Hyaluronidase, acetylcholinesterase inhibiting potential, antioxidant activity, and LC-ESI-MS/MS analysis of polyphenolics of rose (Rosa rugosa Thunb.) teas and tinctures

ABSTRACT

Teas and tinctures prepared from five rose organs were analysed for their antioxidant, anti-hyaluronidase, and acetylcholinesterase inhibiting activity. Moreover, analysis of phenolic acids and flavonoids was carried out by the use of liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). As a result, the presence of 10 phenolic acids and 10 flavonoid glycosides was shown. Significant antioxidant potential (0.26 to 4.25 mmol Trolox/g) and reducing power (1092.04 to 9258.70 mmol Fe²⁺/g) was revealed. Moreover, high anti-hyaluronidase activity demonstrated especially by tinctures and differentiated acetylcholinesterase inhibiting effects were observed. The investigated extracts may be considered as potential sources of phenolics and health-promoting ingredients in everyday diet.

KEYWORDS:

• ABTS
• Acetylcholinesterase inhibitor
• FRAP
• Hyaluronidase inhibitor
• Japanese rose
• Phenolics

Introduction

Herbal extracts and plant-based beverages are natural components of the human daily diet. Apart from their palatability value, they are considered as source of pro-health food ingredients. Many studies have shown a reduced incidence of many chronic diseases in individuals with the diet rich in phytochemicals. This preventive potential appears to be associated to a large extent with antioxidant activity of plant components.^[1] Therefore, new potent sources of antioxidants are still being searched. At the same time, there is also an on-going interest to find effective inhibitors of enzymes involved in aging, inflammatory processes, and leading to degenerative changes. In health-promoting diet, plant products have unquestionable advantages over dietary supplements in supplying of phytochemicals, for example, due to increased bioavailability. It was shown that consumption of plant-based beverages and extracts is one of the most common way of supplying biologically active secondary metabolites.^[2] For this purpose, extracts which are frequently consumed and can be easily obtained in household settings (e.g., teas and tinctures) are particularly suitable.

Rose materials are easily accessible both from wild growing and cultivated plants on a commercial scale (e.g., Rosa rugosa Thunb.). Rose hips and petals have long tradition of being used for medicine and food purposes. Other rose parts (i.e., leaves, roots, and nuts) are rarely utilised. However, they seem to have great therapeutic potential.^[3–6] Previous reports have also showed that all parts of this species can be rich sources of biologically active constituents.^[7–9] Moreover, it was demonstrated that significant amount of active compounds, including flavonoids, tannins, and o-dihydroxyphenols, pass during the short extraction procedures to aqueous and hydroethanolic galenic preparations affecting their biological activity.^[4,10]

Therefore, this study was aimed to investigate phenolic compounds and biological activity of two types (teas and tinctures) of galenic preparations prepared from five different organs of R. rugosa Thunb. (i.e., flowers, leaves, hips, nuts, roots). Their antioxidant, ferric reducing activity as well as hyaluronidase and acetylcholinesterase inhibiting potential will be determined.

Materials and methods

Plant material and preparation of extracts

Flowers, hips, nuts (seeds), leaves, and roots of R. rugosa were collected in Serock (Lublin Voivodeship, Poland) in 2014. Flowers and leaves were picked in July. Pseudo-fruits (hips) were collected in August, and nuts were separated from red hypanthium. Roots were collected in October. The plant material was air-dried and powdered according to the European Pharmacopoeia, 6th edition.^[11] Teas (infusions) and tinctures were prepared according to the monographs of the Polish Pharmacopoeia, 6th edition^[12] from all the collected R. rugosa parts. For preparation of infusion, 100 mL of boiling water was poured over 2 g of ground plant material and left to cool; teas were filtered and filled up with distilled water to the volume of 100 mL. For tinctures preparations, 10 g of pulverised plant material was extracted with 50 mL of 70° ethanol and left in a closed glass container for 7 days at 24°C (the mixtures were stirred occasionally three times a day). After this time, tinctures were filtered and filled up with 70° ethanol to the measured volume. All extracts were lyophilised in Free Zone 1 apparatus (Labconco, Kansas City, KS, USA), then weighed and stored in the refrigerator. Dry extracts were re-dissolved in the appropriate solvents in order to obtain the solutions of needed concentrations prior to further analysis. All galenic preparations were prepared in duplicate, and the obtained samples were marked with the following symbols: I: infusions (teas); T: tinctures; Fl: flower; Lf: leaf; Hip: hip (pseudo-fruit); Nut: nut (seed); R: root; for example, I-Lf means infusion prepared from leaves.

