Exploring the Pharmacological Potential of Onosma riedliana: Phenolic Compounds and Their Biological Activities

Onosma riedliana Binzet & Orcan, a traditionally used plant species, has been explored for its therapeutic potential in this study. The work presented here is the first report on the phenolic profile and biological activity of this species. Three extracts of varying polarity were prepared, with the methanolic extract containing the highest phenolic content (97.62 ± 0.20 mgGAE/g). Key phenolic compounds identified included pinoresinol, hesperidin, 4-hydroxybenzoic acid, and p-coumaric acid. The methanolic extract exhibited exceptional antioxidant properties, rivaling Trolox as a positive control, primarily attributed to hesperidin and luteolin. Moreover, the ethyl acetate extract demonstrated remarkable inhibition of cholinesterase and tyrosinase enzymes, while the methanolic extract displayed potent activity against carbohydrate hydrolytic enzymes, α-amylase and α-glucosidase. Again, phenolic compounds were shown to be responsible for the inhibition of cholinesterases and tyrosinase, but not for α-amylase and α-glucosidase. These findings underscore Onosma riedliana’s potential for incorporation into diverse pharmaceutical formulations, given its multifaceted bioactivity. Supplementary Information The online version contains supplementary material available at 10.1007/s11130-023-01131-0.


Introduction
Throughout history, medicinal and aromatic plants have played a crucial role in treating a wide range of disorders.Phenolic compounds are found to be the major carriers of plants therapeutic properties.Numerous studies have highlighted the protective role of plant phenolics in degenerative diseases, including cardiovascular issues, cancer, diabetes, inflammation, and more [1].The Onosma genus comprises

Materials and Methods
The materials and methods section is presented as supplementary material.

Chemical Composition
The yields of extracts obtained from O. riedliana varied among the three solvents used, with the ethyl acetate extract yielding 2.03%, the methanolic extract 0.41%, and the aqueous extract 9.28%.The selection of solvents for extracting active compounds from medicinal and aromatic plants depends on several factors, including the plant type, the specific plant part to be extracted, and the nature of the bioactive compounds.To capture a broader spectrum of phenolic compounds, this study employed three solvents with varying polarities.As per the spectrophotometric assessments of total phenolic and flavonoid content in O. riedliana extracts (Fig. 1), the methanolic extract exhibited significantly higher levels of these compounds compared to the other two extracts.Specifically, the methanolic extract contained 97.62 ± 0.20 mg GAE/g of phenolic compounds and 54.98 ± 0.05 mg RE/g of flavonoids (Fig. 1).In comparison to other Onosma species, O. riedliana showcased similar levels of both phenolic and flavonoid compounds as O. aucheriana [18], yet significantly higher levels than in other species within the genus [5][6][7][8][9][10][11][12][13][14][15][16], where the total phenolic content did not exceed 70 mg GAE/g of extract.
The detailed phenolic composition of the plant extracts was analyzed using LC-MS/MS, and the results are summarized in Table 1.The predominant compound in both the ethyl acetate and methanolic extracts was the tetrahydrofuran lignan pinoresinol, with levels of 2992.0 ± 38.9 and 73628.2± 1256.9 μg/g of extract, respectively.This phenol, also found in some Brassicae vegetables, sesame seeds, and olives, possesses strong antioxidant [17] and hypoglycemic [18], and antibacterial properties [19].It's worth noting that while pinoresinol is present in other Onosma species, it is typically found at significantly lower levels.The aqueous extract contained the lowest amounts of flavonoids, whereas the methanolic extract was particularly rich in hesperidin, with a concentration of 32455.3 ± 27.7 μg/g.Hesperidin, primarily found in citrus species, also stood out as the main flavonoid in the ethyl acetate extract of O. riedliana (Table 1).Furthermore, other Onosma species, such as O. trapezuntea and O. rigidum [6], O. nana [7], O. papillosa [10], O. stenoloba [14], and O. mollis [16], have been found to contain significant levels of this flavonoid.

Antioxidant Activity
To assess the antioxidant potential of O. riedliana more comprehensively, various assays were employed, each based on distinct mechanisms of action.These included reducing stable molybdenum (phosphomolybdenum assay), ferric (FRAP assay), and cupric (CUPRAC assay) cations, as well as reducing stable radicals (DPPH and ABTS assays) and the ability to chelate ferrous ions.The results are summarized in Table 2 and expressed as IC 50 values (mg/mL), indicating the concentration needed to scavenge, reduce, or chelate 50% of the potential oxidant.Among the O. riedliana extracts investigated, the methanolic extract exhibited the most potent antioxidant activity across all six assays.In many studies, different antioxidant assays relying on various reaction mechanisms can lead to challenging and inconsistent result interpretation and comparison.To address this issue, relative antioxidant capacity indices (RACI) have been introduced.These indices offer a numerical scale that integrates multiple chemical methods, enabling the

