Intermolecular Interaction of Glycyrrhizin with Cholesterol 180 D O I: 1 0. 15 82 6/ ch im te ch .2 02 0. 7. 4. 08 Yakovishin L. A., Grishkovets V. I. Chimica Techno Acta. 2020. Vol. 7, no. 4. P. 180–185. ISSN 2409–5613 Intermolecular Interaction of Glycyrrhizin with Cholesterol L. A. Yakovishina*, V. I. Grishkovetsb a Sevastopol State University, 33 University Str., Sevastopol, 299053, Russia b V. I. Vernadsky Crimean Federal University, 4 Vernadsky Ave., Simferopol, 295007, Russia *email: chemsevntu@rambler.ru Abstract. The 1:1 molecular complex of licorice triterpene glycoside glycyrrhizic acid (in the form of monoammonium salt) with cholesterol was obtained in 80% aque- ous isopropyl alcohol for the first time. The complexation was studied by 13C NMR, UV, and ATR IR-Fourier spectroscopy. The hydrogen bonds and hydrophobic interactions are formed in the molecular complex. Keywords: triterpene glycosides; licorice; glycyrrhizin; glycyrrhizic acid; cholesterol; mo- lecular complex Received: 27.10.2020. Accepted: 09.12.2020. Published:30.12.2020. © Yakovishin L. A., Grishkovets V. I., 2020 Introduction Glycyrrhizin (glycyrrhizic acid, 3-О-β-D-glucuronopyranosyl-(1→2)- О-β-D-glucuronopyranoside of 18β-gly- cyrrhetinic acid, GA; Fig. 1) is the domi- nant triterpene saponin from licorice roots Glycyrrhiza glabra L. and Glycyr- rhiza uralensis Fisch. (Fabaceae) [1, 2]. GA has anti-inflammatory, antioxidative, antiviral, anticancer, hypocholesterolem- ic, and hepatoprotective properties [1–3]. The most important derivative of GA is its monoammonium salt (monoammonium glycyrrhizinate, glycyram, GC; Fig. 1). GC is used as an anti-inflammatory, hepato- protective, antiallergic, mineralocorticoid, and antitussive drug [2–4]. Some biological properties of saponins explain their molecular complexation with sterols [1, 2, 5–7]. GA increases perme- ability and reduces the  elastic modulus of cell membranes [8]. On the other hand, recent spectrophotometric titration did not confirm the complexation of GC with cholesterol (Chol; Fig. 1) and 1,2-dipalmi- toylphosphatidylcholine [9]. The authors of  this paper have been suggested that the  presence of  11-oxo group in  the  GC aglycone part prevents its complexation. In  order to  consider the  possibility of complexation of GC with Chol in vari- ous media, we studied their intermolecular interaction in aqueous isopropyl alcohol by NMR, IR, and UV spectroscopy. Experimental GC (purity ≥95% by HPLC) was pur- chased from Calbiochem. Chol and other chemicals in  the  highest grade of  purity were obtained from Sigma-Aldrich. 181 The complex of Chol with GC was pre- paratively obtained by liquid-phase meth- od. For this purpose, 1 mmol of the sub- stances was mixed with 50  mL of  80% aqueous isopropyl alcohol (v/v). The ob- tained mixture was incubated at 50 °C for 1.5 h with continuous stirring. The organic solvent was removed by vacuuming. UV spectra of Chol solutions (0.50  · ·  10–4 M = const) with different concen- trations of GC (0, 0.125 · 10–4, 0.25 · 10–4, 0.50 · 10–4, 1.0 · 10–4 M) were recorded on a LEKI SS2110UV spectrophotometer us- ing a quartz cuvette (l = 1 cm) at 25 °C. The  IR spectra were recorded on a Simex FТ-801 IR-Fourier spectrometer in the 4000–550 cm–1 region (spectral reso- lution 4 cm–1; 50 scans) using the universal optical attenuated total reflection (ATR) accessory with diamond crystal plate. IR spectrum of GC (ν, cm–1): 3197 (ОН, NH), 2928 (СН), 2907 (СН), 2868 (СН), 1719 (С=О), 1708 (С=О), 1641 (С(11)=О, C=C), 1587 (СОО–), 1451 (СН), 1425 (NH4 +), 1416 (СОО–), 1387 (СН), 1357 (СН), 1318 (СН), 1260 (СН), 1211 (СН), 1162 (С–О–С, С–ОН), 1037 (С–О–С, С– ОН), 980 (=CH), 946 (СН), 918 (monosac- charide ring), 880 (СН), 818 (СН), 793 (СН), 694 (СН), 687 (СН), 679 (=CH), 663 (ОН). IR spectrum of  Chol (ν, cm–1): 3403 (ОН), 