**Selectivity Tuning by Natural Deep Eutectic Solvents (NADESs) for Extraction of Bioactive Compounds from** *Cytinus hypocistis***—Studies of Antioxidative, Enzyme-Inhibitive Properties and LC-MS Profiles**

**Gokhan Zengin 1, María de la Luz Cádiz-Gurrea 2,\*, Álvaro Fernández-Ochoa 2,\*, Francisco Javier Leyva-Jiménez 3,4, Antonio Segura Carretero 2, Malwina Momotko 5, Evren Yildiztugay 6, Refik Karatas 1, Sharmeen Jugreet 7, Mohamad Fawzi Mahomoodally 7,8,9 and Grzegorz Boczkaj 10,11**


**Abstract:** In the present study, the extracts of *Cytinus hypocistis* (L.) L using both traditional solvents (hexane, ethyl acetate, dichloromethane, ethanol, ethanol/water, and water) and natural deep eutectic solvents (NADESs) were investigated in terms of their total polyphenolic contents and antioxidant and enzyme-inhibitive properties. The extracts were found to possess total phenolic and total flavonoid contents in the ranges of 26.47–186.13 mg GAE/g and 0.68–12.55 mg RE/g, respectively. Higher total phenolic contents were obtained for NADES extracts. Compositional differences were reported in relation to antioxidant potential studied by several assays (DPPH: 70.19–939.35 mg TE/g, ABTS: 172.56–4026.50 mg TE/g; CUPRAC: 97.41–1730.38 mg TE/g, FRAP: 84.11–1534.85 mg TE/g). Application of NADESs (choline chloride—urea 1:2, a so-called Reline) allowed one to obtain the highest number of extracts having antioxidant potential in the radical scavenging and reducing assays. NADES-B (protonated by HCl L-proline-xylitol 5:1) was the only extractant from the studied solvents that isolated a specific fraction without chelating activity. Reline extract exhibited the highest acetylcholinesterase inhibition compared to NADES-B and NADES-C (protonated by H2SO4 L-proline-xylitol 5:1) extracts, which showed no inhibition. The NADES extracts were observed to have higher tyrosinase inhibitory properties compared to extracts obtained by traditional organic solvents. Furthermore, the NADES extracts were relatively better inhibitors of the diabetic enzymes. These findings provided an interesting comparison in terms of total polyphenolic content yields, antioxidant and enzyme inhibitory properties (cholinesterase, amylase, glucosidase, and tyrosinase) between traditional solvent extracts and NADES extracts, used as an alternative. While the organic solvents showed better antioxidant activity, the NADES extracts were found to have some other improved properties, such as higher total phenolic content and enzyme-inhibiting properties, suggesting

**Citation:** Zengin, G.; Cádiz-Gurrea, M.d.l.L.; Fernández-Ochoa, Á.; Leyva-Jiménez, F.J.; Carretero, A.S.; Momotko, M.; Yildiztugay, E.; Karatas, R.; Jugreet, S.; Mahomoodally, M.F.; et al. Selectivity Tuning by Natural Deep Eutectic Solvents (NADESs) for Extraction of Bioactive Compounds from *Cytinus hypocistis*—Studies of Antioxidative, Enzyme-Inhibitive Properties and LC-MS Profiles. *Molecules* **2022**, *27*, 5788. https://doi.org/10.3390/ molecules27185788

Academic Editor: Nour Eddine Es-Safi

Received: 23 August 2022 Accepted: 4 September 2022 Published: 7 September 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

functional prospects for their use in phytonutrient extraction and fractionation. The obtained results could also be used to give a broad overview of the different biological potentials of *C. hypocistis*.

**Keywords:** NADES; total polyphenolic content; antioxidants; enzyme inhibition; functional food; natural medicine; Alzheimer cholinesterase inhibitors

### **1. Introduction**

The genus *Cytinus*, composed of endophytic parasitic plants (family: *Cytinaceae*), bears eight recognised species distributed around two centres of diversity: one in southern Africa and Madagascar, and one in the Mediterranean region [1].

Indeed, folkloric medicine has dedicated substantial consideration to this genus. These plants have been used traditionally for treating dysentery, including their ability to soothe inflammations of the eyes and throat. Some ethnobotanical reviews have also noted the use of *Cytinus* juice as an astringent, a haemostatic, and a tonic substance. They are also used as a scar-healing agent, whereby the scalp pulp is applied daily on corns and calluses, skin and swollen mucous membranes as an astringent and anti-inflammatory therapy [1,2].

