Isolation and identification of antioxidant phenylpropanoids from the fruit peel of Akebia trifoliata koidz

Abstract

In the course of screening for antioxidant agricultural products in Shizuoka Prefecture, Japan, we found that Akebia trifoliata Koidz fruit had a high activity. Furthermore, we found that the peel of A. trifoliata showed remarkable antioxidant activity compared to other parts of the fruit. We isolated seven known phenylpropanoids, including caffeoylquinic acids from the peel of A. trifoliata: 5-O-caffeoylquinic acid (1), 4-O-caffeoylquinic acid (2), 3-O-caffeoylquinic acid (3), 4,5-di-O-caffeoylquinic acid (4), 3,5-di-O-caffeoylquinic acid (5), calceolarioside B (6), and (4-hydroxyphenyl)-ethyl-6-O-(E)-caffeoyl-β-d-glucopyranoside (7). These isolated compounds were found to contribute to the antioxidant activity of the peel, with 6 exhibiting the strongest activity amongst them. Quantitative analyses showed that 6 is abundant in the peel of A. trifoliata. Therefore, the peel of A. trifoliata can be effectively used as a functional material with antioxidant activity.

Introduction

Oxidation in foods and biological systems is known to cause damage to food and human health. In particular, biochemical oxidative stresses in cells and tissues induce DNA mutations, protein oxidation, and lipid oxidation, and have been associated with many degenerative diseases such as arteriosclerosis and cancer (Kim et al., 2015; Adwas et al., 2019; Pisoschi et al., 2021). To solve this problem, many studies have focused on investigating novel antioxidant compounds from natural products (Pohl and Lin, 2018), since such compounds are thought to reduce the risk of development of these degenerative diseases. In recent years, much attention has been given to the natural antioxidants in fruits and vegetables. Various fruits and vegetables such as citrus fruits, berries, apples, and leafy greens are known to contain efficient antioxidants (Jideani et al., 2021).

Following this trend, we screened for the antioxidant activity of native crops, vegetables, and fruits produced in Shizuoka Prefecture, Japan (Kosugi et al., 2022). We found that Akebia trifoliata Koidz fruit has high antioxidant activity. A. trifoliata is a perennial tree belonging to the Akebiaceae family. Its fruits are produced from June to September, and are oblong (approximately 8 cm in diameter and 15 cm in length). The color of the fruit changes from yellow to brown and light purple to bluish when it ripens (Mimaki et al., 2003). The dried stem of A. trifoliata is used in Chinese herbal medicines. Some studies have been conducted on the constituents of the stem of A. trifoliata, and triterpenes, phenylpropanoids, and other constituents were reported to have been found there in (Gao and Wang, 2006; Maciąg et al., 2021).

A. trifoliata is cultivated in the western region of Shizuoka Prefecture, and its flesh has long been used locally as food; however, few studies have been conducted on its peel. Therefore, the purpose of this study is to evaluate the function of phenolic compounds in A. trifoliata peels, to determine its effective use as a functional material. In this study, we found antioxidant activity in the ethanol extract of the A. trifoliata peel, and discussed the components that contributed to the antioxidant activity.

Materials and Methods

Materials    5-O-Caffeoylquinic acid (1) and 4-O-caffeoylquinic acid (3) were purchased from Funakoshi (Tokyo, Japan). 3-O-Caffeoylquinic acid (2) and Folin-Ciocalteu reagent were purchased from Kanto Chemicals (Tokyo, Japan). 2,2′-Azobis (2-amidinopropane) dihydrochloride (AAPH), 2,2′-azinobis (3-ethylbenzothiazole-6-sulfonic acid) (ABTS), L-ascorbic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), gallic acid, and 2,4,6-tri-2-pyridil-1,3,5-triazine (TPTZ) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). 4,5-Di-Ocaffeoylquinic acid (4), 3,5-di-O-caffeoylquinic acid (5), and 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid (Trolox) were purchased from Sigma-Aldrich (St. Louis, MO, USA). A. trifoliata fruit samples cultivated in Hamamatsu, Shizuoka, Japan, were collected in October 2018 (Fig. 1). After which they were separated into peel, pulp, and seed and each sample was freeze-dried for 24 h.

