Structure–Activity Relationship of Oligomeric Flavan-3-ols: Importance of the Upper-Unit B-ring Hydroxyl Groups in the Dimeric Structure for Strong Activities

Proanthocyanidins, which are composed of oligomeric flavan-3-ol units, are contained in various foodstuffs (e.g., fruits, vegetables, and drinks) and are strongly biologically active compounds. We investigated which element of the proanthocyanidin structure is primarily responsible for this functionality. In this study, we elucidate the importance of the upper-unit of 4–8 condensed dimeric flavan-3-ols for antimicrobial activity against Saccharomyces cerevisiae (S. cerevisiae) and cervical epithelioid carcinoma cell line HeLa S3 proliferation inhibitory activity. To clarify the important constituent unit of proanthocyanidin, we synthesized four dimeric compounds, (−)-epigallocatechin-[4,8]-(+)-catechin, (−)-epigallocatechin-[4,8]-(−)-epigallocatechin, (−)-epigallocatechin-[4,8]-(−)-epigallocatechin-3-O-gallate, and (+)-catechin-[4,8]-(−)-epigallocatechin and performed structure–activity relationship (SAR) studies. In addition to antimicrobial activity against S. cerevisiae and proliferation inhibitory activity on HeLa S3 cells, the correlation of 2,2-diphenyl-l-picrylhydrazyl radical scavenging activity with the number of phenolic hydroxyl groups was low. On the basis of the results of our SAR studies, we concluded that B-ring hydroxyl groups of the upper-unit of the dimer are crucially important for strong and effective activity.


Introduction
Currently, there is great interest in the strong biological activity of compounds commonly found in some foods. Such compounds have been considered safe functional compounds. Polyphenols are found in various plants such as vegetables and fruits and are consumed regularly [1,2]. Among polyphenol compounds, proanthocyanidins such as oligomeric flavan-3-ols and condensed tannins are known to be strongly bioactive compounds. It is widely believed that proanthocyanidins have a beneficial impact on health; therefore, they are included in various health foods, and scientific investigations of proanthocyanidins are becoming increasingly important. However, the structure-activity relationship (SAR) of proanthocyanidins is not well understood because, in many cases, they are obtained as mixtures of various analogues. Thus, purification of each compound is difficult. Therefore, synthesis is considered the most suitable method for clarifying the SAR of proanthocyanidins. Following contribution by Kozukowski et al. [3][4][5], many studies on proanthocyanidin synthesis and their biological properties were demonstrated [6][7][8][9][10][11][12][13][14].
We have also reported a simple, versatile, stereoselective and length controlled synthetic method for various procyanidins, a member of the proanthocyanidin class, which have two hydroxyl groups on the B-ring. We demonstrated that galloyl modification of the hydroxyl groups of flavan-3-ols enhanced their biological activities [15][16][17][18]. We further reported the synthesis of semi-acetylated analogues 1-3 of procyanidin B1, a dimeric flavan-3-ol, and discussed their inhibitory activities against HeLa S3 cell proliferation ( Figure 1). The lower-unit acetylated procyanidin B1 (2) strongly inhibited proliferation of HeLa S3 cells, whereas procyanidin B1 (1) and the upper-unit acetylated analog 3 showed no inhibitory activity [19]. These results indicated that the upper-unit of dimeric flavan-3-ol is critical for biological activity. More recently, we elucidated that there is poor correlation between the inhibitory activity of HeLa S3 cell proliferation and 2,2-diphenyl-l-picrylhydrazyl (DPPH) radical scavenging activity [20]. In addition, we proved that the stereochemistry of the 3-hydroxyl group of flavan-3-ol-3,5-di-O-gallate is important for inhibitory activity against HeLa S3 cell proliferation [20]. Here, we describe further investigations. Synthetic studies of dimeric flavan-3-ols, (−)-epigallocatechin-3-O-gallate (4), (−)-epigallocatechin (5), and (+)-catechin (6) as well as SAR studies were performed ( Figure 2). In addition to the inhibitory activity assay against HeLa S3 cell, antimicrobial activity against Saccharomyces cerevisiae is also demonstrated. Oligomeric flavan-3-ols consisting of 4, 5 and 6 are isolated from fermented foods such as beer, wine, etc. Flavan-3-ols and proanthocyanidins are known as antimicrobial active agents against various microorganisms, including yeast [21]. Although numerous studies about the biologically effect on yeast of plant extracts which are mixtures of various polyphenol compounds have been reported [22,23], there is little information allowing a detailed SAR study. Our results suggested that increases in the number of phenolic hydroxyl groups in the entire molecule correlate poorly with biological activities. In addition, we confirmed the importance of the upper-unit for the functionality of dimeric flavan-3-ols.

