme inhibitory activities of ethyl acetate (EtOAc), methanol (MeOH), and water extracts of Onosma mollis DC. MeOH extract was richer in both phenolics and flavonoids than other extracts (44.06 mg GAEs/g and 41.57 mg QEs/g, respectively). The findings obtained from the results of the chromatographic analysis also supported the results of the spectrophotometric analysis. The MeOH extract was the richest in terms of most of the phytochemicals screened. Apigenin 7-glucoside, luteolin 7-glucoside, rosmarinic acid, vanillic acid, and pinoresinol were over 1000.0 μg/g in MeOH extract. The extract in question showed the highest activity in phosphomolybdenum, DPPH, and ABTS radical scavenging and CUPRAC and FRAP reducing power activity assays (2.01, 3.33, 2.30, 1.48, and 0.79 mg/ml, respectively). The water extract presented the highest activity in the ferrous ion chelating assay (1.01 mg/ml). While EtOAc extract showed high activity in acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and α-glucosidase inhibitory activity tests (1.11, 1.49, and 1.07 mg/ml, respectively), MeOH extract showed significant efficacy in tyrosinase and α-amylase inhibitory activity assays (2.94 and 2.08 mg/ml, respectively). There was a high correlation between the total phenolics/flavonoids of the extracts and their antioxidant activities (correlation coefficients were over 0.9). In addition, the phytochemicals mentioned above were found to contribute significantly to the antioxidant activity. It was concluded that a more detailed analysis should be done to determine the compounds responsible for the enzyme inhibitory activities of the extracts.


Introduction
Plants and plant-based nutrients are an indispensable part of the human diet. Many researchers agree that, in addition to fruits and vegetables, many medicinal and aromatic plants that we consume in our daily lives serve as an additional source of natural antioxidants [1][2][3][4]. Studies have shown that plant-derived flavonoids, some alkaloids, phenolic compounds, some diterpenoids, and sulfur compounds have a high degree of antioxidant activity [4][5][6].
ese compounds help protect our body from the harmful effects of these compounds by eliminating oxidant substances that occur due to both normal metabolic reactions and some environmental conditions (gamma, UV, and X-ray radiation, etc.). erefore, plants continue to be of great interest as additional sources of antioxidant compounds [4,5,[7][8][9][10].
Alzheimer's disease (AD) is a critical neurodegenerative disorder affecting older people [11]. Various strategies have been developed to treat this health problem. Some of these strategies are targeting amyloid-beta (Aβ) aggregation and modulating enzymes responsible for metabolizing the amyloid precursor protein (APP) or tau phosphorylation [12]. e first pharmacological treatment method approved by the US Food and Drug Administration (FDA) in AD treatment is the use of cholinesterase (ChE) inhibitors. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) hydrolyze acetylcholine (ACh), impairing signal transduction. is situation is considered among the fundamental reasons for the progression of AD [13]. Some ChE inhibitors such as galantamine, rivastigmine, donepezil, or tacrine have been shown to reduce symptoms in AD patients. However, these agents exhibit some unwanted side effects that have limited their use [14][15][16].
erefore, researchers are screening plants for the discovery of new and alternative ChE inhibitors.
Melanin is a heteropolymer synthesized by the catalytic activity of tyrosine in melanosomes. Researchers have reported that metal ions TRP-1 and TRP-2 play a critical role in melanin synthesis [17][18][19][20]. However, the most crucial task in the melanogenesis process is fulfilled by tyrosinase. e use of tyrosinase inhibitors to treat hyperpigmentation disorders due to excessive melanin synthesis is considered a critical treatment approach. Tyrosinase inhibitors are also frequently used in skin whitening applications and are extremely important for the cosmetic industry. Over the last decades, researchers have been conducting intensive research on plants to discover new and effective tyrosinase inhibitors [21][22][23][24][25][26][27][28].
Diabetes is a severe disease that affects a large number of people worldwide [29,30]. It is evaluated in two categories, Type 1 or Type 2. e leading cause of type 2 diabetes is the imbalance between the transfer of blood sugar to tissues and insulin secretion. Postprandial hyperglycemia is one of the most important reasons for Type 2 diabetes to develop [31]. In the treatment of Type 2, the plasma glucose level is tried to be kept under control. One of the ways to achieve this is to stimulate insulin secretion through medical intervention or diet [30,32,33]. Another solution is to slow down the digestion of starch and lower the glucose concentration, which is the end product of digestion. us, the amount of glucose absorbed from the small intestine is reduced [33]. is strategy is more practical and economical than controlling insulin secretion [30]. Digestive enzymes such as α-amylase and α-glucosidase must be inhibited to limit glucose production [34][35][36]. In addition to the biological activities documented above, the researchers also analyze the plants for their antidiabetic activity.
Onosma, one of the important genera of Boraginaceae, consists of approximately 150 species distributed worldwide. Literature records show that only 10% of these species are reported in terms of their chemical constituents and biological activity potential. ese reports indicate that Onosma species are rich in shikonins and onosmins. ese species have long been used traditionally for respiratory tract infections, abdominal pain, wound treatment, burns, and constipation. Onosma species are also used to color foods, medicinal preparations, and wool because of the dye contained in the root [37] [38][39][40][41][42]. ere is also a report in the literature on the antioxidant activity of methanol (MeOH) extract from the aerial parts of O. mollis [43]. However, in the report in question, ethyl acetate (EtOAc) and water extracts, which include nonpolar compounds, were not evaluated, and the enzyme inhibitory activity of O. mollis was not included.
is study aimed to determine the chemical composition, antioxidant, and enzyme inhibitor activities of ethyl acetate (EtOAc), methanol (MeOH), and water extracts obtained from the aerial parts of Onosma mollis DC.
Aerial parts of the plants were used as the study material to obtain solvent extracts. Details of the extraction procedure can be found in the supplementary file.

