Structure Elucidation of Procyanidins Isolated from Rhododendron formosanum and Their Anti-Oxidative and Anti-Bacterial Activities

Rhododendron formosanum is an endemic species distributed in the central mountains of Taiwan. In this study, the biological activities of major procyanidins isolated from the leaf extract of R. formosanum were investigated. Four compounds, including two procyanidin dimers, procyanidin A1 (1) and B3 (2), and two procyanidin trimmers, procyanidin C4 (4) and cinnamtannin D1 (5), were isolated and identified on the basis of spectroscopic data. The structure of a new procyanidin dimer, rhodonidin A (3), was elucidated by 2D-NMR, CD spectrum and MS. The procyanidin trimmers and rhodonidin A are reported for the first time in Ericaceae. The biological activities of these procyanidins were evaluated using anti-bacterial and anti-oxidative assays. Only the new compound 3 demonstrated strong anti-bacterial activity against Staphylococcus aureus at an MIC value of 4 μg/mL. All compounds showed pronounced antioxidant activities and the activities are enhanced as the amount of OH groups in procyanidins increased. In conclusion, the pleiotropic effects of procyanidins isolated from the leaves of R. formosanum can be a source of promising compounds for the development of future pharmacological applications.


Introduction
Procyanidins are widely distributed throughout the plant kingdom. The evidences linked procyanidins with organoleptic characteristics, plant defense mechanisms, and potential health benefits were reported [1][2][3]. Among plant secondary metabolites, procyanidins are most liable to oxidation and their activity is closely related to plant defense systems against oxidative stress. Moreover, reports of several assays in vitro demonstrate potential interactions with biological functions, including antimicrobial [4], anti-proliferation [5], enzyme inhibiting [6], antioxidant, and radical-scavenging properties [1,2]. Typical condensed procyanidins exist as oligomers containing from two to five or six catechin or epicatechin units and as more condensed polymers. However, the structures of procyanidins, particularly larger polymeric procyanidins, are poorly understood.
Rhododendron formosanum is an endemic species distributed in the central mountains of Taiwan at elevations from 1500 m to 2500 m. Previously, 18 hydrophobic compounds and two isomeric epoxysitosterols have been isolated and their allelopathic activities were also evaluated [7,8]. Recently, the anti-lung cancer activity of the pentacyclic triterpenoids isolated from R. formosanum was reported [9]. Moreover, the hydrophilic compounds responsible for allelopathic phenomenon were also identified by HPLC methods and the major chemical components of the leaves extract of R. formosanum were identified as (−)-catechin [10]. Catechin was further transformed into protocatechuic acid in the soil by microbes in the rhizosphere [11].The successful stabilization of R. formosanum is due to the synergistic phytotoxic effects of protocatechuic acid and (−)-catechin. Although the major chemicals in the leaves of R. formosanum have been investigated prominently, the structures of condensed procyanidins containing catechins or epicatechins units are still unknown.
The aim of this study was to isolate and elucidate the structure of procyanidins from the leaf extract of R. formosanum. The biological activities, including antibacterial and antioxidative activities, were also examined.

Identification of Isolated Procyanidins
Chemical structures of compounds 1-5 were illustrated in Figure 1 6 Hz, H-4), the meta-coupled doublets at 5.95, 6.06 (each d, J = 2. 4 Hz, H-6, H-8), a residual one aromatic proton singlet at δH 6.08 (s, H-6′), and two AMX systems in the aromatic region (δH 6.5-7.5) due to rings B and E confirmed the A-type procyanidin. This doubly linked dimeric structure was also supported by the one acetal carbon at δC 100.3 in its 13 C-NMR spectrum. A large value of 8-10 Hz for J2,3 indicates a catechin unit (2,3-trans), and a small value of 2 Hz or a broad singlet indicates an epicatechin unit (2,3-cis). The signal widths and observable couplings J2,3 and J3,4 in 1 indicated the presence of epicatechin and catechin units. In addition, two flavanol units of A-type procyanidins must possess either (2α, 4α) or (2β, 4β) double interflavanyl bonds. The positive Cotton effect at 220-250 nm ( Figure 2) of CD spectrum of compound 1 allowed assignment of absolute configuration of C-4 as R [12,13], thus deciding the 2β,4β-configuration for compound 1. Comparison of the 1 H-and 13 C-NMR spectroscopic data with the literature established compound 1 as procyanidin A1 (Figure 1), previously isolated from peanut skins [14].  Compound 2 showed a molecular ion with m/z 577.1 in negative-ion modes, indicating that it was a B-type procyanidin dimer. Two AMX systems in the aromatic region (δH 5.8-6.9) with large coupling constants in the region of δH 4.5-3.7 (H-2/H-3/H-4) and the 13 C-NMR spectrum of two carbon signals at 82.4 and 83.9 corresponding to C2 of C and F rings, two catechin units can be identified. The position of the interflavan bond was determined by HMBC data. CD measurements revealed a negative Cotton effect in the diagnostic wavelength region (220-240 nm), reflecting α-orientation of the 4-flavanyl substituents ( Figure 2). Because of rotational and heterocyclic ring conformational heterogeneity in dimeric procyanidins, the proton NMR spectrum of compound 2 exhibited two distinct sets of resonances showing the presence of two rotamers in an approximate 2:1 ratio. Comparison of the 1 H-and 13 C-NMR spectroscopic data with the literature established compound 2 as procyanidin B3 (Figure 1) [15].  The C-8′ involvement in the interflavan lineage was construed from the HMBC correlations, which permitted us to assign the C-8a′ and the C-5′ carbon atoms. The observation of the HMBC correlation from H-13 to C-8′ also confirmed the linkage between C-14 and C-8′ ( Figure S2). In addition, IR spectrum at 1843 and 1714 cm −1 also confirmed the ketonoic structure of C-12 ( Figure S5). According to the data of 1 H-and 13 C-NMR (Table 1) and 2D NMR (HSQC, HMBC), compound 3 is similar to dehydrodicatechin A, a (+)-catechin derivative which had been obtained by enzymatic oxidation [16] and isolated from the roots of Rosa laevigata [17] and Quercus ilex [18]. However, the NOE correlation between H-2 and H-10′ ( Figure S6) indicated the 3D structure of 3 is a compact and not extended form.

