Sanguiins—Promising Molecules with Broad Biological Potential

Compounds of natural origin, an infinite treasure of bioactive chemical entities, persist as an inexhaustible resource for discovering new medicines. In this review, we summarize the naturally occurring ellagitannins, sanguiins, which are bioactive constituents of various traditional medicinal plants, especially from the Rosaceae family. In-depth studies of sanguiin H-6 as an antimicrobial, antiviral, anticancer, anti-inflammatory, and osteoclastogenesis inhibitory agent have led to potent drug candidates. In addition, recently, virtual screening studies have suggested that sanguiin H-6 might increase resistance toward SARS-CoV-2 in the early stages of infection. Further experimental investigations on ADMET (absorption, distribution, metabolism, excretion, and toxicity) supplemented with molecular docking and molecular dynamics simulation are still needed to fully understand sanguiins’ mechanism of action. In sum, sanguiins appear to be promising compounds for additional studies, especially for their application in therapies for a multitude of common and debilitating ailments.


Introduction
Most of the discovered drugs are either drugs of natural origin or synthetic derivatives of natural compounds. Thus, a multidisciplinary approach to drug discovery and molecular diversity from natural product sources needs to be combined to provide the best solution to the problems with drug discovery and development [1,2]. Plants are known to be a rich source of pharmacologically active secondary metabolites divided into structural chemical classes [3,4]. One of the pharmacologically valuable classes of phytoconstituents are ellagitannins (ETs), and belonging to them, sanguiins. ETs, water-soluble phenolics, are esters of hexahydroxydiphenic acid and a polyol, usually β-D-glucose or quinic acid [5][6][7]. ET compounds demonstrate an enormous structural variability connected with various possibilities for the linkage of hexahydroxydiphenic residues with the glucose moiety and particularly by their easy susceptibility to creating dimeric and oligomeric derivatives [8]. The polyphenol-protein system and its interactions may underlie the medicinal properties exhibited by members of the ETs family. Fruits and nuts are rich sources of ellagitannins and are important in the human diet due to their properties as micronutrients [9,10]. Due to the limited bioavailability of ellagitannins, as orally administered and the metabolic chemical changes as a result of their transit through the gastrointestinal tract, comprising of hydrolysis and gut microbiota metabolism, the activity of the produced metabolites also needs to be taken into consideration [3].

