Silviridoside: A New Triterpene Glycoside from Silene viridiflora with Promising Antioxidant and Enzyme Inhibitory Potential

A new triterpene glycoside, silviridoside, was isolated from the aerial parts of Silene viridiflora (Caryophyllaceae) using different chromatographic techniques. The structure of silviridoside was comprehensively elucidated as 3-O-β-D-galacturonopyranosyl-quillaic acid 28-O-β-D-glucopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→3)]-β-D-fucopyranosyl ester by one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HR-MS). Silviridoside showed promising antioxidant activity in different antioxidant assays such as 2,2-diphenyl-1-picrylhydrazyl (DPPH) (2.32 mg TE/g), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (1.24 mg TE/g), cupric-reducing antioxidant capacity (CUPRAC) (9.59 mg TE/g), ferric-reducing antioxidant power (FRAP) (5.13 mg TE/g), phosphomolybdenum (PHD) (0.28 mmol TE/g), and metal-chelating (MCA) (6.62 mg EDTA/g) assays. It exhibited a good inhibitory potential on acetylcholinesterase (AChE) (2.52 mg GALAE/g), butyrylcholinesterase (BChE) (7.16 mg GALAE/g), α-amylase (0.19 mmol ACAE/g), α-glucosidase (1.21 mmol ACAE/g), and tyrosinase (38.83 mg KAE/g). An in silico evaluation of the pharmacodynamic, pharmacokinetic, and toxicity properties of silviridoside showed that the new compound exhibited reasonable pharmacodynamic and pharmacokinetic properties without any mutagenic effect, but slight toxicity. Thus, it could be concluded that silviridoside could act as a promising lead drug for pharmaceutical and nutraceutical developments to combat oxidative stress and various disorders, but a future optimization is necessary.


Introduction
Free radicals, naturally produced within the human body, trigger many adverse effects and cause oxidative damage in proteins, lipids, and genetic material, which is counteracted by the human antioxidant system. An uncontrolled balance between the antioxidant defense and the production of free radicals results in undesirable side effects [1]. Antioxidants from natural products can restore this balance, reducing oxidative stress and its undesired health effects such as nervous disorders and hyperglycemia [2][3][4][5]. Nowadays, enzyme inhibitors are recognized as targets to treat a variety of diseases, including cancer, diabetes, hypertension, cardiac disorders, and Alzheimer's [6]. Cholinesterase inhibitors are widely used to treat neurodegenerative disorders such as Alzheimer's disease, which is characterized by an irreversible neurological pattern [7,8]. In addition, tyrosinase is crucial for the formation of melanin, which leads to the overproduction of skin pigments and the appearance of dark spots in different skin areas [9,10]. The inhibition of α-amylase and α-glucosidase is widely used to reduce postprandial glucose levels and, consequently, to control postprandial hyperglycemia in diabetic patients [11][12][13]. The discovery of novel enzyme inhibitors could open a new window for treating diseases.
Natural product-based drugs are highly recommended all over the globe due to their safer properties with a reasonably high activity compared with synthetic drugs [14]. Natural products provide a wide array of secondary metabolites to which their promising biological activity is also attributed [15]. Silene is a genus of flowering plants in the Caryophyllaceae family with about 700 species that are spread in Eurasia, America, and Africa [16]. Silene viridiflora L. natively grows in Turkey; Central, West, and South Europe; East Asia; and Crimea-Siberia [17]. Previous phytochemical studies of S. viridiflora resulted in the isolation and identification of several ecdysteroids [18], lipids [19], essential oils [20], carbohydrates, and microelements [21]. Among the isolated ecdysteroids from S. viridiflora, the principal active component is 20-hydroxyecdysone, which has shown immunomodulatory, cytoprotective, and adaptogenic potential [22,23].
Continuing the search for structurally unique and biologically active compounds, the aerial parts of S. viridiflora were investigated in this work. As a result, one new triterpene glycoside-denominated silviridoside was isolated using different chromatographic techniques, comprehensively elucidated using advanced spectroscopic techniques such as oneand two-dimensional nuclear magnetic resonance (1D and 2D NMR) and high-resolution mass spectrometry (HR-MS) and tested in different antioxidant and enzyme inhibition assays. In addition, an evaluation of its ADMET characteristics and a TOPKAT (Toxicity Prediction using Komputer-Assisted Technology) prediction were performed in silico using Discovery Studio 4.5 software (Accelrys Inc., San Diego, CA, USA) to predict its pharmacodynamic, pharmacokinetic, and toxicity properties.

