(aS)-Glucosciadopitysin, a New Biflavonoid Glycoside from the Leaves of Ginkgo biloba and Osteogenic Activity of Bioflavonoids (2025)

Keywords: Ginkgo biloba, NMR analysis, electronic circular dichroism (ECD) calculations, mesenchymal stem cell, alkaline phosphatase

2.1. Isolation of Compounds from the Leaves of G. biloba

The dried leaves of G. biloba were extracted with 80% aqueous MeOH to obtain a crude MeOH extract. The extract was then solvent-partitioned with four organic solvents to give four main fractions, among which the hexane-soluble fraction was subjected to analysis using column chromatography techniques. Medium-pressure liquid chromatography (MPLC) and high-performance liquid chromatography (HPLC) were conducted for the phytochemical investigation of the hexane-soluble fraction, with LC/MS analysis combined with our in-house UV library database. The phytochemical investigations resulted in the isolation of flavonoids (1–6) (Figure 1), including a new biflavonoid glycoside termed (aS)-glucosciadopitysin (1).

Compound 1 was isolated as a yellowish plate, and the HR-ESIMS data of 1 demonstrated the protonated molecular ion peak [M + H]⁺ at m/z 743.1977 (calculated for C₃₉H₃₅O₁₅⁺, 743.1970) in the positive-ion mode, which was compatible with the molecular formula of C₃₉H₃₄O₁₅ (Figure S1). Detailed interpretation of the ¹H NMR data (Figure S3), combined with HSQC (Figure S6 and Table 1) revealed the presence of two sets of flavonoid skeleton at H-6/C-6 [δ_H 6.35 (1H, d, J = 2.0 Hz)/δ_C 97.9], H-8/C-8 [δ_H 6.66 (1H, d, J = 2.0 Hz)/δ_C 91.9], H-3/C-3 [δ_H 6.79 (1H, s)/δ_C 103.4], H-5′/C-5′ [δ_H 7.35 (1H, d, J = 8.5 Hz)/δ_C 111.1], H-2′/C-2′ [δ_H 8.08 (1H, d, J = 2.0 Hz)/δ_C 131.2] and H-6′/C-6′ [δ_H 8.16 (1H, dd, J = 8.5, 2.0 Hz)/δ_C 128.1], as well as H-3′′/C-3′′ [δ_H 6.75 (1H, s)/δ_C 102.6], H-6′′/C-6′′ [δ_H 6.82 (1H, s)/δ_C 98.5], H-3′′′,H-5′′′/C-3′′′,C-5′′′ [δ_H 6.92 (2H, d, J = 9.0 Hz)/δ_C 114.1], and H-2′′′,H-6′′′/C-2′′′,C-5′′′ [δ_H 7.59 (2H, d, J = 9.0 Hz)/δ_C 127.6]. Three sets of the methoxy group were also assigned at 7-OCH₃ [δ_H 3.87 (3H, s)/δ_C 55.0], 4′-OCH₃ [δ_H 3.81 (3H, s)/δ_C 54.9] and 4′′′-OCH₃ [δ_H 3.80 (3H, s)/δ_C 54.5]. Peaks within the δ_H 3.25–3.92 range, along with an anomeric proton resonating at H-1⁗ [δ_H 5.19 (1H, d, J = 7.5 Hz)] and a corresponding carbon signal at δ_C 100.1, were assigned as part of a sugar moiety, which was confirmed through ¹H-¹H COSY correlation (Figure S4), revealing correlations of H-1⁗/H-2⁗/H-3⁗/H-4′′′′/H-5⁗/H-6⁗. The chemical shifts in the corresponding carbon signals for the sugar moiety and the coupling constant (J = 7.5 Hz) of the anomeric proton at δ_H 5.19 matched well with previously reported data [22], confirming the sugar moiety as β-glucopyranose.