Natural Occurrence of Sanguiins
Among various phenolic compounds isolated from the Rosaceae family, tannins and related compounds seem to have a leading position. It is known that plants previously used in folk medicine represent a suitable beginning to discover new potent drugs to treat various human disorders [15]. Sanguiins (Figure 1), naturally occurring ET, have been isolated chiefly from Rubus species and are used as a traditional drug to cure, e.g., diarrhea, menstrual pain, menopause disorders, liver diseases, aphtha, gingivitis, as well as fever, angina, enteritis, hepatitis, concretion, eczema, rheumatism, enterocolitis, bronchitis, prostate disorders, pain, cold, cough, and fever (Table 1) [16,17]. Moreover, SH6 seems to be the most widespread within plants of the Rubus and is present in 22 species of this genus. Furthermore, the largest number of isolated and identified types of sanguiins, including SH2, SH4, SH5, SH6, and SH11, are found in Rubus coreanus [18]. Besides the Rubus genus, sanguiins and their isomers are found and reported in Alchemilla vulgaris, Alchemilla mollis [19], Duchesnea indica [20], Euphorbia fischeriana [21], Fragaria vesca, Fragaria ananassa [22], Punica granatum [23], Terminalia calamansanai [24], as well as in Sanguisorba officinalis [25], and Sanguisorba tenuijolia var. alba [18]. isolated chiefly from Rubus species and are used as a traditional drug to cure, e.g., diarrhea, menstrual pain, menopause disorders, liver diseases, aphtha, gingivitis, as well as fever, angina, enteritis, hepatitis, concretion, eczema, rheumatism, enterocolitis, bronchitis, prostate disorders, pain, cold, cough, and fever (Table 1) [16,17]. Moreover, SH6 seems to be the most widespread within plants of the Rubus and is present in 22 species of this genus. Furthermore, the largest number of isolated and identified types of sanguiins, including SH2, SH4, SH5, SH6, and SH11, are found in Rubus coreanus [18]. Besides the Rubus genus, sanguiins and their isomers are found and reported in Alchemilla vulgaris, Alchemilla mollis [19], Duchesnea indica [20], Euphorbia fischeriana [21], Fragaria vesca, Fragaria ananassa [22], Punica granatum [23], Terminalia calamansanai [24], as well as in Sanguisorba officinalis [25], and Sanguisorba tenuijolia var. alba [18]. Among all sanguiins detected in plant material, only part of them was quantitatively analyzed. The place of harvest displays a relevant role in the amount of isolated sanguiins. For example, in Rubus fruticosus fruits, the range of detected SH6 is 135.04-547.48 mg/100 g of d.w. (dry weight) [26] and in Rubus idaeus shoots, 170.9-633.1 mg/100 g of d.w of the extract [27]. Following that, sanguiins content depends on fruits' ripeness, harvest time, climate, geographic location, and mineral nutrition [10,28]. It is reported that in Rubus and Fragaria species, ellagitannins content represents a range of 50% to 80% of all phenolic compounds [10,29]. In this review, the list of plants that produce sanguiins and their reported traditional uses are tabulated in Table 1. Among all sanguiins detected in plant material, only part of them was quantitatively analyzed. The place of harvest displays a relevant role in the amount of isolated sanguiins. For example, in Rubus fruticosus fruits, the range of detected SH6 is 135.04-547.48 mg/100 g of d.w. (dry weight) [26] and in Rubus idaeus shoots, 170.9-633.1 mg/100 g of d.w of the extract [27]. Following that, sanguiins content depends on fruits' ripeness, harvest time, climate, geographic location, and mineral nutrition [10,28]. It is reported that in Rubus and Fragaria species, ellagitannins content represents a range of 50% to 80% of all phenolic compounds [10,29]. In this review, the list of plants that produce sanguiins and their reported traditional uses are tabulated in Table 1.

Chromatographic Techniques for the Analysis of Sanguiins
Chromatography displays a crucial role in the analysis of chemical compound mixtures. As a method for the separation and analysis of extracts and fractions from plants, it provides the possibility of qualitative and quantitative determination of the test substance with high resolution [67]. Chromatographic techniques and analysis conditions for detection, quantitative determination, and isolation of sanguiins and their isomers are given in Table 2.

Biological Potential of Sanguiins
Sanguiins, as one of the subgroups of polyphenolic ellagitannins, exhibit various pharmacological activities due to having different chemical structures. They possess a broad spectrum of pharmacological features such as anticancer, anti-inflammatory, antioxidant, osteoprotective, estrogenic, antibacterial, antifungal, and antiviral (including SARS-CoV-2), as shown in Table 3. Various in vivo and in vitro investigations on sanguiins, especially on sanguiin H-6, have elucidated their medicinal characteristics and mechanisms of action [68,69].    S. aureus inhibition: reduction in the growth from 10 9 CFU/mL to 10 3 CFU/mL 2.

