Georgian Scientists/ქართველი მეცნიერები ტ. 7 N 2, 2025 467 Georgian Scientists ქართველი მეცნიერები Vol. 7 Issue 2, 2025 https://doi.org/10.52340/gs.2025.07.02.43 Biologically Active Polyethylene Glycol-Based Multiple-Catechol-Containing Biopolymer Poly[3-(3,4-dihydroxyphenyl)glyceric acid] from Different Medicinal Plants of Boraginaceae Family Vakhtang Barbakadze 1, * ID, Maia Merlani 1, ID, Lali Gogilashvili 1, Lela Amiranashvili 1, Karen Mulkijanyan 2, ID V.B. – Research Scientist, v.barbakadze@tsmu.edu, tel. 595 53 15 09 M.M.– Head of the Department, Principal Research Scientist, m.merlani@tsmu.edu, tel. 599 761 117 L.G. – Senior Research Scientist, l.gogilashvili@tsmu.edu, tel. 599 347 042 L.A. – Senior Research Scientist, l.amiranashvili@tsmu.edu, tel. 577 723 144 K.M. – Head of the Department, Principal Research Scientist. k.mulkijanyani@tsmu.edu, 555 235 829 1Department of Plant Biopolymers and Chemical Modification of Natural Compounds, Tbilisi State Medical University, I.Kutateladze Institute of Pharmacochemistry, Tbilisi 0159, Georgia 2Department of Preclinical Pharmacological Research, Tbilisi State Medical University, I.Kutateladze Institute of Pharmacochemistry, Tbilisi 0159, Georgia *Correspondence v.barbakadze@tsmu.edu Abstract High molecular weight (>1000 kDa or >500 kDa) water-soluble preparations (HMPs) from Symphytum asperum, S. caucasicum, S. officinale, S. grandiflorum, Anchusa italica, Cynoglossum officinale, Borago officinalis, and Paracynoglossum imeretinum (Boraginaceae) were obtained. The main chemical constituent of these HMPs is the first and only representative of a previously unreported class of natural polyethers — a novel poly[oxy-1-carboxy-2-(3,4-dihydroxyphenyl)ethylene], also known as poly[3- (3,4-dihydroxyphenyl)glyceric acid] (P-DGA). The structure elucidation of P-DGA was performed using data from various nuclear magnetic resonance (NMR) techniques, including liquid-state 1H, 13C NMR, two-dimensional (2D) homonuclear gCOSY, two-dimensional (2D) heteronuclear 1H/13C gHSQCED, two-dimensional (2D) DOSY (Diffusion-ordered spectroscopy), and solid-state 13C NMR spectra. The polyoxyethylene (polyethylene glycol) (PEG) chain serves as the backbone of this biopolymer, with a residue of 3-(3,4-dihydroxyphenyl)glyceric acid functioning as the repeating unit. The 3,4-dihydroxyphenyl (catechol) and carboxyl groups consistently substitute for two carbon atoms in the PEG backbone chain. Hence, P-DGA represents a unique class of natural polyethers. Each repeating trifunctional structural unit of P-DGA contains two phenolic hydroxyl groups in the ortho position and one carboxyl group. The multifunctionality of P-DGA likely explains its wide spectrum of biological activities, including anti-complementary, antioxidant, anti-inflammatory, burn and wound healing, antimicrobial, and anti-cancer properties. https://orcid.org/0000-0002-8716-909X https://orcid.org/0000-0002-0237-6699 https://orcid.org/0000-0001-9360-3422 mailto:v.barbakadze@tsmu.edu mailto:m.merlani@tsmu.edu mailto:l.gogilashvili@tsmu.edu mailto:l.amiranashvili@tsmu.edu mailto:k.mulkijanyani@tsmu.edu Georgian Scientists/ქართველი მეცნიერები ტ. 7 N 2, 2025 468 Keywords: natural polyethers; poly[3-(3,4-dihydroxyphenyl)glyceric acid]; Boraginaceae; catechol; polyethylene glycol. 1. Introduction Herbs can be seen as biosynthetic chemical laboratories that produce numerous chemical compounds. Chemicals with medicinal benefits are known as “active ingredients” or “active principles.” Plant-derived products, also referred to as herbal or phytotherapeutic products, demonstrate a broad therapeutic spectrum. The bioactive constituents of herbal products, such as alkaloids, flavonoids, terpenes, and polyphenols, can interact with biological systems, leading to the discovery of novel drugs that provide therapeutic benefits [1-4]. Plants used in traditional medicine have endured the test of time. Medicinal plant extracts can contain hundreds or thousands of bioactive compounds in varying abundances, making it a significant challenge to identify the compounds responsible for specific biological activities [5]. Phytochemicals are categorized into primary and secondary metabolites based on their roles in plant metabolism. Primary metabolites essential for plant survival include carbohydrates, amino acids, proteins, lipids, purines, and pyrimidines found in nucleic acids. In contrast, secondary metabolites consist of other chemical compounds produced from metabolic pathways that diverge from the primary metabolic routes [6]. A wide variety of secondary metabolites intrigues scientists, particularly due to their unique pharmacophores and medicinal properties. Polymers of natural origin are known as “biopolymers”—macromolecules typically produced by living systems, including plants, animals, and microorganisms. In recent years, there has been a growing trend toward the use of more biopolymers in the development of various food and medical products [7, 8]. Compared to