NEW 2,5-BIS(2-ETHYLHEXYL)- PYRROLO[3,4-c]PYRROLE-1,4(2H,5H)-DIONE-2,2’-BIPYRIDINE-BASED CO-POLYMER, SYNTHESIS, PHOTOPHYSICAL PROPERTIES AND RESPONSE TO METAL CATIONS Chimica Techno Acta LETTER published by Ural Federal University 2022, vol. 9(1), No. 20229101 eISSN 2411-1414; chimicatechnoacta.ru DOI: 10.15826/chimtech.2022.9.1.01 1 of 3 New molecular complex of ammonium glycyrrhizate with rutin L.A. Yakovishin a* , V.D. Ratnikov a , P.I. Bazhan a, G.P. Zaitsev b a: Sevastopol State University, 299053 University st., 33, Sevastopol, Russia b: All-Russian National Research Institute of Viticulture and Winemaking “Magarach” of RAS, 298600 Kirova st., 31, Yalta, Russia * Corresponding author: chemsevntu@rambler.ru This short communication (letter) belongs to the MOSM2021 Special Issue. © 2021, The Authors. This article is published in open access form under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). Abstract A new 1:1 molecular complex of triterpene glycoside ammonium glycyrrhizate (GC) with flavonoid glycoside rutin (Rut) was obtained in aqueous ethanol. The stability constant (9.70.2)104 (mol/L)–1 was calculated for the complex via isomolar curves. The complexa- tion was studied by UV- and ATR IR-Fourier spectroscopy and a method of isomolar series. The hydrogen bonds and hydrophobic in- teractions are formed in the molecular complex. A preliminary anti- oxidant activity assessment of the complex was made. Keywords triterpene glycosides ammonium glycyrrhizate rutin molecular complex antioxidant capacity Received:03.11.2021 Revised: 21.12.2021 Accepted: 21.12.2021 Available online: 11.01.2022 1. Introduction Rutin (Rut; Fig. 1) is one of the most famous flavonols and glycosides [1]. Its aglycone is quercetin. Found in different plants, Rut has P-vitamin activity and exhibits antimicro- bial, antioxidant, anti-inflammatory, antidiabetic, anti- spasmodic, antisclerotic, diuretic, and anticancer effects [1]. Rut has a low solubility in water and limited mem- brane permeability [1]. The therapeutic effect of Rut is limited due to its bioavailability [1]. The study [2] showed that the solubility and bioavailability of bioactive com- pounds can be significantly increased by their molecular complexation with triterpene glycosides. Triterpene sapo- nins are amphiphilic multidentate ligands that are capable of molecular complexation with both polar and non-polar fragments of other molecules [2]. Glycyrrhizic acid (GA) and its monoammonium salt (am- monium glycyrrhizate, glycyram, GC; Fig. 1) are widely used as complexing agents [2–6]. GA is the main triterpene sapo- nin of licorice roots [2]. GA increases permeability of cell membranes [5]. Previously, we studied the molecular com- plex of quercetin with GC [6]. However, molecular complexes of Rut with GC have not been described. This article reports the preparation of a new molecular complex of GC with Rut. 2. Experimental GC (purity ≥95% by high-performance liquid chromatography (HPLC)) was purchased from Calbiochem. Other chemicals of the highest grade of purity were obtained from Sigma-Aldrich. The isomolar series were prepared by mixing 10– 4 mol/L solutions of GC and Rut in 70% aqueous ethanol (v/v) with continuous stirring at 25 °C for 40 min. Spec- troscopic 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 