The planar structure of 1 was fully elucidated with the assistance of ¹H-¹H COSY and HMBC analysis (Figure 2 and Figure S7). The following correlations helped to confirm the presence of one flavonoid moiety: ¹H-¹H COSY correlation of H-5′/H-6′, and HMBC correlations of H-3/C-2, C-4, C-10, and C-1′; H-6/C-5, C-8, and C-10; H-8/C-6, C-7, C-9, and C-10; H-2′/C-1′, C-4′, and C-6′; H-5′/C-1′ and C-3′; and H-6′/C-2′ and C-4′. Furthermore, the following correlations allowed us to confirm the presence of another flavonoid moiety: ¹H-¹H COSY correlation of H-2′′′/H-3′′′, and HMBC correlations of H-3′′/C-2′′, C-4′′, C-10′′, and C-1′′′; H-6′′/C-5′′, C-7′′, C-8′′, and C-10′′; H-2′′′/C-4′′′ and C-6′′′; and H-3′′′/C-1′′′, C-4′′′, and C-5′′′. The linkage of two flavonoid substructures was identified between C-3′ and C-8′′, which was verified by the HMBC correlation of H-2′/C-8′′. The positions of the three methoxy groups were confirmed by HMBC correlations: 7-OCH₃ (δ_H 3.87)/C-7; 4′-OCH₃ (δ_H 3.81)/C-4′; and 4′′′-OCH₃ (δ_H 3.80)/C-4′′′. The carbon linked to the sugar moiety was confirmed at C-7′′ via the HMBC correlation of H-1⁗/C-7′′. Consequently, the complete planar structure of 1 was determined as shown in Figure 1.

To assign the absolute configuration of sugar moiety for compound 1, acid hydrolysis followed by a chemical derivatization reaction was performed to determine the D/L configuration of β-glucopyranose (Figure S8) [24]. β-Glucopyranose obtained from the acid hydrolysis of compound 1 and standard of β-D-glucopyranose underwent a chemical reaction to obtain thiocarbamoyl-thiazolidine derivatives. Their retention times were compared using LC/MS analysis, which revealed that β-glucopyranose from compound 1 was identified as D-form, as they showed the same retention time (t_R = 19.5 min).

Compound 1 possesses flavone residues linked at 3′ and 8′′, forming a heterodimeric biaryl system. The steric hindrance of 4′-OCH₃ and 7′′-OGlc in the biaryl system results in a high rotational energy barrier for compound 1, leading to atropisomerism [25]. Practically, two biflavonoid glycosides with different axial chirality from Malus hupehensis Pamp. (Rosaceae) exhibited opposite cotton effects [26]. To determine the absolute configuration and axial chirality of compound 1, quantum chemical calculations for ECD simulations were performed. ECD data were simulated for two isomers, (aS)-1 and (aR)-1, and the calculated ECD data were compared with the experimental ECD spectrum of 1 (Figure 3). As a result, the experimental ECD data matched with the calculated ECD data of (aS)-1, indicating that the absolute configuration and axial chirality of compound 1 were definitively determined to be (aS). Accordingly, the chemical structure of compound 1, including the absolute configuration and axial chirality, was determined (Figure 3) and designated as (aS)-glucosciadopitysin.

The other isolated compounds were identified as ginkgetin (2) [27], isoginkgetin (3) [27], kaempferol 3-O-β-D-(6′′-p-coumaroyl)glucopyranosyl(1→2)-α-L-rhamnopyranoside (4) [28], 3-[5,7-dihydroxy-2-(4-methoxyphenyl)-4-oxo-4H-1-benzopyran-8-yl]-4-methoxybenzoic acid (5) [29] and apigenin (6) [30] through a comparison of their NMR spectra with the reported data and MS data from their LC/MS analysis (Figures S9-S18, Tables S1 and S2).

2.2. The Regulatory Effects of Compounds 1–6 on MSC Differentiation into Adipocytes and Osteoblasts

To assess the regulatory effects of the isolated compounds 1–6 on MSC differentiation into adipocytes and osteoblasts, the mouse mesenchymal stem cell line C3H10T1/2 was exposed to a concentration of 20 µM of compounds 1–6 during adipogenic or osteogenic differentiation. While the six isolated compounds showed no significant impact on the adipocyte differentiation process of MSCs, compound 2 exhibited a notable enhancement of osteogenic differentiation at a concentration of 20 μM, surpassing the effects observed with the other isolated compounds (Figure 4).

