Molecular Complexation of Hederasaponin C with Cholesterol in Aqueous Isopropyl Alcohol 150 D O I: 1 0. 15 82 6/ ch im te ch .2 02 0. 7. 4. 02 Yakovishin L. A., Grishkovets V. I., Korzh E. N., Golovchenko I. V., Nagirnyak A. A. Chimica Techno Acta. 2020. Vol. 7, no. 4. P. 150–153. ISSN 2409–5613 Molecular Complexation of Hederasaponin C with Cholesterol in Aqueous Isopropyl Alcohol L. A. Yakovishina*, V. I. Grishkovetsb, E. N. Korzha, I. V. Golovchenkoa, A. A. Nagirnyaka 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 ivy triterpene glycoside hederasaponin C (HedC) with cholesterol (Chol) was obtained in aqueous isopropyl alcohol. The stability constant of (3.3 ± 0.7) · 106 (mol/L)–1 was calculated for the complex. The complexa- tion was studied by UV- and ATR IR-Fourier spectroscopy, and method of isomolar series. The hydrogen bonds and hydrophobic interactions are formed in the molecular complex. Keywords: triterpene glycosides; hederasaponin C; cholesterol; molecular complex Received: 06.08.2020. Accepted: 07.12.2020. Published:30.12.2020. © Yakovishin L. A., Grishkovets V. I., Korzh E. N., Golovchenko I. V., Nagirnyak A. A., 2020 Introduction T r i t e r p e n e g l y c o s i d e h e d e r a s a p o n i n C ( h e d e r a g e n i n 3 - O -α- L- rham nopy r ano s y l - ( 1→2 ) - O -α- L - a r a b i n o p y r a n o s y l - 2 8 - О -α- L - r h a m n o p y r a n o s y l - ( 1→ 4 ) - О -β - D - g l u c o p y r a n o s y l - ( 1→6 ) - О -β- D - glucopyranoside, HedC; Fig.  1) was discovered in the most species of the ivy genus Hedera  L. (Araliaceae Juss.) [1]. HedC is the dominant ivy saponin. HedC was also founded in plants of various spe- cies of Kalopanax, in Aralia elata, Acan- thopanax sieboldianus and Schefflera oc- tophylla [1]. H H OH H H 1 3 5 6 11 12 13 15 9 10 8 14 17 22 24 25 26 27 18 19 20 21 23 Chol 1 3 12 23 28 16 20 30 9 18 O O OH OH O OH OH OH CH3 O COOR OH 1'' 2' 5' HedC Fig. 1. Structures of HedC (R = ←βGlcp-(6←1)-βGlcp-(4←1)-αRhap) and Chol 151 HedC is the component of antitussive drugs Prospan, Hedelix and other contain- ing Hedera helix L. leaves [1]. A character- istic feature of triterpene glycosides is their ability to form molecular complexes with sterols [1–4]. The complexation of sapo- nins with sterols is responsible for hemolyt- ic, antitumor, ichthyotoxic, molluscicidal, antifungal, hypocholesterolaemic, and em- bryotoxic activity of triterpene glycosides [1, 2]. On the other hand, it was reported that some triterpene glycosides do not form a molecular complex with cholesterol (Chol; Fig. 1) [3]. The interaction of HedC with Chol has been studied by spectrophotometric titra- tion in aqueous ethanol [3] and isomolar series [4]. A preparation of molecular com- plex of Chol with HedC and bisdesmoside ivy triterpene glycoside hederacoside B mixture in aqueous ethanol and its analysis by planar chromatography [2] was previ- ously reported. To  study the  complexation of  HedC with Chol in various media we examined their interaction in 80% aqueous isopropyl alcohol. Experimental HedC was preparatively isolated from leaves of Hedera canariensis Willd. (Arali- aceae Juss.) by column chromatography on SiO2 and its structure was confirmed using chemical and physical methods [5]. The  isomolar series were prepared by mixing 10–4 mol/L solutions of HedC and Chol in 80% aqueous isopropyl alcohol (v/v) at 25 °C for 40 min with continuous stirring. Spectroscopic analysis of isomolar series was performed on a LEKI SS2110UV spectrophotometer using a quartz cuvette (l = 1 cm) at  25  °C.  Stability constant of the complex was calculated according to the A. K. Babko method based on iso- molar curves [4, 6]. The  complex of  Chol with HedC was preparatively obtained by  liquid- phase method. For this purpose, 1 mmol of the substances was mixed with 50 mL of  80% aqueous isopropyl alcohol (v/v). The  obtained mixture was incubated at 50 °C for 1.5 h with continuous stirring. The  organic solvent was removed under reduced pressure. Synthesized complex was analyzed by IR spectroscopy. The  IR spectra were recorded on the  Simex FТ-801 IR-Fourier spectrom- eter in the 4000–550 cm–1 region (spectral resolution 4 cm–1; 50 scans) using ATR ac- cessory with diamante crystal plate. IR spectrum of  HedC (ν, cm–1): 3333 (ОН), 2930 (СН), 2907 (СН), 2878 (СН), 1734 (С=О), 1624 (C=C), 1451 (СН), 1433 (СН), 1417 (СН), 1387 (СН), 1357 (СН), 1342 (СН), 1319 (СН), 1260 (СН), 1230 (СН), 1201 (СН), 1050 (С–О–С, С–ОН), 1024 (С–О–С, С–ОН), 979 (=CH). 