the isomolar curves [5, 7]. The complex of Rut with GC was preparatively obtained by the liquid-phase method. For this purpose, 1 mmol of the substances was mixed with 50 mL of 70% aqueous ethanol (v/v). The obtained mixture was incubated with continuous stirring at 50 C for 1.5 h. The organic solvent was removed by vacuuming. The synthesized complex was analyzed by IR spectroscopy. The IR spectra were recorded on a Simex FТ- 801 IR-Fourier spectrometer (Russia) in the 4000–550 cm–1 region (spectral resolution 4 cm–1; 25 scans) using the ATR accessory with a diamante crystal plate. IR spectrum of Rut (, cm–1): 3415 (OH), 3343 (OH), 2954 (CH), 2914 (СН), 2847 (СН), 1656 (С=О), 1596 (C=CAr), 1572 (C=CAr), 1552 (C=CAr), 1502 (C=CAr), 1453 (C=CAr, СН), 1426 (СН), 1405 (С–ОН), 1360 (С–ОН, СН), 1313 (CH), 1294 (С–О–С, С–ОН), 1234 (С–О–С, С–ОН), 1203 (С–О–С, С–ОН), 1168 (С–О–С, С–ОН), 1149 (С–О–С, С–ОН), 1123 (С–О–С, С–ОН), 1093 (С–О–С, С–ОН), 1059 (С–О–С, С–ОН), 1041 (С–О–С, С–ОН), 1013 (С–О–С, С–ОН), 999 (С–О–С, С–ОН), 967 (СН), 943 (СН), 910 (monosaccharide ring), 880 (СН), 848 (СН), 826 (СН), 807 (СН), 794 (СН), 727 (СН), 719 (СН), 707 (СН), 688 (СН), 655 (ОН), 629 (СН), 594 (СН). http://chimicatechnoacta.ru/ https://doi.org/10.15826/chimtech.2022.9.1.01 https://orcid.org/0000-0002-8728-7950 https://orcid.org/0000-0002-6673-5435 https://orcid.org/0000-0001-6416-8417 http://creativecommons.org/licenses/by/4.0/ Chimica Techno Acta 2022, vol. 9(1), No. 20229101 LETTER 2 of 3 O OOH OH O OH OH O CH2 OH OH OH O CH3 OH OHOH O 1 3 2 45 7 6 8 1' 3' 5' 2' 4'6' O HOOC O O OH OH COOH OH O OH OH COO O H NH 4 1 3 9 12 11 18 20 7 28 Rut GC Fig. 1 Structures of Rut and GC IR spectrum of GC (, cm–1): 3212 (ОН, NH), 2944 (CH), 2928 (СН), 2911 (CH), 2862 (СН), 1726 (С=О), 1710 (С=О), 1692 (С=О), 1642 (С(11)=О, C=C), 1588 (СОО–), 1453 (СН2, СН3), 1428 (NH4+), 1414 (СОО–), 1390 (СН), 1358 (СН), 1350 (СН), 1324 (CH), 1304 (СН), 1279 (CH), 1258 (СН), 1211 (СН), 1162 (С–О–С, С–ОН), 1032 (С–О–С, С–ОН),980 (=CH), 946 (СН), 918 (monosaccharide ring), 879 (СН), 868 (CH), 818 (СН), 786 (СН), 749 (CH), 692 (СН), 684 (СН), 677 (=CH), 657 (ОН). IR spectrum of the complex of Rut with GC (, cm–1): 3284 (ОН, NH), 2956 (CH), 2919 (СН), 2850 (CH), 1730 (С=ОGC), 1715 (С=ОGC), 1682 (С=ОGC), 1639 (С=ОRut), 1584 (СОО–, C=CAr), 1509 (C=CAr), 1451 (C=CAr, СН), 1424 (NH4+), 1411 (СОО–), 1360 (С–ОН, СН), 1301 (С–О–С, С–ОН, CH), 1275 (С–О–С, С–ОН, СН), 1235 (С–О–С, С–ОН), 1201 (С–О–С, С–ОН), 1168 (С–О–С, С–ОН), 1073 (С–О–С, С–ОН), 1047 (С–О–С, С–ОН), 1030 С–О–С, С–ОН), 982 (=CH), 929 (СН), 878 (СН), 806 (СН), 786 CH), 717 (СН), 691 (СН), 655 (OH), 598 (СН). Antioxidant activity was studied on a Photochem ana- lyzer (Analytik Jena AG, Germany). Determination of anti- oxidant capacity of lipid soluble compounds (ACL) and water soluble compounds (ACW) was carried out accord- ing to the manufacturer's standard protocols. ACW = 161.2 nM and ACL = 157.3 nM (in terms of trolox for 49.5 mg/L solutions of Rut–GC complex in 70% aque- ous ethanol (v/v)). 