synthetic polymers, biopolymers offer several advantages, such as biocompatibility, biodegradability, abundant renewable sources, and the ability to metabolize in the human body without releasing toxic or harmful products [8, 9]. The United States Food and Drug Administration (US FDA) has approved many biopolymer-based products. Biopolymers are made up of repeating units of monomers, such as sugars, amino acids, or fermentative products like aliphatic polyesters. These biopolymers may possess different functional groups: hydroxyl, amino, amide, carboxyl, phosphate, and phenolic, which impart various biological activities. Biopolymers are generally classified into three groups: polysaccharides, proteins, and polynucleotides [7, 8]. Another significant group of plant-derived compounds is phenolics, which provide numerous beneficial effects on human health due to their antioxidant, anti-inflammatory, antithrombotic, antiallergenic, anticancer, antifungal, antimicrobial, and antiatherosclerosis properties [10]. Phenolic compounds are widespread secondary metabolites produced in plants through the shikimate, pentose phosphate, and phenylpropanoid pathways [10, 11]. They consist of hydroxybenzenes that contain an aromatic ring with one or more hydroxyl substituents. These compounds can be divided into two main groups: simple phenols and polyphenols, based on the number of phenol units in their structure [10- 12]. The antioxidant activity, the most notable property of phenolic compounds, relates to their structure, specifically the number and position of hydroxyl groups in relation to carboxyl groups [10]. Various mechanisms have been described regarding the effects of phenolic compounds on health: they Georgian Scientists/ქართველი მეცნიერები ტ. 7 N 2, 2025 469 reduce the expression of inflammatory factors, increase brain-derived neurotrophic factor expression, modulate the gut microbiota, decrease cancer cell proliferation, and enhance apoptosis induction [10, 12]. Plant phenols range from simple phenolic molecules to highly polymerized compounds. Phenolic phytochemicals are also classified into two main categories: (i) water-soluble (e.g., phenolic acids, hydrolyzable tannins (ellagitannin), phenylpropanoids, flavonoids, and quinones) and (ii) water- insoluble (e.g., condensed tannins, lignins, and cell wall-bound hydroxycinnamic acids) [12]. The covalent conjugation of phenolics with other biomolecules (proteins, polysaccharides, fatty acids, etc.) and electrostatic interactions with metal ions (silver, gold, iron, zinc, etc.) enhance their functionality and applications [12]. Among a wide variety of phenolics, catechol (ortho-dihydroxybenzene) derivatives and their corresponding ortho-quinones represent an important class that is widespread in many living organisms, including mussels, sandcastle worms, geckos, insects, and squids. They play a crucial role in numerous biological processes and functions. Due to their unique chemical and physico-chemical properties, the remarkable biological activity of catechols encourages scientists to develop countless advanced multifunctional materials with outstanding and fascinating properties through the synergistic combination of catechols and polymers [13]. Among the catechol-containing natural products that have attracted considerable attention for their diverse biological activities and low toxicity are hydroxycinnamic acid derivatives, such as caffeic acid [14]. The Boraginaceae family includes approximately 2000 species worldwide, mainly in Europe and Asia. The therapeutic effects of these plants are attributed to the presence of various biologically active compounds, such as naphthoquinones, flavonoids, terpenoids, and phenols, which display antimicrobial, antitumor, antiviral, anti-inflammatory, cardiotonic, contraceptive, and antiplatelet activities. However, these plants are also high in hepatotoxic pyrrolizidine alkaloids, which significantly restrict their use. Nevertheless, applying Boraginaceae plants as a poultice for wounds remains acceptable. Despite the beneficial qualities of Boraginaceae plants, their medical application is still controversial [15-17]. Ether bonds are found in a wide variety of natural products, mainly secondary metabolites, including lipids, oxiranes, terpenoids, flavonoids, polyketides, and carbohydrate derivatives or aromatic polymers such as lignin [18]. In the latter case, peroxidases initiate the radical coupling of monolignols to lignin, yielding ether links between two aromatic rings or between an aromatic ring and an aliphatic moiety [19]. However, reports concerning biopolymers containing aliphatic ethers as repeating units are sparse. Within the field of pharmacologically active biopolymers, the area of stable natural polyethers seems relatively new and rather attractive. This review aims to consolidate knowledge about the isolation, fractionation, detection, and structural elucidation of the first representative of a unique class of natural