Antimicrobial Activity against S. cerevisiae
Antimicrobial activity against S. cerevisiae is shown in Figure 5. Upper-unit epigallocatechin compounds 7 and 8 exhibited strong inhibitory activity against S. cerevisiae proliferation. It should be noted that activity comparison between compound 7 and reference procyanidin B1 (1) revealed that the three hydroxyl groups on the B-ring in the upper-unit are very important. Contrary to our expectations, it is clear from the activity between 8 and 9 that the galloyl moiety at the lower-unit resulted in decreased activity. Surprisingly, compound 10, which had the same building unit (number of hydroxy groups) as compound 7, showed no inhibitory activity of proliferation against S. cerevisiae. These data reveal that the existence of upper-unit hydroxyl groups in the dimeric structure is important for strong activities. Furthermore, microbial activity against S. cerevisiae showed no correlation to DPPH radical scavenger ability, which suggests that this antimicrobial activity is due to another mechanism.

Cervical Epithelioid Carcinoma Cell Line, HeLa S3 Proliferation Inhibitory Activity
The inhibitory activities of the synthetic dimeric flavan-3-ols against HeLa S3 cell proliferation are shown in Figure 6. Similar to antimicrobial activity against S. cerevisiae, compound 10 showed low activity relative to other synthesized compounds 7-9. Compared to compound 10, the significance of the upper epigallocatechin unit of 7 is clear. Furthermore, the huge difference of activity between 1 and 7 indicates the importance of the third hydroxyl group on the B-ring. In addition, this activity was not enhanced by the introduction of a galloyl moiety to the lower-unit. Among the tested compounds, compound 7, 8 and 9 showed comparable inhibitory activity, which indicates that the structure of the lower-unit is not significant. . *** p < 0.001, ** p < 0.005 vs. DMSO-treated groups, asterisks indicate * p < 0.001 in Student's t test.

DPPH Radical Scavenging Activity
DPPH radical scavenging activity was measured with general procedure [28]. A solution of DPPH radical in EtOH (30 μM, 1.0 mL) was added to 1 μL of the synthesized each compound in DMSO, and incubated at 30 °C for 30 min (n = 6). The scavenging activity was estimated with a microplate reader (Filter Max F5 multi-mode microplate reader; Molecular Devices, Downingtown, PA, USA) to measure the OD at 515 nm. Negative controls, the samples that 1 μL of DMSO added to the 1.0 mL of EtOH were prepared at the same time. And the absorbance values converted into the percentage radical scavenging activity as follows: [(absorbance of the control − absorbance of the sample)/absorbance of the control] × 100. VE (vitamin E) was used as the standard sample.

Antimicrobial Activity against S. cerevisiae
Overnight culture of S. cerevisiae was diluted to OD = 0.25 with culture medium, and 200 μL of aliquot was transferred to each well in a 96-well plate. Then 1 μL of the DMSO solution of compound was added in each well to a final concentration of 50 μM, and the growth of the S. cerevisiae at 27 °C with constant shaking was monitored by measuring absorbance at 600 nm every 15 min using an incubation reader, HiTS (Scinics, Itabashi, Japan). The results were obtained as growth curves as well as raw data. DMSO was used as negative controls. As negative controls, medium alone well and DMSO added to the well were prepared at the same time.

Inhibitory Activity of HeLa S3 Cell Proliferation
10 4 cells per well with 100 μL of medium in a 37 °C incubator equilibrated with a 5% CO2: 95% humidified air atmosphere. D-MEM (Dulbecco's Modified Eagle's Medium; Gibco ® (Life Technologies, Grand Island, NY, USA) supplemented with 5% fetal calf serum and 1% Pen-Strep; Invitrogen™ (Life Technologies). After 24 h of incubation, 1 μL of synthesized six compounds in DMSO were added (final 100 μM) and incubated for 48 h. As negative controls, medium alone well and DMSO added to the well were prepared at the same time. After the medium was removed and the cell was washed with PBS, 90 μL of new medium and 10 µL of the MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5 mg/mL) was added to each well and incubated at 37 °C for 2.5 h. After incubation the reaction medium was removed and 100 μL of DMSO was added to each well and mix thoroughly with the pipette.
Following which, viable cells were assessed using a microplate reader (Filter Max F5 multi-mode microplate reader; Molecular Devices) to measure the OD at 570 nm.

Conclusions
In conclusion, we have synthesized four dimeric flavan-3-ol derivatives to clarify the importance of the upper-unit for biological activities. Our results support the assumption that the hydroxyl groups on the B-ring of the upper-unit are essential for the antimicrobial activity against S. cerevisiae and HeLa S3 cell proliferation inhibitory activity. Furthermore the correlation of antimicrobial activity against S. cerevisiae, proliferation inhibitory activity on HeLa S3 cells and of 2,2-diphenyl-l-picrylhydrazyl radical scavenging activity with the number of phenolic hydroxyl groups was low. Further synthesis and biological works to explain these mechanisms are now underway, since much more research using various analogs is needed to completely understand the SAR of these compounds.