The Spectrum of Pharmacological Actions of Syringetin and Its Natural Derivatives—A Summary Review

Mono- and poly-O-methylated flavonols and their glycoside derivatives belong to the group of natural plant polyphenols with a wide spectrum of pharmacological activities. These compounds are known for their antioxidant, antimutagenic, hepatoprotective, antidiabetic, and antilipogenic properties. Additionally, they inhibit carcinogenesis and cancer development. Having in mind the multidirectional biological activity of methylated flavonols, we would like to support further study on their health-promoting activities; in this review we summarized the most recent reports on syringetin and some of its structural analogues: laricitrin, ayanin, and isorhamnetin. Natural sources and biological potential of these substances were described based on the latest research papers.


Introduction
Polyphenolic compounds, which are commonly found in fruits, wine, and herbs, constitute a group of over 8000 substances. Among them we can find flavones and their hydroxylated analogues flavonols [1,2]. The health-promoting properties of flavonoids arise from their cyclic structures with conjugated double bonds, and from the presence of various functional groups in the aromatic rings. Methylation of free hydroxyl groups in flavonoids greatly enhances the metabolic stability and increases membrane transport, facilitating absorption and improving oral bioavailability of flavonoids [3]. Methylated and methoxylated flavones and chalcones showed potent anticancer [4][5][6], cytoprotective [7], and neuroinflammatory effects [8]. Some of them are already in the clinical trial phase [9]. Furthermore, there are studies suggesting that methylated forms of flavonoids have higher metabolic stability, bioavailability, and biological activity than non-methylated ones.
Looking at the chemical structure, syringetin is an O-methylated flavonol of plant origin. It is found, among others, in red wine. In the literature there are many reports on the health-promoting properties of syringetin. The compound exhibits a wide range of biological activities that include strong antioxidant, anticancer, antidiabetic, and antiinflammatory properties and thus it may be beneficial to protect against neurodegenerative disorders such as Parkinson's and Alzheimer's diseases. The objective of this paper is to review the data on the possible role of syringetin in the treatment and prevention of human diseases. We summarized the current knowledge of the structure, sources, and bioactivities of syringetin and its derivatives.

Syringetin
Generally, methylated flavonoids demonstrate better physiological properties than non-methylated ones. Methylation of free phenolic hydroxyls provides the derivatives that are not susceptible to glucuronic acid or sulfate conjugation, which results in higher metabolic stability of the compounds. Methylation also affords much better transport of ayanin Figure 1. Chemical structures of syringetin, laricitrin, isorhamnetin, and ayanin.
Syringetin is a dimethyl myricetin derivative, which contains free hydroxyl groups at the C-2′ and C-4′ positions in ring B. It is found in red grapes, in Lysimachia congestiflora [11], and in Vaccinium ashei (blueberry) [12]. It is also one of the phenolic compounds found in wine [13,14] and in the edible parts of jambolan fruits (Syzygium cumini (L.) skeels) [15].
The first mention of syringetin (MW 346, C17H14O8) isolated from plants Soymida febrifuga dates back to 1972 [16]. It was first isolated as yellow needles with a m.p. of 288 °C. The extraction of the root heartwood with chloroform yielded also dihydrosyringetin (m.p. 228 °C), which does not contain a double bond between C-2 and C-3.
The presence of syringetin in the form of glucuronides was observed among others in the blueberry fruits [17]. In plants flavonoids occur most often attached to sugars. Glycosylation of flavonoids is carried out by the plant glycosyltransferase, the enzyme catalyzing the attachment of a sugar molecule into an aglycone, resulting in glycoside synthesis. Glycosides of syringetin were observed in such plants as Caragana jubata (Pall.) [18,19] and Artiplex halimus L. [20]. Syringetin-3-O-glucoside was also found in the skin of wine grapes such as Cabernet Sauvignon, Merlot, Syrah, and Marselan [21], needles of Larix Syringetin is a dimethyl myricetin derivative, which contains free hydroxyl groups at the C-2 and C-4 positions in ring B. It is found in red grapes, in Lysimachia congestiflora [11], and in Vaccinium ashei (blueberry) [12]. It is also one of the phenolic compounds found in wine [13,14] and in the edible parts of jambolan fruits (Syzygium cumini (L.) skeels) [15].
The first mention of syringetin (MW 346, C 17 H 14 O 8 ) isolated from plants Soymida febrifuga dates back to 1972 [16]. It was first isolated as yellow needles with a m.p. of 288 • C. The extraction of the root heartwood with chloroform yielded also dihydrosyringetin (m.p. 228 • C), which does not contain a double bond between C-2 and C-3.