OH
The three-dimensional structure of compound 3 was obtained using ChemBio3D software and the MM2 force field. In the compact form of (−)-catechin dimer, correlation peaks are observed between H-2 and H-10′, H-10′ and H-13, and H-2 and H-13, for which the interatomic distance measured on the minimized structure are 3.76 Å, 2.98 Å, and 3.32 Å, respectively ( Figure 3). In the extended structure of (+)-catechin dimer, the NOE correlation could not be observed because the interatomic distances are all over the detection limited (5 Å). Moreover, circular dichroism is a powerful tool for establishing the absolute configuration of flavonoids and procyanidin. A positive Cotton effect at 280 nm indicated a 2S configuration while the negative Cotton effects in the 240 nm region indicated 3R absolute configurations, respectively ( Figure 2) [19]. The 2S, 3R configuration was also suggested by the negative optical rotation of 3. Taking the NOE interactions into consideration, the data of circular dichroism defined the (−)-catechin unit with 2S and 3R absolute configurations. Thus, the name of compound 3 is given as rhodonidin A (Figure 1). 14, m) attributable to the H-3 atoms, along with a set of signals due to the H-2 atoms of confirmed the one epicatechin with two catechin units. The 13 C-NMR spectrum of compound 4 exhibited two C-2 signals at δC 82.0 and 83.5 due to catechin units and one C-2 signal at δC 76.1 consistent with an epicatechin unit. The spectroscopic data indicated the lineages between units were connected at position C-4 of unit I/II to C8 of unit II/III, which were confirmed by HMBC correlations between H-4 and C-7′, C-8′, and C-9′ and between H-4′ and C-7′′, C-8′′, and C-9′′, respectively. The CD spectrum of 4 showed a positive Cotton effect at 220-250 nm (Figure 2), demonstrated a β-orientation of 4-flavanyl linkage. According to the data of 1 H-and 13 C-NMR and 2D NMR (HSQC, HMBC, COSY, NOESY), compound 4 is defined as procyanidin C4 [20].

Antibacterial Activity
As shown in Table 2, the antibacterial activities of compounds 1-5 were tested against eight bacterial pathogens by minimum inhibitory concentration (MIC) or minimum bactericidal concentration (MBC) methods. Only procyanidins dimer (compound 1-3) displayed antibacterial activities against S. aureus. None of the procyanidins trimer showed pronounced antibacterial activities against all tested pathogens. In addition, only compound 1 demonstrated medium antimicrobial activities against L. monocytogenes and B. cereus. None of the bactericidal activities of isolated compounds against H. pylori were observed in this study.
Previous studies revealed a moderate antibacterial activity for certain procyanidins against Streptococcus pyogenes, Bacillus cereus, Klebsiella pneumoniae, and Proteus vulgaris at concentrations <100 µg/mL [24]. The determination of MIC against S. aureus gave a value of 100 µg/mL for procyanidin B2 [25], a procyanidin dimer with two epicatechin units linked with 4β-8 interfavan bond. In this study, procyanidin A1 (1) and B3 (3) generated anti gram-positive bacteria activities at MIC values of 64 µg/mL. All these results indicated procyanidin dimers displayed moderate antimicrobial activity against certain pathogens. Structure modification of procyanidins, such as rhodonidin A (3), may increase the antibacterial ability against S. aureus. In Asia, S. aureus is the leading cause of food-born pathogen. Thus, assessing potential antibacterial agent, such as rhodonidin A, and its antibacterial mechanism against S. aureus is a hot area of investigation.

