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. 2025 Mar 13;15(1):18. doi: 10.1007/s13659-025-00499-7

Asprecosides A–J, ten new pentacyclic triterpenoid glycosides with cytotoxic activity from the roots of Ilex asprella

Yuwei Wu 1,2, Baihui Zhang 1,2, Wenxian Li 2, Lihua Peng 3, Weilin Qiao 3, Wei Li 1,2,✉, De-an Guo 1,2,✉
PMCID: PMC11906957  PMID: 40080319

Abstract

Phytochemical study of the n-BuOH extract of Ilex asprella resulted in the discovery of ten new pentacyclic triterpenoid glycosides, comprising nine ursane-type glycosides (1−9) and one oleanane-type glycoside (10), along with seven known compounds (11−17). Compound 1 is the first reported 19,22-epoxy ursane triterpenoid glycoside, whereas 4 and 5 are rare examples of ursane triterpenoid glycosides containing a 28,19-lactone group. The structural characterization of these compounds was achieved using spectroscopic and chemical techniques, as well as single-crystal X-ray analysis. Compounds 7, 12, 15, and 17 exhibited moderate cytotoxic activities against H1975 and HCC827 cancer cells.

Graphical abstract

graphic file with name 13659_2025_499_Figa_HTML.jpg

Supplementary Information

The online version contains supplementary material available at 10.1007/s13659-025-00499-7.

Keywords: Ilex asprella, Pentacyclic triterpenoid glycoside, Ursane, Oleanane, Cytotoxic activity

Introduction

Pentacyclic triterpenoid glycosides represent a large class of secondary metabolites predominantly found in higher plants, consisting of diverse triterpenoid aglycones and glycosyl moieties [1, 2]. Common pentacyclic triterpenoid aglycones mainly include oleanane-type, ursane-type, and friedelane-type (6/6/6/6/6 carbocyclic ring system), as well as lupane-type (6/6/6/6/5 carbocyclic ring system) [3]. The structural variability resulting from various functional modifications of triterpenoid aglycones endows these compounds with notable biological properties [4, 5]. In recent years, numerous pentacyclic triterpenoid glycosides exhibiting remarkable bioactivities have been identified, as exemplified by the PTP1B inhibitors gymlatinosides GL2 and GL3 [6], the neuroprotective medicagoside A [7], the β-glucuronidase inhibitor astraoleanoside H [8], and the cytotoxic compound ilekudinoside B n-butyl ester [9].

Ilex asprella (Hook. & Arn.) Champ. ex Benth (Aquifoliaceae) is a deciduous shrub and predominantly distributed in southern regions of China, particularly in Guangdong, Hunan, and Guangxi provinces [10]. The roots of I. asprella are extensively utilized in traditional Chinese medicine to treat of headache, cough, and pharyngitis, etc. [10]. Earlier phytochemical studies indicated that triterpenoids and their glycosides are characteristic components of this plant [11–17], with some exhibiting antiviral and cytotoxic activities. As part of ongoing research to identify structurally diverse terpenoids from medicinal plants [18, 19], nine new ursane triterpenoid glycosides (1−9), one new oleanane glycoside (10), and seven known analogues (11−17) were isolated from the roots of I. asprella (Fig. 1). Compound 1 represents the first 19,22-epoxy ursane triterpenoid glycoside, whereas 4 and 5 are rare ursane triterpenoid glycosides with a 28,19-lactone group. This study presents the isolation, structural characterization, and cytotoxic properties of these triterpenoid glycosides (1−17).

Fig. 1.