The presence of syringetin in the form of glucuronides was observed among others in the blueberry fruits [17]. In plants flavonoids occur most often attached to sugars. Glycosylation of flavonoids is carried out by the plant glycosyltransferase, the enzyme catalyzing the attachment of a sugar molecule into an aglycone, resulting in glycoside synthesis. Glycosides of syringetin were observed in such plants as Caragana jubata (Pall.) [18,19] and Artiplex halimus L. [20]. Syringetin-3-O-glucoside was also found in the skin of wine grapes such as Cabernet Sauvignon, Merlot, Syrah, and Marselan [21], needles of Larix decidua [22], blueberries [23], and fruits of Embelia ribes [24]. The presence of syringetin-3-O-glucoside and syringetin-3-O-acetylglucoside was confirmed for the first time in Cabernet Sauvignon grape and wines by Wang et al. in 2003 [25].
Syringetin was identified also in various types of Tunisian figs belonging to the Smyrna-type Ficus carica varieties known as Kholi, Tchich Asal, Himri, and Bidhi [46].
Glycoside derivatives of syringetin may be also obtained as a result of biotransformation of myricetin in the culture of P. americana cells. According to the study by Fujitaka et al. [47] myricetin was glucosylated, then methylation of the glucosylated product at 3and 5 positions occurred in a 2-day biotransformation process to provide syringetin-3-O-β-D-glucoside.

Pharmacological Effects of Syringetin
O-Methylated flavonols attract a great interest within the research community as active substances with anticancer potential [48,49]. Anticancerogenic properties of these compounds arise from their antioxidant properties, their ability to change activities of some enzymes and to modulate certain biosynthetic pathways [50][51][52]. According to the research by Gómez-Alonso et al. [53], syringetin alone as well as a mixture of flavonols composed of quercetin, myricetin, laricitrin, and syringetin (45:35:10:10) are capable to inhibit the proliferation of colorectal epithelial adenocarcinoma cells Caco-2 (ECACC). An exposure to syringetin (50 µM) induced a dose-dependent reduction in cyclin D1 and COX-2 levels, which is up-regulated in many cancers. Syringetin is capable to inhibit the growth of cancer cells both via the induction of cell cycle arrest in the G 2 /M phase and the initiation of apoptosis. In the cells exposed to syringetin, there was a 16.7% reduction in the proportion of cells in G 0 /G 1 and a 16.1% increase in the proportion in G 2 /M.
There are some hypotheses that syringetin may be used to prevent bone metastasis in patients with lung cancer. Tsai et al. [54] reported that syringetin may suppress in vitro osteoclastogenesis mediated by osteoblasts in human lung adenocarcinoma (A549 and CL1-5) in a dose-dependent manner. Induction of differentiation by syringetin is associated with the increased activation of SMAD1/5/8 and extracellular signal-regulated kinase 1/2 (ERK1/2).
Bando et al. [55] hypothesized that syringetin can be employed for the development of novel effective radiosensitizers. The study confirmed that syringetin effectively enhanced radiosensitivity of cancer cells (H1299 and C3H/MCA clone 15) compared with normal ones (HFL-III and C3H/10T1/2), through enhancement of the caspase-3-mediated apoptosis pathway.
Among the substances of plant origin there is a search for promising compounds that would help in prevention and treatment of metabolic diseases that negatively affect digestion and absorption of nutrients, e.g., carbohydrates. The effect of flavonoids on the cellular metabolism of lipids is a good reason to use them as dietary therapeutic agents in cancer treatment [56]. Many plant flavonoids demonstrate antidiabetic properties [57]. The most promising substances are subjected to clinical trials [58]. According to Wu et al. [59] syringetin belongs to alpha-glucosidase (AGH) inhibitors; therefore, in the future it may be used in the treatment of diabetes so as to inhibit absorption of carbohydrates and reduce postprandial glycemia. The established IC 50 value for syringetin (36.8 µM) was lower than for the antidiabetic drug acarbose, which was used as a positive control. Moreover, syringetin and isorhamnetin bound to hexose (syringetin 3-O-hexoside and isorhamnetin 3-O-hexoside) are natural ingredients of Cercis chinensis Bunge fruits, contributing to their high α-glucosidase inhibitory activity (IC 50 = 11.94 ± 1.23 µg/mL), higher than for acarbose [60]. There is a hypothesis that syringetin 3-O-hexoside, being an ingredient of Lilium species, is responsible for high antioxidant activity of methanolic extracts of their bulbs [61].
