Phytochemical Study on Seeds of Paeonia clusii subsp. rhodia—Antioxidant and Anti-Tyrosinase Properties

In this study, the black fertile (BSs) and the red unfertile seeds (RSs) of the Greek endemic Paeonia clusii subsp. rhodia (Stearn) Tzanoud were studied for the first time. Nine phenolic derivatives, trans-resveratol, trans-resveratrol-4′-O-β-d-glucopyranoside, trans-ε-viniferin, trans-gnetin H, luteolin, luteolin 3′-O-β-d-glucoside, luteolin 3′,4′-di-O-β-d-glucopyranoside, and benzoic acid, along with the monoterpene glycoside paeoniflorin, have been isolated and structurally elucidated. Furthermore, 33 metabolites have been identified from BSs through UHPLC-HRMS, including 6 monoterpene glycosides of the paeoniflorin type with the characteristic cage-like terpenic skeleton found only in plants of the genus Paeonia, 6 gallic acid derivatives, 10 oligostilbene compounds, and 11 flavonoid derivatives. From the RSs, through HS-SPME and GC-MS, 19 metabolites were identified, among which nopinone, myrtanal, and cis-myrtanol have been reported only in peonies’ roots and flowers to date. The total phenolic content of both seed extracts (BS and RS) was extremely high (up to 289.97 mg GAE/g) and, moreover, they showed interesting antioxidative activity and anti-tyrosinase properties. The isolated compounds were also biologically evaluated. Especially in the case of trans-gnetin H, the expressed anti-tyrosinase activity was higher than that of kojic acid, which is a well-known whitening agent standard.


Introduction
Paeonia is the sole genus in Paeoniaceae plant family, distributed within Europe and Asia, and is named after Paeon, a student of the Greek father of medicine Asclepius. The plant has been cultivated in China since the Han dynasty, and is known as the "king of flowers" and/or "flowers of richness and honor" [1]. It is known commercially for its ornamental value due to its impressive and often fragrant flowers. Since antiquity, the use of peonies has been reported in traditional medicine for the treatment of epilepsy conditions, as described by Dioscorides in the 1st century A.D. [2]. In Traditional Chinese Medicine, peonies have been used for the treatment of gynecological problems, cramps, and pain and have been used for their antioxidant, anti-inflammatory, analgesic, and antimicrobial properties [3][4][5].
So far, hundreds of secondary metabolites have been isolated from the various peony species, which have been classified into the following chemical categories: monoterpene glycosides, stilbenes, triterpenes, tannins, steroids, flavonoids, and phenols [3]. Paeoniflorin, a water-soluble monoterpene glycoside, has been observed in all peony species examined to date. It has not been found in any other genus beyond that of peonies and is therefore a chemo-taxonomic indicator of the genus, and it is also suspected to be responsible for some of the medicinal properties of the plant [6][7][8]. Trans-gnetin H, a resveratrol (3,5,4 -trihydroxystilbene) trimer that is usually found in plants of the Paeoniaceae and

Identification of Secondary Metabolites
The BSs of P. clusii subsp. rhodia were studied phytochemically through UHPLC-HRMS analysis, and 33 metabolites were tentatively identified from the methanolic extract ( Figure S1), including 6 monoterpene glycosides of the paeoniflorin type with the characteristic cage-like terpenic skeleton found only in plants of the genus Paeonia, 6 gallic acid derivatives, 10 oligostilbene compounds, and 11 flavonoid derivatives ( Table 1).
The GC-MS analysis of the RS dichloromethane extracts led to the detection of 11 substances, whereas nine substances were detected in the pentane extract (Table 2). Furthermore, the HS-SPME analysis led to the identification of 10 metabolites, and the main metabolites for all of these analyses were myrtanal and nopinone ( Table 2).

Total Phenolic Content (TPC)
The methanolic extracts of the BSs and RSs exhibited high phenolic contents: 204.62 ± 4.0 mg GAE/g extract for the BS extract and 177.59 ± 7.5 GAE/g extract for the RS extract.

DPPH Assay
The antioxidant properties of both seeds' methanolic extracts and of selected isolated compounds were determined using the DPPH assay and the results are displayed in Table 3 as percentages of DPPH inhibition in three different concentrations. Two-way analysis of variance (ANOVA) was used to evaluate results (p-value summary: p < 0.0001) and the significance of differences between means was determined by means of Tukey's post hoc test. Multiple-comparisons differences at p < 0.05 were regarded as statistically significant. Values expressed are means ± S.D. of three parallel measurements. Superscripts A to C and a to g denote significant differences within rows and columns, respectively, and multiple comparison analysis resulted in statistical significance levels of p < 0.05.