polyethers, which exhibits a broad spectrum of biological activity. This activity originates from the routine assessment results of the anticomplementary and antioxidant activities of crude polysaccharides isolated from S. aspeum and S. caucasicum. Georgian Scientists/ქართველი მეცნიერები ტ. 7 N 2, 2025 470 2. Materials and Methods 2.1. Bioactivity-guided Isolation of the Main Chemical Constituent of Water-soluble High-molecular Preparations (HMPs) from Various Genera and Species of the Boraginaceae Family Medicinal plants, including Symphytum asperum (SA), S. caucasicum (SC), S. officinale (SO), S. grandiflorum (SG), Anchusa italica (AI), Cynoglossum officinale (CO), Borago officinalis (BO), and Paracynoglossum imeretinum (PI) (Boraginaceae), are widely distributed across the Caucasus, particularly in Georgia. The raw plant materials — roots or stems — were cut into small pieces, air- dried, and ground. Lipids, pigments, and low-molecular-weight compounds (monosaccharides, phenolics, etc.) were extracted using Soxhlet extraction with chloroform, methanol, and acetone. The hot-water extraction of the pretreated materials was followed by dialysis, resulting in crude water- soluble polysaccharides with immunomodulatory activity, including anticomplementary and antioxidant activities [20]. A general strategy involving the fractionation of bioactive crude water- soluble polysaccharides and the isolation of active principles was used to identify new or novel compounds responsible for that activity, utilizing both i) bioactivity-guided and ii) structure-directing isolation [21]. Consequently, the fractionation of crude polysaccharides was carried out using molecular mass-based ultrafiltration (UF) under a nitrogen pressure of 3 atmospheres in a stirred ultrafiltration cell. Various membranes with cut-off values of 10 kDa, 100 kDa, 500 kDa, and 1000 kDa were employed. During the UF process, the fractionation was monitored by evaluating anticomplementary and antioxidant activities and measuring total sugars and UV absorption at 286 nm in both the solutions and effluents. Fractions containing molecules with relative molecular masses (Mr) above 1000 kDa or 500 kDa exhibited higher anticomplementary and antioxidant activities than crude polysaccharides. These active fractions had low carbohydrate content and showed strong UV absorption at 286 nm, unlike high carbohydrate content fractions below 500 kDa, which exhibited neither anticomplementary nor antioxidant activity. Thus, the removal of most ballast bio-inactive polysaccharides and the acquisition of water-soluble high-molecular-weight preparations (HMPs) greater than 1000 or 500 kDa has been facilitated by UF (Scheme 1, fraction “C”) [20, 22]. In addition, the UF enabled the complete removal of hepatotoxic and carcinogenic pyrrolizidine alkaloids with an average weight of 340 Da, characteristic of Boraginaceae family species [23]. Georgian Scientists/ქართველი მეცნიერები ტ. 7 N 2, 2025 471 Scheme 1. Isolation of HMPs from SA, SC, SO, SG, AI, CO, BO, and PI. Fractions “A” and “B” represent neutral glucofructan and pectin-type acidic arabinogalactan, respectively [24]. HMPs with a low carbohydrate concentration (approx. 20%) and strong UV absorption at 286 nm displayed significant anti-complementary activity and antioxidant properties. However, sugars do not absorb light in the typical UV range due to their lack of chromophores. The monosaccharide composition of HMPs notably differed from that of the initial crude polysaccharide extracts. HMPs included rhamnose, arabinose, mannose, glucose, galactose, uronic acids, and minimal amounts of fructose. Subsequently, HMPs underwent gel chromatography on a Sepharose 2B column. Equal volumes of eluents were examined for the inhibition of human complement activity, total sugar content, and UV absorption at 286 nm. The elution profile showed anti-complementary activity that coincided with a UV absorption pattern at 286 nm but did not correspond with the carbohydrate elution. To characterize an immunomodulatory component of HMPs, it was treated with skin powder. The modulatory effect on human complement significantly decreased, while also reducing absorption in the UV range. The absorption of active substances by skin powder, along with the UV spectrum of these compounds, exhibited an absorption maximum at 286 nm, attributable to phenolic substances, which suggested the presence of a phenolic moiety in the structure of the active compounds. Based on this data, we hypothesized that the isolated bioactive compounds are phenolic polymers, and their presence in crude polysaccharides can be explained by their similar hydrophilicity [20]. Georgian Scientists/ქართველი მეცნიერები ტ. 7 N 2, 2025 472 2.2. The Elucidation of the Structure of the Main Chemical Constituent of HMPs from Various Genera and Species within the Boraginaceae Family The elucidation of the structure of a main chemical constituent of HMPs from SA, SC, SO, SG, AI, CO, BO, and PI was based