Fig. 1

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Chemical structures of compounds 1−17

Results and discussion

Asprecoside A (1) was isolated as colorless needles, and its molecular formula C37H56O10 was determined by the HRESIMS ion peak at m/z 659.3803 [M − H]− (calcd for C37H55O10−, 659.3801). The 1H NMR data of 1 (Table 1) exhibited characteristic signals, including six methyl singlets (δH 0.86, 0.93, 0.99, 1.28, 1.30, and 1.32), a methyl doublet [δH 0.91 (d, J = 6.7 Hz)], a methoxy group [δH 3.76 (s)], seven oxygenated methines [δH 3.42 (1H, dd, J = 11.7, 4.3 Hz), 4.43 (1H, d, J = 5.5 Hz), 5.03 (1H, d, J = 7.8 Hz), 4.11 (1H, dd, J = 9.0, 7.8 Hz), 4.28 (1H, t, J = 9.0 Hz), 4.49 (1H, dd, J = 9.7, 9.0 Hz), and 4.62 (1H, d, J = 9.7 Hz)], and an olefinic proton [δH 5.42 (1H, t, J = 3.3 Hz)]. The 13C NMR and DEPT data (Table 2) of 1 revealed the presence of 37 carbon resonances, including a carboxyl group (δC 178.7), a methoxycarbonyl group (δC 171.2 and 52.4), a trisubstituted double bond (δC 137.3 and 126.6), an anomeric carbon (δC 107.7), an oxygenated sp3 tertiary carbon (δC 90.4), five sp3 quaternary carbon, ten sp3 methines (six oxygenated), eight sp3 methylenes, and seven methyls. The above NMR data indicated that the compound exhibited the primary structural characteristics of ursane triterpenoid glycosides and closely resembled ilexasoside A (14) [13], a previously reported triterpenoid glycoside, except that the sp3 methylene (CH2-22: δH 2.17 and 2.07; δC 38.5) in 14 was replaced by an oxygenated methine group (δH 4.43; δC 82.6) in 1, as confirmed by the 1H−1H COSY correlation between H2-21 and H-22. Additionally, two heavily deshielded O-bearing carbons (C-19: δC 90.4; C-22: δC 82.6) implied the existence of an epoxy bridge connecting C-19 and C-22, which was further substantiated by the HMBC correlation of H-22/C-19. Comprehensive analysis of 2D NMR data enabled the establishment of the gross structure of 1, as illustrated in Fig. 2.

Table 1.

1H NMR data of compounds 1−5 (500 MHz, J in Hz, δ in ppm)