Chemicals

Standards of phenolic acids and 2,2ʹ-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), potassium persulphate, 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid (Trolox), ferric chloride, hyaluronidase from bovine testes type I-S, Streptococcus equi hyaluronic acid, methyl indole-3-carboxylate, 5,5′-dithiobis(2-nitrobenzoic acid (DTNB), acetylthiocholine iodide, sodium phosphate buffer pH 7.0 were purchased from Sigma-Aldrich Fine Chemicals (St. Louis, MO, USA). Standards of flavonoids were from ChromaDex (Irvine, USA). Ethanol was from Avantor Performance Materials (Gliwice, Poland). All the chemicals were of analytical grade. LC grade water was prepared using a Millipore Milli-Q purification system (Millipore, Bedford, MA, USA). LC grade acetonitrile and methanol were from Merck (Darmstadt, Germany).

LC-ESI-MS/MS analysis of phenolics

Phenolic acids and flavonoid glycosides contents were determined by ultra-high-pressure liquid chromatography-mass spectrometry using method previously described by Nowak et al.^[13] Analyses were performed on a Acquity TQD tandem quadrupole mass spectrometer with an electrospray ion source operated in negative ionisation mode and coupled to an Acquity UPLC System (Waters Corporation, Milford, MA, USA). Chromatographic separations of phenolic acids were carried out on an Acquity HSS T3 column (1.0 × 100 mm; 1.8 µm; Waters Corporation, Milford, CT, USA) using 0.1% aqueous formic acid (solvent A) and acetonitrile (solvent B). The solvent A concentration was changed as follows: 0 min (95%); 0.8 min (90%); 2.0 min (85%); 3.6 min (79%); 5.3 min (73%); 7.6 min (50%); 8.6 min (5%); 10.0 min (95%); and finished at 11 min. Column temperature was 30°C, the injection volume was 3 µL, the flow rate was 0.06 mL/min. The other parameters were as follows: capillary voltage 2.4 kV, source temperature 110°C, desolvation temperature 350°C, desolvatation gas (nitrogen) 500 L/h. Separations of flavonoids were carried out on a Acquity BEH C18 (2.1 × 100 mm; 1.7 µm; Waters Corporation, Milford, CT, USA). The column was maintained at 50°C at a constant flow rate of 0.04 mL/min. The sample injection volume was 5 μL. Gradient elution program with solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile) was used. The solvent A concentration was changed as follows: 0 min (87%); 0.5 min (87%); 6.0 min (85.5%); 8.5 min (60%); 9.5 min (60%); 9.6 min (87%), and finished at 11.6 min. The other ESI-MS parameters were as follows: capillary voltage 2.8 kV, source temperature 120°C, desolvation temperature 350°C, desolvatation gas 1000 L/h.

Triplicate injections were made for each standard solution and sample. Compounds were quantified using the external standard method by employing peak areas. Calibration curves were generated for injections of known concentrations of standard solutions at several concentration levels (0.05–15 ng/μL). The correlation coefficients of all calibration curves were R² > 0.9987. The limits of detection (LOD) and quantification (LOQ) were determined at a signal-to-noise ratio of 3:1 and 10:1, respectively, by injecting a series of dilute solutions with known concentrations. Detailed LC-MS/MS conditions for all analytes are given in the online supplementary information (SI) (Table 1).

Table 1. Content of phenolic acids in different beverages prepared from R. rugosa organs (mean values of three replicate assays with standard deviation).