Antioxidant and Anti-Inflammatory Activities
One of the best-shown properties of polyphenols, and following that, sanguiins, is the potential antioxidant effect. Most references mention sanguiin H-6 as the primary compound having antioxidant activity, e.g., its influences on stress and oxidative damage were investigated. The production of peroxynitrite (ONOO-) was induced by the administration of lipopolysaccharide (LPS), followed by the induction of ischemia and reperfusion [88]. It was revealed that receiving SH6 before induction of oxidative damage could reduce the adverse effects associated with the release of ONOO-and enhance the improvement of injured kidney function [72]. Another chemical compound belonging to the sanguiins group that exhibits antioxidant activity is SH11. An examination of the protective effect of SH11 isolated from Sanguisorbae radix and its mechanism against glutamate-induced death in HT22 murine hippocampal cells exposed a significant reduction in glutamine-induced reactive oxygen radicals' accumulation and calcium ion influx [74]. Furthermore, ellagitannins from the berries of the Rubus family, including dimeric SH6 and SH10, function both as radical scavengers (in a DPPH test) and as antioxidants toward lipid oxidation in food emulsions (studied in bulk and emulsified methyl linoleate, in human low-density lipoprotein in vitro) [75]. The impact of sanguiins on the inflammation process was investigated by measuring their effect on rat neutrophils' chemotaxis. SH11 and SH6 effectively inhibited the cytokine-induced neutrophil chemoattractant migration process by 10.7% and 33%, respectively, in comparison with the control. Additionally, the study showed no toxic effect of sanguiin on neutrophils [70]. Furthermore, at a concentration of 2.5 µM, SH6 completely inhibited the release of IL-8 induced by tumor necrosis factor α and interleukin-1β and inhibited TNFα stimulated NF-κB transcription [71]. SH6 caused a concentration-dependent reduction in nitrite production, regression in induced NO synthase (iNOS) activity, and an increase in cell viability. Moreover, SH6 showed an apparent scavenging effect for NO generated from sodium nitroprusside (NO donor) [76].

Osteoclastogenesis Inhibitory Activity
In a subsequent in vitro study, the action of Rubus parvifolius L. and its main component, SH6, was tested as the inhibitor of osteoclastogenesis and bone resorption. Sanguiin influence was based on the reduction in osteoclast differentiation and bone resorption, a decrease in the production of reactive oxygen species, as well as the inhibition of the nuclear translocation of the nuclear factor of activated T cells cytoplasmic-1 (NFATc1), c-Fos, and nuclear factor-κB. Additionally, sanguiin reduced the levels of NFATc1, cathepsin K, c-Src, and inhibited in vivo TNF-α-mediated osteoclastogenesis [47].

Antibacterial Activity
The growing resistance of bacteria to currently used antibiotics is a growing problem in current medicine [89]. Increasingly emerging research on sanguine antibacterial properties gives hope for the discovery of antibacterial agents with the lack of unpleasant side effects. Examination of the antibacterial activity of fruits of selected Rubus species and compounds (SH6 and ellagic acid) against selected Gram-negative and Gram-positive bacteria allowed assessment of their usefulness in the fight against microorganisms. The results showed that SH6 was active against Streptococcus A (MIC = 0.5 mg/mL), Streptococcus pneumoniae (MIC = 0.5 mg/mL), Corynebacterium diphtheriae (MIC = 0.03 mg/mL), Bacillus subtilis (MIC = 0.5 mg/mL), Clostridium sporogenes (MIC = 0.06 mg/mL), Staphylococcus aureus (MIC = 0.25 mg/mL), Staphylococcus epidermidis (MIC = 0.125 mg/mL), and Moraxella catarrhalis (MIC = 0.5 mg/mL) [27].

Antifungal Activity
Moreover, Rubus ulmifolius fruit extract was tested as an antifungal agent. It was proved that the extract containing SH6 exhibited fungistatic activity against Candida albicans. The minimum inhibitory concentration was 5 mg/mL. Unfortunately, the extract did not show any fungicidal activity, achieving a result of >20 mg/mL [78].