3. Results and discussion The composition of the complex of GC with Rut was de- termined by the isomolar series method (Figs. 2, 3). This method gave a molar ratio 1.0 at 258 nm, which corre- sponded to a 1:1 complex (Fig. 3). In addition, the isomolar curve shows a clear minimum at 361 nm at a 1:1 ratio of components (Fig. 3). Such ratio was obtained for complex- es of GA and GC with several drugs [2]. However, a differ- ent composition (1:2) was found in the complex of GC with quercetin [6]. Due to hypsochromic shift, the absorption maxi- mum of the solutions decreases from 258 to 252 nm (Fig. 2). Stability constant of the complex (KGC–Rut = (9.70.2)104 (mol/L)–1) was calculated based on the isomolar curves at 258 nm by A.K. Babko method. The previ- ously obtained 1:1 molecular complexes of different bioactive com- pounds with GC had stability constants of 103–105 (mol/L)–1 [2]. ATR FT-IR spectra of GC and Rut complex show low- frequency shifts of the absorption band of stretching vi- brations of О–Н bonds in Rut from 3415 and 3343 cm–1 to 3284 cm–1. Fig. 2 Absorption curves of isomolar series of solutions at 25 °C Chimica Techno Acta 2022, vol. 9(1), No. 20229101 LETTER 3 of 3 0 0.5 1 0 1 2 3 4 5 6 7 8 9 10 c (GC)/c (Rut) DА 258 -0.04 0 0.04 0.08 0.12 0 1 2 3 4 5 6 7 8 9 10 c (GC)/c (Rut) DА 361 (a) (b) Fig. 3 Optical density change DА as a function of component ratio of isomolar series at 258 nm (a) and at 361 nm (b) Such changes in the spectra confirm the formation of hydrogen bonds. IR spectrum of complex shows certain changes related to stretching vibrations, absorption bands of С–О bonds in С–О–С and С–ОН groups, for example 1059→1047 cm–1 for Rut, 1162→1168 cm–1 for GC. The presence of low- frequency shifts of C=O stretching vibrations, absorption band is indicative of С=О groups of Rut and GC involve- ment in hydrogen bonding: 1656→1639 cm–1 for Rut and 1692→1682 cm–1 for GC. In addition, the IR spectra show shifts of the main ab- sorption bands of CH bonds, stretching vibrations, which can be caused by hydrophobic interactions in the complex. Their presence explains the stability of the molecular complex of GC with Rut. Rut, GA and some complexes of GA have antioxidant activi- ty [1, 8, 9]. For example, the antioxidant capacity of complexes of uracil derivatives with GA was studied in several oxidative systems, where they showed a higher activity than ionol [8]. A preliminary study of Rut–GC complex antioxidant ac- tivity was performed. At the same time, the analysis of the complex antioxidant capacity (in terms of trolox) showed an increase of ACW by 7.31%, but a slight decrease of ACL by 2.73% in comparison with the Rut standard. 4. Conclusions A joint molecular complex of triterpene and flavonoid gly- cosides was obtained for the first time. The composition of the complex of Rut with GC is 1:1. The complex has suffi- cient stability and is formed by hydrogen bonds (C=OGC…H–ORut and C=ORut…H–OGC) and hydrophobic con- tacts. Complexation of Rut with GC can improve its bioa- vailability and membrane permeability, and expand the spectrum of biological activity. Acknowledgements This study was carried out with the experimental equip- ment of the Sevastopol State University (project PR/807- 42/2017). Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References 1. Gullón B, Lú-Chau TA, Moreira MT, Lema JM, Eibes G. Rutin: a review on extraction, identification and purification methods, biological activities and approaches to enhance its bioavaila- bility. 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