Tyrosinase Inhibitory Activity
The tyrosinase inhibitory activity of the studied methanolic extracts of BSs and RSs and of selected isolated compounds is presented in Table 4. One-way ANOVA was used to compare IC50 mean values (p < 0.0001) and two-way ANOVA (p-value summary: p < 0.0001) followed by Tukey's post hoc test was used to evaluate the significance of differences between means of the inhibition activity percentages. A p-value < 0.05 was considered an indicator of statistical significance. Values expressed are means ± S.D. of three parallel measurements. Superscripts A to C and a to d denote significant differences within rows and columns, respectively, and multiple comparison analysis resulted in statistical significance levels of p < 0.05.

Discussion
Monoterpenes and monoterpene glucosides are predominant in Paeonia species, according to data in the literature [40]. In this study, six monoterpene glycosides were characterised, including paeoniflorin, galloyl-paeoniflorin, dibenzoyl-paeoniflorin, methyl-debenzoyl-paeoniflorin, albiflorin, and one that was not further identified, referred to in Table 1 as a paeoniflorin isomer. Paeoniflorin is commonly found in the Paeoniaceae family and it is considered a chemotaxonomic marker for this family, having been reported in P. lactiflora, P. suffruticosa, P. emodi, P. osii, and P. anomala [35], as well as in the roots of the Greek species P. clusii subsp. clusii [14,15].
To date, stilbenes have been isolated only from the seeds of Paeonia species and appear to be resveratrol oligomers. In this study, 10 oligostilbene compounds were identified: transand cisresveratrol, as well as their dimers trans and cis-ε-viniferin and two trimers in the form of trans-gnetin H isomers, along with their glucosides (trans and cis-resveratrol hexosides, trans and cis-ε-viniferin hexosides). Resveratrol and its oligomers have attracted the interest of researchers as potential therapeutic agents for various diseases due to the wide variety of biological and pharmacological activities they have presented [41][42][43][44]. For this reason, extensive research has been undertaken to find natural stilbene agents, which have so far been reported in plant families such as Dipterocarpaceae, Vitaceae, Cyperaceae, Gnetaceae, Fabaceae (Leguminosae), Paeoniaceae, and Apiaceadoraceae [45,46]. In the family Paeoniaceae, they have been found only in the seeds of peony species such as P. suffruticosa, P. lactiflora [3,9], P. rockii [47], and other paeony trees of Chinese origin, and only in P. officinalis [36] from Europe.
A number of gallic derivatives from Paeonia species have been observed previously. The results of this study indicated that six of them, including gallic acid, methyl gallate, glucogallin, trigalloyl glucose, tetragalloylglucose, and di-O-galloyl-β-D-glucopyranose, were detected in the studied Greek plant. These compounds have been very recently reported only once in the seed kernels and hulls of P. lactiflora Pall. [21]. Furthermore, benzoic acid has been previously detected in several peony species [35].
In addition to the above constituents, 11 flavonoids, including kaempferol and luteolin and their glucosides, as well as two tetrahydroxyflavone hexosides, were present in the black seeds of the studied peony species. Furthermore, in the chemical category of flavonoids, luteolin and its glucosides (luteolin 3 -O-β-D-glucopyranoside and luteolin 3 ,4 -di-O-β-D-glucopyranoside) were isolated and structurally determined, whereas luteolin-glucopyranosides were isolated for the first time in peonies and in the whole Paeoniaceae family.
Furthermore, many of the compounds which were identified in the non-polar extracts of RS through GC-MS and HS-SPME, such as nopinone and myrtanal, which were the most abundant compounds, as well as cisand trans-myrtanol, E-myrtenol, myrtenal, β-pinene oxide, camphene, α-methylbenzyl alcohol, and phellandral, have been found in previous studies in several peonies, mainly in their roots and flowers [14,[48][49][50] but here they were detected for the first time in seeds and especially in RSs (red seeds) from this genus.
Additionally, among the identified metabolites, luteolin and other flavonoid derivatives, such as kaempferol glucoside and di-glucoside, were found to possess significant DPPH radical scavenging activity in the study presented in [51], suggesting that a high flavonoid content (kaempferol-and luteolin-glycosides) is directly related to strong antioxidant properties [52]. In addition, polyphenols such as gallic acid and methyl gallate were previously reported to be more effective in scavenging free-radicals than the known antioxidant compound α-tocopherol [53]. Stilbenes such as resveratrol dimers (trans-and cis-ε-viniferin) and trimers (trans-gnetin H) exhibited the strongest antioxidant activity among the stilbenes referred to in the literature [10]. Moreover, resveratrol showed moderate activity which was higher than that of its glycosides trans and cis-resveratroloside, suggesting that its glucosylation decreased its antioxidant activity, probably due to its low affinity to biological lipid membranes [10,54]. Paeoniflorin and monoterpenes with pinane structures have been found not to inhibit free-radical DPPH [53], which was also confirmed by our results. Therefore, the correlation between the synergistic, antagonistic, and antioxidant activities of phenolic substances in peonies should be further examined in the future.
No previous studies have been conducted on the antioxidant activity of P. clusii subsp. rhodia, but after comparing our results with those presented for other peonies in the literature, we concluded that the activity of this subspecies is significantly higher than that reported for other subspecies. Specifically, Sevim and coworkers [55] reported that seed extracts from seven herbaceous peony species in Turkey exerted low to moderate radical scavenging activity (below 50% at 2000 µg/mL) against DPPH. In another study [56], the authors concluded that the antioxidant capacity of seed coats of Paeonia ranged from 66.56 mg/g to 82.85 mg/g (expressed as GAE mg per 1 g DW). Therefore, it seems that the studied red seeds (RSs) from Greek peonies may serve as a good source for extracts with high antioxidant capacity (84.0 ± 2.1 at 100 µg/mL), but further investigation is suggested and required.
Regarding the isolated compounds, luteolin (IC50 = 13.2 ± 3.5 µg/mL) was characterized as a strong antioxidant in relation to gallic acid (IC50 = 4.2 ± 0.1 µg/mL), which is confirmed by the literature [57]. In addition, trans-resveratrol showed higher activity than its glycoside and trans-ε-viniferin, whereas trans-gnetin H showed low inhibition, a fact that is also confirmed by the literature [58,59]. Finally, paeoniflorin showed a negative ability to inhibit free-radical DPPH, as was also confirmed in the literature by Picerno et al. [53] and Wu et al. [60], and according to Matsuda et al. [61], the presence of the galloyl group is essential for the radical scavenging effect. Another study comparing the antioxidant effects of oxypaeoniflorin and paeoniflorin reported that oxypaeoniflorin has strong antioxidant potential in comparison to paeoniflorin although they have very similar chemical structures [62]. Therefore, the pinane structure, combined with the lack of a galloyl group and the lack of a hydroxyl group in its aromatic ring, further weaken the antioxidant potential of paeoniflorin as it has no free electrons to contribute.