No 1a 2a 3b 4a 5a
1α 0.79, m 0.88, m 1.01, m 0.81, m 0.84, m
1β 1.45, m 1.42, m 1.64, m 1.57, m 1.59, m
2α 2.17, m 2.17, m 1.80, m 2.20, m 2.19, m
2β 1.88, m 1.86, m 1.69, m 1.90, m 1.91, m
3 3.42, dd (11.7, 4.3) 3.42, dd (11.7, 4.3) 3.17, dd (11.6, 4.5) 3.40, dd (11.8, 4.5) 3.39, dd (11.7, 4.3)
5 0.81, m 0.83, br d (10.7) 0.81, br d (10.9) 0.76, m 0.77, br d (11.8)
6α 1.49, m 1.54, m 1.57, m 1.51, m 1.51, m
6β 1.25, m 1.29, m 1.45, m 1.29, m 1.24, m
7α 1.39, m 1.35, m 1.51, m 1.35, m 1.23, m
7β 1.49, m 1.53, m 1.34, m 1.44, m 1.45, m
9 1.58, dd (9.1, 8.3) 1.74, m 1.72, m 1.38, m 1.49, m
11 1.90, m α 1.97, m 1.99, m α 1.41, m α 1.42, m
β 1.90, m β 1.18, m β 1.32, m
12 5.42, t (3.3) 5.58, t (3.3) 5.34, t (3.4) α 1.87, m α 2.09, td (14.7, 5.2)
β 2.58, m β 2.78, br d (14.7)
15α 1.19, m 1.27, m 1.05, m 1.20, m 1.60, m
15β 1.68, m 2.53, td (13.2, 3.0) 1.83, m 1.72, m 1.66, m
16α 2.24, m 1.75, m 1.61, m 1.59, m 2.24, m
16β 2.48, m 2.73, br d (13.2) 1.73, m 2.03, m 2.28, m
18 3.31, s 3.98, br s 3.85, br s
20 1.91, m 2.28, m 2.05, m 2.20 m
21α 1.37, m 1.80, m 1.61, m 1.34, m 1.78, m
21β 2.30, dd (12.4, 8.1) 2.10, dt (12.3, 3.9) 1.66, m 2.02, m 2.32, m
22 4.43, d (5.5) 4.51, dd (11.7, 3.9) α 1.55, m α 1.54, m 4.18, br dd (4.6, 3.1)
β 2.21, td (13.0, 5.3) β 1.71, m
23 1.32, s 1.32, s 1.05, s 1.33, s 1.32, s
24 0.99, s 0.98, s 0.86, s 0.98, s 0.99, s
25 0.86, s 0.82, s 0.98, s 0.78, s 0.83, s
26 0.93, s 1.05, s 0.86, s 0.77, s 0.87, s
27 1.28, s 1.37, s 1.13, s 1.17, s 1.49, s
29 1.30, s a 5.27, s a 5.28, s 1.67, s 1.79, s
b 5.13, s b 5.09, s
30 0.91, d (6.7) 1.18, d (6.4) 1.40, s 0.85, d (7.1) 1.33, d (7.1)
1′ 5.03, d (7.8) 5.02, d (7.8) 4.39, d (7.8) 5.04, d (7.8) 5.04, d (7.8)
2′ 4.11, dd (9.0, 7.8) 4.10, dd (9.0, 7.8) 3.23, dd (9.2, 7.8) 4.12 dd (9.0, 7.8) 4.12, dd (9.0, 7.8)
3′ 4.28, t (9.0) 4.28, t (9.0) 3.35, t (9.2) 4.29, t (9.0) 4.30, t (9.0)
4′ 4.49, dd (9.7, 9.0) 4.49, dd (9.7, 9.0) 3.51, dd (9.8, 9.2) 4.51, dd (9.7, 9.0) 4.51, dd (9.7, 9.0)
5′ 4.62, d (9.7) 4.62, d (9.7) 3.83, d (9.8) 4.63, d (9.7) 4.64, d (9.7)
OMe-6′ 3.76, s 3.76, s 3.77, s 3.76, s 3.76, s
OH-22 6.91, d (4.6)
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aIn pyridine-d5, bin CD3OD

Table 2.

