Characterization of flavonoid and quinic acid derivatives from Calystegia soldanella with antioxidant activities

The nuclear magnetic resonance (NMR) spectra were generated using Varian VNMRS 500 MHz FT-NMR spectrometer (Varian, Palo Alto, CA, USA). The high-performance liquid chromatography (HPLC; Waters, Miliford, MA, USA) with a PDA detector and C₁₈ column (Cosmosil, Kyoto, Japan) was analyzed using extract and isolated compounds. MPLC (Biotage, Uppsala, Sweden) and preparative-HPLC (Shimadzu, Kyoto, Japan) were performed to isolation and purification. The HPLC of elution solvents were used for the HPLC grade (ACN, Water, MeOH, J. T. Baker^®, Phillipsburg, NJ, USA) and Mass elution solvents were used by mass grade (ACN, MeOH, Supelco, Bellefonte, PA, USA). The HR-ESI-MS spectra were measured on a SCIEX X500R Q-TOF LC-MS/MS spectrometer (SCIEX, Framingham, MA, USA).

Aerial parts of C. soldanella were collected from Ujeong-eup, Hwaseong-si, Gyeonggi-do, Korea in September 2019. A voucher specimen (NP-0682) was kept in the National Biodiversity institute of Korea.

The aerial parts of C. soldanella (150 g) were cut and extracted three times with 70% EtOH at room temperature for 8 h. The 70% EtOH extract was successively partitioned with n-hexane, chloroform, ethyl acetate, and n-butanol. The most active portion (n-BuOH-soluble layer) was chromatographed by MPLC eluting with an increasing MeOH in water to give seven fractions (S1–S7). The mixed fraction was further followed by Sephadex LH-20 column chromatography (eluent: MeOH) to yield two compounds 5 (27.6 mg), 6 (2.7 mg), and nine subfractions (S1–S9). The subfraction 5 was finally isolated using preparative-HPLC with increasing solvents (2:8/MeOH:Water → 5:5/MeOH:Water) to obtain four compounds 1 (9.6 mg), 2 (1.2 mg), 3 (2.9 mg), and 4 (14.5 mg). The subfraction 8 was further divided with preparative-HPLC eluted with increasing solvents (3:7/MeOH:Water → 65:35/MeOH:Water) in water to obtain two compounds 7 (5.0 mg) and 8 (3.7 mg).

Chlorogenic acid (1): HR-ESI-MS (negative) m/z 353.0878 [M-H]^–, calcd for C₁₆H₁₇O₉ 353.0873; ¹H NMR (500 MHz, CD₃OD): δ7.56 (1H, d, J = 15.9 Hz, H-7’), 7.05 (1H, d, J = 1.8 Hz, H-2’), 6.95 (1H, dd, J = 8.2, 1.8 Hz, H-6’), 6.77 (1H, d, J = 8.2 Hz, H-5’), 6.27 (1H, d, J = 15.9 Hz, H-8’), 5.34 (1H, m, H-3), 4.17 (1H, m, H-5), 3.73 (1H, m, H-4), 2.20/2.06 (2H, m, H-2), 2.18/2.05 (2H, m, H-6); ¹³C NMR (125 MHz, CD₃OD): δ177.5 (C-7), 168.8 (C-9’), 149.7 (C-4’), 147.2 (C-7’), 146.9 (C-3’), 127.9 (C-1’), 123.1 (C-6’), 116.5 (C-5’), 115.3 (C-8’), 115.2 (C-2’), 76.4 (C-1), 73.7 (C-4), 72.1 (C-3), 71.6 (C-5), 39.0 (C-6), 38.4 (C-2) (Supplementary Figs. S1-S3).