According to the literature, methylated flavonoids may modulate aging-or stressrelated pathways in the nematode. Büchter et al. [64] documented that syringetin enhanced the life span of C. elegans by 35.7%. They also analyzed the effect of methylated derivatives, including syringetin, on the accumulation of lipofuscin, which is the autofluorescent age pigment, consisting of molecular aggregates of highly oxidized proteins and lipids. Syringetin caused even stronger reduction (46.1%) in the lipofuscin fluorescence than myricetin (33.1%).
The studies by Kakorin's team [18] showed that the lyophilized aqueous extract of Caragana jubata, containing mono-and diglycosides of O-methylated flavonols (syringetin and syringetin-3-rhamnoside) had moderate antimicrobial activity against pathogenic microorganisms, such as Gram-positive (Staphylococcus aureus 209-P ATCC 6538) and Gramnegative bacteria (Escherichia coli ATCC 25922, Proteus vulgaris ATCC 6896, Pseudomonas aeruginosa ATCC 9027), yeasts (Candida albicans ATCC 10231), and mycelial fungi (Microsporum canis 352) at the concentration from 4.000 µg/ml to 4.500 µg/mL. Earlier studies by the same research team [19] showed that the content of phenolic compounds was stable during the long-term storage of the dried raw material.
Furthermore, El-Asar et al. [20] isolated two O-methylated flavonols from the aerial parts of Atriplex halimus L. They were identified as syringetin 3-O-β-D-rutinoside and syringetin 3-O-β-D-glucopyranoside, which may act as immunomodulators. The results of the anti-inflammatory studies showed that both compounds markedly increased the levels of cytokines and pro-inflammatory mediators. What is more, the glucopyranoside derivatives possessed also good antimicrobial activity with inhibition zone diameter from 10 mm for Ac. baumanii to 19 mm for S. pyogenes. The zone diameter of 17 mm was observed for S. aureus, and E. faecalis. 3-O-β-D-rutinoside was active against S. aureus, S. pyogenes, E. faecalis, and C. albicans, with the inhibition zone from 10 mm to 15 mm. Syringetin 3-O-β-D-glucopyranoside was identified also in fresh leaves of Eucalyptus maideni [65].
Flavonoids, including methoxy derivatives of 3-hydroxyflavonol have also antiviral properties. The anti-respiratory syncytial virus (RSV) activity of syringetin isolated from Hovenia dulcis was reported for the first time by Xu et al. [26]. The study was performed Nutrients 2022, 14, 5157 5 of 12 using the RSV A2 strains, which were cultivated in HEp-2 cells. The substances were tested using the cytopathic effect reduction assay (CPE) in comparison with the positive control ribavirin (IC 50 = 51.23 ± 10.25 µM).
The best results were noted for two structural analogues of syringetin: kaempferol (IC 50 = 64.69 ± 5.24 µM) and myricetin (IC 50 = 130.21 ± 8.68 µM). The substituents, such as hydrogen, hydroxyl, and methoxy groups in the B ring of the compounds affected their anti-RSV activities. Syringetin was inactive in this assay. The 50% cytotoxic concentration (CC 50 ) calculated as the concentration of a tested compound required to reduce cell viability by 50% (compared with untreated cells, incubated with the medium only) was estimated through the OD values and for syringetin it was over 100 µM.
Grewal et al. [66] subjected syringetin to molecular docking studies in order to investigate its binding interactions with eight anti-Alzheimer's drug targets. In the in silico study syringetin showed strong binding interactions and complementary orientation pattern in the binding site of all the targets involved in pathogenesis of AD. The evaluated parameters included molecular weight (MW), distribution coefficient (log D), partition coefficient (log P), water solubility (log Sw), topological polar surface area (tPSA), hydrogen bond donors (HBDs), hydrogen bond acceptors (HBAs), solubility (mg/L), and number of rotatable bonds (NRBs).
In addition, another research revealed that some other flavonoid compounds, such as non-methylated syringetin analogues, are beneficial for the treatment of Alzheimer's disease. As it was described by Ramezani et al. [67], myricetin protects hippocampal CA3 pyramidal neurons and improves learning and memory impairments in rats with Alzheimer's disease.
It has been reported that certain flavonoids are activators of the rat vitamin D receptor (rVDR), which except for acting as the major regulator of vitamin D and calcium homeostasis, controls also the expression of genes involved in the transport, bioactivation, and detoxification of endogenous substances and xenobiotics, including drugs. As it was demonstrated by Lau et al. [68], syringetin is not an agonist of rat vitamin D receptor (rVDR), mouse VDR (mVDR), or human VDR (hVDR), as judged by cell-based and in silico evidence. Syringetin does not activate the rVDR, mVDR, or hVDR in HEK-293 and HepG2 cells transfected with the corresponding receptor expression plasmid and either the secreted phosphoprotein 1 (Spp1) or cytochrome P450 24A1 (CYP24A1) reporter plasmid compared with the respective empty vector control group transfected with one or the other reporter plasmid and treated with tested flavonol.