on data obtained from UV, IR, and various NMR spectroscopy techniques in both liquid and solid states (Figs. 1-14, Tables 1-5). The absorption maxima observed in the UV spectrum of HMPs from SA, SC, SO, SG, AI, CO, BO, and PI were found at 212 nm, 236 nm (shoulder), 282 nm (shoulder), and 286 nm (H2O, max, nm), which can be attributed to substituted phenols. Typically, catechol exhibits an absorbance peak of around 270–290 nm [25]. The IR spectra of HMPs from SA, SC, SO, SG, AI, CO, BO, and PI were recorded using KBr (KBr, υ, cm-1). They exhibited bands at 3425 (OH), 2924 (CH), 1605 (ionized carboxyl), 1512 and 1443 (aromatic C=C), 1404 and 1219 (phenols), 1265, 1080, and 1018 (R-O-R'), 872 (C-H in the aromatic ring with one isolated hydrogen atom), and 818 cm-1 (C-H in the aromatic ring with two neighboring hydrogen atoms) [26]. The spectrum contains absorption bands characteristic of phenol-carboxylic acids. The elucidation of the main chemical constituents of HMPs from SA, SC, SO, CO, PI (Figs. 1-6 and 8, Tables 1-3) [27-33] and SG, AI, BO (Figures 9-14, Tables 4 and 5) [34-36] was performed at 80 °C using various techniques in liquid-state and solid-state NMR spectroscopy. According to the data from the liquid-state 13C NMR spectrum (Figure 1) of HMPs derived from SA, SC, SO, CO, and PI, nine distinct signals corresponding to the carbon atoms of the substituted phenylpropionic acid fragment are observed (Figure 4). Interestingly, the signals from the carbohydrate components are nearly unobservable in the HMP spectra (Figure 1), likely due to their diverse monosaccharide composition, which includes rhamnose, arabinose, mannose, glucose, galactose, uronic acids, and only trace amounts of fructose. The broadened signal at 175.4 ppm is assigned to the carboxyl group in the compound. Figure 1. The 13C NMR spectrum of HMPs from SA, SC, SO, CO, and PI at 80 °C. The attached proton test (APT) technique [37] revealed (Figure 2) that five signals are assigned to CH groups and four signals to the non-protonated carbon atoms. The two signals with chemical Georgian Scientists/ქართველი მეცნიერები ტ. 7 N 2, 2025 473 shifts of 78.2 and 80.4 ppm correspond to oxygen-bound protonated aliphatic carbon atoms. Six signals were assigned to aromatic carbon atoms (protonated atoms at 117.4, 118.6, and 122.3 ppm and non- protonated atoms at 131.5, 143.8, and 144.7 ppm) (Figure 2) [27-29]. Figure 2. The proton attachment test (APT) spectrum of HMPs from SA, SC, SO, CO, and PI at 80 °C. The 1H NMR spectrum of HMPs from SA, SC, SO, CO, and PI displays four signals at 4.9, 5.3, 7.1, and 7.2 ppm, with one of these (7.1 ppm) showing double the intensity (Figure 3). Unfortunately, the spin-spin coupling constants could not be determined due to the broadening of these signals. Figure 3. The 1H NMR spectrum of HMPs from SA, SC, SO, CO, and PI at 80 °C. The simulated 13C NMR spectrum was calculated using the ACD/CNMR Version 1.1 program (Advanced Chemistry Development Inc., Canada) (Table 1, Figure 4) [38-40]. Table 1. Chemical shifts of resonances in the 13C NMR spectrum of HMPs from SA and SC. Experimental 175.4 144.6 143.8 131.5 122.3 118.6 117.4 80.4 78.2 Calculated* 175.1 145.5 145.1 128.8 118.3 115.7 114.4 82.9 77.2 * Calculated values for the 3-(3,4-dihydroxyphenyl)glyceric acid residue of the corresponding polyether are provided for comparison. Georgian Scientists/ქართველი მეცნიერები ტ. 7 N 2, 2025 474 Figure 4. A hypothetical representation of the repeating unit of the biopolymer derived from SA and SC, which includes the 3-(3,4-dihydroxyphenyl)glyceric acid residue. The two-dimensional (2D) heteronuclear 1H/13C gHSQCED spectrum reveals the following correlations between protons and carbon atoms at 4.9/80.4, 5.3/78.2, 7.1/118.6, 7.1/122.3, and 7.2/117.4 ppm (Figure 5, Table 2). The hydroxyl groups at positions 3 and 4 of the phenyl ring were confirmed by the 1D-NOE experiment conducted in a different mode. Pre-irradiation of the aliphatic proton at position 1 (Figures 5, 6, and Table 2), with a chemical shift of 5.3 ppm, provided a NOE for two aromatic protons with chemical shifts of 7.1 (3%) and 7.2 (1%) ppm. Therefore, both ortho-positions in the dihydroxyphenyl ring were occupied by protons. The differing NOE values for these two protons, along with their distinct chemical shifts in the 1H NMR spectrum and the varying positions of resonances of the corresponding carbons in the 13C NMR spectrum, ruled out the possibility of symmetrical bis-meta- substitution of the aromatic ring by two hydroxy groups [27-29]. Based on the data about signal assignments for 13C and 1H NMR spectra (Figures 1-4, Table 2), the correlations observed in the 2D heteronuclear 1H/13C gHSQCED (Figure 5, Table 2), and in the 2D homonuclear gCOSY (Figure 6), the main chemical component of HMPs derived from the roots and stems of SA, SC, SO, CO, and PI is poly[3-(3,4-dihydroxyphenyl)glyceric acid] (P-DGA) (Figure 7A- B). Consequently, P-DGA represents a unique class of