13C NMR data (125 MHz) of compounds 1–10

No 1a 2a 3b 4a 5a 6a 7a 8a 9b 10a
1 39.3 39.1 39.8 39.5 39.5 39.4 39.1 39.6 40.3 38.8
2 27.0 27.0 27.0 27.2 27.1 27.1 27.0 27.1 27.2 26.9
3 89.5 89.5 91.1 89.4 89.4 89.5 89.6 89.5 90.6 89.5
4 39.9 39.9 40.2 40.0 40.0 39.9 39.9 39.9 40.2 39.9
5 56.3 56.3 57.1 56.2 56.1 56.3 56.2 56.3 57.2 56.2
6 18.8 18.8 19.3 18.8 18.8 18.7 19.0 18.8 19.2 19.0
7 34.5 33.7 34.2 35.4 35.9 34.6 33.8 35.7 35.8 33.5
8 39.6 40.1 40.5 42.7 42.4 40.1 40.8 39.9 40.2 40.5
9 48.5 48.3 48.9 52.3 51.8 48.5 48.0 48.6 49.2 48.5
10 37.2 37.3 38.0 37.7 37.7 37.2 37.3 37.1 37.8 37.4
11 24.0 24.2 24.6 22.4 22.1 23.9 24.3 23.6 24.2 24.5
12 126.6 128.8 129.3 26.6 27.7 127.6 128.5 125.4 127.2 124.1
13 137.3 138.2 138.3 137.1 141.3 138.9 140.2 141.1 139.9 144.8
14 42.2 43.8 43.6 43.5 43.5 44.7 43.0 46.4 45.7 42.7
15 27.0 29.1 29.6 28.3 28.6 28.5 29.3 29.5 29.8 29.2
16 28.9 19.7 26.3 27.0 23.6 18.7 19.7 27.5 35.9 21.1
17 59.8 55.5 49.6 48.9 55.2 53.4 54.9 57.4 49.5 53.1
18 55.5 53.4 48.7 133.5 130.5 54.1 55.8 135.0 134.5 46.4
19 90.4 153.5 154.1 91.4 92.2 129.5 72.9 133.1 137.0 81.5
20 44.1 35.8 71.0 40.2 39.6 123.4 41.0 35.1 35.7 36.8
21 37.1 39.6 35.6 27.4 36.8 39.0 36.3 38.0 27.5 38.3
22 82.6 74.7 32.7 32.5 72.8 72.5 75.4 72.3 32.4 72.0
23 28.5 28.5 28.5 28.4 28.4 28.6 28.5 28.6 28.6 28.5
24 17.3 17.3 17.0 17.2 17.2 17.4 17.2 17.4 17.1 17.2
25 16.4 15.9 16.1 17.3 17.2 16.3 15.8 16.6 16.7 15.7
26 17.7 17.5 17.6 19.2 19.0 18.5 17.4 18.8 18.6 17.7
27 21.9 27.1 26.5 25.0 21.9 22.7 25.4 22.6 22.3 25.6
28 178.7 179.3 181.3 178.5 178.2 178.9 180.1 178.7 180.5 180.9
29 16.6 110.9 113.3 23.3 23.4 17.5 27.3 17.5 19.8 26.3
30 19.9 19.7 29.2 15.8 18.5 20.8 17.0 21.4 19.1 29.5
1′ 107.7 107.7 107.1 107.8 107.7 107.7 107.7 107.7 107.4 107.7
2′ 75.8 75.8 75.3 75.8 75.8 75.8 75.8 75.8 75.4 75.8
3′ 78.2 78.3 77.5 78.3 78.3 78.3 78.3 78.3 78.0 78.2
4′ 73.5 73.5 73.2 73.6 73.6 73.5 73.6 73.5 71.2 73.6
5′ 77.5 77.6 76.6 77.7 77.6 77.6 77.6 77.6 66.7 77.6
6′ 171.2 171.2 171.4 171.3 171.3 171.2 171.2 171.2 171.3
OMe 52.4 52.4 52.8 52.4 52.4 52.4 52.4 52.4 52.4
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aIn pyridine-d5, bin CD3OD

Fig. 2.

Fig. 2

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Key 1H−1H COSY and HMBC correlations of 1−10

The relative configuration of 1 was partially elucidated through the analysis of NOESY spectra and coupling constant data. The β-configuration of the glycosyl group was confirmed based on the coupling constant of its anomeric proton (d, J = 7.8 Hz, H-1′). The observed NOESY correlations (Fig. 3) of H-3/H-5, H-5/H-9, H-9/H3-27 revealed that these protons, together with Me-27, were cofacial and were arbitrarily assigned α-orientations. Consequently, the correlations of H-11β/H3-25 and H3-26 indicated that Me-25 and Me-26 were β-oriented. Furthermore, the β-orientations of H-18 and H-20 were supported by the NOE correlations of H-18/H-12 and H-20. Nevertheless, due to the absence of useful NOESY signals, the configurations of the other stereocenters could not be determined. Ultimately, the complete structure of 1, including its absolute configuration, was confirmed through single-crystal X-ray diffraction analysis (Fig. 4), with a Flack parameter of − 0.02(3).

Fig. 3.

Fig. 3

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Key NOESY correlations of 1−10

Fig. 4.