Due to the correlation between the endocrine and the osteoarticular systems, interactions of methylated flavonols and their impact on bone formation were investigated. Hsu et al. [69] demonstrated that syringetin significantly induces differentiation in MC3T3-E1 mouse calvaria osteoblasts and in human fetal osteoblastic 1.19 cell line. The results indicate that syringetin stimulates osteoblast differentiation at various stages, from maturation to terminally differentiated osteoblasts. Induction of differentiation by syringetin is associated with increased bone morphogenetic protein-2 (BMP-2) production and increased activation of SMAD1/5/8 and extracellular signal-regulated kinase 1/2 (ERK1/2).
The research on methylated myricetin derivatives (laricitrin, syringetin, and myricetin trimethyl ether) revealed that these compounds strongly enhance the life span of C. elegans and increase the stress resistance of the nematode. The pro-longevity effect was dependent on DAF-16.
Büchter et al. [64] used methylated derivatives of myricetin (laricitrin and syringetin) to examine whether the free OH groups in ring B are necessary for the life span extending effect. Such as in the case of myricetin, all the methylated derivatives extended the life span, reduced oxidative stress (DCF), and reduced the accumulation of lipofuscin. In contrast to myricetin, the methylated derivatives strongly increased the resistance against thermal stress. What is more, treatment with the methylated compounds induced a much stronger nuclear localization of the DAF-16 transcription factor (FoxO homologue).

Laricitrin
One of the structural analogues of syringetin is the monomethylated myricetin named laricitrin. The compound also possesses an interesting biological activity. It is a dietary flavonoid derivative, which can be found for example in grapes, red wine, Rhododendron luteum [70], Carpobrotus edulis [71], leaves of Psidum litorale [72], Ginkgo biloba [73], and needles of Larix sibirica Ledb. The content of laricitrin and other health-promoting compounds in vine can be increased by means of malolactic fermentation (MLF) with the help of recombinant Pediococcus acidilactici BD16 (fsc+/ech+) strains [74].
Chang et al. [75] documented that laricitrin inhibited progression of lung cancer cells induced by benzo(a)pyrene (BaP) in the lung cancer tumor microenvironment. In the study, human lung adenocarcinoma cell lines (H1395, H1975, H2087, and HCC2935) were used. Laricitrin can be also an efficacious immunoadjuvant and has a synergistic effect when combined with chemotherapy. This compound potentiated the anticancer activity of cisplatin in mouse models [76]. Being an ingredient of aqueous extracts of Asparagus officinalis L., laricitrin-O-glucoside contributes to its high antitumor activity against breast cancer cells (NIH/3T3, MDA-MB-231, and MCF-7). The most sensitive to laricitrin proved MCF-7 cell line (expressing HR + /HER2 − phenotype), where a significant reduction in proliferation was observed, along with the cell cycle arrest and low levels of apoptosis [77]. Anticancer activity of laricitrin may be associated among others with inhibition of the breast cancer resistance protein (BCRP/ABCG2) [78].

Isorhamnetin
According to Mattivi et al. [79], isorhamnetin is one of the main flavonols contained in grapes. In red grapes the reported content of this compound was 3.89%, whereas in white ones the reported content was 1.74%. Interestingly, laricitrin and syringetin were missing in all white varieties, indicating that the enzyme flavonoid 3',5'-hydroxylase is not expressed in white grape varieties. In red grapes the content of 3.22% and 5.65% was determined for syringetin and laricitrin, respectively. Isorhamnetin in the form of rutoside (isorhamnetin-3-O-rutinoside) is found in beverages with yerba mate (Ilex paraguariensis). It has been proved that being an ingredient of drinks, yerba mate inhibits fat absorption in vivo by inhibition of pancreatic lipase activity. Therefore, it may be used to stabilize body weight and the overall lipid metabolism [80]. A new derivative of isorhamnetin, namely isorhamnetin-3-O-(2"-O-galloyl)-β-D-glucopyranoside, was identified in the petals of R. rugosa, which are used in various cosmetic products [81]; whereas isorhamnetin glycosides found in extracts of various Spiraea species (S. media, S. hypericifolia, S. salicifolia L.) in high probability are responsible for the antiviral activity of these extracts against viruses of influenza A and B [82]. Glycoside derivatives of isorhamnetin (isorhamnetin-3-O-β-D-glucoside) were prepared by biotransformation of quercetin in the culture of Phytolacca americana [47]. In turn, the stepwise bioconversion of rutin by Eurotium amstelodami BSX001 leads to aglycones such as quercetin, kaempferol, and isorhamnetin [83]. Moreover, isorhamnetin is one of the main ingredients of a common food seasoning Zanthoxylum schinifolium, known for its special aroma [84], and in fermented vegetables such as pickled chayote [85]. An in vitro study showed that isorhamnetin may be used as a potential therapeutic compound against COVID-19 in the future [86].