Antiviral Activity (Including SARS-CoV-2)
Viruses, as pathogenic microorganisms, show significant genetic variability and the ability to mutate. Often, they do not show signs of infection at first. Currently, an increasing number of drug-resistant strains, as well as the toxicity of previously known drugs, force researchers to develop new antiviral substances [90]. In recent months, the entire world has been severely affected by the SARS-CoV-2 pandemic, which has led scientists to focus their attention on potential candidates against its eradication. More and more recent research conducted worldwide shows that sanguiins may be a potential candidate in the fight against viral diseases, including COVID-19 [91,92]. One of the studies predicted that SH6 is a compound that binds very well to the S1 and S2 subunits of the SARS-CoV-2 virus spine, which is responsible for entering the host cells and causing infection. SH6 showed the best binding energy among all tested compounds in the molecular docking assay. Additionally, SH2, also mentioned in the study, showed a lower result than the one mentioned above. Moreover, sanguiin has been proposed to act not only against the spike subunits of the SARS-CoV-2 virus [93]. Another molecular docking examination of polyphenolic compounds against the SARS-CoV-2 virus M pro protease revealed that SH6 had the best result of all tested compounds in the in silico model [80]. Moreover, the study performed by S. Luo et al. concerned the verification of bacterial neuraminidase inhibitory properties by nine compounds isolated from mock strawberry (Duchesnea indica Andr.). SH4 exhibited significant inhibitory activity in an in vitro model, which offers potential for its use as a new antiviral substance [20].

Anticancer Activity
Additionally studied features of sanguiins are their potential anticancer activity. Several investigations on SH6 have explained its anticancer effect due to its promising competency in inhibiting DNA topoisomerases I and II. Moreover, the compound acted as a blocker to HeLa cells. It inhibited their growth at an effective dose of 12 µM and also had a dose-dependent effect on intracellular topoisomerase activity. SH6 also exhibited significant antiangiogenic potential [82]. A study by Lee S. et al. on HT1080 human fibrosarcoma cells showed that this compound blocked KDR/Flk-1-Fc binding to VEGF165 in a dose-dependent manner. Moreover, the compound obstructed the VEGF-induced proliferation of HUVEC cells (IC 50 ca. = 7.4 µg/mL) but was not active against HT1080 human fibrosarcoma cells [83].
The potential antitumor properties of sanguiins were also tested on PRMI-7951 melanoma cells. A moderate selective cytotoxicity was shown by SH2, SH6, and SH11 with ED 50 results of 0.44, 0.5, and 5.0 µg/mL, respectively [68]. Furthermore, anticancer activity was tested with SH4 isolated from Terminalia calamansanai leaves against large tumor cells lines, including human promyelocytic leukemia HL-60 cells. The compound induced a decrease in human poly (ADP-ribose) polymerase [79] (PARP) associated with the cleavage of procaspase-3 and exhibited strong activation of proapoptotic caspase-3 in HL-60 cells. It is worth mentioning that SH4 does not affect healthy cells, suggesting this compound is selective against cancer cells [24]. In another examination, SH6 was responsible for modulating the Smad 2/3 signaling pathway by TGF-β1, increasing the expression of the epithelial marker E-cadherin, repressing the expression of Snail and the mesenchymal marker N-cadherin during TGF-β1-induced EMT (epithelial-mesenchymal transition), and regulating the expression of EMT-dependent genes induced by TGF-β1. In summary, SH6 inhibits the migration and invasion of A549 lung cancer in vitro by inhibiting TGF-β1 induction of EMT [84].
Moreover, SH6 showed a large number of antiproliferative, antimigration, and cytotoxic effects against human breast carcinoma cells. A study performed by Berdowska et al. proved that the tested compound exhibited an inhibitory effect on adriamycin-resistant cells (MCF-7/Adr) [85]. It also showed antimetastatic properties in MDA-MB-231 cells by reducing the expression of vascular endothelial growth factor (VEGF), phosphorylated Akt, and kinase 1/2 (ERK1/2) regulated by extracellular signals [86]. In addition, SH6 increased the ratio of Bax to Bcl-2 in both MCF-7 and MDA-MB-231 cells [79].
SH6 was also studied for its activity against A2780 human ovarian carcinoma cells. The tested compound induced an antiproliferative effect and a morphological change similar to apoptotic cell death but did not arrest the cancer cell cycle. Moreover, SH6 showed an early apoptotic effect, caspase activation, PARP cleavage, activation of mitogen-activated protein kinases (MAPKs), especially p38, and an increase in truncated p15/BID [87].