Determination of the Phenolic Compositions of the
Extracts. Details of the spectrophotometric and chromatographic methods are given in the Supplementary file [44,45].

Statistical Analysis.
e details of the statistical analysis are presented in the Supplementary file. Table 1 shows the yields of extracts from O. mollis. e yield of the water extract was found to be higher than the others (14.9%). e yields of MeOH and EtOAc extracts were 4.58 and 3.91%, respectively. e phytochemical composition data of the extracts obtained by spectrophotometric method are also given in Table 1. e total amount of phenolic compounds of the extracts was found to be higher than the total amount of flavonoid compounds. e total amounts of phenolic and flavonoid compounds of the MeOH extract were 44.06 mg GAEs/g and 41.57 mg QEs/g, respectively. e EtOAc extract was extremely poor in these compounds (20.43 mg GAEs g and 1.69 mg QEs/g, respectively). e results of the chromatographic analysis are presented in Table 2. Apigenin 7-glucoside, luteolin 7-glucoside, rosmarinic acid, vanillic acid, and pinoresinol were found in MeOH extract at a concentration above 1000 μg/g. In addition, the MeOH extract was found to contain significant amounts of apigenin (679.43 μg/g), luteolin (581.55 μg/g), and 4-hydroxybenzoic acid (553.68 μg/g). On the other hand, vanillic acid and pinoresinol were higher concentrations in water extract than other phytochemicals (896.81 and 841.68 μg/g, respectively). Compared to the others, the EtOAc extract was the poorest in terms of phytochemicals screened. Apigenin 7-glucoside in this extract stood out with its concentration (752.01 μg/g) than other phytochemicals. Table 3 shows the antioxidant activities of the extracts. MeOH   extract showed higher activity than others in all the tested assays except for the ferrous ion chelating activity. e activity data exhibited by the MeOH extract in phosphomolybdenum, DPPH and ABTS radical scavenging, CUPRAC, and FRAP tests were 2.01, 3.33, 2.30, 1.48, and 0.79 mg/ml, respectively. e water extract exhibited higher ferrous ion chelating activity test activity than other extracts (1.01 mg/ml). It was followed by MeOH and EtOAc extracts, respectively (2.40 and 2.58 mg/ml, respectively). None of the extracts exhibited as high antioxidant activity as trolox and EDTA used as positive control agents. Table 4 shows the AChE, BChE, tyrosinase, α-amylase, and α-glucosidase inhibitory activity data. e results were given as IC 50 and the equivalent of galantamine, kojic acid, and acarbose. None of the extracts exhibited as high antioxidant activity as the positive control agents. e EtOAc extract exhibited 1.11 and 1.49 mg/ml activity in the AChE and BChE inhibitory activity tests, respectively. It was followed by the MeOH extract (1.41 and 6.72 mg/ml, respectively). e water extract exhibited relatively weak inhibitory activity (11.54 and >20.00 mg/ml, respectively). In the case of tyrosinase inhibitory assay, the MeOH extract showed the highest activity (2.94 mg/ml). It was followed by EtOAc and water extracts, respectively (3.66 and 4.54 mg/ml, respectively). In the α-amylase inhibitory activity test, the MeOH extract showed the highest activity (2.08 mg/ml), while the α-glucosidase inhibitory activity test resulted in the superiority of the EtOAc extract (1.07 mg/ml). e water extract showed weaker inhibition in both activity tests than the others (16.88 and 4.12 mg/ml, respectively).