Phenolic acids (μg/mg of dry extract and *mg per serving ± SD)	I-Fl	I-Lf	I-Hip	I-Nut	I-Rt	T-Fl	T-Lf	T-Hip	T-Nut	T-Rt
Caffeic acid	–	0.04 ± 0.00	–	0.02 ± 0.00	–	–	0.04 ± 0.00	–	0.09 ± 0.00	–
	–	*0.09 ± 0.00	–	*0.01 ± 0.00	–	–	*0.03 ± 0.00	–	*0.01 ± 0.00	–
Gentisic acid	0.11 ± 0.00	–	0.02 ± 0.00	–	–	–	0.02 ± 0.00	–	–	–
	*0.33 ± 0.02	–	*0.08 ± 0.00	–	–	–	*0.01 ± 0.00	–	–	–
Protocatechuic acid	0.38 ± 0.02	0.08 ± 0.00	0.05 ± 0.00	–	0.01 ± 0.00	0.07 ± 0.00	0.16 ± 0.01	0.06 ± 0.00	0.07 ± 0.00	0.01 ± 0.00
	*1.14 ± 0.09	*0.18 ± 0.01	*0.20 ± 0.01	–	*0.01 ± 0.00	*0.06 ± 0.00	*0.12 ± 0.01	*0.08 ± 0.01	*0.01 ± 0.00	*0.00 ± 0.00
Gallic acid	11.35 ± 0.47	2.05 ± 0.09	0.16 ± 0.01	–	0.63 ± 0.03	0.67 ± 0.03	0.98 ± 0.05	0.10 ± 0.01	0.25 ± 0.01	0.22 ± 0.01
	*34.05 ± 0.86	*4.64 ± 0.19	*0.62 ± 0.04	–	*0.55 ± 0.03	*0.66 ± 0.03	*0.73 ± 0.05	*0.14 ± 0.01	*0.04 ± 0.00	*0.08 ± 0.00
Salicylic acid	–	0.03 ± 0.00	–	–	–	–	0.07 ± 0.00	–	–	–
	–	*0.07 ± 0.00	–	–	–	–	*0.05 ± 0.00	–	–	–
Synapic acid	–	–	0.91 ± 0.04	0.05 ± 0.00	–	–	––	–	–	–
	–	–	*3.55 ± 0.15	*0.03 ± 0.00	–	–	–	–	–	–
Ferulic acid	–	–	–	0.04 ± 0.00	–	–	0.02 ± 0.00	–	–	–
	–	–	–	*0.02 ± 0.00	–	–	*0.01 ± 0.00	–	–	–
p-coumaric acid	–	0.03 ± 0.00	–	0.05 ± 0.00	–	–	0.06 ± 0.00	0.01 ± 0.00	0.13 ± 0.01	–
	–	*0.07 ± 0.00	–	*0.03 ± 0.00	–	–	*0.04 ± 0.00	*0.01 ± 0.00	*0.02 ± 0.00	–
3-hydroxybenzoic acid	–	0.04 ± 0.00	–	–	0.11 ± 0.00	–	–	–	–	0.13 ± 0.01
	–	*0.09 ± 0.00	–	–	*0.10 ± 0.00	–	–	–	–	*0.05 ± 0.00
4-hydroxybenzoic acid	–	–	0.30 ± 0.01	–	–	–	0.04 ± 0.00	–	–	–
	–	–	*1.17 ± 0.04	–	–	–	*0.03 ± 0.00	–	–	–
Sum	11.84 ± 0.49	2.27 ± 0.10	1.44 ± 0.07	0.16 ± 0.01	0.75 ± 0.03	0.74 ± 0.03	1.39 ± 0.07	0.17 ± 0.00	0.54 ± 0.02	0.36 ± 0.02
	*35.52 ± 1.36	*5.14 ± 0.22	*5.62 ± 0.25	*0.09 ± 0.00	*0.66 ± 0.03	*0.73 ± 0.03	*1.04 ± 0.06	*0.24 ± 0.02	*0.08 ± 0.00	*0.13 ± 0.00

D: infusion (tea); T: tincture; Fl: flower; Lf: leaf; Hip: pseudo-fruit (hip); Nut: nut (fruit); R: root; One serving = 250 mL for teas and 25 mL for tinctures.