Antioxidative Activity
The antioxidant activities of the isolated procyanidins were measured using the DPPH free radical-scavenging assay and CUPric reducing antioxidant capacity (CUPRAC) method. The results from the DPPH (IC50) method for the standard trolox, (−)-catechin and compounds 1-5 isolated in this study showed values of 61.12, 27.07, 20.89, 8.55, 13.06, 6.26 and 3.29 μg/mL, respectively (Table 3). Cinnamtannin D1 showed lowest IC50 value at 3.29 μg/demonstrating the strongest free radical-scavenging activity in this study. The radical scavenging activity is enhanced as the amount of OH groups in procyanidins increased ( Figure 4A). These observations were in line with the results reported previously [24,26]. Ricardo da Silva et al. stated that it was not the degree of polymerization, but the number of hydroxyl groups that was important for the radical scavenging activity.  In CUPRAC assay, trolox was used as standard chemical for antioxidant activity comparison. B-type procyanidins, such as procyanidin B3 and C4, displayed the highest values of antioxidant activities at 4.87 and 3.48 (TEACs), respectively. In contrast, A-type procyanidins A1 and rhodonidin A showed the lowest value at 1.75 and 1.96 (TEACs), respectively. Our results did not show a pronounced difference in antioxidant activity between total OH groups or the degree of polymerization (data not shown) but a significant increase between the average OH groups/unit with the antioxidant activity ( Figure 4B).

Plant Material
The leaves of Rhododendron formosanum were collected in April and July of 2010 from the study sites in Yuanzui mountain (24°14′6.49′′ N, 120°57′7.29′′ E at 1911 m a.s.l.) in Hopin township of Taichung County, Taiwan.

Total Antioxidant Capacity (TAC)
Pure compounds were tested by using the CUPric Reducing Antioxidant Capacity (CUPRAC) method [28] according to the protocol of QuantiChrom Antioxidant Assay kit (Bioassay Systems, Hayward, CA, USA) [29]. These assays are based on the reduction of Cu 2+ to Cu + by the combined action of all antioxidants (reducing agents) in a sample. The resulting Cu + specifically forms a colored complex with a dye agent (4,4′-dicarboxy-2,2′-biquinoline) and the color intensity at 570 nm is measured as TAC. Briefly, compounds were diluted with distilled water to produce solutions of 0.1, 0.25, 0.5, and 1 mM concentration. The reaction was initiated by the addition of 100 µL mixture of copper sulfate and dye agent with 20 µL of each compound solution. The absorbance at 570 nm was calculated for each concentration relative to a blank absorbance and was plotted as a function of concentration of standard Trolox. At least three independent determinations were performed. The antioxidant activities of purified compounds 1-5 are expressed as TEAC (Trolox Equivalent Antioxidant Activity) values in comparison with TEAC activity of reported reference compounds, catechin (Sigma-Aldrich, USA) and epicatechin (Sigma-Aldrich, USA). Trolox was employed at concentrations ranging from 10-1000 μM to construct a calibration curve. TEAC value is defined as the concentration of standard Trolox solution with equivalent activity to 1 mM concentration solution of purified compound.

Free Radical Scavenging Activity
The free radical scavenging activities of purified compounds were determined according to previous report. Briefly, the reaction for scavenging DPPH radicals was carried out by adding 2 μL sample to 198 μL DPPH solution (100 μM) at 25 °C. The mixture was shaken vigorously and left to stand for 30 min in the dark before measuring the absorbance at 517 nm against a blank. For the radical scavenging activities of procyanidins, EC50 values were calculated as the concentrations (μM) that inhibited 50% of the DPPH radicals in the reaction.

Antibacterial Activity
Eight strains of microorganisms were used: Bacillus cereus (ATCC 9139), Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 35150), Listeria monocytogenes (ATCC 7644), Pseudomonas aeruginosa (ATCC 27853), Salmonella enterica (ATCC 13311), Staphylococcus aureus (ATCC 43300), and Helicobacter pylori (ATCC 700392), which were employed to evaluate the antibacterial assay. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by the broth micro-dilution method according to the guidelines of the Clinical and Laboratory Standards Institute [30]. H. pylori was grown on blood agars under microaerophilic conditions at 37 °C for 48-72 h while other bacteria strains were cultured on nutrient agar (Difco, USA) and incubated at 37 °C for 24 h. Bacterial inoculums were prepared in normal saline and diluted to give a final density of 5 × 10 5 cfu/mL. All compounds were dissolved in DMSO (Sigma, USA) and then in nutrient broth to reach a final concentration of 512 µg/mL. Serial two-fold dilutions were made in a concentration range from 0.25-256 µg/mL. The MIC and MBC were defined as the lowest concentration at which no visible growth occurred in comparison with antibiotics (ampicillin, tetracyclin and metronidazole) as a positive control. Tests were repeated three times for each compound.

Conclusions
Five compounds, including two procyanidin dimers, procyanidin A1 (1) and B3 (2), two procyanidin trimmers, procyanidin C4 (4) and cinnamtannin D1 (5), and one new procyanidin dimer, rhodonidin A (3), have been isolated from the leaves of R. formosanum. Compound 3 demonstrated strong antimicrobial activity against Staphylococcus aureus at MIC value of 4 μg/mL. Compounds 1-5 also showed pronounced antioxidant activities. The pleiotropic effects of procyanidins isolated from the leaves of R. formosanum can be a source of promising compounds for the development of future pharmacological applications.