Fig. 1.

Picture of the fruit of Akebia trifoliata Koidz.

Total polyphenol contents    The total polyphenol contents (TPC) in the A. trifoliata extract were determined using the Folin-Ciocalteu colorimetric method (Singleton et al., 1999). Each extract sample was dissolved in 50% ethanol and a 200 µg/mL solution was prepared. The solution (50 µL) was mixed with 50 µL of 10% (v/v) Folin-Ciocalteu reagent and 50 µL of 10% (w/v) Na₂CO₃ solution. After incubation for 1 h at room temperature, absorbance was measured at 765 nm. The TPC was expressed as gallic acid equivalents (GAE).

DPPH radical scavenging assay    The DPPH radical scavenging assay was performed according to the method by Okumura et al. (2016), with some modifications. The reaction mixture contained 100 µL of 1.0 mM DPPH in ethanol and 100 µL of the test samples. Absorbance was recorded at 517 nm after 30 min of incubation at room temperature in the dark. The extract samples and isolated compounds were evaluated at concentrations of 6.25–100 µg/mL. Trolox was used as the antioxidant standard. Results are expressed as µmol Trolox equivalent (TE) per gram or half maximal inhibitory concentration (IC₅₀) values for radical scavenging activity.

ABTS assay    The ABTS assay was performed according to the method of Re et al. (1999), with some modifications. ABTS was dissolved in water to a concentration of 7 mM. ABTS radical cation was produced by reacting the ABTS solution with 7.35 mM potassium persulfate at room temperature for 12 h in the dark. This solution was diluted with ethanol prior to use. Trolox was used as an antioxidant standard, and each sample was prepared to 12.5–200 µm and 12.5–100 µg/mL concentrations in 50% ethanol. The sample solutions (30 µL) were mixed with 150 µL of the diluted ABTS reagent, and the absorbance was measured at 735 nm after 5 min of incubation at room temperature in the dark. The results were expressed as µmol TE per gram of IC₅₀ values using the percentage decrease with respect to the control values.

FRAP assay    The ferric ions reducing antioxidant power (FRAP) assay was performed according to the method by Benzie and Strain (1996), with some modifications. The reaction solution contained a 300 mM acetate buffer (pH 3.6), 10 mM TPTZ in 40 mM HCl, and 20 mM ferric chloride in water. The working solution for the FRAP assay was incubated at 37 °C until further use. Each sample was dissolved in 50% ethanol (30 µL) and mixed with 150 µL of the FRAP working solution, and the absorbance was measured at 593 nm after 4 min of incubation at room temperature in the dark. Trolox was used as the antioxidant standard. Each extract and isolated compound were evaluated for antioxidant activity at concentrations of 100 µg/mL and 100 µm, respectively. The results were expressed as µmol TE per gram of the extract or µM TE.

Extraction and isolation of components    Freeze-dried peels of A. trifoliata (13 g) were added to 1 L of 70% ethanol and the mixture was stirred at room temperature for 12 h. The solution was then filtered, and the filtrate was concentrated in vacuum to obtain a crude extract (8.4 g). Part of the extract was used for preparative HPLC to isolate the phenolic components. The HPLC systems were column: Capcell Pak UG120 C18 (5.0 µm, ϕ20 × 250 mm; Osaka Soda, Osaka, Japan), solvent: 0.1% trifluoroacetic acid (TFA) in water: 0.1% TFA in acetonitrile = 90:10 or 85:15 or 78:22, detection wavelength of UV: 280 nm. From these we obtained 1 (5.2 mg), 2 (4.1 mg), 3 (0.5 mg), 4 (1.9 mg), 5 (21.5 mg), 6 (1.9 mg), and 7 (1.9 mg).