3337 (ОН), 2929 (CH), 2899 (CH), 2865 (CH), 2848 (СН), 1672 (C=C), 1460 (СН), 1434 (СН), 1377 (СН), 1364 (СН), 1341 (СН), 1333 (СН), 1318 (СН), 1275 (СН), 1268 (СН), 1253 (СН), 1234 (СН), 1220 (СН), 1190 (СН), 1169 (С–ОН), 1132 (С–ОН), 1106 (С–ОН), 1052 (С–ОН), 1022 (С–ОН), 986 (=CH), 953 (СН), 925 (=CH), 881 (СН), 839 (С–С–С), 799 (СН), 738 (СН), 720 (СН), 694 (СН), 687 (СН), 679 (=CH), 662 (ОН). IR spectrum of the complex of GC with Chol (ν, cm–1): 3216 (ОН, NH), 2928 (CH), 2903 (CH), 2863 (CH), 1717 (С=О), 1698 (С=О), 1669 (С=СChol), 1648 (С(11)=О, C=CGC), 1586 (СОО –), 1459 (СН), 1450 (СН), 1433 (СН), 1424 (NH4 +), 1418 (СОО–), 1386 (СН), 1379 (СН), 1362 (СН), 1339 (СН), 1316 (СН), 1277 (СН), 1261 (СН), 1211 (СН), 1163 (С–О–С, С–ОН), 1038 (С–О–С, С–ОН), 1030 (С–О–С, С–ОН), 978 (=CH), 947 (СН), 919 (monosaccharide ring, =CH), 880 (СН), 818 (СН), 795 (СН), 741 (СН), 719 (СН), 692 (СН), 685 (СН), 679 (=CH), 662 (ОН). 13C NMR spectra were recorded on a Bruker WM-250 spectrometer (62.9 MHz for 13С) in С5D5N at 30 °С. NMR spectra are reported in Tables 1 and 2. COOH O O O HOOC HO HO HO O HO O H ROOC OH HO Intermolecular hydrogen bond Hydrophobic interactions3 23 12 28 30 20 17 GA: R=H GC: R=NH4 Chol 1' 6' 1'' 3 19 5 6 21 20 2526 27 18 22 241 11 1 11 17 24 25 27 29 6'' Chol GC .... . Fig. 1. Chemical structures of GA, GC, Chol and schematic representation of the possible orientation of GC and Chol molecules during their intermolecular interaction 182 Results and discussion The intermolecular interaction of GC with Chol was studied by  UV spectros- copy. As the GC concentration increases (at constant Chol concentration), the op- tical density of  their solutions increases (hyperchromic effect) (Fig. 2). The absorp- tion maximum of the solutions increases (bathochromic shift) from 237 to 250 nm. Similar spectral changes were previously noted for molecular complexes of  some triterpene glycosides [10] and cyclodex- trins [11]. The  complex of  Chol with GC was preparatively obtained by  liquid-phase method in  80% aqueous isopropyl alco- hol. The potential centers of intermolecular interactions in the molecules are COOH groups of  GC and OH group of  Chol. The lipophilic nature of the aglycone part of  GC and sterane system with the  hy- drocarbon “tails” in Chol may contribute to hydrophobic contacts between them. The nature of intermolecular interac- tions in the complex was confirmed by ATR FT-IR spectroscopy. Upon the formation of  complex in  the  IR spectra for the  ab- sorption bands of  stretching vibrations of  Chol O–H bonds are observed shifts from 3403 and 3337 cm–1 to  3216 cm–1, and for GC  — from 3197 to  3216 cm–1. We also found a  low-frequency shift of  the  absorption band of  C=O bond in one of GC carboxyl groups at 1708 cm–1 by  10 cm–1. Similar shifts of  the  absorp- tion bands of  C=O bonds in  IR spectra were previously observed during the in- teraction of ivy triterpene glycosides with Chol [12], as well as during the formation of GA and GC complexes [11]. In addition, the band of stretching vibrations of C–O bonds in C–OH for GC at 1037 cm–1 shifts by –7 cm–1 and for Chol at 1169, 1052, and 1022 cm–1 — by –6, –14, and +8 cm–1, re- spectively. IR spectroscopic data indicate about the formation of a hydrogen bond between Chol OH group and C=O group in one of GC carboxyl groups: С=ОGC…Н–ОChol. Fig. 2. UV spectra of Chol solutions (0.50 · 10–4 M = const) with different concentrations of GC: 0 M (curve 1), 0.125 · 10–4 (curve 2), 0.25 · 10–4 (curve 3), 0.50 · 10–4 (curve 4), 1.0 · 10–4 (curve 5) (a) and ATR FT-IR spectra of GC, Chol, and GC–Chol molecular complex (b) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 220 A 1 5 270 λ, nm a b 183 The complexation also causes chang- es in  certain frequencies of  absorption of CH bonds: 2868 → 2863 cm–1 and 2907 → 2903 cm–1 (for GC), and also 2899 → 2903 cm–1, 953 → 947 cm–1, and 799 → 795 cm–1 (for Chol). These facts may indi- cate the presence of hydrophobic contacts between Chol and GC molecules. The location of GC carboxyl group in- volved in  the  interaction with Chol was determined by  13C NMR spectroscopy. The value of the chemical shift of the C-30 atom in  the  carboxyl group of  the  agly- cone portion of  GC remains practically unchanged (Table  1). However, there is a change in the chemical shift of the С-6″ Table 1 13C NMR spectral data for free GC and GC in the molecular complex with Chol (δ, ppm, 0 — TMS, С5D5N, 30 °С) С -a to m G C G C in  c om pl ex w ith C ho l ∆ δ = =  δ G C –C ho l – δ G C С -a to m G C G C in  c om pl ex w ith C ho l ∆ δ = = δ G C –C ho l – δ G C Aglycone part 1 40.03 40.04 0.01 16 26.78 26.75 –0.03 2 26.96 26.96 0 17 32.23 32.23 0 3 89.28 89.30 0.02 18 48.81 48.82 0.01 4 40.14 40.15 0.01 19 41.84 41.89 0.05 5 55.52 55.51 –0.01 20 44.19 44.19 0 6 17.66 17.68 0.02 21 31.72 31.71 –0.01 7 33.05 33.07 0.02 22 38.51 38.51 0 8 43.55 43.58 0.03 23 28.17 28.16 –0.01 9 62.20 62.19 –0.01 24 16.91 16.92 0.01 10 37.29 37.29 0 25 16.79 16.78 –0.01 11 199.62 199.63 0.01 26 18.87 18.87 0 12 128.75 128.75 0 27 23.66 23.65 –0.01 13 169.63 169.63 0 28 28.85 28.85 0 14 45.63 45.61 –0.02 29 28.85 28.85 0 15 26.78 26.75 –0.03 30 179.22 179.21 –0.01 Carbohydrate part GlcUA′ GlcUA″ 1′ 105.10 105.12 0.02 1″ 106.63 106.61 –0.02 2′ 82.82 82.82 0 2″ 76.71 76.74 0.03 3′ 77.15 77.16 0.01 3″ 77.65 77.67 0.02 4′ 73.45 73.47 0.02 4″ 73.51 73.50 –0.01 5′ 78.02 78.00 –0.02 5″ 78.23 78.15 –0.08 6′ 172.92 172.94 0.02 6″ 174.19 174.03 –0.16 184 atom of the carboxyl group of the termi- nal residue of  glucuronic acid (GlcUA″) in the disaccharide fragment GC by –0.16 ppm compared to individual GC (Fig. 1). A smaller effect was also noted on the neigh- boring C-5″ atom (∆δ = –0.08 ppm). In addition, it is noted ∆δ (up to 0.05 ppm) for a  number of  GC aglycone and Chol C-atoms (Tables 1 and 2). The great- est effects were found for some C-atoms in the B–E rings of GC, in the B–D rings, and side chain of Chol, as well as all methyl groups of Chol. These data may indicate about hydrophobic interactions between the aglycone part of GC and Chol (Fig. 1). Conclusions The  results of  this work confirm the molecular complexation between GC and Chol. The interaction is accompanied by bathochromic shift and a hyperchromic effect. The formation of an intermolecular hydrogen bond between OH group at C-3 of Chol and C=O group of terminal glucu- ronic acid residue in the carbohydrate part of GC (C3–О–Н…О=С6″) and hydrophobic contacts were confirmed by 13C NMR and ATR FT-IR spectroscopy. The results of this work can be used to study of mechanisms of biological activity of GA, GC and other saponins. Table 2 13C NMR spectral data for free Chol and Chol in the molecular complex with GC (δ, ppm, 0 — TMS, С5D5N, 30 °С) С -a to m C ho l C ho l i n co m pl ex w ith G C ∆ δ = = δ G C –C ho l – δ C ho l С -a to m C ho l C ho l i n  co m pl ex w ith G C ∆ δ = = δ G C –C ho l – δ C ho l 1 37.92 37.94 0.02 15 24.59 24.62 0.03 2 32.71 32.71 0 16 28.58 28.61 0.03 3 71.35 71.38 0.03 17 56.49 56.52 0.03 4 43.59 43.58 –0.01 18 12.09 12.13 0.04 5 142.07 142.06 –0.01 19 19.68 19.72 0.04 6 121.29 121.31 0.02 20 36.11 36.13 0.02 7 32.31 32.34 0.03 21 19.02 19.06 0.04 8 32.26 32.29 0.03 22 36.57 36.59 0.02 9 50.60 50.62 0.02 23 24.22 24.24 0.02 10 36.99 37.02 0.03 24 39.80 39.83 0.03 11 21.45 21.48 0.03 25 28.29 28.32 0.03 12 40.12 40.15 0.03 26 22.74 22.78 0.04 13 42.60 42.63 0.03 27 22.99 23.03 0.04 14 57.00 57.04 0.04 185 Acknowledgements This work was carried out in the frame of an internal grant of Sevastopol State Uni- versity (identifier 30/06-31). References 1. 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