Plant Material
Fresh black (BSs) and red seeds (RSs) of P. clusii subsp. rhodia (Stearn) Tzanoud. (Figure 1) were obtained from Prophet Elias mountain (798 m in height) on the island of Rhodes (South Aegean, Greece) in April 2020. The sample was identified botanically by Dr Bazos I., Section of Ecology and Systematics, Department of Biology, National and Kapodistrian University of Athens, Greece. The seeds were naturally dried, ground using a laboratory mill, and stored in darkness at room temperature.
Fresh black (BSs) and red seeds (RSs) of P. clusii subsp. rhodia (Stearn) Tzanoud. (Figure 1) were obtained from Prophet Elias mountain (798 m in height) on the island of Rhodes (South Aegean, Greece) in April 2020. The sample was identified botanically by Dr Bazos I., Section of Ecology and Systematics, Department of Biology, National and Kapodistrian University of Athens, Greece. The seeds were naturally dried, ground using a laboratory mill, and stored in darkness at room temperature.  A part of the dried fragmented RSs (500 mg) was extracted with 10 mL of dichloromethane (DCM) and another quantity of 500 mg with 10 mL of pentane C 5 H 12 using ultrasound for 30 min at 25 • C to yield 7.4 mg and 5.1 mg, respectively. These extracts were further analyzed through GC-MS.
A part of the crude RS DCM extract (25 mg) was subjected to preparative TLC with a mixture of toluene:EtOAc 70:30 v/v as a mobile phase to afford benzoic acid (2.1 mg).