Structure determination of components by NMR and MS analyses    Structures of the phenolic compounds isolated by preparative HPLC were determined by NMR and MS. The 1D ¹H (400 MHz) and ¹³C NMR (100 MHz) spectra, and 2D ¹H-¹H COSY, HSQC, and HMBC spectra were recorded on a Bruker AVANCE III 400 spectrometer (Bruker BioSpin, Billerica, MA, USA). The chemical shift values (δ) are reported in ppm, while the coupling constants (J) are reported in Hz. Chemical shifts in ¹H and ¹³C NMR spectra were corrected using the residual solvent signals of methanol-d₄ (δH 3.31, δC 49.0). High-resolution electrospray ionization mass spectra (HR-ESI-MS) were recorded using an Ultimate 3000 liquid chromatography system (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an Orbitrap mass spectrometer (Q-Exactive, Thermo Fisher Scientific). Xcalibur software was used for system control and data analysis (Kurata et al., 2019).

Quantification analysis of isolated compounds using HPLC    Quantification of isolated compounds 1–4 and 6 was performed using HPLC. The 70% ethanol extracts from the peel of A. trifoliata were analyzed using a gradient program with water/acetonitrile (in 0.1% TFA) ratios from 100:0 (0 min) – 0:100 (40 min). The detection wavelength was set at 280 nm to detect the target compounds. The standards used were purchased reagents (1–4) and the isolated compounds (6) to obtain a calibration curve for each compound. The 70% ethanol extracts of the peel of A. trifoliata were analyzed three times, and the standard deviation (SD) was calculated. The limits of detection (LOD) and quantification (LOQ) were determined at signal-to-noise (S/N) ratios of 3 and 10, respectively. A recovery test was performed using the standard addition method.

Statistical analysis    All data are expressed as the mean ± standard deviation. Variants were analyzed using one-way ANOVA followed by Tukey’s test, to test the significance of the differences between means at the 5% level of significance.

Results and Discussion

Total polyphenol contents    The consumption of foods rich in polyphenols has been reported to play a role in the prevention of degenerative diseases such as cancer and cardiovascular diseases (Manach et al., 2004). We divided the peel, pulp, and seed of A. trifoliata collected from Hamamatsu, Shizuoka Prefecture-Japan, and determined their total polyphenol content using the Folin-Ciocalteu coloration method. One gram of each freeze-dried sample was mixed with 10 mL of 70% ethanol for 1 h to obtain extracts. Filtrates from the solutions made from each part of the plant (peel, pulp, and seed) were concentrated in vacuum to obtain 272, 644, and 223 mg of crude extracts, respectively. Table 1 shows the total polyphenol contents of each part. Total polyphenol contents of peel, pulp, and seed of A. trifoliata per one gram of 70% ethanol extracts were 45.7, 5.1, and 4.5 mg GAE, respectively. Therefore, the peels of A. trifoliata are rich in polyphenols.

Table 1. Total polyphenol contents and antioxidant activity of the extracts of each part of Akebia trifoliata Koidz.

Part	Total polyphenol *	Antioxidant activity **
	(mg GAE/g)	DPPH (µmol TE/g)	ABTS (µmol TE/g)	FRAP (µmol TE/g)
Peel	45.7 ± 0.4	228.2 ± 7.8	149.1 ± 1.3	169.6 ± 13.3
Pulp	5.1 ± 0.2	7.9 ± 5.7	15.2 ± 1.2	−1.9 ± 0.1
Seed	4.5 ± 0.1	38.0 ± 7.7	44.1 ± 0.4	4.6 ± 1.6

*  Total polyphenol contents were determined by the Folin-Ciocalteu method.

**  Each value is the mean ± standard deviations (n = 3).

Antioxidant activity of extracts of A. trifoliata    The antioxidant activity of each part of A. trifoliata was evaluated using DPPH, ABTS, and FRAP assays. These assays are simple methods for evaluating antioxidant activity by colorimetry. Table 1 shows the antioxidant activity of each part of A. trifoliata expressed as µmol TE. Peel extracts showed the highest activity in all antioxidant assays. The potent antioxidant activity of the peel is considered to be correlated with its high total polyphenol contents. Other studies have also reported that the antioxidant activity of the fruit peel is stronger than that of its pulp and seed (Singh et al 2002; Guo et al 2003).

Structure determination of isolated compounds    In this study, seven phenolics were isolated by preparative HPLC on the 70% ethanol peel extracts of A. trifoliata. Their structures were determined by MS and NMR analyses (Fig. 2).