Cryptochlorogenic acid (2): HR-ESI-MS (negative) m/z 353.0880 [M-H]^–, calcd for C₁₆H₁₇O₉ 353.0873; ¹H NMR (500 MHz, CD₃OD): δ7.63 (1H, d, J = 15.9 Hz, H-7’), 7.07 (1H, d, J =1.7 Hz, H-2’), 6.97 (1H, dd, J = 8.1, 1.7 Hz, H-6’), 6.78 (1H, d, J = 8.1 Hz, H-5’), 6.37 (1H, d, J = 15.9 Hz, H-8’), 4.86 (1H, m, overlap, H-3), 4.30 (1H, m, H-4), 4.21 (1H, m, H-2), 2.14-2.02 (4H, m, H-1 and 5); ¹³C NMR (125 MHz, CD₃OD): δ177.4 (C-7), 169.0 (C-9’), 149.7 (C-4’), 147.2 (C-7’), 146.9 (C-3’), 128.0 (C-1’), 123.1 (C-6’), 116.6 (C-5’), 115.5 (C-8’), 115.2 (C-2’), 78.9 (C-3), 75.9 (C-6), 69.3 (C-4), 66.4 (C-2), 39.2 (C-5), 38.7 (C-1) (Supplementary Figs. S4-S7).

5-O-(E)-p-coumaroylquinic acid (3): HR-ESI-MS (negative) m/z 337.0924 [M-H]^–, calcd for C₁₆H₁₇O₈ 337.0923; ¹H NMR (500 MHz, CD₃OD): δ7.63 (1H, d, J = 15.9 Hz, H-7’), 7.46 (2H, d, J = 8.5 Hz, H-2’ and H-6’), 6.80 (2H, d, J = 8.5 Hz, H-3’ and H-5’), 6.34 (1H, d, J = 15.9 Hz, H-8’), 5.34 (1H, m, H-3), 4.16 (1H, m, H-5), 3.72 (1H, dd, J = 8.5, 2.6 Hz), 2.22-2.02 (4H, m, H-2 and H-6); ¹³C NMR (125 MHz, CD₃OD): δ179.2 (C-7) 168.8 (C-9’), 161.4 (C-4’), 146.7 (C-7’), 131.3 (C-2’ and 6’), 127.3 (C-1’), 116.9 (C-3’ and 5’), 115.5 (C-8’), 75.8 (C-1), 73.9 (C-4), 72.3 (C-3), 71.9 (C-5), 39.3 (C-2), 38.5 (C-6) (Supplementary Figs. S8-S11).

Rutin (4): HR-ESI-MS (negative) m/z 609.1449 [M-H]^–, calcd for C₂₇H₂₉O₁₆ 609.1456; ¹H NMR (500 MHz, CD₃OD): δ7.67 (1H, d, J = 1.9 Hz, H-2’), 7.63 (1H, dd, J = 8.4, 2.0 Hz, H-6’), 6.87 (1H, d, J = 8.4 Hz, H-5’), 6.38 (1H, d, J = 1.8 Hz, H-8), 6.20 (1H, d, J = 1.8 Hz, H-6), 5.11 (1H, d, J = 7.6 Hz, H-1’’), 4.52 (1H, d, J = 1.0 Hz, H-1’’’), 3.81/3.39 (2H, m, H-5’’), 3.64 (1H, m, H-2’’’), 3.55 (1H, m, H-3’’’), 3.47 (1H, m, H-2’’), 3.45 (1H, m, H-5’’’), 3.44 (1H, m, H-3’’), 3.33 (1H, m, H-5’’), 3.30 (1H, m, H-4’’’), 3.29 (1H, m, H-4’’), 1.12 (3H, d, J = 6.2 Hz, H-6’’’); ¹³C NMR (125 MHz, CD₃OD): δ179.5 (C-4), 166.1 (C-7), 163.0 (C-5), 159.4 (C-9), 158.6 (C-2), 149.9 (C-4’), 145.9 (C-3’), 135.7 (C-3), 123.6 (C-6’), 123.2 (C-1’), 117.8 (C-2’), 116.1 (C-5’), 105.7 (C-10), 104.8 (C-1’’), 102.5 (C-1’’’), 100.0 (C-6), 94.9 (C-8), 78.2 (C-3’’), 77.3 (C-5’’), 75.8 (C-2’’), 74.0 (C-4’’’), 72.3 (H-3’’’), 72.2 (H-2’’’), 71.5 (H-4’’), 69.8 (H-5’’’), 68.6 (H-6’’), 18.0 (H-6’’’) (Supplementary Figs. S12-S14).