Headspace Solid-Phase Microextraction (HS-SPME)
The aroma of the P. clusii subsp. rhodia red seeds was studied through HS-SPME/GC-MS analyses [64]. HS-SPME was performed with Supelco SPME fiber 100 µm PDMS (polydimethylsiloxane coating) and Supelco SPME fiber 75 µm PDMS/DVB (polydimethylsiloxane/divinylbenzene coating) attached to a manual SPME fiber holder (Supelco, Bellefonte, PA, USA). First, the fiber was conditioned in the GC at 250 • C for 30 min and then was inserted into the sample vial. For SPME extraction, 500 mg of the sample in a glass vial (15 mL), closed with a PTFE-coated silicone rubber septum, was used. The temperature in our experiment was set at 60 • C and the vial with the sample was placed on the hotplate for 30 min. After that time, the fiber was exposed to the sample for 30 min at 60 • C and then it was transferred to perform GC-MS analysis. The initial temperature of the column was 55 • C for 2 min with an increase of 5 • C/min until it reached 240 • C, where it remained for 2 min. The total analysis time was 41 min. The substances were identified by comparing the mass spectrum of each substance with those of the Wiley 275 library and the literature.

GC-MS Analysis
The analyses were performed on an Agilent Technologies Gas Chromatography 7820A sysem (Shanghai, China), connected to an Agilent Tecnologies 5977B mass spectrometer (Santa Clara, CA, USA), which worked via EI with an ionization energy of 70 eV. The gas chromatograph was equipped with a split/splitless injector and a 30 m long HP 5MS capillary column with an inner diameter of 0.25 mm and a membrane thickness of 0.25 µm. The temperature in the injection sample was 250 • C and the carrier gas was helium. The following temperature program was used: an initial column temperature of 60 • C was kept constant for 5 min and with an increase of 3 • C/min it reached 280 • C, where it remained for 15 min. The total analysis time was 93 minutes. Identifications were made using the Wiley275 library and bibliographic data.

Analysis through UHPLC-HRMS
Ultra-high-performance liquid chromatography was performed employing a Vanquish UHPLC system (Thermo Fisher Scientific, Germering, Germany) equipped with a binary pump, an autosampler, an online vacuum degasser, and a temperature-controlled column compartment. LC-MS-grade acetonitrile (ACN), methanol (MeOH), and formic acid (FA) were purchased from Fisher Scientific (Thermo Fisher Scientific, Leicestershire, UK) and LC-MS water was produced using a Barnstead MicroPure Water Purification System (Thermo Scientific, Germany). An Accucore Vanquish UPLC C18 (2.1 × 50 mm, 1.5 µm) reverse-phase column (Thermo Scientific, Germany) was used for the analysis. High-resolution mass spectrometry was performed on an Orbitrap Exactive Plus mass spectrometer (Thermo Scientific, Germany). Samples were injected at concentrations of 100 ppm diluted in MeOH/H 2 O at 50:50. The mobile phase consisted of solvents A (aqueous 0.1% (v/v) formic acid) and B (acetonitrile). Different gradient elutions were performed for positive-and negative-ion-mode detection and after optimization of the chromatography the gradient applied was: T = 0 min, 5% B; T = 3 min, 5% B; T = 21 min, 95% B; T = 26 min, 95% B; T = 26.1 min, 5% B; T = 30 min, 5% B. The flow rate was 0.3 mL/min and the injection volume was 5 µL. The column temperature was kept at 40 • C and the sample tray temperature was set at 10 • C. The ionization was performed at HESI, in both positive and negative modes. The conditions for the HRMS for both negative and positive ionization modes were set as follows: capillary temperature, 320 • C; spray voltage, 2.7 kV; S-lense Rf level, 50 V; sheath gas flow, 40 arb. units; aux gas flow, 8 arb. units; aux. gas heater temperature, 50 • C. Analysis was performed using the Fourier transform mass spectrometry mode (FTMS) in the full-scan ion mode, applying a resolution of 70,000, whereas the acquisition of the mass spectra was performed in every case using the centroid mode. The data-dependent acquisition capability was also used at 35,000 resolution, allowing for MS/MS fragmentation of the three most intense ions of every peak exceeding the predefined threshold, applying a 10 s dynamic exclusion window. The normalized collision energy was set at 35. Data acquisition and analysis were completed employing Xcalibur 2.1.