Fig. 4

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X-ray crystal structures of compounds 1 and 2

Asprecoside B (2) was identified with a molecular formula of C37H56O10, based on the HRESIMS data. A comparison of the NMR data of 2 (Tables 1 and 2) with those of 14 revealed key distinctions: the presence of an additional terminal double bond [δH 5.27 and 5.13 (each 1H, s); δC 153.5 and 110.9] in place of the oxygenated sp3 tertiary carbon and a methyl group in 14, along with an oxymethine [δH 4.51 (1H, dd, J = 11.7, 3.9 Hz); δC 74.7] in 2 instead of the methylene in 14. The location of Δ19(29) double bond was confirmed by HMBC, which showed correlations (Fig. 2) from the olefinic protons (δH 5.27 and 5.13, H2-29) to C-18, C-19, and C-20; as well as from H3-30 to C-19. Furthermore, the position of the hydroxy group at C-22 was verified through the HMBC correlations of H-22/C-17 and C-28 and H-18/C-22, alongside the 1H − 1H COSY correlation of H-22/H2-21. The relative configuration of 2 was determined based on analysis of its NOE data. In particular, the NOESY correlations (Fig. 3) of H-18/H-22 and H-22/H-20 indicated the β-configurations of H-20 and H-22. The structure of 2 was further confirmed by single-crystal X-ray diffraction (Fig. 4).

Asprecoside C (3) shared the same molecular formula (C37H56O10) as that of 2, suggesting that they were structural isomers. The 1D NMR spectra of 3 revealed similar structural characteristics found in 2, with the key difference being the hydroxy group relocating from C-22 in 2 to C-20 in 3. This was supported by the HMBC correlations (Fig. 2) from a methyl singlet (δH 1.40, H3-30) to an oxygenated sp3 tertiary carbon (δC 71.0, C-20), as well as the 1H−1H COSY correlation of H2-21/H2-22. The stereochemistry of 3 was assigned to be the same as that of 2 by comparison of their NOE data (Fig. 3).

The molecular formula of asprecoside D (4) was determined to be C37H56O9 based on its HRESIMS data. The 1H and 13C NMR data (Tables 1 and 2) of 4 exhibited a close resemblance to those of 14, except for the presence of a tetrasubstituted double bond instead of the trisubstituted double bond in 14. This suggested that Δ12(13) in 14 shifted to Δ13(18) in 4, which was validated by the HMBC corrections (Fig. 2) from H2-12 and H3-27 to a hydrogen-free sp2 carbon (δC 137.1, C-13) and from H2-12 and H3-29 to the other hydrogen-free sp2 carbon (δC 133.5, C-18). Additionally, the significant downfield shift of C-19 (δC 91.4 in 4; δC 72.6 in 14) and slight upfield shift of C-28 (δC 178.5 in 4; δC 180.8 in 14) suggested that C-19 and C-28 were linked via an O atom, leading to the formation of a five-membered lactone ring. The relative stereochemistry of 4 was established by analysis of its NOESY correlations (Fig. 3).

Asprecoside E (5) exhibited a molecular formula of C37H56O10, containing one additional O atom compared to 4. The 1D NMR spectra of 5 resembled those of 4, except that the sp3 methylene in 4 was substituted by an oxymethine [δH 4.18 (1H, br dd, J = 4.6, 3.1 Hz); δC 72.8], indicating that 5 was a hydroxylated analogue of 4. HMBC correlations (Fig. 2) revealed that the hydroxy group was attached at C-22, as evidenced by signals from OH-22 [δH 6.91, 1H, d (J = 4.6 Hz)] to C-17 and C-22. Especially, the α-orientation of OH-22 was assigned by the NOE correlations (Fig. 3) of OH-22/H3-30 and H-16α as well as H-16α/H3-27.

The molecular formula of asprecoside F (6), C37H56O10, was determined to be identical to that of 2 through HRESIMS analysis ([M − H]− m/z 659.3805). When comparing its 1D NMR data (Tables 2 and 3) of with those of 2, the only structural difference between these two compounds was that Δ19(29) double bond in 2 migrated to Δ19(20) in 6. This was confirmed by the HMBC correlations (Fig. 2) from a vinyl methyl (δH 1.73, H3-29) to C-18, C-19, and C-20 and from the other vinyl methyl (δH 1.70, H3-30) to C-19, C-20, and C-21. Comparison of their NOE data demonstrated that the relative stereochemistry of 6 aligned with that of 2.

Table 3.

