**1. Introduction**

Plants biosynthesize a large number of various phytochemicals that have demonstrated antioxidant and health beneficial properties in numerous studies. Polyphenolics can be defined as a group of heterogeneous biologically active non-nutrients, belonging to the most important and widely investigated class of such phytochemicals [1]. In the last few decades, numerous publications have reported that high consumption of phytochemical-rich foods might reduce the risk of several diseases. Therefore, modifying one's diet by increasing the intake of fruits, vegetables, herbs and spices may be a promising strategy for cancer prevention.

Formation of free radicals and reactive oxygen species (ROS) is a normal process in human cells. However, excessive production of ROS, which may occur due to various exogenous factors, can disturb homeostatic conditions, resulting in oxidative stress, which may largely contribute to the development of chronic health problems, including cancer, inflammation, cardiovascular diseases and aging [2]. Therefore, it has been hypothesized that polyphenolic compounds and other dietary antioxidants, which are abundant in many fruits, vegetables, and botanicals, are essential nutrients protecting against harmful effects of the excessive free radicals [1]. Since ancient times, botanicals have been used in folk medicine to treat various diseases and health disorders, as well as in flavourings, fragrances, and colorants. However, only recently have the health benefits of many medicinal and spicy plants been explained by scientific evidence. For instance, a large number of polyphenolic antioxidants have been identified and characterized in aromatic herbs, spices, and other plant materials. Many of them have been proved as effective antioxidants [3], while their health benefits have been linked to various mechanisms, including the scavenging of harmful free radicals [1]. However, considering a vast number of species in the Plant Kingdom, there are still many poorly investigated plant species, which might be a good source of new natural bioactive substances including strong antioxidants.

Literature survey indicates that some species of the genus *Paeonia* (Paeoniaceae), which is divided into the three sections (*Moutan*, *Oneapia* and *Paeonia*) may be considered poorly studied plants [4]. The section of *Paeonia* consists of 25 species, which are widely distributed throughout temperate Eurasia. Many of them, including *P. <sup>o</sup>*ffi*cinalis*, remain under-investigated until now. For instance, among 262 compounds identified in different anatomical parts of *Paeonia* (terpenoids, tannins, flavonoids, stilbenes, steroids, paeonols, and phenols), only several anthocyanins were reported in *P. o*ffi*cinalis* flowers and one phenolic acid glycoside (1,2,3,6-*tetra*-*O*-galloyl-β-*d*-glucose) in its roots [4].

A large spectrum of bioactive substances found in different *Paeonia* spp. may be responsible for their biological and pharmacological activities. For instance, it has been used in folk medicine to treat epilepsy, liver diseases, diarrhea, and many other disorders [5]. Furthermore, antioxidant, antitumor, anti-inflammatory, anti-microbial, immune system modulation, central nervous, and cardiovascular system protective activities were also reported for *Paeonia* plants [6–10]. *P. lactiflora* roots are among the most traditional Chinese medicines and have been used for their anti-inflammatory, analgesic, blood tonifying, stringent, and menstruation regulation properties [11]. Besides the ornamental value of flowers, the seeds of some other *Paeonia* spp. (*P. lactiflora*, *P. su*ff*ruticosa*) have been considered as a rich source of polyunsaturated oil and proteins for foods [12]. *P. o*ffi*cinalis* has been used mainly for medicinal purposes, e.g., as an antiepileptic and antispasmodic drug, whereas its flowers were used to produce syrup [13].

Considering the rather scarce scientific knowledge reported for *P. <sup>o</sup>*ffi*cinalis*, the purpose of this study was to perform more systematic studies of its extracts, including its phytochemical composition and antioxidant activity, in order to provide more comprehensive data on the potential of *P. o*ffi*cinalis* as a source for functional ingredients for various applications.

#### **2. Materials and Methods**

#### *2.1. Plant Material, Solvents and Chemicals*

*Paeonia o*ffi*cinalis* roots and leaves of blooming plant were harvested at the Kaunas Botanical Garden of Vytautas Magnus University (Kaunas, Lithuania) and dried in a ventilated and protected from direct sunlight room. Dried leaves were separated from stems before further use.

Analytical grade methanol was purchased from StanLab (Lublin, Poland); formic acid (98%), liquid chromatography-mass spectrometry (LS-MS) and high performance liquid chromatography (HPLC) grade acetonitrile, 2,2-azino-*bis*-3-ethylbenzothiazoline-6-sulfonic acid (ABTS, 98%), Folin-Ciocalteu reagent, monopotassium phosphate, gallic acid, 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•, 98%), <sup>2</sup>,2-azo-*bis*-(2-amidinopropane) dihydrochloride (AAPH), picolinic acid, 6-hydroxyl-2,5,7,8-*tetra*-methylchroman-2-carboxylic acid (Trolox), potassium chloride, H2O2, caffeic acid, cobalt (II) fluoride tetrahydrate, NaCl, and sodium phosphate monobasic monohydrate were from Sigma-Aldrich (Darmstadt, Germany); ethanol (99%) were from Scharlau (Barcelona, Spain); microcrystalline cellulose (20 μm) was from Sigma-Aldrich (Steinheim, Germany); sodium phosphate dibasic dihydrate, potassium iodine, and ferric chloride were from Riedel-de-Haen (Seelze, Germany); disodium fluorescein was from TCI Europe (Antwerpen, Belgium); α–amylase, type VI-B (from porcine pancreas), sodium carbonate, quercetin, and 2,7-dichlorofluorescin diacetate (DCFH-DA) were from

Sigma-Aldrich (St. Quentin Fallavier, France); potato starch was from Fluka (Buchs, Switzerland); acarbose was from Bayer Pharma AG (Leverkusen, Germany); epithelial colorectal adenocarcinoma cells (Caco-2) were from DSMZ (Braunschweig, Germany). Trypsin, Roswell Park Memorial Institute (RPMI) 1640, penicillin streptomycin, and heat inactivated fetal bovine serum were obtained from Invitrogen (Gibco, Paisley, UK); phosphate bu ffer saline solution (PBS) was from Sigma-Aldrich (St. Louis, MO, USA). Ultrapure water was produced in a Simplicity 185 system (Millipore, MA, USA).