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). IR spectrum of  the  complex of  HedC with Chol (ν, cm–1): 3316 (ОН), 2930 (СН), 2900 (СН), 2865 (СН), 1732 (С=О), 1670 (C=C), 1625 (C=C), 1458 (СН), 1434 (СН), 1378 (СН), 1363 (СН), 1339 152 (СН), 1316 (СН), 1261 (СН), 1230 (СН), 1199 (СН), 1128 (С–О–С, С–ОН), 1050 (С–О–С, С–ОН), 1024 (С–О–С, С–ОН), 983 (=CH), 958 (СН). Results and discussion The  composition of  the  complex of  HedC with Chol was determined by the isomolar series method. This meth- od gave a molar ratio ≈1.0, which corre- sponded to a 1:1 complex (Fig. 2). Such ratio was obtained for complex of HedC with Chol in 90% and 70% aque- ous ethanol [3, 4], and for complexes of  HedC with several drugs [7]. Stabil- ity constant of  complex (3.3 ± 0.7)  · · 106 (mol/L)–1 was calculated based on iso- molar curves (A. K. Babko method) [4, 6]. The stability constants of complex in aque- ous ethanol were of the order 10–4 [3, 4]. Thus, the  stability constant of  complex formed in 80% aqueous isopropyl alcohol was greater than in aqueous ethanol. As the HedC concentration increases (at constant Chol concentration), the op- tical density of  their solutions increases (hyperchromic effect) (Fig. 3). The absorp- tion maximum of the solutions decreases insignificantly (hypsochromic shift) from 237 to 230 nm. Similar spectral changes were previously observed for molecular complexation of HedC with caffeine [7]. The complexation of HedC with Chol was studied by ATR FT-IR spectroscopy. The  potential centers of  intermolecular interactions in  the  molecules of  HedC and Chol are OH groups. Indeed, upon the formation of complex in the IR spec- tra for the absorption bands of stretching vibrations of O–H bonds in Chol are ob- served shifts from 3403 and 3337 cm–1 to 3316 cm–1, and in HedC — from 3333 to 3316 cm–1. This may indicate to formation of hydrogen bonds between HedC and Chol. 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2 220 260 300 340 380 λ, nm A 1 4 Fig. 3. UV spectra of Chol solutions (0.50 · 10–4 M = const) with different concentrations of HedC: 0 M (curve 1), 0.125 · 10–4 (curve 2), 0.25 · 10–4 (curve 3), 0.50 · 10–4 (curve 4) 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1 0.11 0.12 0.13 0 1 2 3 4 5 6 7 8 9 10 c (HedC) / c (Chol) ∆А 237 Fig. 2. Optical density change DА as a function of component ratio of isomolar series at 237 nm 153 The complexation also causes changes in certain frequencies of absorption of CH bonds. These facts may indicate the pres- ence of  hydrophobic contacts between Chol and HedC molecules in the molecular complex. The presence of hydrophobic in- teractions explains the high stability of trit- erpene glycosides molecular complexes [4]. Conclusions The  1:1  molecular complex of  HedC with Chol has been prepared for the first time in  aqueous isopropyl alcohol. The  presence of  molecular complexa- tion of Chol with HedC has been proved by UV- and ATR IR-Fourier spectroscopy. Intermolecular interaction in the complex is carried out by hydrogen bonds forma- tion and hydrophobic contacts. The results can be used to  explain the  mechanisms of the biological activity of triterpene gly- cosides. Acknowledgements This work was carried out within the framework of an internal grant of Sevastopol State University (identifier 30/06-31). References 1. Hostettmann K, Marston A. Saponins. Cambrige: Cambrige University Press; 1995. 548 р. 2. Tschesche R, Wulff G. Konstitution und eigenschaften der saponine. Planta Medica. 1964;12(3):272–92. doi:10.1055/s-0028–1100180 3. Wojciechowski K, Orczyk M, Gutberlet T, Geue T. Complexation of phospholipids and cholesterol by triterpenic saponins in bulk and in monolayers. Biochim Biophys Acta Biomembr. 2016;1858(2):363–73. doi:10.1016/j.bbamem.2015.12.001 4. Yakovishin LA, Grishkovets VI. Molecular complexes of ivy triterpene glycosides with cholesterol. Khimiya Rastitel’nogo Syr’ya. 2018;4:133–40. doi:10.14258/jcprm.2018043607 5. Grishkovets VI, Sidorov DYu, Yakovishin LA, Arnautov NN, Shashkov АS, Chirva VYa. Triterpene glycosides of Hedera canariensis I. Structures of glycosides L-A, L-B1, L-B2, L–C, L–D, L-E1, L-G1, L-G2, L-G3, L-G4, L-H1, L-H2, and L–I1 from the leaves of Hedera canariensis. Chem Nat Compd. 1996;32(3):360–5. doi:10.1007/BF01372340 6. Babko AK. Phiziko-khimicheskii analiz kompleksnykh soedinenii v rastvorakh [Physico-chemical analysis of complex compounds in the solutions]. Kiev: Izd-vo AN USSR; 1955. 328 p. Russian. 7. Yakovishin LA, Grishkovets  V. I.  Ivy and licorice triterpene glycosides: promis- ing molecular containers for some drugs and biomolecules. Stud Nat Prod Chem. 2018;55:351–83. doi:10.1016/B978-0-444-64068-0.00011–5