Isolation and Structural Elucidation of Compound 1
A phytochemical investigation performed on the methanol extract of the aerial parts of S. viridiflora resulted in the isolation of compound 1, which was further subjected to a comprehensive structural elucidation using several chromatographic and spectroscopic methods. The molecular formula of compound 1 was established as C 54 13 C, and HSQC spectra also revealed a trisubstituted double bond with the olefinic proton at δ H 5.22 ppm and its corresponding carbon at δ C 121.2 ppm as well as a quaternary carbon at δ C 143.2 ppm. In addition, the HSQC spectrum also indicated two oxygenated methine groups at δ H /δ C 3.82/80.2 ppm and δ H /δ C 4.34/72.4 ppm. This information pointed to compound 1 featuring an olean-12-ene skeleton [24]. Another striking feature was the presence of an aldehyde function at δ H /δ C 9.35/207.0 ppm. This aldehyde function was assigned to position 23 based upon the HMBC correlations of methyl protons (CH 3 -24) at δ H 0.98 ppm and the quaternary C-5 carbon at δ C 46.7 ppm. Moreover, a long-range cross-peak from CH 3 -24 to the carbon (C-3) at δ C 80.2 ppm established oxygenation at position 3. The second oxymethine function (δ H /δ C 4.34/72.4) was assigned to position 16 due to COSY correlations with H-15 (δ H 1.64 and 1.26 ppm), which further showed long-range cross-peaks to C-27 (δ C 26.4 ppm). The stereochemistry at the C-3 and C-16 chiral centers was determined via NOESY correlations; a cross-peak from H-3 (δ H 3.82 ppm) to H-5 (δ H 1.25 ppm) led to H-3α whereas a correlation between H-16 (δ H 4.34 ppm) and CH 3 -26 (δ H 0.65 ppm) established a β-orientation of H-16. Based on the above evidence, the skeleton (aglycon part) of compound 1 was established to be the triterpene quillaic acid. This was further confirmed by the COSY, NOESY, HSQC, and HMBC spectra, along with a comparison with the literature data [24].   Tables 1 and 2.
The structure and connection of the sugar moieties were also confirmed via the observed fragmentation pattern of the molecule in a negative ESI-MS/MS mode ( Figure S2). The proposed fragmentation pathway of compound 1 is presented in Figure 2. A loss of glucose gave the fragment a peak at m/z 953 whereas, due to the fragmentation of the trisaccharide unit, a peak at m/z 661 was formed. Further decarboxylation led to a fragment with a peak at m/z 599. Finally, splitting off the galacturonic acid moiety built a fragment with a peak at m/z 405.  The structure and connection of the sugar moieties were also confirmed via the observed fragmentation pattern of the molecule in a negative ESI-MS/MS mode ( Figure S2). The proposed fragmentation pathway of compound 1 is presented in Figure 2. A loss of glucose gave the fragment a peak at m/z 953 whereas, due to the fragmentation of the trisaccharide unit, a peak at m/z 661 was formed. Further decarboxylation led to a fragment with a peak at m/z 599. Finally, splitting off the galacturonic acid moiety built a frag- served fragmentation pattern of the molecule in a negative ESI-MS/MS mode ( Figure S2). The proposed fragmentation pathway of compound 1 is presented in Figure 2. A loss of glucose gave the fragment a peak at m/z 953 whereas, due to the fragmentation of the trisaccharide unit, a peak at m/z 661 was formed. Further decarboxylation led to a fragment with a peak at m/z 599. Finally, splitting off the galacturonic acid moiety built a fragment with a peak at m/z 405.

