Investigation of phenolic compounds at the leaves and shoots Arctostaphylos spp. and their antioxidant and antityrosinase activities

Abstract Comparative phytochemical and pharmacological analysis of leaves and shoots of Arctostaphylos uva-ursi (L.) Spreng. and endemic of Transcaucasia and North Caucasia Arctostaphylos caucasica Lipsch. was carried out. Phenolic compounds in methanol extracts were investigated by HPLC-PDA-ESI-MS. Pharmacological investigation was carried out at the models of generation of superoxide, DPPH, hydroxyl and nitrosyl radicals. The antityrosinase properties were evaluated in the reaction of tyrosinase inhibition using L -tyrosine as a substrate. Five new compounds were identified in the shoots and leaves of A. uva-ursi: 4,6-di-О-galloylglucose; 2,3-di-О-galloylglucose; 1,4,6-tri-О-galloylglucose; 1,2,4,6-tetra-О-galloylglucose; tetra-О-galloylhexose. Thirty-five phenolic compounds and two saponins were identified for the first time in A. caucasica. Water-alcohol extract (70% ethanol) from A. caucasica had the highest antiradical activity. Water extracts from both species had antityrosinase activity and possessed comparable efficiency (IC50 = 36.4 ± 1.2 µg/ml and IC50 = 35,5 ± 2 µg/ml, respectively). GRAPHICAL ABSTRACT