Correlation Coefficients between the Parameters.
e correlation between total phenolic/flavonoid data and activity tests is given in Table 5. e same table also presents the correlation coefficients between biological activity parameters and phytochemicals found in high amounts in the extracts. e correlation coefficients between the total phenolics/flavonoids of the extracts and the results obtained from the phosphomolybdenum, DPPH radical scavenging, CUPRAC, and FRAP assays were over 0.9. According to the data in the table, there was also a high correlation between the antioxidant activities of the extracts with apigenin 7-glucoside, luteolin 7-glucoside, rosmarinic acid, vanillic acid, pinoresinol, and apigenin. However, apart from the correlation between apigenin and tyrosinase inhibitory activity (0.924), no significant correlation was found between the phytochemicals in question and enzyme inhibitory activity.

Discussion
According to the literature research, it was determined that the chemical composition of O. mollis was investigated previously by both spectrophotometric and chromatographic methods [43]. e amounts of phenolic and flavonoid compounds reported in this report were considerably lower than those obtained from the current study. In addition, all of the phytochemicals found in high amounts in the present study were higher than those in the report of Tlili et al. [43]. Although the same plant species were analyzed in both studies, it was seen that the plant species analyzed in these two studies were collected from different altitudes. is situation is thought to cause a difference in climatic conditions and, therefore, in chemical composition.
In many studies on the chemical composition of Onosma species, apigenin 7-glucoside [50][51][52][53], luteolin7glucoside [43,[50][51][52][53], rosmarinic acid [43,[50][51][52][53][54][55], vanillic acid [40,50,53], and pinoresinol [50][51][52] were found to be present in high amounts. erefore, it was understood that the data obtained from the present study support the literature data.   e antioxidant activity of O. mollis was previously reported by Tlili et al. [43]. As in the section above, where chemical composition data were discussed, the antioxidant activity data obtained from the present study were higher than the data in the literature. Tlili et al. [43] reported the antioxidant activity of O. mollis in phosphomolybdenum, DPPH, and ABTS radical scavenging, CUPRAC and FRAP reducing activity, and ferrous ion chelating activity tests as 3.95, 14.22, 8.73, 4.38, 2.53, and 1.95 mg/ml, respectively. e main reason for the difference between the data obtained from the current study and the data published by Tlili et al. [43] is that the polarity gradient of the extracts in the current study is wider. Unlike the previous study, EtOAc and water extracts were also isolated in the current study. While nonpolar phytochemicals are soluble in EtOAc extract, those with high polarity dissolve in water extract. erefore, these phytochemicals exhibit different antioxidant activity than those with moderate polarity (MeOH extract).
Many researchers agree that there is a significant relationship between the chemical composition of plants and their antioxidant activities [56][57][58]. Some reports in the literature showed apigenin 7-glucoside, luteolin 7-glucoside, rosmarinic acid, vanillic acid, and pinoresinol, which were found in high concentrations in the current study, may contribute to antioxidant activity [50][51][52][53]59]. ese literature data support the data obtained from the current study.