Estrogenic Activity
SH6 has also been tested for estrogenic activity against MCF-7 human breast cancer cells. The E-screen examination and the molecular docking analysis showed that the SH6 from Rubus coreanus exhibited the best binding energy of −250,149 kcal/mol. Additionally, at 100 µg/mL, R. coreanus extract significantly stimulated cell proliferation (574.57% ± 8.56%). The study results indicated that SH6 contributed to the estrogenic activity of R. coreanus by activating the ERα coactivator binding site [81].

Neuroprotective Activity
Rubus L. subgenus R. watson, R. brigantinus, and R. vagabundus extracts containing SH2, SH6, and SH10 were tested for their potential neuroprotective properties against SK-N-MC neuroblastoma cells. All digested extracts after 2 and 24 h of preincubation reduced basal ROS production. Rubus brigantinus and R. vagabundus extracts increased the mitochondrial transmembrane potential and the integrity of the cell membrane. Moreover, the extracts increased GSH levels while not changing the GSH/GSSG ratio. It is worth mentioning that there is insufficient evidence for the interaction of brain endothelial cells with polyphenol metabolites, which makes it difficult to determine the level of the passage of the compound across the blood-brain barrier [62].

Clinical Trials
As mentioned above, the efficacy of sanguiins is mainly limited to preclinical studies. However, there has been some research on black raspberry and pomegranate food products in clinical trials. Considering the fact that these products are rich in ellagitannins, it can be concluded that the biological activity may also be connected with the occurrence of sanguiins in the juice from berries and pomegranate. Nevertheless, there is a lack of information on clinical studies that use only sanguiins in medical treatment [44,94,95].

Pharmacokinetics of Sanguiins
Sanguiins, belonging to the ellagitannin group, show similar pharmacokinetics. In vitro studies have shown that ellagitannins are stable in the gastric environment, and in the presence of gastric enzymes, they are not hydrolyzed to ellagic acid. In addition, the absorption of ellagitannins in the stomach is impracticable due to their complex chemical structure. However, free ellagic acid molecules can be absorbed in the stomach. On the other hand, the intestinal environment, together with the gastrointestinal microbiota, creates suitable conditions for their hydrolysis and decomposition into urolithins and their derivatives, which pass through the intestinal wall into the enterohepatic circulation [96]. In addition, in vivo studies have shown that the metabolism of SH6 and SH10 in the liver is partly based on conjugation with glucuronic acid and sulfuric acid, leading to the formation of compounds such as urolithin A-O-glucuronide, urolithin A-sulfate, and urolithin B-3-O-glucuronide. Moreover, urolithins were detected in the unconjugated form. Conjugation of derivatives occurs at different rates and intensities; T max of plasma urolithin glucuronides and sulfates is achieved in the vast majority of compounds 24 h after administration. Ultimately, conjugated and unconjugated compounds are excreted in the urine at varying intervals, up to 48 h after ingestion. Further in vivo clinical studies linked to full pharmacokinetic analysis are necessary to fully determine the participation of urolithins in the therapeutic effects of ellagitannin-rich plants [3,97,98].

Conclusions
The isolation and structure determination, accompanied by the measurement of the diverse pharmacological activities of each isolated sanguiin, has brought about a marked change in the concept of these compounds as active components of medicinal plants. In summary, sanguiins, especially sanguiin H-6, show evidence of promising action in various biological contexts, particularly in respect of their anticancer, antiradical, and antiviral properties. Apart from that, further studies involving drug delivery may improve the effectiveness of these compounds toward the drug target sites. Furthermore, it is worth considering performing a supplementary survey on their metabolism and toxicology patterns with molecular docking and molecular dynamics simulation to understand their mechanisms of action fully.

Conflicts of Interest:
The authors declare no conflict of interest. bone marrow-derived macrophages NFATC1T max nuclear factor of activated T cells 1time take to reach maximum concentration