Total Phenolic Content (TPC)
The total phenolic content of the samples was determined by means of the Folin-Ciocalteu method [64]. In a 96-well plate, 25 µL of extracts with different concentrations (4, 2, 1 mg/mL) or standard solutions of gallic acid (2.5, 5, 10, 12.5, 20, 25, 40, 50, 80, 100 g/mL), both diluted in DMSO, were added to 125 µL of a Folin-Ciocalteu solution (10%), followed by the addition of 100 µL of 7.5% sodium carbonate. The plate was incubated for 30 min in darkness at room temperature. The absorbance at 765 nm was measured using a TECAN Infinite m200 PRO multimode reader (Tecan Group, Männedorf, Switzerland). All measurements were performed in triplicate, the mean values were plotted on a gallic acid calibration curve, and the total phenolic content was expressed as mg equivalents to gallic acid (GAE) per gram of dry extract.

DPPH (2,2-DiPhenyl-1-PicrylHydrazyl) Assay
The antioxidant activity of the samples was evaluated based on DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity according to the literature [65]. For the DPPH assay, the methanol extract and the isolated compounds (1, 2, 3, 4, 5, and 6) (concentration of stock solution: 4 mg/mL) were prepared using dimethylsulfoxide (DMSO) as a solvent. Ten (10) µL of each sample were mixed with 190 µL of DPPH solution (12.4 mg/100 mL in ethanol) in a 96-well plate and then subsequently incubated, at room temperature, for 30 min in darkness. Finally, the absorbance was measured at 517 nm using an Infinite M200 Pro TECAN photometer (Tecan Group, Männedorf, Switzerland). All evaluations were performed in triplicates, whereas gallic acid was used as positive control (IC50 = 4.2 ± 0.1 µg/mL). The percentage inhibition of the DPPH radical for each dilution was calculated using the following formula: . All the samples were tested at final concentrations of 200 µg/mL, 100 µg/mL, and 50 µg/mL, whereas for the most active of them (DPPH inh. >70% at concentration C = 50 µg/mL), the IC50 value was calculated.

Tyrosinase Inhibition
The methanolic extract, as well as compounds 3, 4, and 5, were investigated for their ability to inhibit the oxidation of L-DOPA (L-3,4-dihydroxyphenylalanine) to dopaquinone and subsequently to dopachrome by the enzyme tyrosinase [19]. The fractions were initially dissolved in DMSO (10 mg/mL) and subsequently diluted in the proper concentration in phosphate buffer, 1/15 M (NaH 2 PO 4 /Na 2 HPO 4 ), pH 6.8. Final concentrations of DMSO in the well did not exceeded 3%. In 96-well plates, 80 µL of phosphate buffer 1/15 M (NaH 2 PO 4 /Na 2 HPO 4 ), pH 6.8, 40 µL of sample in the same buffer and 40 µL mushroom tyrosinase (92 Units/mL) in the same buffer, were mixed. The contents of each well were incubated for 10 min at 25 • C, before 40 µL of 2.5 mM L-DOPA were added into the same buffer. After incubation at 25 • C for 5 min, the absorbance at 475 nm of each well was measured. Samples were evaluated at 75 µg/mL, 150 µg/mL, and 300 µg/mL, in triplicate, and blank samples for every fraction were also measured, whereas kojic acid was used as a positive control. The percentage inhibition of tyrosinase activity was calculated using the

Statistical Analysis
All in vitro tests were performed in triplicate and values are expressed as mean values ± S.D. of the three independent experiments, calculated in Excel. Then, statistical analysis was performed on the means of the triplicates using GraphPad Prism Version 8.3.0, applying one-way ordinary analysis of variance (ANOVA) for tyrosinase IC50 and two-way ANOVA for the rest of the experiments.

Conclusions
The black fertile seeds (BSs) and the red unfertile seeds (RSs) of the Greek endemic Paeonia clusii subsp. rhodia were studied and nine (9) phenolic derivatives were isolated, whereas thirty-three (33) metabolites were identified from BSs through UHPLC-HRMS. The total phenolic content of BSs was extremely high, as well as the anti-tyrosinase activity. The high antioxidant activity expressed by the extracts could suggest that the studied extract could be useful for the treatment of the effects of oxidative stress, which can lead to several skin disorders. In addition to presenting the chemical analysis of Greek Paeonia seeds and their abundant resveratrol derivatives, in this study we have also provided new ideas for the comprehensive development and application of Paeonia seeds in the field of skincare, such as dermatology and cosmetology. Due to the increasing demand for natural ingredients from peony species, quantitative methods of analysis for stilbenes, as well as further studies towards dermal absorption, metabolism, and safety of use, will be required for the potential application of such extracts and components in the industry of "eco cosmetics". Furthermore, the quantification of stilbene derivatives in seeds of wild-growing peony species in Greece will be planned in the near future.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.