Enzyme Inhibitory Activity
The exploration of O. riedliana's potential as a source of enzymatic inhibitors aligns with the growing interest in natural remedies for different disorders and the development of safer and more sustainable treatment options.The results presented in Table 3 highlight the differential inhibitory activities of the three O. riedliana extracts against various enzymes.The ethyl acetate extract exhibited the lowest IC 50 values for the inhibition of cholinesterases, α-amylase, and α-glucosidase, while the methanolic extract showed the most favorable outcomes in inhibiting tyrosinase.Additionally, the results are expressed as equivalents of positive probes used in these assays, such as galanthamine (mg GAL/g), kojic acid (mg KA/g), and acarbose (mg AC/g).In this context, the ethyl acetate extract displayed the highest values, except for the inhibition of tyrosinase.Similar findings were reported for O. tauricum var.tauricum [8], O. gigantea [11], and O. pulchra [11], where ethyl acetate extracts demonstrated superior activities against these comparison of antioxidant capacity without being limited to a specific mechanism.RACI values for the methanolic extract of O. riedliana are notably high (Table 2), falling within the range of the most potent antioxidant plant-based foods [20].However, there are limited reports on RACI values for other Onosma species, which generally exhibited lower RACI values than O. riedliana.For instance, methanolic extracts of O. aucheriana, O. frutescens, and O. sericea had RACI values of -0.49, -0.34, and 0.89, respectively [13], while O. ambigens exhibited a value of 0.90 [14].
The correlation matrix, presented in Fig. 2, illustrates the relationships between the antioxidant activity of three O. riedliana extracts, and the levels of the most abundant phenolic compounds (> 100.00 μg/g) present in these extracts.Remarkably, all the phenolic compounds, with the exception of 2,5-dihydroxybenzoic and 3,4-dihydroxyphenylacetic acids, exhibited notably high correlations with both the radical scavenging assays and the assays related to reducing stable cations.When considering correlation data from other Onosma species, it becomes evident that flavonoids tend to yield higher Pearson correlation coefficients compared to phenolic acids.This suggests that flavonoids are primarily responsible for the extracts' ability to scavenge free radicals and reduce stable cations.Among these compounds, luteolin and its glycoside, hesperidin, as well as quercetin, kaempferol, and apigenin, play Fig. 2 Correlation matrix between antioxidant activity and the most abundant phenolic compounds in the extracts of Onosma riedliana P-Mo, phosphomolybdenum assay; FeCH, ferrous ion chelating assay; PCATHA, protocatechuic acid; 34DHPA, 3,4-dihydroxyphenylacetic acid; CGA, chlorogenic acid; 25DHBA, 2,5-dihydroxybenzoic acid; 4HBA, 4-hydroxybenzoic acid; VA, vanillic acid; CA, caffeic acid; SyA, syringic acid; VAL, vanillin; pCA, p-coumaric acid; FA, ferulic acid; LUTG, luteolin-7-glucoside; HESP, hesperidin; RA, rosmarinic acid; HYPER, hyperoside; PINOR, pinoresinol; LUT, luteolin for butyrylcholinesterase or the two carbohydrate hydrolytic enzymes, α-amylase and α-glucosidase.These findings align with previous studies by Baltaci et al. [5] and Sarikukcu et al. [14], which conducted docking analyses and found that flavonoids like hesperidin and hyperoside had strong binding activity for both cholinesterases and tyrosinase, while their activities were comparatively lower for other enzymes.To summarize, phenolic-rich plant extracts of O. riedliana showed pharmacological potential as inhibitors of enzyme cholinesterase, which activity is related with neurodegenerative diseases.Also, these enzymes.The Pearson correlation matrix between enzyme inhibitory activities and the most abundant phenolic compounds in O. riedliana extracts (Fig. 3) suggests that all flavonoids, including luteolin, luteolin-7-glucoside, hesperidin, and hyperoside, as well as phenolic acids (excluding 3,4-dihydroxyphenylacetic and 2,5-dihydroxybenzoic acids), exhibit a highly significant correlation with the inhibitory activity of the extracts against the enzyme tyrosinase.Furthermore, the results indicate that phenolic compounds are primarily responsible for the inhibition of the enzyme acetylcholinesterase but not extract could be used as natural plant-based tyrosinase inhibitors, that normally are used for the prevention of severe skin diseases [9,11,15].

Conclusions
This paper presents the first-ever exploration of the phenolic profiles and biological activities of three extracts derived from Onosma riedliana, a plant belonging to a genus with a long history of therapeutic use for diverse disorders.The primary phenolic compounds identified in these extracts include lignan pinoresinol, flavonoid hesperidin, and phenolic acids, specifically 4-hydroxybenzoic and p-coumaric acids.The antioxidant potential of Onosma riedliana was assessed using six distinct assays.The methanolic extract displayed the highest antioxidant potency against oxidative agents, primarily attributed to the presence of flavonoids such as hesperidin and luteolin.Additionally, the ethyl acetate extract demonstrated notable inhibitory activity against two enzymes, cholinesterase and tyrosinase, while the methanolic extract exhibited remarkable efficacy against two carbohydrate hydrolytic enzymes, α-amylase and α-glucosidase.Statistical analysis of the data suggests that phenolic compounds, particularly flavonoids, are responsible for the inhibition of cholinesterases and tyrosinase, though they do not influence α-amylase and α-glucosidase.In conclusion, the findings presented herein underscore the potential of Onosma riedliana as a valuable source of bioactive natural products.Its diverse array of compounds highlights its suitability for various pharmaceutical formulations, indicating its significance in the realm of herbal medicine and natural product-based therapeutics, mainly in the treatment of different neurodegenerative diseases.

Fig. 1
Fig. 1 Total phenolic and flavonoid contents of Onosma riedliana extracts.Values indicated by the same superscripts do not significantly differ after Tukey's hoc test at a 5% significance level

Table 2
Antioxidant activity of Onosma riedliana extractsThe values indicated by the same superscripts within the same row do not differ significantly after Tukey's post hoc test at a 5% significance level.IC 50 , the concentration of the extract required to reduce or scavenge or chelate 50% of potential oxidant a 27.99 ± 0.35 b 25.43 ± 0.20 b RACI -− 0.63 ± 0.00 b 1.23 ± 0.03 a − 0.61 ± 0.01 b