1H NMR data of compounds 6−10 (500 MHz, J in Hz, δ in ppm)

No 6a 7a 8a 9b 10a
1α 0.79, m 0.85, m 0.82, m 1.03, m 0.86, m
1β 1.46, m 1.43, m 1.50, m 1.73, m 1.39, m
2α 2.15, m 2.14, m 2.16, m 1.84, m 2.13, br d (11.4)
2β 1.87, m 1.86, m 1.85, m 1.68, m 1.86, m
3 3.40, dd (11.7, 4.3) 3.37, dd (11.7, 4.2) 3.42, dd (11.7, 4.3) 3.15, dd (11.8, 4.1) 3.36, dd (10.8, 2.8)
5 0.80, m 0.84, m 0.83, m 0.81, br d (11.2) 0.81, m
6α 1.49, m 1.50, m 1.49, m 1.55, m 1.50, m
6β 1.27, m 1.34, m 1.30, m 1.39, m 1.33, m
7α 1.58, m 1.63, m 1.62, m 1.58, m 1.53, m
7β 1.44, m 1.42, m 1.51, m 1.50, m 1.37, m
9 1.48, m 1.78, m 1.47, m 1.44, m 1.81, m
11 1.84, m 2.00, m 1.91, m 1.96, m 1.98, m
12 5.66, t (3.3) 5.59, t (3.2) 5.71, t (3.3) 5.38, t (3.8) 5.57, t (3.7)
15α 1.35, m 1.39, m 1.45, m 1.17, dt (13.7, 3.8) 1.37, m
15β 2.71, m 2.51, m 2.69, td (13.2, 4.6) 1.90, td (13.7, 3.8) 2.29, br t (12.1)
16α 2.04, m 3.02, td (13.1, 6.4) 2.18, td (13.0, 4.3) 1.38, m 2.75, m
16β 2.73, m 2.78, br d (13.1) 2.91, br d (13.0) 2.17, m 2.83, m
18 3.74, s 3.08, s 3.70, br s
19 3.63, br s
20 1.72, m 2.58, m 2.19, m
21α 2.67, dd (17.0, 10.7) 1.90, m 1.99, m 1.36, m 2.49, t (12.0)
21β 2.46, dd (17.0, 5.4) 2.49, m 2.29, m 1.83, m 1.73, m
22 4.62, dd (10.7, 5.4) 4.44, dd (11.6, 4.1) 4.83, dd (9.9, 3.2) α 1.63, m 4.62, dd (12.0, 3.8)
β 1.81, m
23 1.31, s 1.31, s 1.33, s 1.06, s 1.31, s
24 0.97, s 0.98, s 0.98, s 0.86, s 0.98, s
25 0.83, s 0.83, s 0.84, s 1.01, s 0.83, s
26 1.06, s 1.12, s 1.12, s 0.94, s 1.06, s
27 1.25, s 1.80, s 1.26, s 1.00, s 1.72, s
29 1.73, s 1.46, s 1.96, s 1.74, s 1.22, s
30 1.70, s 1.19, d (6.6) 1.28, d (7.1) 1.10, d (7.0) 1.26, s
1′ 5.02, d (7.8) 5.01, d (7.8) 5.03, d (7.8) 4.27, d (7.5) 5.02, d (7.7)
2′ 4.10, dd (9.0, 7.8) 4.10, dd (8.7, 7.8) 4.11, dd (9.0, 7.8) 3.18, dd (8.9, 7.5) 4.11, dd (9.0, 7.7)
3′ 4.28, t (9.0) 4.28, t (8.7) 4.28, t (9.0) 3.28, t (8.9) 4.29, t (9.0)
4′ 4.49, dd (9.7, 9.0) 4.50, dd (9.7, 8.7) 4.50, dd (9.7, 9.0) 3.46, ddd (10.1, 8.9, 5.3) 4.49, t (9.6, 9.0)
5′ 4.62, d (9.7) 4.62, d (9.7) 4.63, d (9.7) α 3.19, dd (11.4, 10.1) 4.64, d (9.6)
β 3.82, dd (11.4, 5.3)
OMe-6′ 3.76, s 3.76, s 3.77, s 3.74, s
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aIn pyridine-d5, bin CD3OD