Isoquercetin (5): HR-ESI-MS (negative) m/z 463.0873 [M-H]^–, calcd for C₂₁H₁₉O₁₂ 463.0877; ¹H NMR (500 MHz, CD₃OD): δ7.71 (1H, brs, H-2’), 7.57 (1H, d, J = 7.3 Hz, H-6’), 6.86 (1H, d, J = 7.3 Hz, H-5’), 6.36 (1H, brs, H-8), 6.17 (1H, brs, H-6), 5.25 (1H, d, J = 6.6 Hz), 3.72/3.59 (2H, m, H-6’’), 3.46 (1H, m, H-2’’), 3.44 (1H, m, H-3’’), 3.35 (1H, m, H-4’’), 3.25 (1H, m, H-5’’); ¹³C NMR (125 MHz, CD₃OD): δ179.5 (C-4), 166.1 (C-7), 163.0 (C-5), 159.0 (C-2), 158.5 (C-9), 149.5 (C-4’), 145.9 (C-3’), 135.7 (C-3), 123.2 (C-6’), 123.1 (C-1’), 117.6 (C-2’), 116.0 (C-5’), 105.7 (C-10), 104.4 (C-1’’), 100.0 (C-6), 94.8 (C-8), 78.4 (C-5’’), 78.1 (C-3’’), 75.8 (C-2’’), 71.5 (C-4’’), 62.6 (C-6’’) (Supplementary Figs. S15-S17).

Nicotiflorin (6): HR-ESI-MS (negative) m/z 593.1501 [M-H]^–, calcd for C₂₇H₂₉O15 593.1507; ¹H NMR (500 MHz, CD₃OD): δ8.07 (2H, d, J = 8.7 Hz, H-2’ and H-6’), 6.89 (2H, d, J = 8.7 Hz, H-3’ and H-5’), 6.40 (1H, brs, H-8), 6.21 (1H, brs, H-6), 5.13 (1H, d, J = 7.4 Hz, H-1’’), 4.51 (1H, brs, H-1’’’), 3.81/3.38 (2H, m, H-6’’), 3.64 (1H, m, H-2’’’), 3.52 (1H, m, H-3’’’), 3.45 (1H, m, H-5’’’), 3.44 (1H, m, H-2’’), 3.42 (1H, m, H-3’’), 3.33 (1H, m, H-5’’), 3.29 (1H, m, H-4’’’), 3.27 (1H, m, H-4’’), 1.12 (3H, d, J = 6.2 Hz); ¹³C NMR (125 MHz, CD₃OD): δ179.5 (C-4), 166.2 (C-7), 163.1 (C-5), 161.6 (C-4’), 159.5 (C-9), 158.6 (C-2), 135.6 (C-3), 132.5 (C-2’ and C-6’), 122.8 (C-1’), 116.2 (C-3’ and C-5’), 105.7 (C-10), 104.7 (C-1’’), 102.5 (C-1’’’), 100.1 (C-6), 95.0 (C-8), 78.2 (C-3’’), 77.3 (C-5’’), 75.9 (C-2’’), 74.0 (C-4’’’), 72.4 (C-3’’’), 72.2 (C-2’’’), 71.5 (C-4’’), 69.8 (C-5’’’), 68.7 (C-6’’), 18.0 (C-6’’’) (Supplementary Figs. S18-S20).