multiple-catechol-containing natural polyethers, specifically, poly[oxy-1-carboxy-2-(3,4-dihydroxyphenyl)ethylene], which is also recognized as a caffeic acid-derived biopolymer. Georgian Scientists/ქართველი მეცნიერები ტ. 7 N 2, 2025 475 Figure 5. Correlations and assignments of the 1H and 13C signals in the 2D heteronuclear 1H/13C gHSQCED spectrum of P-DGA at 80 °C. The 2D homonuclear gCOSY spectrum (Figure 6) showed a cross-peak between the signals at 4.9 and 5.3 ppm, consistent with the coupling between H-1 and H-2 of P-DGA (Figure 6). Figure 6. The 2D homonuclear gCOSY spectrum of P-DGA at 80 °C. The total assignment of signals is given in Table 2. Georgian Scientists/ქართველი მეცნიერები ტ. 7 N 2, 2025 476 Table 2. The signal assignments for the 13C and 1H NMR spectra of P-DGA. The repeating unit of P-DGA С atom no. 13C chemical shifts, C, ppm 1H chemical shifts, H, ppm 1' 1 2 1'' 2'' 3'' 4'' 5'' 6'' 175.4 78.2 80.4 131.5 117.4 144.6 143.8 118.6 122.3 5.3 4.9 7.2 7.1 7.1 A good resolution and the narrow shape of the 13C NMR signals indicate that the P-DGA is a regular polymer. The polyoxyethylene (polyethylene glycol) (PEG) chain serves as the backbone of the polymer molecule. The catechol moieties (3,4-dihydroxyphenyl residues) and carboxyl groups serve as regular substituents on two carbon atoms in the PEG backbone chain of P-DGA (Figure 7A). Figure 7. A - Catechol moieties and carboxyl groups are regular substituents at two carbon atoms in the PEG backbone of P-DGA; B – Poly[oxy-1-carboxy-2-(3,4-dihydroxyphenyl)ethylene] that is Poly[3-(3,4-dihydoxyphenyl)glyceric acid] (P-DGA). To confirm the proposed structure of the poorly water-soluble high-molecular P-DGA from SA, the solid-state 13C NMR spectrum was recorded under magic-angle spinning (MAS) conditions. Two different experiments were conducted to assign the 13C signals to the corresponding atoms in the monomer structure. The 13C{1H} cross-polarization/magic-angle spinning (CP/MAS) NMR experiment is a standard method for spectral assignment of carbon signals. Additionally, a dipolar-dephasing solid- Georgian Scientists/ქართველი მეცნიერები ტ. 7 N 2, 2025 477 state NMR pulse sequence with gated decoupling was utilized [41, 42]. The complete assignment of the signals is detailed in Figure 8 and Table 3. Figure 8. Solid-state 13C{1H} CP/MAS NMR spectrum of P-DGA from SA. a) The spectrum was recorded using a 67.0 microseconds (µs) dipolar dephasing filter with a cross-polarization (CP) time of 3.0 milliseconds (ms). b) The spectrum was recorded with a CP time of 3.0 ms. c) The spectrum was recorded with a CP time of 1.0 ms. Table 3. The assignment of signals in the solid-state 13C{1H} CP/MAS NMR spectrum of P-DGA from SA (c, ppm). С atom no. 13C chemical shift, C, ppm 1,2 1' 1'' 2'',5'',6'' 3'',4'' 80 174 130 118 143 It is important to emphasize that the resolution of solid-state NMR is inferior to that of liquid- state NMR. For nuclei other than H-1, solid-state NMR peaks are generally at least ten times broader than those of liquid-state NMR. Therefore, the applicability of solid-state NMR spectroscopy to studies is somewhat limited [43]. However, the complete assignment of the signals from the solid-state 13C NMR of P-DGA agrees with the results of the liquid-state 13C NMR spectrum (Figure 1). The elucidation of the P-DGA structure from AI, SG, and BO was carried out using a similar approach [33-35, 40, 41]. The 1H NMR, 13C NMR, 2D heteronuclear 1H/13C gHSQCED, 2D homonuclear gCOSY, and solid-state 13C NMR spectra of HMPs from AI, SG, and BO exhibited a complete set of resonances characteristic of poly[3-(3,4-dihydroxyphenyl)glyceric acid] (P-DGA) (Figures 9-13 and Tables 4, 5) [34-36, 41, 42], which are present in the water-soluble high molecular fractions of SA, SC, Georgian Scientists/ქართველი მეცნიერები ტ. 7 N 2, 2025 478 SO, CO, and PI (Figures 1-6, 8 and Tables 1-3) [26-32]. In addition to the aforementioned signals characteristic of the P-DGA from SA, SC, SO, CO, and PI (Figures 1-6, 8 and Tables 1-3) [34-36, 41, 42], the 13C NMR, 1H NMR, 2D heteronuclear 1H/13C gHSQCED, and solid-state 13C NMR spectra of HMPs from AI, SG, and BO displayed the following additional resonances: 54.9 ppm (-OCH3, 13C NMR, Figure 9); 3.85 ppm (-OCH3, 1H NMR, Figure 10); correlation between proton and carbon atoms: 3.85/54.9 ppm (-OCH3/-OCH3, 2D 1H/13C gHSQCED, Figure 11); 54 ppm (-OCH3, solid-state 13C NMR, Figure 13). The 2D homonuclear gCOSY spectrum of HMPs from AI, SG, and BO (Figure 12) displayed a cross-peak between the signals at 4.7 and 5.2 ppm, consistent with the coupling between H-1 and H-2 of P-DGA (Figure 12). The complete signal assignments are presented in Figures 9-13 and Tables 4, 5 [34-36, 41, 42]. The additional signals may be observed in the spectrum of HMPs from AI, SG, and BO due to the presence of the methoxy group. Two non-sharp signals (172.8 and 175.6 ppm, Figure 9) are believed to arise from two carboxyl groups. A resonance in the 13C NMR spectrum at 54.9 ppm, which