Fig. 2.

Structures of the phenylpropanoids isolated from the peels of Akebia trifoliata Koidz: 5-Ocaffeoylquinic aci. (1), 4-O-caffeoylquinic acid (2), 3-O-caffeoylquinic acid (3), 4,5-di-O-caffeoylquinic acid (4), 3,5-di-O-caffeoylquinic acid (5), calceolarioside B (6), and (4-hydroxyphenyl)-ethyl-6-O-(E)-caffeoyl-β-d-glucopyranoside (7).

In the HR-ESI-MS analysis of 1–3, molecular ion peaks at m/z 353.0878 [M − H]⁻, m/z 353.0876 [M − H]⁻, and m/z 353.0875 [M − H]⁻ were obtained for 1, 2 and 3, respectively (Figs. S2, S6, and S10). From these data, 1–3 were found to have the same molecular formula, C₁₆H₁₈O₉ (calculated for C₁₆H₁₇O₉, 353.0873). Compounds 1–3 were deduced to be isomers of each other, as their ¹H NMR profiles were similar (Figs. S3, S7, and S11). Finally, 1–3 were identified as 5-O-caffeoylquinic acid (1), 3-O-caffeoylquinic acid (2), and 4-O-caffeoylquinic acid (3) by comparison with the NMR spectra and HPLC retention times of the authentic compounds. Luo et al. reported the HPLC profile of A. trifoliata peels and the presence of chlorogenic acids (caffeoylquinic acids) in them; however, they did not isolate each compound (Luo et al., 2021). Thus, ours is the first study to isolate 1–3 from A. trifoliata.

Compounds 4 and 5 were isolated as yellow amorphous substances. HR-ESI-MS of 4 and 5 yielded m/z 515.1191 [M − H]⁻ and m/z 515.1181 [M − H]⁻, respectively (Figs. S14 and S17). Their molecular formula was determined to be C₂₅H₂₄O₁₂ by HR-ESI-MS (calculated for C₂₅H₂₃O₁₂, 515.1195). ¹H and ¹³C NMR spectral patterns for 4 and 5 were similar, suggesting that they are isomers of each other. In the ¹H-NMR spectra of 4 and 5 measured in methanol-d₄, signals originating from double bonds or aromatic rings corresponding to 10 protons were observed between δH 6.2–7.6 (Figs. S15 and S18). The integrated values of these signals and the coupling constants suggest the presence of two aromatic rings containing ortho-meta bonds and two double bonds within the structure. Furthermore, from 2D NMR analysis, 4 and 5 were assumed to be dicaffeoylquinic acids. By comparing the NMR patterns and HPLC retention times of the standard compounds isolated in-house from Brazilian propolis (Kumazawa et al., 2003), 4 and 5 were identified as 4,5-di-O-caffeoylquinic acid and 3,5-di-O-caffeoylquinic acid, respectively. According to Sung et al., the dry ripe fruit of A. quinate also contains 4 and 5 (Sung et al., 2015). However, this study represents the first time 4 and 5 are isolated and identified from A. trifoliata peels.

Compound 6 was also identified using MS and NMR analyses. Its molecular formula was determined to be C₂₃H₂₆O₁₁ (observed m/z 477.1403 [M − H]⁻, calculated for C₂₃H₂₅O₁₁, 477.1402) (Fig. S20). The ¹H-NMR spectrum of 6 measured in methanol-d₄ indicates the presence of a double bond or aromatic ring-derived signal corresponding to eight protons observed between δH 6.3–7.6 (Fig. S21). Their δH values: δH 6.62 (d, 8.12), δH 6.67 (d, 1.96), δH 6.78 (d, 8.12), δH 6.87 (d, 8.12, 1.96), and δH 7.03 (s, 1.96) are approximately either 8 or 2 Hz in frequency, suggesting the presence of two aromatic ring protons with ortho and meta coupling. The ¹³CNMR spectrum of 6 showed 23 carbon signals with an aromatic ring-derived signals between δC 116–150, sugarderived signals between δC 64–78, and a carbonyl-derived signal at δC 169.2 (Fig. S22). The conjugated sugar in 6 was determined using thin layer chromatography (TLC) analysis, that is, 6 was hydrolyzed with 2 M HCl at 100 °C for 2 h, and the reaction mixtures were analyzed via TLC (Miyata et al., 2022). The acid hydrolysis of 6 produced the free sugar moiety, which was identified as glucose. Furthermore, in addition to the analysis of 2D NMR such as ¹H-¹H COSY, HSQC, and HMBC spectra, by comparison with spectral data in literature (Liu et al., 2012), 6 was identified as calceolarioside B. Although Wang et al. reported the tentative identification of calceolarioside B from A. trifoliata pericarp using LC-MS/MS analysis (Wang et al., 2019), this compound has not been successfully isolated prior to this study. Here we first isolated and then identified calceolarioside B from A. trifoliata.