Isochlorogenic acid A (7): HR-ESI-MS (negative) m/z 515.1188 [M-H]^–, calcd for C₂₅H₂₃O₁₂ 515.1190; ¹H NMR (500 MHz, CD₃OD): δ7.62 (1H, d, J = 16.0 Hz, H-3’), 7.58 (1H, d, J = 16.0 Hz, H-3’’), 7.07 (2H, s, H-9’ and H-9’’), 6.96 (2H, m, H-5’ and H-5’’), 6.78 (2H, d, J = 8.0 Hz, H-6’ and H-6’’), 6.36 (1H, d, J = 16.0 Hz, H-2’), 6.27 (1H, d, J = 16.0 Hz, H-2’’), 5.42 (1H, m, H-3), 5.40 (1H, m, H-5), 3.97 (1H, m, H-4), 2.32/2.15 (2H, m, H-2), 2.23/2.19 (2H, m, H-6); ¹³C NMR (125 MHz, CD₃OD): δ177.8 (C-7), 169.0 (C-1’), 168.5 (C-1’’), 149.7 (C-7’), 149.6 (C-7’’), 147.3 (C-3’), 147.1 (C-3’’), 146.9 (C-8’ and C-8’’), 128.0 (C-4’), 127.9 (C-4’’), 123.2 (C-5’), 123.1 (C-5’’), 116.5 (C-6’ and C-6’’), 115.7 (C-2’), 115.3 (C-2’’), 115.2 (C-9’ and C-9’’), 74.9 (C-1), 72.8 (C-3), 72.2 (C-5), 70.9 (C-4), 38.0 (C-2), 36.2 (C-6) (Supplementary Figs. S21-S23).

Isochlorogenic acid C (8): HR-ESI-MS (negative) m/z 515.1184 [M-H]^–, calcd for C₂₅H₂₃O₁₂ 515.1190; ¹H NMR (500 MHz, CD₃OD): δ7.60 (1H, d, J = 15.8 Hz, H-3’), 7.51 (1H, d, J = 15.8 Hz, H-3’’), 7.01 (2H, d, J = 1.8 Hz, H-9’ and H-9’’), 6.91 (2H, brt, H-5’ and H-5’’), 6.74 (2H, d, J = 1.8 Hz, H-6’ and H-6’’), 6.29 (1H, d, J = 15.8 Hz, H-2’), 6.19 (1H, d, J = 15.8 Hz, H-2’’), 5.62 (1H, m, H-5), 5.12 (1H, dd, J = 9.0, 2.8 Hz, H-4), 4.37 (1H, m, H-3), 2.22-2.05 (4H, m, H-2 and H-6); ¹³C NMR (125 MHz, CD₃OD): δ177.3 (C-7), 168.6 (C-1’), 168.3 (C-1’’), 149.8 (C-7’ and C-7’’), 147.8 (C-3’), 147.7 (C-3’’), 146.9 (C-8’ and C-8’’), 127.8 (C-4’), 127.7 (C-4’’), 123.2 (C-5’ and C-5’’), 116.5 (C-6’ and C-6’’), 115.2 (C-9’ and C-9’’), 114.8 (C-2’ and C-2’’), 76.4 (C-1), 76.0 (C-4), 69.6 (C-3), 69.2 (C-5), 39.6 (C-6), 38.5(C-2) (Supplementary Figs. S24-S26).

The separation of main peaks was carried out by UPLC-ESI-Q-TOF mass spectrometry using a BEH C₁₈ column (I.D 1.7 μm, 2.1 × 100 mm). The injection amount (10 μL) of samples was injected and eluted with the gradient solvent conditions of line A (water in 0.1% formic acid) and line C (ACN) with a flow rate at 0.4 mL/min. The gradient solvent was performed using the following protocol: (0–2 min/90:10, A:C), (2–35 min/90:10, A:C → 0:100, A:C), (35–38 min/0:100, A:C), (38–38.5 min/90:10, A:C), and (38.5–45 min/90:10, A:C). The mass spectrometer was operated in negative mode using the mass ranging from 100 to 2,000 Da, spray voltage at −4,500 V, declustering potential at −80 V, and collision energy at −25.

2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities were determined according to slightly modified methods. Simply, ABTS solution (7 mM) including potassium persulfate (2.45 mM) was dissolved in water with dark condition. The sample (10 μL) and ABTS solution (190 μL) were mixed and the absorbance was recorded at 734 nm. The dissolved DPPH solution and the sample were mixed and performed at ultraviolet (UV) absorbance at 517 nm (Kim et al., 2022, 2020).