correlates with the 1H resonance at 3.85 ppm (Figures 9-12), suggests the presence of methoxy groups are present in carboxylic acid methyl esters. Consequently, the signal at 175.6 ppm is attributed to a carboxylic acid group, while the signal at 172.8 ppm is assigned to carboxyl groups in the form of methyl esters (upfield shifted) (Figures 9-12 and Tables 4, 5) [34-36]. Approximately 70% of the current carboxyl groups are methyl esterified (MeO: 13C, 54.9 ppm; 1H, 3.85 ppm). The extent of methyl esterification was calculated by comparing the integral intensity of the methyl ester signal (3.85 ppm, 0.5 H) to that of the aliphatic proton signal at H-1 (5.2 ppm, 0.7 H) in a 1H experiment that included a WATERGATE water suppression routine. The presence of methoxy groups at C3'' and C4'' in the aromatic ring is excluded, as there are no downfield shifts of the C 3'' (145.3 ppm) and C 4'' (144.5 ppm) signals, which could result from substituting the C3'' and C4'' hydroxy groups with methyl groups. Figure 9. The 13C NMR spectrum of P-DGA from AI, SG, and BO was recorded at 80 °C. Georgian Scientists/ქართველი მეცნიერები ტ. 7 N 2, 2025 479 Figure 10. The 1H NMR spectrum of P-DGA from AI, SG, and BO at 80 °C. Figure 11. The 2D 1H/13C gHSQCED spectrum of P-DGA from AI, SG, and BO at 80 °C. Georgian Scientists/ქართველი მეცნიერები ტ. 7 N 2, 2025 480 Figure 12. The 2D COSY spectrum of P-DGA from AI, SG, and BO at 80 °C. Figure 13. Solid-state 13C{1H} CP/MAS NMR spectrum of methylated carboxylic groups in P-DGA of SG. a) The spectrum is recorded using a 67.0 microseconds (µs) dipolar dephasing filter and a cross- polarization (CP) time of 3.0 milliseconds (ms); b) The spectrum is recorded with a CP time of 3.0 ms; c) The spectrum is recorded with a CP time of 1.0 ms. Georgian Scientists/ქართველი მეცნიერები ტ. 7 N 2, 2025 481 Table 4. The signal assignments of the 13C and 1H NMR spectra of methylated carboxylic groups in P- DGA (c, ppm) from AI, SG, and BO. The repeating unit of P-DGA; R=H, CH3 С atom no. 13C chemical shift, C, ppm 1H chemical shift, H, ppm 1' 1' 1 2 1'' 2'' 3'' 4'' 5'' 6'' 175.6 (COOH) 172.8 (COOCH3) 54.9 (OCH3) 78.8 81.0 132.2 118.0 145.3 144.5 119.2 123.0 3.85 (OCH3) 5.2 4.7 7.2 7.1 7.1 Furthermore, the 2D DOSY experiment produced similar diffusion coefficients for both the methylated and non-methylated signals. Both sets of signals occupied the same horizontal range (Figure 14). This indicates a comparable molecular weight (same order of magnitude) for methylated and non- methylated polymers [36-38]. This was further supported by graphical representations of the intensity decay of the 1H signals from aromatic H-2 and aliphatic H-1 at 7.2 and 5.2 ppm (Figures 15a and 15b, respectively), as well as from the methoxy group at 3.85 ppm (Figure 15c). These three 1H signals exhibited essentially the same curve shape. In contrast, the resonance from residual water at 4.35 ppm (Figure 15d) displayed a different decay pattern (faster diffusion) (Figure 15) [34-36]. Georgian Scientists/ქართველი მეცნიერები ტ. 7 N 2, 2025 482 Table 5. The signal assignments of the solid-state 13C{1H} CP/MAS NMR spectrum for methylated carboxylic groups of P-DGA (C, ppm) from SG. Figure 14. The 2D DOSY experiment of P-DGA from AI was conducted at 80 °C. С atom no. 13C chemical shift, C, ppm 1 2 1' 2' (OCH3) 1'' 2'',5'',6'' 3'',4'' 72 78 174 54 130 118 143 Georgian Scientists/ქართველი მეცნიერები ტ. 7 N 2, 2025 483 Figure 15. Intensity decay of 1H signals at 7.2 ppm (Ar H-2) (a), 5.2 ppm (H-1) (b), 3.85 ppm (OMe) (c), and 4.35 ppm (residual water) (d). Units: X-axis in s/cm²; Y-axis in relative intensity (dimensionless). Thus, based on the data regarding the various techniques of NMR spectroscopy, the main structural component of HMPs isolated from AI, SG, and BO is similar to that from SA, SC, SO, CO, and PI — regular poly[3-(3,4-dihydroxyphenyl)glyceric acid] (P-DGA), with a 3-(3,4- dihydroxyphenyl)glyceric acid residue serving as the repeating unit (Figures 1-6, 8-13 and Tables 1-5). However, most of the carboxylic groups in the P-DGA from AI, SG, and BO are methylated, unlike those in P-DGA from SA, SC, SO, CO, and PI (Figures 9-14 and Tables 4, 5). It is important to emphasize that P-DGA was detected in neither the leaves of SA, SC, SO, CO, AI, SG, BO, and PI [27-36, 44] nor in the stems of AI [34]. 