Some studies have also pointed out these plants' beneficial potential and suggested their antimicrobial effects over a range of bacterial strains and antioxidant activities [2–4]. Furthermore, they have been highlighted as good sources of biologically active ingredients of cosmeceutical interest [2,3,5]. In fact, their biological activities have been correlated with their high tannin content. For instance, hydrolysable tannins were found to be the active cytotoxic compounds identified in three *Cytinus* taxa and were assessed against a wide variety of cancer cell lines [6]. In another study, the tested extract of *C. hypocistis* was found to exhibit anti-inflammatory activity and effective cytotoxicity againcst tumour cells, while it showed the lowest cytotoxicity on a non-tumour cell line, and interestingly, hydrolysable tannins and flavonoids were also identified as the main groups in the extract [7].

Plants possess a diverse range of such bioactive constituents. However, their availability strongly depends on the extraction techniques used, among other factors. Even though to date numerous methods have been developed and upgraded, there is still a need to achieve a standardised solution with high consideration for the extraction of bioactive compounds from plants [8].

In addition, there is growing pressure to investigate alternative solvents that retain the technological advantages of organic solvents while posing less risk to human health and the environment. Deep eutectic solvents (DESs) and their specialised form obtained from compounds of natural origin—natural DESs (NADESs)—have shown the most promise in the field of green chemistry because they are abundant, inexpensive, recyclable, and appealing for a wide range of applications (food, cosmetic, and pharmaceutical). DESs have already proved to have several advantages in separation science, especially in terms of unusual selectivity useful in chromatography [9,10] and extraction [11,12], as well as membrane processes [13,14]. Many studies have effectively employed NADES extraction to gain high-quality extracts from numerous plants, including medicinal plants [15–17]. NADESs' potential biological activity, bioavailability, and the availability of a variety of solvent combinations for its preparation are also intriguing characteristics. Therefore, NADES extraction is a cutting-edge technique that has piqued the interest of researchers and already exhibited great promise in the extraction and isolation of bioactive compounds from plants [17].

Therefore, the aim of the present study is to compare characteristics of extracts obtained by means of NADESs with classic organic solvents. For this purpose, the *Cytinus hypocistis* (L.) L. extracts were analysed in respect to their LC-MS profiles, the total phenolic (TPC) and flavonoid (TFC) contents, and antioxidant as well as enzyme-inhibitive properties.

#### **2. Results and Discussion**

#### *2.1. Phytochemical Profiles*

Natural products are important sources for drug development. Thus, it is of crucial importance to develop effective methods to extract and isolate these bioactive products. Indeed, the lab-intensive and laborious extraction and isolation processes have been a major challenge in the application of natural products in drug development. There is an urgent need to develop efficient and selective methods for this [18]. In this respect, different types of solvents have been widely used for the extraction of phytochemicals, whereby dried plant powders are used to extract bioactive phytochemicals and remove the Interference of water concomitantly. The solvents used for the extraction of biomolecules from plants are chosen based on the polarity of the desired solute. For example, a solvent with the same polarity as the solute will effectively dissolve the solute. Several solvents can be used sequentially to limit the number of analogous compounds in the desired yield [19].

Solubility, bioavailability, and stability are all factors in the pharmacological efficacy of plant extracts and their bioactive principles. Natural deep eutectic solvents (NADESs) are considered as green solvents to enhance the extraction performance of plant metabolites [16]. As functional liquid media, NADESs can dissolve both natural and synthetic substances with low water solubility. Hence, they are alternative candidates for applications with some organic solvents, as well as ionic liquids [20], indicating the enormous potential for NADESs to be utilised in the development of pharmaceutical formulations, such as nutraceuticals derived from plant-based metabolites [16]. Thus, in this study, both traditional solvents and NADESs were used to prepare *C. hypocistis* extracts and to compare their overall performance in terms of their bioactive content yields and biological activities.

In the phytochemical studies, the investigation of the polyphenol content present in plant extracts is an important part of assessing their biological properties. In this study, the extracts were found to possess TPC and TFC in the range of 26.47–186.13 mg GAE/g and 0.68–12.55 mg RE/g, respectively. Interestingly, the extracts obtained by NADESs yielded higher TPC (167.57–186.13 mg GAE/g), followed by ethyl acetate, water, ethanol, and ethanol/water extracts (123.51–127.83 mg GAE/g). On the other hand, the dichloromethane and hexane extracts yielded low TPC. In the TFC assay, the highest yield was obtained by ethyl acetate and ethanol/water extracts, followed by water and ethanol extracts, while the least TFC was yielded by hexane, NADES-C, and dichloromethane extracts (Figure 1, Table S1). The variation in total polyphenol content clearly varied with the polarity of the solvents used. However, not only the polarity of the solvents, but also other parameters such as pH, extraction time, methods, and temperature can affect the extraction yield and total phenolic content [21–23].