Considering the data in Table 2, it is thought that apigenin 7-glucoside in EtOAc extract may contribute to cholinesterase inhibitory activity. ere are no reports in the literature that this phytochemical has cholinesterase inhibitory activity. However, some reports indicate that some Onosma species containing high amounts of apigenin 7glucoside exhibit significant cholinesterase inhibitory activity [50][51][52][53]. erefore, it is considered that both the AChE and BChE inhibitory activities of the compound in question should be evaluated in future studies.
As can be seen from the section, where the chemical compositions of the extracts were discussed, the MeOH extract was found to be rich in apigenin 7-glucoside, luteolin 7-glucoside, rosmarinic acid, vanillic acid, and pinoresinol. us, these phytochemicals were thought to be possible compounds contributing to activity. To have more convincing information about the tyrosinase inhibitory activity potential of these compounds, it is helpful to look at some literature data. A study conducted by Aghraz et al. [60] reported that Bubonic imbricatum and Cladanthus arabicus exhibited significant tyrosinase inhibitory activity, and both aglycon and glycosidic forms of apigenin were among the main components. In another study conducted by Sarikurkcu et al. [50], the MeOH extract of O. ambigens exhibited higher tyrosinase inhibitory activity than other extracts and was rich in apigenin 7-glucoside and luteolin 7-glucoside. ere are also some reports in the literature that plant extracts rich in rosmarinic acid [61][62][63], vanillic acid [64,65], and pinoresinol [66] show significant tyrosinase inhibitory activity.
It is thought that the superiority of the MeOH extract in the α-amylase inhibitor activity test may be due to the compounds indicated in Table 1 and also expressed in the sections above. ere are some reports in the literature that these compounds themselves or extracts rich in these compounds have high α-amylase inhibitor activities. In a study conducted by Witkowska-Banaszczak et al. [67], it was reported that Succisa pratensis rich in luteolin and apigenin in glycosidic forms exhibited significant α-amylase inhibitory activity. In another study carried out by Uysal et al. [61], it was reported that the α-amylase inhibitor activity of Salvia ceratophylla might be due to luteolin and rosmarinic acid. In a study in which the α-amylase inhibitory activities of different Brassica oleracea subspecies collected from northcentral Italy were analyzed, it was reported that vanillic acid could be one of the compounds contributing to the activity [68]. However, in a study conducted by Mwakalukwa et al. [69], it was reported that the α-amylase and α-glucosidase inhibitory activity of pinoresinol was weak.
ere is also evidence in the literature that vanillic acid exhibits more than 40% inhibitory activity on glucosidase [70]. is finding is consistent with the data obtained from the current study.
To the best of our knowledge, AChE, BChE, and α-glucosidase inhibitory activity of O. mollis have not previously been reported elsewhere. erefore, data presented on these parameters could be assumed as the first report for the literature.

Conclusion
In this study, the in vitro antioxidant and enzyme inhibitory activities of the extracts obtained from the aerial parts of O. mollis were analyzed. It was concluded that the extracts have antioxidant activity comparable to positive control agents, and therefore, they could be assumed as one of the sources rich in antioxidants. In addition, it was concluded that MeOH and EtOAc extracts exhibited α-amylase and α-glucosidase inhibitory activity comparable to acarbose and, therefore, could be the source of new and alternative compounds in the treatment of diabetes. However, it is thought that more detailed analysis should be carried out to determine the phytochemicals responsible for the activity in these extracts.

Data Availability
e data used to support the findings of this study are included within the article.