Anti-hyaluronidase activity

The ability of the galenic preparations to inhibit hyaluronidase (Hyal) was evaluated spectrophotometrically by the modified method of Yus et al.^[14] The activity was determined on the basis of precipitation of undigestive hyaluronic acid (HA) with albumin. A series of different extract concentrations in 10% water ethanol solution were prepared. Sodium phosphate buffer (50 µL, 50 mM, pH 7.0; with 77 mM NaCl and 1 mg/mL of albumin), 50 µL of enzyme (30 U/mL of acetate buffer, pH 4.5), and 11 µL of sample were incubated at 37°C for 10 min. Then, 50 µL of HA (0.3 mg/mL of acetate buffer pH 4.5) was added and incubated at 37°C for 45 min. The undigested HA was precipitated with 1 mL acid albumin solution made up of 0.1% bovine serum albumin in 24 mM sodium acetate and 85 mM acetic acid. The mixture was kept at room temperature for 10 min, the absorbance of the reaction mixture was measured at λ = 600 nm using the microplate reader Synergy^TM HT (Bio-Tek, USA). Methyl indole-3-carboxylate, naringenin, and escin were used as the positive controls, the absorbance in the absence of enzyme was used as the blind control. All assays were performed in triplicate. The percentage of inhibition was calculated as

where Ats is absorbance of the tested sample, Aes is absorbance of the enzyme + substrate sample, and Abs is absorbance of the blind sample. Next, IC₅₀ values, which represent the concentrations of the extract that cause 50% inhibition, were determined by linear regression analysis.

Acetylcholinesterase inhibiting activity

Samples were tested for acetylcholinesterase (AChE) inhibiting activity according to the method of Yang et al.^[15] Briefly, 140 μL of 50 mM sodium phosphate buffer (pH = 7.5), 20 μL of sample solution (30 mg/mL), and 15 μL (0.05 IU) of enzyme solution were mixed and incubated at 4°C for 20 min. Then, 10 μL of 10 mM DTNB was added and the reaction was started by adding 10 μL of 75 mM acetylthiocholine iodide. After incubation time (20 min at 37°C), the absorbance of reaction solution was measured immediately at 405 nm in 96-well microplate reader (Bio-Tek). A positive control was prepared by adding 20 μL of physostigmine (the final concentration in the reaction mixture was 15 μg/195 μL) instead of 20 μL sample solution. Blanks were set up by adding 20 μL of buffer solutions instead of sample. Experiment control was set up by adding 15 μL buffer solutions instead of 15 μL enzyme solution in order to deduct sample background. All reactions were carried out in triplicate. The inhibition value (%) was calculated by the following equation:

where A_K is absorbance of enzyme + substrate, A_F is absorbance of a blank positive control, A_B is absorbance of sample solution, and A_S is absorbance of an experimental control.

ABTS radical cation scavenging activity

The antioxidant capacity of galenic preparations was measured using TEAC (Trolox equivalent antioxidant capacity) assay, which was performed according to the method described by Re et al.^[16] The results were expressed as milli-moles of Trolox equivalent per gram of the extract.

Ferric-reducing antioxidant power (FRAP) assay

The FRAP assay was performed as previously described by Arfan et al.^[17] FRAP values were expressed as micro-moles of Fe²⁺ equivalents per gram of extract using the calibration curve of Fe²⁺.

Statistical analysis

The extracts were assayed in triplicate in each test. Data were expressed as mean ± standard deviation of the independent measurements. Statistical analysis was performed by use of the Statistica 6.0 and Excel. Significant differences were calculated according to the Duncan’s multiple range test. Differences at the level of 5% were considered statistically significant.