The most recent literature reports indicate that being an ingredient of a novel drug Bushao Tiaozhi Capsule (BSTZC), isorhamnetin may help to regulate the lipids level in the blood, and therefore may be used in the treatment of hyperlipidemia [87]. In addition, an in vivo study on rats showed that isorhamnetin delivered as one of the ingredients of Corinthian currant (dried fruits) easily comes across the blood-brain barrier [88].
The low-density lipoprotein (LDL)-antioxidant activity of ayanin isolated from leaves and stems of Plectranthus hadiensis var. tomentosus was reported with the IC 50 = 53.7 µM by Ji et al. [94]. In addition, Ahn et al. [82] revealed that ayanin markedly down-regulated the PMA+A23187-induced synthesis of interleukin-6 (IL-6) in HMC-1 cells without cytotoxicity, with the IC 50 value of 17.8 µM (montelukast served as a positive control with the IC 50 = 8.7 µM).
Moreover, ayanin was found to be a highly potent inhibitor of breast cancer resistance protein (BCRP) encoded by the ABCG2 gene, showing only slightly lower activity than Ko143, the most potent ABCG2 inhibitor known so far [96]. The authors hypothesized that consumption of flavonoids with high inhibitory activity can change pharmacokinetics and levels of drugs that are BCRP substrates. The exchange of the 3-methoxy group for a hydroxyl one, acting also as a hydrogen bond donor, resulted in a decrease in the activity, underlining the potential role of the hydrogen bond acceptor 3-OCH 3 for the interaction with BCRP.
To sum, mono-and poly-O-methylated flavonols and their glycoside derivatives belonging to the group of natural plant polyphenols may be used to treat various diseases as natural antioxidants, anti-inflammatory, or anticancer substances. Unfortunately, biological activity of many glycosylated methylflavonols, including syringetin derivatives, have not been studied so far. Similarly, the metabolism of syringetin in microorganisms is not wellknown. Molecular and biochemical mechanisms of action of syringetin and its derivatives also require further investigation, as well as their effect on the enzymes that are responsible for maintaining homeostasis in the human body. Such studies may lead to promising preclinical and clinical research results, and may contribute to development of innovative and safe prodrugs based on the structure of syringetin.
The preclinical data collected in this article unequivocally indicate that syringetin has health-promoting properties. The mechanisms of its pharmacological actions are related to inhibition of cancer cell proliferation, modulatory effects on the key enzymes responsible for development of metabolic diseases, and the enzymes responsible for maintaining homeostasis in the human body and interactions with cell receptors. Syringetin may have beneficial effect on alleviation of side effects associated with the oncological treatment, chemo-and radiotherapy. According to the current state of knowledge, no negative effects of syringetin have been noticed. Nevertheless, despite many studies that have been carried out so far, further in vitro and in vivo analyses are needed, and most of all, syringetin should be subjected to clinical trials so as to assess its direct effects on human organism. These allow the safe use of syringetin in medicine.

Conclusions
Methoxylated flavonoids are widely found in many plants, including fruits, vegetables, or seeds, where they are usually bound to sugars. Depending on the chemical structure of the substrates, the nature and positions of substituents in the aromatic rings, the number and nature of glycosidic bonds, and finally the presence of both free and methylated hydroxyl groups, they have different pharmacological effects. They may be used to treat various diseases as natural antioxidants, anti-inflammatory, or anticancer substances. Unfortunately, biological activity of many glycosylated methylflavonols, including syringetin derivatives, have not been studied so far. Similarly, the metabolism of syringetin in microorganisms is not well-known. Molecular and biochemical mechanisms of action of syringetin and its derivatives also require further investigation, as well as their effect on the enzymes that are responsible for maintaining homeostasis in the human body. Such studies may lead to promising preclinical and clinical research results, and may contribute to development of innovative and safe prodrugs based on the structure of syringetin.