When compound 2 was introduced into the culture medium of MSCs undergoing osteogenic differentiation, ranging from 1 to 10 μM concentrations, the intensity of alkaline phosphatase (ALP) staining increased proportionally with the escalating concentration of compound 2, comparable to the active positive control, oryzativol A (Figure 5). This observation suggests that the process of osteogenic differentiation is facilitated in a dose-dependent fashion by compound 2. MSCs cultured in an osteogenic differentiation medium containing compound 2 exhibited a noticeable increase in the expression of osteopontin (OPN) and alkaline phosphatase (ALP), markers typically associated with bone and tooth formation (Figure 5). Osteogenic differentiation of MSCs was induced in the osteogenic differentiation medium, followed by mRNA analysis on the 10th day of culture. This analysis revealed a proportional increase in the expression levels of osteogenic markers ALP and OPN in response to varying concentrations of compound 2. At the highest concentration of 10 μM, the gene expression of ALP and OPN was, respectively, 13.9-fold and 3.8-fold higher compared to the untreated negative control group, which is comparable to 12.5-fold and 4.0-fold in the positive control group, oryzativol A.

We discovered that ginkgetin (2) shows promise for promoting osteogenic differentiation. Our findings suggest that the position of the methoxy group can be critical for this effect. Specifically, the presence of the methoxy group at the C-7 position appears to play a significant role in osteogenic differentiation, as evidenced by the comparison of ginkgetin (2) and isoginkgetin (3). Similarly, previous reports on structure–activity relationships have suggested that the number and position of methoxy groups in biflavonoids can influence polarity modification and the ability to establish hydrogen bonds with target proteins [31]. Additionally, we observed that the functional group attached at C-7′′ is also able to affect osteogenic differentiation activity, as demonstrated in the comparison of compounds 1 and 2. This insight provides a rationale for the potential usefulness of G. biloba extract in promoting osteogenic differentiation, given that ginkgetin (2) is a prominent component of G. biloba [32].

3.1. General Experimental Procedures

Optical rotations were measured using a Jasco P-2000 polarimeter (Jasco, Easton, MD, USA). Ultraviolet (UV) spectra were acquired on an Agilent 8453 UV-visible spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). Electronic circular dichroism spectra were measured on a Jasco J-1500 spectropolarimeter (Jasco). Infrared (IR) spectra were recorded with a Bruker VERTEX 70 FT-IR spectrometer (Bruker, Karlsruhe, Germany). Nuclear magnetic resonance (NMR) spectra were recorded with a Bruker AVANCE III HD 850 NMR spectrometer with a 5 mm TCI CryoProbe operating at 850 MHz (¹H) and 212.5 MHz (¹³C) (Bruker, Karlsruhe, Germany), with chemical shifts given in ppm (δ) for ¹H and ¹³C NMR analyses, and they were referenced to solvent peaks of CD₃OD at 3.310 ppm for ¹H and 49.000 ppm for ¹³C (Cambridge Isotope Laboratories, Tewksbury, MA, USA). The HR-ESI-MS data were obtained with an Agilent 6545 Q-TOF LC/MS spectrometer using an EclipsePlus C18 95 Å column (50 × 2.1 mm, 1.8 μm; flow rate: 0.3 mL/min; Agilent Technologies, Santa Clara, CA, USA). LC/MS analysis was performed on an Agilent 1200 Series HPLC system equipped with a diode array detector and 6130 Series ESI mass spectrometer using an analytical Kinetex C18 100 Å column (100 × 2.1 mm, 5 μm; flow rate: 0.3 mL/min; Phenomenex, Torrance, CA, USA). MPLC was carried out using a Yamazen Smart Flash AKROS system (Yamazen Corporation, Osaka, Japan) with an ODS-SM column (16.5 × 3.0 cm, 50 μm; flow rate: 20 mL/min; Yamazen Corporation) for reversed-phase MPLC. HPLC was performed using a Waters 1525 Binary HPLC pump with a Waters 996 Photodiode Array Detector (Waters Corporation, Milford, CT, USA) and a HECTOR C18 column (250 × 21.2 mm, 5 μm; flow rate: 5 mL/min; RStech, Chungcheongbuk-do, Cheong-Ju, Republic of Korea). Semi-preparative HPLC was performed using a Shimadzu Prominence HPLC System with SPD-20A/20AV Series Prominence HPLC UV-Vis detectors (Shimadzu, Tokyo, Japan) with a Phenomenex Luna Phenyl-Hexyl column (250 × 10 mm, 10 μm; flow rate: 2 mL/min; Phenomenex). Silica gel 60 (230-400 mesh; Merck, Darmstadt, Germany) was used for column chromatography. The packing material for molecular sieve column chromatography was Sephadex LH-20 (Pharmacia, Uppsala, Sweden). Precoated silica gel F₂₅₄ plates and RP-C₁₈ F_254s plates (Merck) were used for thin-layer chromatography (TLC). Spots were detected after TLC under UV light or by heating after spraying with anisaldehyde sulfuric acid.