The total polyphenol content (TPC) in C. soldanella was measured using the Folin-Denis colorimetric method according to some modifications. Simply, samples (20 μL) were mixed with Folin-Denis agent (100 μL) for 1 min at 40°C. The mixtures were contained to a Na₂CO₃ solution and reacted for 15 min at 40°C. The reaction solutions were performed by the microplate reader using UV absorbance at 765 nm. The total flavonoid content (TFC) was recorded using an aluminum chloride colorimetric assay with slight modifications. The calibration curve was prepared by stock solution (5 mg/mL) and then diluted to concentrations ranging from 31.25−500 μg/mL. Briefly, 50 μL of the diluted samples were separately mixed with 150 μL (MeOH), 10 μL (10% aluminum chloride), 10 μL (1 M potassium acetate), and 280 μL (distilled water). After mixing, the solutions were incubated at room temperature for 30 min and measured at 415 nm (Hassan & Al Aqil, 2013; Sant’Ana et al., 2014).

Intracellular ROS formation was investigated using a 2’-7’-dichlorodihydrofluorescein diacetate (DCFH-DA) assay. Briefly, Raw264.7 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM). Cells (3 × 10⁵ cells/mL) were conducted with compound 8 for 1 h and then lipopolysaccharide (LPS; 1 μg/mL) was treated each well plate for 24 h at 37°C. Then, the cells were washed and added serum-free DMEM including DCFH-DA (10 μM) in incubation (37°C and 30 min). The mixed solutions were washed and recorded using a flow cytometer (BD Bioscience, San Jose, CA, USA) (Oh et al., 2023).

All statistical analyses were conducted using IBM SPSS software (Version 22.0, IBM, Armonk, NY, USA). The differences were considered as statistically significant at p < 0.05.

The investigation of anti-oxidative metabolites from 70% EtOH extract of C. soldanella resulted in the isolation of eight compounds (1–8) including three flavonoid derivatives: rutin (4), isoquercertin (5), and nicotiflorin (6) and five quinic acid derivatives: chlorogenic acid (1), crytochlorogenic acid (2), 5-O-(E)-p-coumaroylquinic acid (3), isochlorogenic acid A (7), and isochlorogenic acid C (8), respectively (Fig. 1). The chemical structures were determined by spectroscopic analysis (HR-ESI-MS and NMR spectra) with comparison of reported in the literature (Ganbaatar et al., 2019; Lee et al., 2010; Ma et al., 2022; Pan et al., 2019; Tao et al., 2012).

Fig. 1. Chemical structures of 1 to 8. (1) chlorogenic acid; (2) cryptochlorogenic acid; (3) 5-O-(E)-p-coumaroylquinic acid; (4) rutin; (5) isoquercetin; (6) nicotiflorin; (7) isochlorogenic acid A; (8) isochlorogenic acid C.

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To confirm main components, the 70% EtOH extract (sample concentration: 1 mg/mL) of C. soldanella was analyzed using HR-ESI-Q-TOF mass spectrometry. The peaks (1–8) showed molecular ions based on negative mode of masses consistent with those of isolated compounds (Precursor ions: m/z 1: 353.0878; 2: 353.0880; 3: 337.0924; 4: 609.1449; 5: 463.0873; 6: 593.1501; 7: 515.1188; 8: 515.1184 [M-H]^–). The retention time, molecular formula, error, and characterization of peaks (1–8) are shown in (Table 1; Fig. 2).

Fig. 2. HR-MS profile using negative mode of 70% EtOH extract of Calystegia soldanella.

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Chemical antioxidant assays (ABTS and DPPH radicals scavenging assays, TPC, and TFC protocols) are broadly used for the primary screening of antioxidant activities. Furthermore, these assays are simple and inexpensive. Polyphenols and flavonoids are divided into various moieties related with attachment of phenol rings. These derivatives exhibit potent antioxidant activities. The results are shown (Table 2). The extract of C. soldanella in TPC and TFC revealed that the values are 13.7 ± 0.9 mg GAE/g and 16.2 ± 0.3 mg QE/g, respectively. Furthermore, the amounts of partitioned portions (n-hexane, EtOAc, and n-BuOH- soluble layers) were varied, ranging from 28.6 ± 0.2 to 30.6 ± 0.5 mg GAE/g and from 44.5 ± 3.1 to 72.4 ± 2.9 mg QE/g in TPC and TFC assays. The remaining water-soluble portion exhibited lower values (8.1 ± 0.2 mg GAE/g in TPC, and 10.3 ± 0.1 mg QE/g in TFC) compared with partitioned portions. In addition, the 70% EtOH extract of C. soldanella were evaluated for its scavenging activity against ABTS and DPPH radicals. The 70% EtOH extract of C. soldanella displayed moderate anti-oxidant activities with IC₅₀ values of 167.5 ± 9.4 (ABTS radical) and 379.8 ± 9.8 μg/mL (DPPH radical), respectively. The most active portion (n-BuOH-soluble layer) in ABTS and DPPH radical scavenging assay showed the IC₅₀ values of 69.1 ± 10.0 and 120.6 ± 4.9 μg/mL, respectively. However, other separated portions (n-Hexane, EtOAc, and water-soluble portions) showed no antioxidant activity in the concentrate with an IC₅₀ value of 150 μg/mL (Table 3). Therefore, the main components of the n-BuOH-soluble layer of C. soldanella were isolated and purified to identify the active anti-oxidative compounds.