2.3. Proposing Multiple Hydrogen Bonds between P-DGA and the Residual Polysaccharides Leads to Supramolecular Association, Resulting in a Multiple-hydrogen-bonded Supramolecular Polymer We could not fully achieve the separation of P-DGA and residual polysaccharides through ultrafiltration. On the one hand, this suggests that P-DGA and the residual polysaccharides have similar molecular masses, being of the same order of magnitude. On the other hand, this is likely due to the formation of multiple hydrogen bonds between P-DGA and the residual polysaccharides, resulting in a supramolecular associate (multiple-hydrogen-bonded supramolecular polymer). Additionally, P- DGA was subjected to gel chromatography on the Sepharose 2B column. The anticomplementary activity by the alternative pathway (AP) eluted together with the UV-286 nm absorption pattern, indicating that it coincided with P-DGA but does not align with the carbohydrate’s elution curve. The elution peak of the polysaccharides has shifted away from the AP and P-DGA peaks. Thus, this phenomenon presumably excludes the covalent binding of residual polysaccharides to P-DGA, which is responsible for the anticomplementary activity [20, 31]. Georgian Scientists/ქართველი მეცნიერები ტ. 7 N 2, 2025 484 Biopolymers are often impure. They may bind to polysaccharides, proteins, phenolics, etc., substances through either covalent or non-covalent bonds. When highly directional non-covalent interactions replace the covalent bonds that hold the monomeric units of a macromolecule together, supramolecular polymers are formed. Supramolecular chemistry, also referred to as “chemistry beyond the molecule", studies the function and structure of supramolecular entities, i.e., supermolecules arising from the intermolecular binding. Combining supramolecular chemistry and polymer science has given rise to a promising class of nanomaterials called supramolecular polymers. Unlike classical (covalent) polymers, supramolecular polymers are ordered self-assembled nanostructures formed by non-covalent bridging of monomeric units. The diverse applications of supramolecular polymers range from electronics to medicine. The use of supramolecular polymers for intracellular protein delivery, bone regeneration scaffolds, and drug delivery, among other biomedical therapies, is attributed to their versatility and the ability to modulate their physical and mechanical properties. Notably, the application of supramolecular amphiphilic scaffolds in regenerative medicine reveals the promising biomedical applications of these well-ordered systems [45, 46]. This new generation of biopolymers is created through non-covalent interactions such as hydrogen bonding, hydrophobic interactions, π-π electron stacking through the aromatic (arene-arene, Ar···Ar) structures, π–π electron stacking of aromatic-carbonyl (arene-carbonyl, Ar···C═O) structures, π–π electron stacking of carbonyl-carbonyl (C═O···C═O) structures, cation–π interactions, coordination with metal oxide surfaces, and with metal ions to form supramolecular polymers [47, 48]. Catechol, the ortho isomer of 1,2-dihydroxybenzene, can bind to mucins by forming hydrogen bonds due to the hydroxyl groups at the ortho position. A significant advantage of catechol is its availability for conjugation with bioactive molecules; it can be easily oxidized to the quinone form, which is highly reactive toward numerous functional groups, including thiol and amino groups, via Michael addition or Schiff base reactions. Catechol moieties are suitable for conjugation with metals and metal oxides (i.e., Ag+, Fe3+, etc.) through coordination bonds. Furthermore, the unique combination of hydroxyl and phenolic groups can promote π-π stacking and π-cation interactions [48]. Thus, according to the literature data, the two hydroxyl groups of the catechol moieties of P- DGA can form hydrogen bonds with the hydroxyl groups of residual polysaccharides. Proposing multiple hydrogen bonds between P-DGA and the residual polysaccharides leads to a supramolecular association, resulting in a multiple-hydrogen-bonded supramolecular polymer. 2.4. Proposed Biosynthetic Pathway and Acid Hydrolysis of P-DGA No information on the biosynthesis of such a polymer in plants is available. However, from the chemical perspective, this process can be viewed as the epoxidation of the double bond in caffeic acid, followed by the polymerization of the resulting epoxide (Scheme 2) [28, 29]. Georgian Scientists/ქართველი მეცნიერები ტ. 7 N 2, 2025 485 Scheme 2. Proposed biosynthetic pathway of P-DGA. Acid hydrolysis (2M CF3COOH at 121°C for 2 h) of P-DGA produced a dark brown or black water-insoluble material. This phenomenon can be explained by the formation of a three-dimensional “lignin-like” compound due to the intermolecular oxidative cross-linking (coupling) between catechol moieties in different macromolecules of [20]. Further research should clarify the physiological functions of these polyethers in plants and demonstrate whether their biosynthesis is a unique characteristic of the Boraginaceae family or if such compounds are also produced in other plants [28, 29]. 