**1. Introduction**

The use of plants for the treatment of human diseases is a centuries-old tradition, based on phytotherapy research as well as on ethnopharmacological knowledge. Recently, the use of herbal medicines applied for the prevention and/or preservation of health covers a central role in modern medicine related to the fact that these plant-derived materials avoid the classical side effects of synthetic drugs. Additionally, there are benefits of their long-term historic use—safety, accessibility, and efficacy with a wide range of therapeutic actions [1]. *Galium* is a well-known genus with many medicinal representatives that are rich sources of iridoid glycosides [2–4], flavonoids [5], anthraquinones [6], and small amounts of essential oils and vitamin C [7]. Recent studies showed the antibacterial, antifungal, antiparasitic, and antioxidant activities of representatives of this plant genus [7,8].

*G. verum* L., also known as Lady's Bedstraw, is an herbaceous perennial plant, native to Europe and Asia, and used commonly in many countries' folk medicine for a large variety of treatments. The dried plants' aerial parts were used to stuff mattresses, and the flowers were also used to coagulate milk for cheese production [9]. The cut and dried aerial parts of the plant, '*Herba gallii verii*', are used for homeopathic purposes. These are still used for exogenous treatment of psoriasis or as a tea with diuretic effect for the cure of pyelitis or cystitis [10]. Moreover, *G. verum* L. has been used as a diuretic for bladder and kidney irritation, externally for poorly healing wounds, as well as for epilepsy and hysteria in Montenegro's traditional medicine [11]. Regarding Turkish folk medicine, it has been used for its diuretic, choleretic, antidiarrheal, and sedative effects [4]. In Romania, the plant is used in traditional medicine mainly for its diuretic, depurative, laxative, sedative, and antirheumatic effects. Additionally, in the Romanian traditional medicine, several *Galium* species are used as components of different cosmetic formulations [12]. *G. album* Mill., the "white bedstraw" or "hedge bedstraw", is an herbaceous annual plant, cited in traditional Albanian pharmacopoeias and folk medicine for healing wounds and gingival inflammations [13]. *G. rivale*, *G. pseudoaristatum*, and *G. purpureum* (syn. *Asperula purpurea*) are less-known species, and to the extent of our knowledge, they have not been investigated ye<sup>t</sup> in terms of chemical composition and antioxidant capacity, nor in terms of enzyme inhibitory potential.

Generally, the use of different extraction procedures on plant-derived material yields different biological activities. In this field, the availability of an efficient, fast, exhaustive, and reproducible extraction procedure allows obtaining a standardized starting material for food additives, nutraceuticals, and phytoformulations. For the extraction of bioactive compounds from *Galium* maceration in methanol [7] or ethanol [14], percolation in methanol [8], and ultrasound-assisted extraction [12] were applied, wherein the extraction time was varied from 30 min to one week. In order to reduce the extraction time and retain or increase the extraction efficiency, new extraction methods are required.

Liquid phase microextraction techniques are positioned as 'green' chemistry methodologies, which require small amounts of organic solvents. In order to make the procedures more environmentally-friendly, ionic liquids (ILs) or natural deep eutectic solvents (NADESs) are frequently used. Comparing ILs with NADESs, more advantages are on the side of NADESs due to their natural original (the main components can be sugars and organic acids), which may vary depending on analysis purpose, making them nontoxic, biodegradable, and incombustible. In comparison with NADESs, most ILs are toxic, have low biodegradability, and have high cost. Either IL or NADES can have high viscosity, so their extracts are limited for direct analysis using HPLC or GC systems [15–18].

Regarding biological activities, in the current work, a key enzyme was considered in order to further evaluate the extracts. Particularly, pigmentation is one of the most obvious phenotypical characteristics in the natural world. Between the pigments, melanin is one of the most widely distributed and is found in bacteria, fungi, plants, and animals. Melanins are heterogeneous polyphenol-like biopolymers with complex structure and color varying from yellow to black. The synthesis of melanin plays an important role in skin color and pigmentation. Tyrosinase, a copper-containing mono-oxygenase, is a key enzyme in melanin biosynthesis [19]. Skin disorders, such as melasma (facial pigmentation), scarce, and freckles, are related to excessive melanin biosynthesis. Thus, tyrosinase inhibitors are used to control or treat pigmentation disorders and are widely used in the cosmetic industry. In fact, some tyrosinase inhibitors, such as kojic acid and hydroquinones, are nowadays commercially produced, but they can present severe side effects, such as skin inflammation [20]. Hence, in recent years, more attention has been paid to the use of natural plant extracts as a safe and alternative source of tyrosinase inhibitors for cosmetic purposes.

In the present study, following our research on innovative microextraction procedures [21–27], different microextraction procedures were examined for the analysis of phenolic compounds in *G. verum* aerial parts, and then applied for the determination of the phenolic pattern of four other *Galium* species (*G. album*, *G. rivale*, *G. pseudoaristatum*, and *G. purpureum*). As an alternative, the microwave-assisted extraction (MAE) technique was used as a reference method [27–29]. To the best of our knowledge, it is the first time that microextraction techniques have been applied for the recovery and the establishment of phenolic compounds in *Galium* species.

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