Results and discussion

Characterisation of phenolic constituents by LC-ESI-MS/MS

Qualitative and quantitative analyses of phenolic constituents in rose galenic preparations were interesting and important from chemopreventive and nutritional viewpoint. Ten phenolic acids, including six gallic acids (i.e., gallic, protocatechuic, salicylic, gentisic, 4-hydroxybenzoic, 3-hydroxybenzoic acid) and four cinnamic acid derivatives (synapic, caffeic, ferulic, and p-coumaric acid) were detected (Table 1). The presence of gallic and protocatechuic acid was revealed in almost all galenic preparations. The content of the remaining acids varied considerably. In majority of extracts, gallic acid was the prevailing one. Its largest amount was contained in the petal tea (34.05 mg/serving). Galenic preparations prepared from leaves were the most abundant in terms of the number of detected phenolic acids. Their greatest number (eight phenolic acids) has been detected in T-Lf, where the presence of salicylic, caffeic, gentisic, ferulic, p-coumaric, and 4-hydroxybenzoic was also shown. Rose hip tea, in turn, possessed the highest synapic and 4-hydroxybenzoic acid contents. In all cases, quantities of phenolic acids contained in a typically administered serving (250 mL of tea and 25 mL of tincture) were found to be higher in water extracts than in the corresponding tinctures. These differences are of course related to the different consumed volumes of both types of extracts. However, results were also affected by nature and characteristics of the material and the efficiency of polyphenol extraction caused by different preparation conditions (i.e., solvent, temperature, plant material to solvent ratio).

Particularly large quantity of phenolic acids was found in tea from petals (35.52 mg per serving). Several times smaller amount was demonstrated in I-Lf and I-Hip (5.14 and 5.62 mg per serving, respectively). Teas from nuts and roots, in turn, contained considerably lower quantities (0.09 and 0.66 mg, respectively), comparable to those found in tinctures (0.08–1.04 mg per serving). The presence of all compounds revealed in this study in extracts prepared from petals, hips, and roots was previously reported in these plant parts.^[6,8,13,18] On the other hand, R. rugosa leaf and nut chemical composition has been poorly studied so far. Therefore, this is the first report where caffeic, gentisic, protocatechuic, gallic, salicylic, p-coumaric, 3-hydroxybenzoic, 4-hydroxybenzoic acid in R. rugosa leaves, and caffeic, protocatechuic, gallic, synapic, ferulic, p-coumaric acid in R. rugosa nut extracts were qualitatively and quantitatively determined.

As regards flavonoids, the presence of six quercetin glycosides, three kaempferol glycosides, and one apigenin glycoside was demonstrated (Table 2). Their quantities per serving were quite varied, and total amount ranged from 0.03 to 4.48 mg. Generally, quercetin derivatives, for example, rutin, hyperoside, isoquercetin, and quercitrin were predominant. In most cases, the doses of flavonoids taken in a typical serving of tincture were found to be higher than in tea from a given plant part. Among the extracts, preparations from leaves and flowers have showed the biggest diversity and contents of flavonoid glycosides. The highest quantities of flavonoids were present in T-Lf (4.48 mg), which contained almost all analysed glycosides (except for spireoside). Moreover, apigenin-7-O-glucoside has been detected only in T-Lf. Slightly lower levels of glycosides have been found in petal tincture (3.41 mg). Relatively high amount has been also revealed in I-Hip and I-Lf (1.09 and 1.56 mg per serving, respectively). The lowest flavonoid contents, in turn, were in extracts prepared from nuts and roots (from 0.03 to 0.17 mg per serving).

Table 2. Content of flavonoid glycosides in different R. rugosa teas and tinctures (mean values of three replicate assays with standard deviation).