3. Conclusion The bioactivity-guided fractionation of water-soluble high-molecular preparations (HMPs) from Boraginaceae species — Symphytum asperum, S. caucasicum, S. officinale, S. grandiflorum, Anchusa italica, Cynoglossum officinale, Borago officinalis, and Paracynoglossum imeretinum — revealed that the key constituent of these HMPs is the inaugural representative of a novel class of natural polyethers: poly[oxy-1-carboxy-2-(3,4-dihydroxyphenyl)ethylene], known as poly[3-(3,4- dihydroxyphenyl)glyceric acid] (P-DGA). The PEG chain serves as the backbone of this biopolymer, with a residue of 3-(3,4-dihydroxyphenyl)glyceric acid acting as the repeating unit. The 3,4- dihydroxyphenyl (catechol) and carboxyl groups regularly substitute for two carbon atoms in the chain. The structure of P-DGA was elucidated using data from various NMR techniques, including liquid- state 1H and 13C NMR, homonuclear 2D gCOSY, heteronuclear 2D 1H/13C gHSQCED, 2D DOSY, and solid-state 13C NMR spectra. The synergistic combination of catecholic groups with the PEG main chain of P-DGA highlights the diverse and fascinating biological activity of P-DGA [13]. P-DGA exhibited immunomodulatory (anticomplementary) [20, 31, 49], antioxidant [20, 22, 29, 49, 50], anti-inflammatory [22, 31, 49], wound and burn healing [51, 52], anticancer [53], and antimicrobial [54] activities. Each repeating structural unit of the regular biopolymer P-DGA is tri-functional, comprising two catechol vicinal hydroxyl groups and one carboxyl group, likely responsible for its broad spectrum of biological activities. Incorporating catechol moieties into macromolecules enhances the exceptional therapeutic properties [10, 12, 55-57]. Conversely, the polymeric component — the PEG backbone — of P-DGA protects catecholic moieties from rapid degradation and ensures sustained biological efficacy. Consequently, pyrrolizidine alkaloid-free preparations of P-DGA may hold potential pharmaceutical value and are recommended for external and internal medical applications. Georgian Scientists/ქართველი მეცნიერები ტ. 7 N 2, 2025 486 4. The Future-directed Plan In the future, we plan to conduct the next review, which will present a detailed description of the biological activities and the structure-bioactivity relationship of P-DGA based on the literature data concerning catechol-derived natural and synthetic polymers. Abbreviations The following abbreviations are used in this manuscript: AI Anchusa italica APT Attached proton test BO Borago officinale CO Cynoglossum officinale CP Cross-polarization CP/MAS Cross-polarization/magic-angle spinning DOSY Diffusion-ordered spectroscopy gCOSY Gradient-Selected Correlation Spectroscopy gHSQCED Gradient Heteronuclear Single Quantum Coherence and Double Exclusion HMPs High-molecular-weight preparations IR Infrared kDa Kilodalton NMR Nuclear magnetic resonance P-DGA Poly[3-(3,4-dihydroxyphenyl)glyceric acid] PEG Polyethylene glycol PI Paracynoglossum imeretinum ppm Parts per million SA Symphytum asperum SC Symphytum caucasicum SG Symphytum grandiflorum SO Symphytum officinale UF Ultrafiltration UV Ultraviolet Conflicts of Interest: The authors declare no conflicts of interest. Georgian Scientists/ქართველი მეცნიერები ტ. 7 N 2, 2025 487 References 1. Folashade, K.O.; Omoregie, E.H.; Ochogu, A.P. Standardization of herbal medicines. Intern. J. 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C 2021, 131, 112515 (1–14). https://doi.org/10.1016/j.msec.2021.112515 Georgian Scientists/ქართველი მეცნიერები ტ. 7 N 2, 2025 492 Boraginaceae-ს ოჯახის სხვადასხვა სამკურნალო მცენარის ბიოლოგიურად აქტიური პოლიეთილენგლიკოლის საფუძველზე მრავალკატექოლშემცველი ბიოპოლიმერი პოლი[3-(3,4-დიჰიდროქსიფენილ)გლიცერინის მჟავა] ვახტანგ ბარბაქაძე, მაია მერლანი, ლალი გოგილაშვილი, ლელა ამირანაშვილი, კარენ მულკიჯანიანი რეზიუმე: მიღებულია Boraginaceae-ს ოჯახის სხვადასხვა სამკურნალო მცენარედან Symphytum asperum-ის, S. caucasicum-ის, S. officinale-ის, S. grandiflorum-ის, Anchusa italica-ს, Cynoglossum officinale-ს, Borago officinalis-ის და Paracynoglossum imeretinum-ის წყალში ხსნადი მაღალ მოლეკულური (>1000 kDa ან >500 kDa) პრეპარატები (მმპ). ამ მმპ-ების ძირითადი ქიმიური შემადგენელი ნაწილია ბუნებრივი პოლიეთერების აქამდე უცნობი კლასის პირველი და ერთადერთი წარმომადგენელი - ახალი პოლი[ოქსი-1-კარბოქსი-2-(3,4- დიჰიდროქსიფენილ)ეთილენი], რომელიც ასევე ცნობილია როგორც პოლი[3-(3,4- დიჰიდროქსიფენილ)გლიცერინის მჟავა] (პ-დგმ). პ-დგმ-ს სტრუქტურის გარკვევა განხორციელდა ბირთვული მაგნიტური რეზონანსის (ბმრ) სხვადასხვა ტექნიკის მონაცემების გამოყენებით, მათ შორის თხევად ფაზაში 1H, 13C ბმრ, ორგანზომილებიანი (2D) ჰომონუკლეარული gCOSY, ორგანზომილებიანი (2D) ჰეტერონუკლეარული 1H/13C gHSQCED, ორგანზომილებიანი (2D) DOSY (დიფუზიური ორგანიზებული სპექტროსკოპია) და მყარ ფაზაში 13C ბმრ სპექტრები. პოლიოქსიეთილენის (პოლიეთილენგლიკოლის) (პეგ) ჯაჭვი წარმოადგენს ამ ბიოპოლიმერის ხერხემალს, ხოლო 3-(3,4-დიჰიდროქსიფენილ) გლიცერინის მჟავას ნაშთი ფუნქციონირებს როგორც განმეორებადი ერთეული. 3,4-დიჰიდროქსიფენილის (კატექოლი) და კარბოქსილის ჯგუფები არიან რეგულარული ჩამნაცვლებლები პეგ-ის ჯაჭვში. ამრიგად, პ-დგმ წარმოადგენს ბუნებრივი პოლიეთერების უნიკალურ კლასს. პ-დგმ-ის თითოეული განმეორებადი სამფუნქციური სტრუქტურული ერთეული შეიცავს ორ ფენოლის ჰიდროქსილის ჯგუფს ორთო პოზიციაში და ერთ კარბოქსილის ჯგუფს. პ-დგმ-ს მრავალფუნქციურობა, სავარაუდოდ, ხსნის მის ბიოლოგიური აქტიურობის ფართო სპექტრს, მათ შორის ანტიკომპლემენტურ, ანტიოქსიდანტურ, ანთების საწინააღმდეგო, დამწვრობისა და ჭრილობების შეხორცების, ანტიმიკრობულ და კიბოს საწინააღმდეგო თვისებებს.