Biological Evaluation of Compound 1
The antioxidant and enzyme inhibitory properties of silviridoside were investigated by different spectrophotometric assays. The results are summarized in Table 3, showing that silviridoside exhibited moderate antioxidant properties. The compound was more active in the DPPH radical scavenging assay, showing an antioxidant activity of 2.32 mg TE/g compared with the ABTS radical scavenging assay (1.24 mg TE/g). The compound also had a greater cupric-reducing power (estimated at 9.59 mg TE/g) compared with the ferric-reducing power (5.13 mg TE/g). The results for the phosphomolybdenum and metal-chelating assays were 0.28 mmol TE/g and 6.62 mg EDTA/g, respectively. In accordance with our results, other authors have reported significant antioxidant effects of triterpene glycosides [25][26][27].

Biological Evaluation of Compound 1
The antioxidant and enzyme inhibitory properties of silviridoside were investigated by different spectrophotometric assays. The results are summarized in Table 3, showing that silviridoside exhibited moderate antioxidant properties. The compound was more active in the DPPH radical scavenging assay, showing an antioxidant activity of 2.32 mg TE/g compared with the ABTS radical scavenging assay (1.24 mg TE/g). The compound also had a greater cupric-reducing power (estimated at 9.59 mg TE/g) compared with the ferric-reducing power (5.13 mg TE/g). The results for the phosphomolybdenum and metalchelating assays were 0.28 mmol TE/g and 6.62 mg EDTA/g, respectively. In accordance with our results, other authors have reported significant antioxidant effects of triterpene glycosides [25][26][27]. Enzyme inhibition represents one of the most popular subjects in the pharmaceutical industry [28]. In this study, the enzyme inhibition properties of silviridoside were tested against cholinesterases (AChE and BChE), tyrosinase, amylase, and glucosidase. The compound exhibited inhibitory effects against all the tested enzymes. The anticholinesterase ability (AChE and BChE) was 2.52 and 7.16 mg GALAE/g, respectively. The compound also showed a good tyrosinase inhibitory property (38.83 mg KAE/g). The α-amylase and α-glucosidase inhibitory potentials, which reflected the possible antidiabetic properties of this compound, were 0.19 and 1.21 mmol ACAE/g, respectively. Significant inhibitory abilities of several triterpene glycosides have been reported in the literature [29,30]; thus, we hope that our results are useful for novel applications in pharmaceutical areas.

In Silico Evaluation of the Pharmacodynamic, Pharmacokinetic, and Toxicity Properties of Silviridoside
An in silico evaluation of the pharmacodynamics and pharmacokinetics of silviridoside was performed using the ADMET (absorption, distribution, metabolism, excretion, and toxicity) protocol. Silviridoside was very soluble and showed moderate human intestinal absorption, which placed it within the 99% confidence limit absorption ellipses, as illustrated in Figure 3. In contrast, it showed an unpredictable blood-brain barrier (BBB) penetration and was thus allocated outside the 99% confidence limit BBB ellipses (Figure 3). Silviridoside also revealed less than 90% plasma protein binding (PPB) and a slight toxicity to the liver, but no cytochrome P450 2D6 inhibition. Importantly, the new compound showed optimum cell permeability (polar surface area (PSA) < 140 A • and atom-based log P98 (Alog P98) < 5). The Toxicity Prediction using Komputer-Assisted Technology (TOPKAT) prediction revealed that silviridoside caused no mutagenicity in the Ames mutagenicity test and was non-carcinogenic to female National Toxicology Program (NTP) rats; unfortunately, it caused certain carcinogenic effects in male NTP rats. It also showed a rat oral LD50 and a chronic rat lowest observed adverse effect level (LOAEL) of 1.116 and 0.075 g/kg.bw, respectively. Furthermore, silviridoside showed a mild dermal irritation and a severe ocular irritant effect; thus, it should be handled and used cautiously. The in silico evaluation of the pharmacodynamic, pharmacokinetic, and toxicity properties of silviridoside are summarized in Table 4.