Flavonoids (μg/mg of dry extract and *mg per serving ± SD)	I-Fl	I-Lf	I-Hip	I-Nut	I-Rt	T-Fl	T-Lf	T-Hip	T-Nut	T-Rt
Quercetin-3-l-arabinofuranoside (avicularin)	0.01 ± 0.00	0.21 ± 0.03	0.01 ± 0.00	0.01 ± 0.00	0.01 ± 0.00	0.02 ± 0.00	0.19 ± 0.01	0.01 ± 0.00	0.15 ± 0.01	0.01 ± 0.00
	*0.03 ± 0.00	*0.48 ± 0.05	*0.04 ± 0.00	*0.01 ± 0.00	*0.01 ± 0.00	*0.02 ± 0.00	*0.14 ± 0.01	*0.01 ± 0.00	*0.02 ± 0.00	*0.00 ± 0.00
Kaempferol-3-O-rutinoside	0.01 ± 0.00	0.02 ± 0.00	0.02 ± 0.00	–	–	0.12 ± 0.01	0.25 ± 0.01	0.01 ± 0.00	0.05 ± 0.00	0.01 ± 0.00
	*0.03 ± 0.00	*0.05 ± 0.00	*0.08 ± 0.01	–	–	*0.12 ± 0.01	*0.19 ± 0.01	*0.01 ± 0.00	*0.01 ± 0.00	*0.00 ± 0.00
Quercetin-3-O-rutinoside (rutin)	0.01 ± 0.00	0.10 ± 0.01	0.05 ± 0.00	0.01 ± 0.00	–	0.32 ± 0.04	0.63 ± 0.03	0.07 ± 0.00	0.10 ± 0.00	0.05 ± 0.00
	*0.03 ± 0.00	*0.23 ± 0.02	*0.20 ± 0.02	*0.01 ± 0.00	–	*0.32 ± 0.04	*0.47 ± 0.02	*0.10 ± 0.01	*0.01 ± 0.00	*0.02 ± 0.00
Quercetin-3-O-galactoside (hyperoside)	0.04 ± 0.00	0.09 ± 0.00	0.10 ± 0.01	0.01 ± 0.00	0.01 ± 0.00	0.82 ± 0.06	1.50 ± 0.08	0.16 ± 0.02	0.05 ± 0.00	0.11 ± 0.00
	*0.12 ± 0.01	*0.20 ± 0.01	*0.39 ± 0.03	*0.01 ± 0.00	*0.01 ± 0.00	*0.81 ± 0.06	*1.12 ± 0.07	*0.22 ± 0.03	*0.01 ± 0.00	*0.04 ± 0.00
Quercetin-3-O-glucoside (isoquercitrin)	0.08 ± 0.01	0.17 ± 0.02	0.10 ± 0.01	0.02 ± 0.00	0.01 ± 0.00	1.57 ± 0.09	2.26 ± 0.10	0.17 ± 0.02	0.29 ± 0.05	0.18 ± 0.02
	*0.24 ± 0.03	*0.38 ± 0.04	*0.39 ± 0.03	*0.01 ± 0.00	*0.01 ± 0.00	*1.55 ± 0.08	*1.68 ± 0.06	*0.24 ± 0.03	*0.04 ± 0.00	*0.07 ± 0.00
Quercetin-4ʹ-O-glucoside (spireoside)	0.03 ± 0.00	0.01 ± 0.00	–	–	–	–	–	–	–	–
	*0.09 ± 0.01	*0.02 ± 0.00	–	–	–	–	–	–	–	–
Kaempferol-3-O-(6″-O-(E)-p-coumaroyl)-glucoside (tiliroside)	0.02 ± 0.00	0.01 ± 0.00	–	–	–	0.17 ± 0.01	0.45 ± 0.02	0.03 ± 0.00	–	–
	*0.06 ± 0.00	*0.02 ± 0.00	–	–	–	*0.17 ± 0.01	*0.34 ± 0.02	*0.04 ± 0.00	–	–
Apigenin-7-O-glucoside	–	–	–	–	–	–	0.01 ± 0.00	–	–	–
	–	–	–	–	–	–	*0.01 ± 0.00	–	–	–
Kaempferol-3-O-glucoside (astragalin)	–	–	–	–	–	–	0.03 ± 0.00	–	0.02 ± 0.00	–
	–	–	–	–	–	–	*0.02 ± 0.00	–	*0.00 ± 0.00	–
Quercetin-3-O-rhamnoside (quercitrin)	0.02 ± 0.00	0.09 ± 0.01	–	0.01 ± 0.00	–	0.43 ± 0.05	0.69 ± 0.08	–	0.09 ± 0.00	0.10 ± 0.01
	*0.06 ± 0.00	*0.20 ± 0.02	–	*0.01 ± 0.00	–	*0.42 ± 0.05	*0.51 ± 0.06	–	*0.01 ± 0.00	*0.04 ± 0.00
Sum	0.22 ± 0.02	0.69 ± 0.08	0.28 ± 0.03	0.06 ± 0.00	0.03 ± 0.00	3.46 ± 0.26	6.01 ± 0.34	0.45 ± 0.04	0.74 ± 0.07	0.46 ± 0.04
	*0.66 ± 0.05	*1.56 ± 0.14	*1.09 ± 0.10	*0.05 ± 0.00	*0.03 ± 0.00	*3.41 ± 0.25	*4.48 ± 0.25	*0.63 ± 0.06	*0.11 ± 0.01	*0.17 ± 0.01

Abbreviations as in Table 1.