The molecular formula of 7 was determined to be C₂₃H₂₆O₁₀ (observed m/z 461.1450 [M − H]⁻, calculated for C₂₃H₂₅O₁₀, 461.1453) (Fig. S24). Although the ¹H-NMR spectrum for 7 was very similar to that of 6, the coupling constant of the doublet signal at δH 6.66 for 7 was 8.52 Hz, whereas that (the signal at δH 6.67) for 6 was 1.96 Hz (Fig. S25). Furthermore, the integration value of δH 6.66 for 7 was 2, whereas that for 6 was 1. From these results, it was determined that the C-3 position of 7 is not substituted by a hydroxyl group, unlike in the case for 6. The sugar moiety in 7 was identified as glucose in a similar manner for 6. Thus, 7 was identified as (4-hydroxyphenyl)-ethyl-6-O-(E)-caffeoyl-β-d-glucopyranoside. Although 7 has previously been identified in the stem of A. trifoliata (Gao and Wang, 2006), this study is the first in which it was isolated from its peel.

Antioxidant activity of isolated compounds    All phenylpropanoids (1–7) isolated from the peel of A. trifoliata were evaluated for antioxidant capacity using DPPH, ABTS, and FRAP assays (Table 2). The purchased standards of 1–5 were used for each assay. l-Ascorbic acid was used as the positive control. DPPH and ABTS assays showed IC₅₀ values for each compound, whereas the FRAP assay showed antioxidant capacity as TE. As shown in Table 2, all the compounds showed antioxidant activity equivalent to or higher than that of the positive control. This high antioxidant activity of caffeoylquinic acids has been previously reported in literature (Liu et al., 2020; Rojas-González et al., 2022).

Table 2. Antioxidant activity of the isolated compounds from the peel of Akebia trifoliata Koidz.

Compound	Antioxidant activity *
	DPPH IC50 (µM)	ABTS IC50 (µM)	FRAP ** (µM TE)
1	26.0 ± 0.4a	27.2 ± 0.6a	96.3 ± 2.3a
2	20.3 ± 2.9b	26.2 ± 0.8a	110.2 ± 1.4a
3	25.5 ± 0.2a	29.6 ± 0.2a	94.3 ± 1.3a
4	12.3 ± 0.2c	11.8 ± 0.1b	234 ± 2.9b
5	11.6 ± 0.2c	17.0 ± 0.4c	138 ± 3.8c
6	12.6 ± 1.9c	12.0 ± 0.3b	218 ± 3.3b
7	23.6 ± 2.1a	30.0 ± 0.3a	70.8 ± 1.2d
L-ascorbic acid	21.5 ± 1.0b	28.0 ± 0.4a	96.2 ± 3.9a

*  Each value is the mean ± standard deviations (n = 3).

Values that are followed by different letters within each column are significantly different (p < 0.05).