3.2. Plant Material

The leaves of Ginkgo biloba were collected at the arboretum of Sungkyunkwan University, Suwon, Korea, in August 2016. The plant material was authenticated by one of the authors (K. H. Kim). A voucher specimen of the material (UHY-2016) was deposited in the herbarium of the School of Pharmacy, Sungkyunkwan University, Suwon, Republic of Korea.

3.3. Extraction and Isolation

Air drying of G. biloba leaves was performed in a room at approximately 25 °C with humidity levels between 20 and 30%. The drying process lasted for a week and was conducted without direct sunlight exposure. Dried leaves of G. biloba (1.7 kg) were extracted with 80% aqueous MeOH (3.0 L × 3) and filtered. The extract was concentrated under vacuum using a rotary evaporator to gain a crude MeOH extract (293.8 g). The extract was suspended in distilled water (700 mL) and solvent-partitioned with hexane, dichloromethane (CH₂Cl₂), ethyl acetate (EtOAc), and n-butanol (n-BuOH), and then, four fractions were gained: hexane-soluble (31.2 g), CH₂Cl₂-soluble (5.5 g), EtOAc-soluble (7 g), and n-BuOH-soluble fractions (37.4 g). The hexane-soluble fraction (31.2 g) was subjected to silica gel column chromatography, employing a gradient solvent system of CH₂Cl₂/MeOH (100:1 to 1:1). Subsequently, the column was eluted with MeOH to yield six fractions (H_A–H_F). Fraction H_D (5.6 g) was fractionated by MPLC on the Universal ODS-SM column with a gradient solvent system of MeOH/H₂O (65% MeOH to 75% MeOH) to afford four subfractions (H_D1–H_D4). Subfraction H_D1 (171 mg) was subjected to silica gel column chromatography, employing a gradient solvent system of CH₂Cl₂/MeOH (20:1 to 1:1) to obtain eight subfractions (H_D11–H_D18). Subfraction H_D13 (9.9 mg) was purified by semi-preparative HPLC with an isocratic solvent system of MeCN/H₂O (27% MeCN; acetonitrile) to yield compound 6 (t_R = 61.2 min, 0.5 mg). Subfraction H_D2 (87.6 mg) was separated using preparative reversed-phase HPLC with a gradient solvent system of MeOH/H₂O (67% MeOH to 100% MeOH) to obtain four subfractions (H_D21–H_D24). Subfraction H_D24 (40.5 mg) was purified by semi-preparative HPLC with an isocratic solvent system of MeCN/H₂O (48% MeCN) to yield compounds 2 (t_R = 39.5 min, 0.9 mg) and 3 (t_R = 42.5 min, 0.8 mg). Fraction H_E (4.3 g) was fractionated by Sephadex LH-20 column chromatography using a solvent system of 100% MeOH to yield four subfractions (H_E1–H_E4). Subfraction H_E3 (110.1 mg) was separated using preparative reversed-phase HPLC with a gradient solvent system of MeOH/H₂O (40% MeOH to 90% MeOH) to afford five subfractions (H_E31–H_E35). Subfraction H_E33 (7.0 mg) was purified using semi-preparative HPLC with an isocratic solvent system of MeCN/H₂O (27% MeCN) to isolate compound 4 (t_R = 21.2 min, 0.7 mg). Subfraction H_E34 (13.1 mg) was purified using semi-preparative HPLC with an isocratic solvent system of MeCN/H₂O (45% MeCN) to obtain compound 5 (t_R = 23.8 min, 1.3 mg). Subfraction H_E35 (30.7 mg) was purified using semi-preparative HPLC with an isocratic solvent system of MeCN/H₂O (48% MeCN) to obtain compound 1 (t_R = 25.6 min, 1.2 mg). The scheme of isolation process is described in Figure S19.