Majority of compounds found in petals and all flavonoids revealed in hips were previously reported.^[7,13,18,19] However, the present study is the first one to report spireoside in petals, kaempferol in roots, and avicularin, kaempferol-3-O-rutinoside, hyperoside, spireoside, tiliroside, and quercitrin in rugosa rose leaves. Moreover, all the flavonoids found in nut extracts were qualitatively and quantitatively determined for the first time.

Anti-hyaluronidase activity

Hyaluronidase inhibiting potential of extracts was tested and compared with activity of standards, that is, escin, naringenin, and methyl indole-3-carboxylate. Results are presented in Table 3. It was revealed that teas from flowers, leaves, and roots and all tinctures strongly affect hyaluronidase’s activity (IC₅₀ 0.66–3.40 mg/mL). Inhibiting activity was not observed for teas prepared from hips and nuts. Root tincture (T-Rt), flower tincture (T-Fl), and leaf infusion (I-Lf) were found to be the most effective hyaluronidase inhibitors (IC₅₀ 0.66–0.80 mg/mL). Their potential was comparable with that presented by methyl indole-3-carboxylate. Slightly higher IC₅₀ values, similar to that of naringenin, were obtained for T-Lf and I-Rt (1.04 and 1.79 mg/mL, respectively). The lowest anti-hyaluronidase activity was demonstrated by nut tincture, hip tincture, and flower infusion. It is worth emphasizing that all active extracts acted stronger than escin (IC₅₀ 8.14 mg/mL), which is known for its anti-exudative, anti-oedemigenous activity, and used in products for increased capillary permeability and venous circulatory disorders.^[20] To the best of our knowledge, our study is the first to report anti-hyaluronidase activity of extracts from R. rugosa organs.

Table 3. Anti-hyaluronidase potential (IC₅₀), acetylcholinesterase (AChE) inhibiting activity, total antioxidant activity (TEAC), and reducing power of R. rugosa teas and tinctures.

Sample	IC50 (mg/mL)	AChE inhibition (%)	TEAC (mmol Trolox/g)	Reducing power/FRAP (μmol Fe^Citation2+/g)
Teas
I-Fl	3.23 ± 0.15	61.63 ± 0.04	2.16 ± 0.01	4810.55 ± 80.02
I-Lf	0.80 ± 0.03	n.o.	3.15 ± 0.22	7567.51 ± 27.22
I-Hip	n.o.	39.00 ± 0.03	0.52 ± 0.01	1517.27 ± 20.17
I-Nut	n.o.	53.65 ± 0.55	0.89 ± 0.06	1776.38 ± 31.47
I-Rt	1.79 ± 0.07	n.o.	3.20 ± 0.12	5994.51 ± 53.64
Tinctures
T-Fl	0.78 ± 0.04	n.o.	1.91 ± 0.09	4997.78 ± 14.00
T-Lf	1.04 ± 0.03	n.o.	2.84 ± 0.10	9258.70 ± 24.88
T-Hip	3.40 ± 0.12	38.30 ± 0.61	0.26 ± 0.00	1092.04 ± 10.34
T-Nut	2.39 ± 0.09	15.15 ± 0.12	1.19 ± 0.03	2161.60 ± 25.37
T-Rt	0.66 ± 0.02	n.o.	4.25 ± 0.05	7310.34 ± 187.34
Standards
Methyl indole-3-carboxylate	0.49 ± 0.02	n/a	n/a	n/a
Escin	8.14 ± 0.29	n/a	n/a	n/a
Naringenin	1.28 ± 0.04	n/a	n/a	n/a
Physostigmine	n/a	89.4	n/a	n/a

Mean values of three replications ± SD; n.o.: activity was not observed; n/a: not applicable; other abbreviations as in Table 1. Physostigmine was tested at a concentration of 15 µg/195 µL of the reaction mixture.