**  Each compound was evaluated at 100 µM.

All the compounds isolated in this study had a caffeoyl group, suggesting that the catechol structure contributed to the antioxidant activity. The catechol structure has been reported to exhibit radical scavenging activity by donating hydrogen and oxidizing it to semiquinone and quinone (Bors and Michel, 2002; Choe and Min, 2009). Since this reaction is believed to proceed in the same manner for reactive oxygen species present in living organisms, a high antioxidant capacity of the compounds with catechol moiety in foods is also expected. Among caffeoylquinic acids 1–5, the antioxidant activity of dicaffeoylquinic acids (4 and 5) was higher than that of monocaffeoylquinic acids (1–3). Furthermore, the antioxidant activity of 6 with dicaffeoyl groups was also higher than that of 7 with the monocaffeoyl group. The results showed that the compounds with high antioxidant activity were those with two catechol moieties in their structure, confirming the importance of the catechol moiety in antioxidant activity.

Antioxidant activity of caffeoylquinic acids and their related compounds in cultured cells or animal experiments has also been studied (Li et al., 2018; Liu et al., 2020; Magaña et al., 2021). For example, it has been reported that oxidative stress-induced secretion of interleukin-8 (IL-8) and mRNA expression was significantly inhibited by 5-caffeoylquinic acid in human intestinal epithelial Caco-2 cells (Zhao et al., 2008). Further, 5-caffeoylquinic acid diminished the mRNA expression of macrophage inflammatory protein 2 in a dextran sulfate sodium-induced colitis in mice (Shin et al., 2015). It is believed that the anti-inflammatory mechanism of caffeoylquinic acids is the inhibition of the activation of the protein kinase D (PKD)-nuclear factor kappa-light-chainenhancer of activated B cells (NF-κB)-IL-8 signaling pathway by scavenging intracellular reactive oxygen species (Shin et al., 2017). Hence, caffeoylquinic acids and their related compounds possessing the catechol group might be helpful in contributing to preventing inflammatory diseases.

Quantitative analysis    Quantitative analysis was performed for compounds (1–4 and 6) for which independent peaks were detected using HPLC (Fig. 3). The contents of 5 and 7 could not be determined because of the overlapping of their peaks. The contents are shown as the content per gram of 70% ethanol extract of A. trifoliata peel (Table 3). Values are expressed as the means of triplicate analyses of each sample. A calibration curve was plotted for each compound and tested for linearity. Good linearity was observed for compounds between the peak areas and concentrations over the test range (r > 0.998). LOD and LOQ values for each compound were 0.2 and 0.7 µg/mL, respectively. From the recovery test, the percentage recovery value of 1 was found to be approximately 102%; hence, correction procedures were not performed for these results.

Fig. 3.

HPLC profile for the 70% ethanol extract of Akebia trifoliata Koidz peel.

Table 3. Contents of the isolated compounds in Akebia trifoliata Koidz.

Compound	Contents *
	(mg/g of extract)	(mg/g of dry weight)
1	4.3 ± 1.3a	1.2 ± 0.4a
2	6.3 ± 2.4b	1.8 ± 0.8b
3	2.9 ± 1.0c	0.8 ± 0.3a
4	1.1 ± 0.3d	0.3 ± 0.1c
6	12.3 ± 0.7e	3.4 ± 0.2d

*  Each value is the mean ± standard deviations (n = 3).

Values that are followed by different letters within each column are significantly different (p < 0.05).

Burdock (Arctium lappa L.), which is rich in chlorogenic acids, has been reported to contain approximately 1.7 to 7.9 mg/g dry weight of these compounds (Wang et al., 2001). In addition, apples have been reported to contain 0.2 mg/g of chlorogenic acids in their dry peel (Awad et al., 2000). In comparison with the results from literature, it was confirmed that A. trifoliata peels contain large amounts of chlorogenic acids. These findings suggest that A. trifoliata peels could be a useful source of chlorogenic acids. Similarly, A. trifoliata peels contained high amounts of 6 (3.4 mg/g of dry weight). It has been reported that ash wood is rich in phenolic compounds and 6 is present in 74.8 µg/g of the seasoned wood extract of Fraxinus excelsior (Sanz et al., 2012). Therefore, 6 is also a characteristically abundant compound in A. trifoliata peels.

Conclusions

In this study, seven phenylpropanoids were isolated and identified from the peels of A. trifoliate. The antioxidant activity of the isolated compounds was evaluated using DPPH, ABTS, and FRAP assays, and all showed high antioxidant activity. In particular, 6 had the highest antioxidant activity. Quantitative analyses showed that 6 is abundant in A. trifoliata peels. Therefore, A. trifoliata peels can effectively be used as functional material with antioxidant activity.