3.4. Acid Hydrolysis and Absolute Configuration Determination of the Sugar Moiety of 1

An HPLC-UV-based method was used to confirm the absolute configuration of the sugar moiety of 1 [24]. Compound 1 (0.5 mg) was hydrolyzed by 1 N HCl (1 mL) at 80 °C for 1 h, and solvent partitioned with CH₂Cl₂ (1 mL) to separate the aglycone and sugar. The aqueous layer was neutralized by repeated evaporation using a vacuum evaporator and dissolved in anhydrous pyridine (0.5 mL) with the addition of L-cysteine methyl ester hydrochloride (0.5 mg). After the reaction mixture was heated at 60 °C for 1 h, o-tolyl isothiocyanate (30 μL) was added, and the mixture was kept at 60 °C for 1 h. The reaction product, amounting to 30 μL, was evaporated using a vacuum evaporator and dissolved in 300 μL MeOH. Then, 10 μL of the sample was injected and analyzed by LC/MS with a gradient solvent system [MeOH/H₂O, 0% MeOH → 80% MeOH gradient system (0–31 min), 100% MeOH (31–41 min), and 0% MeOH (42–52 min)]. The sugar moiety from 1 was identified as β-D-glucose based on a comparison of the retention time with the standard β-D-glucose subjected to the derivatization (t_R: 19.5 min).

3.5. Electronic Circular Dichroism (ECD) Calculations

Initial conformational searches were performed for compound 1 in the MMFF94 force field using the MacroModel (version 2021-4, Schrödinger LLC, New York, NY, USA) programme with a mixed torsional/low-mode sampling method, in which a gas phase with a 20 kJ/mol energy window and 10,000 maximum iterations were employed. The Polak–Ribiere conjugate gradient (PRCG) algorithm was established with 10,000 maximum iterations and a 0.001 kJ (mol $Å$)⁻¹ convergence threshold on the root-mean-squared gradient to minimize conformers. The proposed conformers (found within 5 kJ/mol in the MMFF force field) were distinguished by their axial chirality and selected for geometry optimization using TmoleX 4.3.2, with density functional theory settings of B3-LYP/6-31+G (d,p) [33].

ECD calculations were conducted for each conformer of (aS)-1 (10 conformers) and (aR)-1 (10 conformers) at the theoretical level and basis sets. The calculated ECD spectra were simulated by superimposing each transition, where σ is the band width at a height of 1/e, and ΔE_i and R_i are the excitation energy and rotatory strength for transition i, respectively [33]. In this study, the value of σ was 0.2 eV. The excitation energies and rotational strengths of the ECD spectra were calculated based on the Boltzmann populations of conformers, and ECD visualization was performed using SigmaPlot 14.0.

$$∆ϵ\left(E\right)={\displaystyle \frac{1}{2.297\times {10}^{-39}}}{\displaystyle \frac{1}{\sqrt{2\pi \sigma }}}\sum _A^i∆E_i{R}_i{e}^{[-{\left(E-∆E_i\right)}^2/{\left(2\sigma \right)}^2]}$$