General Experimental Procedures
The NMR experiments were performed using a Bruker Avance II 400 spectrometer (resonance frequencies of 400.13 MHz for 1 H and 100.63 MHz for 13 C) equipped with a 5 mm broadband observe probe head with z-gradients at room temperature and standard Bruker pulse programs. The chemical shifts were presented in parts per million (δ/ppm) and referenced to residual solvent signals (DMSO-d 6 : 2.49 ppm for 1 H and 39.6 ppm for 13 C). The coupling constants (J) were reported in Hz. The HR-ESI-MS spectra were recorded on an Orbitrap XEVO G2 Xs QToF mass spectrometer (Waters Inc.) coupled to a UPC 2 HPLC system (Waters Inc.). Chromatographic separation was performed on a 2-PIC column at 45 • C (2-Picolylamine, Taurus series, Waters Inc., Milford, MA, USA)) using a gradient of supercritical CO 2 and MeOH with 25 mM ammonium hydroxide. For the data analysis, MassLynx software V4.2 was used. Silica gel (100/200 mesh, Tianjin Sinomed Pharmaceutical, Tianjin, China) and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Sweden) were used as the stationary media for the column chromatography. Thin layer chromatography (TLC) was performed on aluminum plates pre-coated with silica gel 60 F254 (Merck, Germany).

Plant Materials
The aerial parts (flowers, leaves, and stems) of S. viridiflora were collected from the botanical field of the Institute of the Chemistry of Plant Substances (Tashkent, Uzbekistan). The taxonomic authentication was accomplished by Dr. A. Nigmatullaev at the Department of Herbal Plants of the ICPS. The voucher specimen of the plant was deposited in the departmental herbarium under the code 2017/087. The plant material was air-dried and powdered before use.

Extraction and Isolation
The air-dried aerial organs (4 kg) of S. viridiflora were ground and then extracted with CH 3 OH (20 L × 3). The extract was condensed to 1 L, diluted with an equal amount of H 2 O, and left overnight. The resulting precipitate was filtered off. The CH 3 OH was evaporated. The aqueous solution was extracted with CHCl 3 (3 L) and butanol (1 L). The solvents were evaporated in a vacuum to obtain a butanol (168 g) fraction. The dried butanol fraction (80 g) was subjected to silica gel column chromatography (CC) (column size: 11 cm × 90 cm) and the fraction was eluted by CHCl 3 /MeOH gradients with the polarity increasing to 20% MeOH. Each fraction (200 mL) was analyzed by TLC and the fractions with similar TLC patterns were combined to obtain the main fractions (Fr. A-C). Fraction C (3.9 g) was chromatographed over a silica gel column and eluted stepwise by  Tables 1 and 2 and Supplementary Materials Figures S1-S11.

In Silico Evaluation of Silviridoside
An in silico evaluation of the pharmacodynamics and pharmacokinetics of silviridoside was undertaken according to the ADMET protocol (absorption, distribution, metabolism, excretion, and toxicity) using Biovia Discovery Studio software (Accelrys Inc., San Diego, CA, USA). The ADMET parameters included human intestinal absorption, plasma protein binding (PPB) prediction, blood-brain barrier (BBB) penetration, aqueous solubility, hepatotoxicity level, and inhibition of cytochrome P450 (2D6). The toxicity properties were determined using the TOPKAT protocol where the Ames mutagenicity, rat chronic LOAEL (lowest observed adverse effect level), and rat oral LD 50 , together with ocular and skin irritant effects and carcinogenicity on male and female NTP (National Toxicology Program) rats, were selected as the toxicity descriptors [32,33].

Statistical Analysis
The results of each assay were reported as the means ± SD (n = 3) of three parallel measurements. The calculation for each in vitro assay was performed using GraphPad version 9.2.

Conclusions
In the current study, a new triterpene glycoside-silviridoside-was isolated from the aerial parts of Silene viridiflora. The structure of the new compound was comprehensively identified using different spectroscopic techniques, comprising HR-ESI-MS and 1D and 2D NMR spectroscopy. Different antioxidant assays such as 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), cupric-reducing antioxidant capacity (CUPRAC), ferric-reducing antioxidant power (FRAP), phosphomolybdenum (PHD), and metal-chelating (MCA) assays were performed, and confirmed a pronounced activity as an antioxidant. In addition, silviridoside revealed a potent inhibition of enzymes such as acetylcholinesterase (AChE), butyrylcholinesterase (BChE), α-amylase, and tyrosinase. Moreover, an in silico evaluation of the pharmacodynamic, pharmacokinetic, and toxicity properties of silviridoside showed that the new compound exhibited reasonable pharmacodynamic and pharmacokinetic properties without a mutagenic effect, but with a slight toxicity. Thus, it could be concluded that silviridoside is a promising candidate for pharmaceutical and nutraceutical drug developments.