Ellagitannin-Lipid Interaction by HR-MAS NMR Spectroscopy

Abstract

Ellagitannins have antimicrobial activity, which might be related to their interactions with membrane lipids. We studied the interactions of 12 different ellagitannins and pentagalloylglucose with a lipid extract of Escherichia coli by high-resolution magic angle spinning NMR spectroscopy. The nuclear Overhauser effect was utilized to measure the cross relaxation rates between ellagitannin and lipid protons. The shifting of lipid signals in 1H NMR spectra of ellagitannin-lipid mixture due to ring current effect was also observed. The ellagitannins that showed interaction with lipids had clear structural similarities. All ellagitannins that had interactions with lipids had glucopyranose cores. In addition to the central polyol, the most important structural feature affecting the interaction seemed to be the structural flexibility of the ellagitannin. Even dimeric and trimeric ellagitannins could penetrate to the lipid bilayers if their structures were flexible with free galloyl and hexahydroxydiphenoyl groups.

Keywords: E. coli; HR-MAS-NMR; UPLC-DAD-MS; interaction; lipid membrane; tannins.

Figure A1

¹H NMR chemical shift deltas (Δδ, ppm) of E. coli lipid extracts in the presence of vescalagin 2, geraniin 6, chebulagic acid 7, chebulinic acid 8, punicalagin 9, oenothein B 10, and oenothein A 12. The tannin numbering refers to Figure 1 and lipid proton assignations refer to Figure 5. Values are presented as Δδ (n = 1).

Figure A2

¹H NMR chemical shift deltas (Δδ, ppm) of E. coli lipid extracts in the presence of three different weighed amounts of tellimagrandin II 4.

Figure A3

Cross relaxation rates of the aromatic protons (cross peak 1 (a), cross peak 2 (b), cross peak 3 (c), and cross peak 4 (d)) of three different weighed amounts of tellimagrandin II 4 against different lipid protons with a mixing time of 0.1 s. The lipid proton assignations refer to Figure 5 and cross peak labels are defined in Section 3.3.

Figure A4

Cross relaxation rates of the aromatic protons (cross peak 1 (a), cross peak 2 (b), cross peak 3 (c), and cross peak 4 (d)) of three different weighed amounts of tellimagrandin II 4 against different lipid protons with a mixing time of 0.3 s. The lipid proton assignations refer to Figure 5 and cross peak labels are defined in Section 3.3.

Figure 1

Chemical structures of 12 ellagitannins and pentagalloylglucose studied for their interaction with lipids: tellimagrandin I 1, vescalagin 2, casuarictin 3, tellimagrandin II 4, pentagalloylglucose 5, geraniin 6, chebulagic acid 7, chebulinic acid 8, punicalagin 9, oenothein B 10, sanguiin H-6 11, oenothein A 12, and lambertianin C 13. DHHDP = dehydrohexahydroxydiphenoyl, G = galloyl, HHDP = hexahydroxydiphenoyl, chebuloyl = modified dehydrohexahydroxydiphenoyl, NHTP = nonahydroxytriphenoyl.

Figure 2

¹H NMR spectra of the E. coli lipid extract with assigned proton signals at 25 °C: (a) measured in MeOD-d4 with 600 MHz, (b) measured in D₂O with 400 MHz HR-MAS probe along with (c) a highlighted ppm range of 3.7–4.7. Example structures of the lipids with labels are presented in Figure 5. * Possible distortion in the HR-MAS spectra caused by water presaturation masks some signals detected in the 600 MHz spectra.

Figure 3

¹H NMR chemical shift deltas (Δδ, ppm) of E. coli lipid extracts in the presence of tellimagrandin I 1, casuarictin 3, tellimagrandin II 4, pentagalloylglucose 5, sanguiin H-6 11, and lambertianin C 13. The tannin numbering refers to Figure 1 and lipid proton assignations refer to Figure 5. Values are presented as Δδ (average values and standard error, n = 4).

Figure 4

Cross relaxation rates of the aromatic protons (cross peaks 1–4 refer to Section 3.3 for definitions) of tellimagrandin I 1, casuarictin 3, tellimagrandin II 4, pentagalloylglucose 5, sanguiin H-6 11, and lambertianin C 13 against different lipid protons with mixing times of (a) 0.1 s and (b) 0.3 s. The tannin numbering refers to Figure 1 and lipid proton assignations refer to Figure 5. Cross peak labels are defined in Section 3.3. Values are presented as s⁻¹ (average values and standard error, n = 4).

Figure 5

Example structures of the known components of the commercial E. coli lipid extract, L-α-phosphatidylethanolamine (PE), L-α-phosphatidylglycerol (PG), and cardiolipin (CA), with labeling on the PE lipid to illustrate the NMR assigned protons. The length and precise structure of the fatty acid chains (CH₂) have not been determined.