3.6. Cell Culture and Differentiation

The C3H10T1/2 cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Hyclone Laboratories Inc., Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) (Hyclone) and antibiotics (100 U/mL of penicillin and 100 μg/mL of streptomycin, Hyclone) at 37 °C in a 5% CO₂ humidified atmosphere. For adipocyte differentiation, confluent cells were cultured in DMEM supplemented with 10% FBS, 1 μM dexamethasone (Sigma, St. Louis, MO, USA), 0.5 mM isobutyl-1-methylxanthine (Sigma), 5 μg/mL insulin (Sigma), and 10 μM troglitazone for 48 h. Subsequently, the cells were transferred to DMEM containing 10% FBS, 5 μg/mL insulin, and 10 μM troglitazone and further incubated for an additional 3 days. During adipogenesis, 20 µM of compounds 1–6 was added to the cells, and resveratrol (20 µM) was used as a positive control. Resveratrol (CAS No. 501-36-0) was purchased from Sigma-Aldrich (Cat. No. R5010). For osteogenic differentiation, cells reaching confluence were cultured in DMEM supplemented with 10% FBS, 100 U/mL of penicillin, and 100 μg/mL of streptomycin, along with 10 mM β-glycerophosphate (Sigma-Aldrich) and 50 μg/mL ascorbic acid (Sigma-Aldrich), for 9 to 12 days. The medium was refreshed every 3 days. During osteogenic differentiation, 20 µM of compounds 1–6 was added to the cells, and 5 µM oryzativol A was used as a positive control.

3.7. Oil Red O Staining

The cultured cells were rinsed with phosphate-buffered saline and fixed in 10% neutral-buffered formalin at room temperature for 1 h. Subsequently, the cells were stained with a 0.5% filtered stock solution of Oil Red O in isopropanol (Sigma, Saint Louis, MO, USA). After staining, the intracellular triglyceride content was assessed by redissolving the stained cells in isopropanol, followed by a measurement of absorbance at a wavelength of 520 nm.

3.8. Alkaline Phosphatase (ALP) Staining

The cultured cells were rinsed with 2 mM MgCl₂ solution. Subsequently, they were incubated with ALP buffer (composed of 100 mM Tris-HCl, pH 9.5; 100 mM NaCl; 10 mM MgCl₂; and 0.05% Tween-20) for 15 min. Following this, the cells were incubated in ALP buffer supplemented with 0.4 mg/mL of nitro-blue tetrazolium (Merck) and 0.2 g/mL of 5-bromo-4-chloro-3-indolyl phosphate (Merck). After washing with 0.5 mM ethylenediaminetetraacetic acid, the cells were fixed with 10% neutral-buffered formalin for 1 h.

3.9. ALP Activity

To assess ALP activity, an alkaline phosphatase assay kit (ab83369; Abcam, Cambridge, MA, USA) was employed. Cell lysates obtained from the differentiated osteogenic cells were treated with a p-nitrophenyl phosphate (p-NPP) solution at 25 °C for 1 h in the dark. Following cessation of the reaction, the absorbance of the samples was measured at 405 nm.

3.10. mRNA Isolation and Real-Time Polymerase Chain Reaction (PCR)

RNA extraction from the cells was performed using the NucleoZOL reagent (NucleoZOL; Macherey-Nagel GmbH & Co., KG, Düren, Germany). Subsequently, complementary DNA (cDNA) was synthesized from 0.5 μg of total RNA utilizing a ReverTraAce qPCR reverse transcription (RT) Master Mix Kit (FSQ-201; Toyobo, Japan) with random primers. The resulting cDNA was then combined with the amplification mixture comprising the Thunderbird SYBR qPCR Mix (Toyobo) along with the specified primers.

Alkaline phosphatase (ALP): forward 5′-CAAGGATGCTGGGAAGTCCG-3′ and reverse 5′-CGGATAACGAGATGCCACCA-3′; osteopontin (OPN): forward 5′-CTGGCAGCTCAGAGGAGAAG-3′ and reverse 5′-CAGCATTCTGTGGCGCAAG-3′.

3.11. Statistical Analysis

Each sample was tested in triplicate, and the test was repeated three times. Data are presented as the mean ± standard deviation. One-way analysis of variance was used to identify statistically significant differences between the control and test groups.

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Supplementary Materials

Data Availability Statement

Data are contained within the article and Supplementary Materials.