*2.4. Enzyme Inhibitory Effects*

Low levels of the neurotransmitter acetylcholine, oxidative stress, and inflammation in the central nervous system (CNS) are hallmarks of Alzheimer's disease (AD), a progressive neurodegenerative disease. To date, patients diagnosed with AD are only offered enzyme inhibitors (acetylcholinesterase/butyrylcholinesterase, or AChE/BChE) for treatment [40]. Hence, as mechanism of pharmacological action, these cholinesterase inhibitors are able to modify cholinergic signalling by disrupting the degradation of acetylcholine [41].

All extracts, except NADES-B and NADES-C extracts, possessed anti-AChE activity (7.32–15.16 mg GALAE/g). Interestingly, the highest anti-AChE activity was revealed for NADES-A. It is particularly important observation, as components of NADES-A choline chloride and urea—are nontoxic; thus, such obtained extracts could be further used without removal of NADES. On the other hand, only the extracts prepared in the traditional way, with solvents hexane, ethyl acetate, dichloromethane, ethanol, and ethanol/water, displayed anti-BChE potential (1.39–2.13 mg GALAE/g). The water extract and the NADES extracts showed no anti-BChE activity. It follows from the ionic nature of the used NADESs, which in this case was not favourable for extraction. The ethanol extract demonstrated relatively higher BChE inhibitory effect compared to the other extracts (Figure 3).

**Figure 3.** Cholinesterase inhibitory effects (**A**), amylase and glucosidase inhibitory effects (**B**), tyrosinase inhibitory effects (**C**), Pearson's correlations between total bioactive compounds and enzyme inhibitory assays (*p* < 0.05) (**D**). na: not active. Different letters in column for same assays indicate significant differences in the extracts (*p* < 0.05).

Tyrosinase is the rate-limiting enzyme in melanin synthesis. Melanin is synthesised in human melanocytes when tyrosine is hydroxylated to l-DOPA, which is then oxidised to dopaquinone and polymerises to form melanin. Melasma, melanoma, and freckles are just some of the dermatological conditions that can develop when melanin production increases too rapidly. However, tyrosinase in plant-based foods oxidises phenolic compounds into quinones. The former reacts with amino acids and proteins to produce brown/black pigments, a process known as enzymatic browning, which is one of the most pressing problems in the food industry and the source of 50 percent of the industry's economic losses. In addition, browning reduces the food's nutritional value and safety because it leads to the loss of vitamin C, antioxidants, and other nutrients, and can even lead to the production of antinutritional and toxic substances. Consequently, tyrosinase inhibition is seen as an efficient method for preventing hyperpigmentation in the pharmaceutical industry and delaying enzymatic browning, which is helpful in the food industry [42].

In the current investigation, all the studied extracts were found to possess antityrosinase activity (49.14–153.97 mg KAE/g). However, NADES-A exhibited the highest inhibitory activity against tyrosinase, while the hexane extract displayed the lowest (Figure 3). On this basis, it is clear that polar NADES-A, as well as other polar solvents, should be preferred for the extraction of bioactive compounds responsible for tyrosinase activity.

Interestingly, in the study by Zucca et al. [2], the ethanolic extract of *C. hypocistis* showed the highest tyrosinase inhibition activity, compared to cyclohexane and water extracts, probably due to being predominantly rich in polyphenols, in most part hydrolysable tannins. *C. hypocistis* aerial part extract was also reported to show tyrosinase inhibition of 80%, when tested at 50 μg/mL [43]. Furthermore, a linear correlation was obtained between enzymatic activities and increasing TPC and TFC. The reason could be a specific class of polyphenols acting against tyrosinase through a competitive inhibition mechanism, thus interfering with the biological function of tyrosinase, which is a polyphenoloxidase [43].

The inhibition of the carbohydrate-digesting enzymes alpha-glucosidase and alphaamylase is an important strategy for controlling blood glucose levels in patients with Type 2 diabetes and borderline diabetes, because it significantly reduces the postprandial rise in blood glucose [44]. Even though drugs such as voglibose, acarbose, and miglitol are commercially available as those enzymes' inhibitors and are also used in practice, they produce undesirous effects such as abdominal discomfort, bloating, and diarrhoea.

In addition, many chronic diseases such as diabetes are associated with oxidative stress, during which reactive oxygen species (O2 −, H2O2 and OH−) are generated. The role of free radicals in the onset and development of diabetes has also been established. Therefore, compounds that possess both antidiabetic and antioxidant properties without causing serious side effects would be of great value [45].

In the present investigation, all extracts were found to inhibit both carbohydrate-hydrolysing enzymes (Amylase: 0.35–2.54 mmol ACAE/g; glucosidase: 0.93–2.20 mmol ACAE/g). Remarkably, the NADES extracts were found to be better inhibitors of amylase and glucosidase compared the other extracts (Figure 3). This could be due to the higher TPC in the NADES extracts, and this fact was also confirmed by Pearson's correlation analysis (Figure 3). In fact, it has been previously suggested that phenolics are involved in the modulation of the activity of starch digestive enzymes [46].

#### *2.5. Data Mining*

To gain more insight into the tested extracts and biological activity assays, we performed PCA analysis. The results are given in Figure 4. Firstly, we examined the relationship between the tested extracts based on the biological activity results. We obtained a good distribution, and the tested extracts were very well-separated based on the biological activity results. Two components (PC1: 47.3% and PC2: 33.8) accounted for 811% of the total components. Two extracts (hexane and dichloromethane) exhibited the lowest biological abilities and were distributed in the same axis. In addition, polar extracts (ethanol, ethanol/water, water) and NADES-A had similar biological abilities and were placed in the

same group. The PCA plot also confirmed a strong correlation between total flavonoid and antioxidant properties, which were very close to each other on the PCA plot. In addition to biological activity results, we investigated the similarities/differences of the tested extracts based on their chemical profiles. Two components were used in the analysis to determine the distribution of the tested solvents (PC1: 60.1% and PC2: 18.8%). In Figure 4b, the used nonpolar and polar solvents and NADESs were clearly separated, and these results were very similar to the distribution from the biological activity results. Taken together, we concluded that there is a good connection between chemical compounds and biological activities of *Cytinus* extracts.

**Figure 4.** Principal component analysis between tested extracts and biological activities (**a**). Distribution of the tested extracts in principal component analysis by using chemical compound peak areas (**b**).