To search anti-oxidative compounds from the n-BuOH-soluble portion of C. soldanella, Compounds (1−8) were isolated through purification and isolation, which is followed by column chromatography techniques. The anti-oxidant activities of compounds (1−8) showed IC₅₀ values ranging from 12.4 μM to > 200 μM and 37.9 μM to > 200 μM, respectively. The most active compound 8 (isochlorogenic acid C) exhibited the IC₅₀ values of 12.4 ± 0.1 μM (ABTS radical) and 37.9 ± 0.5 μM (DPPH radical). Compounds 7 and 8 displayed higher activities than ascorbic acid (IC₅₀ values: 24.4 ± 0.4 μM/ABTS radical and 74.4 ± 0.7 μM/DPPH radical). Furthermore, compound 5 (isoquercetin) also showed potent antioxidant activities (ABTS and DPPH radicals) with IC₅₀ values of 27.4 ± 0.6 and 47.0 ± 1.9 μM, successively (Table 3). In particular, we confirmed that two main classes were flavonoid and quinic acid derivatives from the extract of C. soldanella. Increasingly, compound 5 showed higher antioxidant effect than compound 6, indicating that is contributed to the existence of the hydroxyl unit at C ring in the flavone moiety. Furthermore, the attachment of an additional sugar moiety at glucose unit in isoquercetin slightly decreased the antioxidative activity compared with that of compound 5. Therefore, the attachment of the hydroxyl unit at C ring in flavone moiety plays a role for antioxidant activity. In addition, the presence of coumaroyl moiety in compound 3 considerably decreased its antioxidant activity compared with caffeoyl moiety in compounds 1 and 2. Furthermore, the additional attachment of a caffeoyl moiety (compounds 7 and 8) at quinic acid moiety significantly enhance the antioxidant activity than compounds 1 and 2. Therefore, these results considered that the existence of a caffeoyl moiety and the attachment between quinic acid and an additional caffeoyl moiety play important role for the antioxidant activity.

DCFH-DA is one of the most commonly used dyes for the direct detection of intracellular ROS levels. Therefore, we used flow cytometric analysis to measure the effect of compound 8 on the intracellular ROS levels in LPS-stimulated RAW 246.7 cells. As shown in Fig. 3, compound 8 did not show the cytotoxicity at the concentration of 50, 100, and 200 μM. The results displayed that the LPS-treated group shifted rightward compared with LPS non-treated group. However, the compound 8 and ascorbic acid treated groups were shifted at leftward compared with the LPS-treated group. In particular, compound 8 decreased intracellular ROS levels at the concentrate of 50, 100, and 200 μM. Additionally, we presented DCF-DA fluorescence as fold changes in mean fluorescence intensity (MFI) compared to the non-treated group. The compound 8−treated groups significantly decreased MFI compared to the non-treated group, Therefore, compound 8 showed the antioxidant effects against intracellular ROS production.

Fig. 3. Cytotoxicity of 8 at the concentration of 25-200 uM (A); Effect of 8 on intracellular reactive oxygen species production in lipopolysaccharide-treated (LPS-treated) RAW264.7 (B). ^#p < 0.05 vs. the non-treated group; ^*p < 0.05 vs. the sample treated group.

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