Acetylcholinesterase inhibiting potential

Rose galenic preparations were evaluated for their ability to affect acetylcholinesterase activity. It was found that T-Fl and extracts prepared from leaves and roots had no effect on enzyme’s activity (Table 3). The other extracts demonstrated acetylcholinesterase inhibiting potential, however at the relatively high concentration. Flower tea (I-Fl) was found to be the most potent acetylcholinesterase inhibitor (61.63% enzyme inhibition), followed by tea from nuts (I-Nut, 53.65%). Both rose hip extracts showed similar, moderate effect (38.3% and 39%). T-Nut demonstrated the lowest inhibiting potential.

Antiradical activity

Previous study revealed that extracts from aerial and underground parts of R. rugosa possess strong capability to scavenge DPPH^• radical. This activity was found to be closely related to phenolics’ concentration.^[4] However, significant quantities of lipophilic substances, such as carotenoids, tocopherols, and fatty acids in nuts and hips, and presence of terpenoids in roots were also reported.^[4,7,21,22] Therefore, it was decided to evaluate total antioxidant activity of galenic preparations using ABTS radical (more suitable to analyse both hydrophilic and lipophilic antioxidants).

As a result, very high antioxidant potential of root and leaf extracts was shown (Table 3). Particularly strong was the activity of T-Rt (4.25 mmol Trolox/g). It could be related to the considerable amount of tannins and o-dihydroxyphenols found in this plant part.^[4] The other extracts from roots and leaves presented TEAC in the range from 2.84 to 3.20 mmol Trolox/g. Slightly lower ability to scavenge ABTS radical was observed for flower galenic preparations (1.91–2.16 mmol/g). The weakest antioxidant potential was presented by both hip extracts (0.26 and 0.52 mmol Trolox/g, for T-Hip and I-Hip, respectively). Nut extracts have showed moderate radical scavenging power. Similar order in decreasing of antioxidant activity was observed in our previous study conducted with DPPH radical.^[4] However, ABTS scavenging assay has revealed larger differences between activity of hip and nut extracts.

Ferric-reducing antioxidant power

All samples were also analysed for their ferric reducing power. The FRAP values are presented in Table 3. Generally, tinctures presented higher reducing activity in comparison with teas obtained from analogous plant organs. The only exception is T-Hip, which showed lower potential than I-Hip (1092.04 and 1517.27 μmol Fe²⁺/g, respectively). Both hip extracts were also the weakest from all preparations. Slightly higher activity was presented by nut extracts (1776.38 and 2161.60 μmol Fe²⁺/g). Also in the case of FRAP assay, galenic preparations prepared from petals have demonstrated the average activity, several times higher than the activities of hip and nut samples. Again, the highest potential was showed by leaf and root extracts, T-Lf in particular (9258.70 μmol Fe²⁺/g). High antioxidant potential of roots, leaves, and petals is probably associated with high o-dihydroxyphenols and flavonoids levels revealed in these organs in the previous study.^[4]

Conclusion

Although some reports revealed that R. rugosa is a potential source of biologically active substances, it seems to be still unknown, underestimated, and underused medicinal plant. Teas and tinctures prepared from petals and hips are already well known, used, and tasty preparations. Our study demonstrated that galenic preparations prepared from other organs could be rich sources of natural radical scavengers and other health beneficial substances which play an important role in everyday healthy diet. Water and ethanolic extracts from leaves, petals, and roots were found to be excellent sources of antioxidants. Moreover, the possibility of using R. rugosa extracts as effective hyaluronidase inhibitors seems to be new and very promising issue. We hope that study findings will contribute to increased knowledge on the therapeutic potential and a wider use of different rose organs. Part of them may be obtained as post-harvest or post-production residues (e.g., roots, nuts) contributing to the comprehensive utilisation of plantation resources or production materials.

Funding

This work was financially supported by the Medical University of Lublin (grant No. MNmb 53).

Supplemental data

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Additional information

Funding

This work was financially supported by the Medical University of Lublin (grant No. MNmb 53).