Introduction
Genus Arctostaphylos according the data base The Plant List in 2021 includes 75 species (The Plant List 2021). Two species of genus Arctostaphylos grow at the area of Russian Federation: Arctostaphylos uva-ursi (L.) Spreng. and relict endemic of Transcaucasia and North Caucasia Arctostaphylos caucasica Lipsch. (Czerepanov 1995;Takhtadjan 2009), for which there are not any data about chemical content and pharmacological activity. Arctostaphylos uva-ursi (L.) Spreng. is included in national pharmacopoeias of different countries as diuretic and antiseptic agent due to presence total amount phenologlycosides, the dominant among which is arbutin (Linnenbrink and Kraus 1986;Kubo et al. 1990). Analysis of literature data showed that Arctostaphylos uva-ursi (L.) Spreng. is a rich source for its chemical content including not only phenologlycosides (Sticher et al. 1979;Matsuda et al. 1992;Alam et al. 2011), but flavonoids, phenylpropanoids, galloylglycosides and catechins (Olennikov and Chekhirova 2013), polysaccharides (Olennikov and Nazarova 2009), iridoids (Jahodar et al. 1978), saponins (Takada et al. 2010;Caligiani et al. 2013), essential oil (Radulovi c et al. 2010), macro-and microelements (Afanasyeva and Ayushina 2018). It is known that in the folk medicine bearberry leaves are used for their antihelmintic, astringent, sedative, haemostatic, adaptogenic properties, and normalize metabolism (Budancev and Lesiovskaya 2001). Several scientists detected antibacterial (Larsson et al. 1993), nephrolytic (Grases et al. 1994), diuretic (Beaux et al. 1999), depigmenting (Matsuda et al. 1992), anti-inflammatory (Kubo et al. 1990), antidiabetic (Swanston-Flatt et al. 1989), neuroprotective (Chandler et al. 2010), antioxidant (in vitro) (Mohd Azman et al. 2016) and antiproliferative activities (Amarowicz and Pegg 2013). Also it is important to control quality of A. uva-ursi leaves and products from its. For this purpose HPLC-PDA-ESI-MS can be used. It is a modern analytical method using to get fingerprint identification or to get away from column and preparative chromatography and investigated crude extract without additional purification (Gallo et al. 2013).
Due to high rule of tyrosinase in the melanogenesis inhibitors of this enzyme often are used in cosmetic purposes as depigmentation agents or for therapy myeloleucosis (Furue and Tsuji 2019). Also the prevention of oxidative reactions is a potential target of pathogenic therapy of wide range of pathological conditions (Zhang et al. 2015).
So the aim of this work was investigation of phenolic complex in the leaves and shoots of both species of genus Arstostaphylos and evaluation of direct antioxidant and antityrosinase activities (Mapunya et al. 2012, Marcocci et al. 1994, Mensor et al. 2001, and Winterbourn et al. 1975).
The complex of pharmacological tests of the analysed dry residues allowed us to establish that the most pronounced antiradical activity has the extract from A. caucasica obtained by extraction with 70% alcohol. IC 50 in all cases for this compound was comparable with the reference trolox solution. Less indirect antioxidant activity was characterized by extracts that were obtained by extracting raw materials with ethyl alcohol 40% and 95% concentration, and the level of activity did not depend on the type of raw materials that was used (Table S4). Dry residues of aqueous extracts from A. caucasica and A. uva-ursi did not show significant antiradical activity.
The antiradical activity of the test extracts may be a background for their use in the correction of oxidative stress. It has been established that oxidative stress is part of the pathogenesis of numerous diseases, such as atherosclerosis, hypertension, diabetes mellitus, chronic heart failure, Alzheimer's disease and some types of cancer. It is known that the main trigger of oxidative stress is the hyperproduction of free radicals (ROS), represented by superoxide and hydroxide radicals, as well as reactive forms of nitrogen (nitrosyl radical, peroxonitrite). Having a non-specific effect on the cell, ROS are extremely cytotoxic agents (Poprac et al. 2017). At the same time, as a number of numerous studies show, the suppression of ROS production or their effects is the leading method of treating oxidative stress. The group of direct scavengers of ROS stands out, which combines both synthetic and natural objects; moreover, plant extracts occupy a leading position (Parham et al. 2020). The obtained results suggest the presence of ROS scavengers in ethanol extracts, as evidenced by the value of IC 50 in the assessment of superoxide, hydroxyl, and nitrosyl antiradical activity. Also, in addition to direct antioxidant activity, the ethanol extracts under study may be characterized by the presence of reductive properties, which is confirmed by the data of the DPPH test (Sirivibulkovit et al. 2018). The high antioxidant properties of the studied extracts are probably associated with the presence of a large number of polyphenolic compounds.
An assessment of the anti-tyrosinase properties was shown that the extracts from A. caucasica and A. uva-ursi obtained by water extraction demonstrate the most pronounced tyrosinase-inhibiting properties similar to kojic acid (Table S4). At the same time, ethanol extracts did not have anti-tyrosinase activity. The obtained data open up certain prospects for the use of aqueous extracts from the therapy of a number of diseases, the basis of which is abnormal melanogenesis due to the high content of arbutin.
Melanogenesis is a complex process represented by a complex cascade of enzymatic reactions, in which the determining role is assigned to proteins related to tyrosinase TYRP1 and TYRP2-multifunctional copper-containing metalloenzymes that catalyze the reaction that limits the rate of melanin synthesis. Therefore, it can be assumed that tyrosinase inhibition may be one of the approaches to correcting skin hyperpigmentation. Among tyrosinase inhibitors, hydroquinone and kojic acid are the most well-known, the use of which in clinical practice is limited by a significant number of side effects. Thus, hydroquinone is recommended to be used only in combination with isotretinoin (in the gel form), which significantly increases the drug load on the patient (Chandra et al. 2012). Kojic acid is a natural metabolite produced by fungi that inhibits tyrosinase. Kojic acid is characterized by high biocompatibility, which, however, does not increase the effectiveness and does not contribute to reducing the toxicity of the compound (Saeedi et al. 2019). Thus, medicines obtained on the basis of water extracts of A. uva-ursi and A. caucasica can be a safer alternative to existing depigimenting agents.

Conclusions
The comparative study of chromatographic profiles both species of bearberry, A. uvaursi and A. caucasica were carried out also as chromatographic profile of extracts from leaves and shoots A. caucasica were obtained at the first time. Thus, medicines obtained on the basis of water extracts of A. uva-ursi and A. caucasica can be a safer alternative to existing depigimenting agents. At the same time, water-alcohol extraction based on raw materials A. caucasica and A. uva-ursi can have a wide range of therapeutic applications due to the presence of anti-radical activity.