Acknowledgements    We thank Shinsuke Noyori, Hamamatsu Fruit Park Tokinosumika, Hamamatsu, Shizuoka Prefecture, Japan, for providing the A. trifoliata fruit samples.

Conflict of interest    There are no conflicts of interest to declare.

Supplemental material

Table S1. Physico-chemical properties of 1.

Appearance	White powder
Molecular formula	C16H18O9
HRESIMS (m/z)	Found : 353.0878 [M − H]−
	Calcd : 353.0873 [M − H]−

Fig. S1.

Structure of 1.

Fig. S2.

HRESI-MS spectrum of 1.

Fig. S3.

¹H NMR spectrum of 1 (400 MHz, CD₃OD).

Fig. S4.

¹H NMR spectrum of 5-O-caffeoylquinic acid standard (400 MHz, CD₃OD).

Table S2. Physico-chemical properties of 2.

Appearance	Yellow amorphous
Molecular formula	C16H18O9
HRESIMS (m/z)	Found : 353.0876 [M − H]−
	Calcd : 353.0873 [M − H]−

Fig. S5.

Structure of 2.

Fig. S6.

HRESI-MS spectrum of 2.

Fig. S7.

¹H NMR spectrum of 2 (400 MHz, CD₃OD).

Fig. S8.

¹H NMR spectrum of 3-O-caffeoylquinic acid standard (400 MHz, CD₃OD).

Table S3. Physico-chemical properties of 3.

Appearance	White powder
Molecular formula	C16H18O9
HRESIMS (m/z)	Found : 353.0875 [M − H]−
	Calcd : 353.0873 [M − H]−

Fig. S9.

Structure of 3.

Fig. S10.

HRESI-MS spectrum of 3.

Fig. S11.

¹H NMR spectrum of 3 (400 MHz, CD₃OD).

Fig. S12.

¹H NMR spectrum of 4-O-caffeoylquinic acid standard (400 MHz, CD₃OD).

Table S4. Physico-chemical properties of 4.

Appearance	Yellow amorphous
Molecular formula	C25H24O12
HRESIMS (m/z)	Found : 515.1191 [M − H]−
	Calcd : 515.1195 [M − H]−

Fig. S13.

Structure of 4.

Fig. S14.

HRESI-MS spectrum of 4.

Fig. S15.

¹H NMR spectra of 4 (A) and 4,5-di-O-caffeoylquinic acid standard (B) (400 MHz, CD₃OD).

Table S5. Physico-chemical properties of 5.

Appearance	Yellow amorphous
Molecular formula	C25H24O12
HRESIMS (m/z)	Found : 515.1188 [M − H]−
	Calcd : 515.1195 [M − H]−

Fig. S16.

Structure of 5.

Fig. S17.

HRESI-MS spectrum of 5.

Fig. S18.

¹H NMR spectra of 5 (A) and 3,5-di-O-caffeoylquinic acid standard (B) (400 MHz, CD₃OD).

Table S6. Physico-chemical properties of 6.

Appearance	Yellow amorphous
Molecular formula	C23H26O11
HRESIMS (m/z)	Found : 477.1403 [M − H]−
	Calcd : 477.1402 [M − H]−

Fig. S19.

Structure of 6.

Fig. S20.

HRESI-MS spectrum of 6.

Fig. S21.

¹H NMR spectrum of 6 (400 MHz, CD₃OD).

Fig. S22.

¹³C NMR spectrum of 6 (100 MHz, CD₃OD).

Table S7. Physico-chemical properties of 7.

Appearance	Yellow amorphous
Molecular formula	C23H25O10
HRESIMS (m/z)	Found : 461.1450 [M − H]−
	Calcd : 461.1453 [M − H]−

Fig. S23.

Structure of 7.

Fig. S24.

HRESI-MS spectrum of 7.

Fig. S25.

¹H NMR spectrum of 7 (400 MHz, CD₃OD).

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