Asprecoside G (7) was assigned a molecular formula of C37H58O11, as determined based on HRESIMS data at m/z 677.3905 [M − H]− (calcd for C37H57O11−, 677.3906). Comparing the NMR data (Tables 2 and 3) of 7 with those of 14 demonstrated their structural resemblance. The primary distinction was the substitution of the sp3 methylene (CH2-22) in 14 with an oxymethine [δH 4.44 (1H, dd, J = 11.6, 4.1 Hz); δC 75.4] in 7, indicating that 7 was a 22-hydroxylated derivative of 14. This was corroborated by the analysis of 2D NMR data (Fig. 2), especially the 1H−1H COSY correlation of the H2-21/H-22. The β-configuration of H-22 was established based on the NOE correlation between H-18 and H-22 (Fig. 3).

Asprecoside H (8) showed a molecular formula of C37H56O10, as proved by the HRESIMS data. Its 1D NMR data resembled those of 7, except for the presence of a tetrasubstituted double bond (δC 135.0 and 133.1) in 8 replacing the sp3 methine at C-18 (δC 55.8) and the oxygenated sp3 tertiary carbon at C-19 (δC 72.9) in 7. This suggested that 8 was a C-18 − C-19 dehydrated derivative of 7, and was further confirmed by the HMBC correlations (Fig. 2) from a vinyl methyl (δH 1.96, H3-29) to C-18 and C-19. The similar NOE correlations observed in 8 and 7 (Fig. 3) indicated that 8 shared the same relative stereochemistry as 7.

The molecular formula of asprecoside I (9) was determined as C35H54O7 by its HRESIMS data at m/z 585.3799 [M − H]− (C35H53O7−, 585.3797). Comparison of its NMR data (Tables 2 and 3) with those of known compound (13) [20] revealed that they shared the same aglycone moiety but a different sugar unit. In the 1H and 13C NMR spectra of 9, the chemical shifts [δH 4.27 (1H, d, J = 7.5 Hz), 3.82 (1H, dd, J = 11.4, 5.3 Hz), 3.46 (1H, ddd, J = 10.1, 8.9, 5.3 Hz), 3.28 (1H, t, J = 8.9 Hz), 3.19 (1H, dd, J = 11.4, 10.1 Hz), and 3.18 (1H, dd, J = 8.9, 7.5 Hz); δC 107.4, 78.0, 75.4, 71.2, and 66.7] indicated the presence of a β-xylosyl group, which was attached to C-3 of the aglycone by the HMBC correlation (Fig. 2) from H-1′ (δH 4.27) to C-3 (δC 90.6). Compound 9 shared the same relative stereochemistry with that of 13 regarding the aglycone moiety, by comparing their 1D NMR and NOESY data, especially the NOE correlations of H-1/H-3, H-3/H-5, H-5/H-9, H-9/H3-27, H3-25/H-11β, H-11β/H3-26. The relative configuration of the chiral center C-20 was specifically determined by quantum chemical calculations of the 1D NMR chemical shifts of aglycone moiety in 9, for both the 20S*-isomer and 20R*-isomer. The improved probability DP4 + analysis of the experimental and calculated 1D NMR data yielded the final score, with 20S*-isomer (100.00%) showing an absolute advantage over 20R*-isomer (0.00%), suggesting a 20S*-configuration for compound 9.

Asprecoside J (10) exhibited an [M − H]− ion peak at m/z 677.3898 (C37H57O11−, 677.3906), indicating a molecular formula of C37H58O11. The NMR data (Tables 2 and 3) of 10 bore a resemblance to those of known oleanane glycoside (11) [21]. The key difference was the substitution of the sp3 methylene in 11 by an oxymethine [δH 4.62 (1H, dd, J = 12.0, 3.8 Hz); δC 72.0] in 10, identifying 10 as a hydroxylated derivative of 11. The HMBC correlations (Fig. 2) of H-22/C-20, C-21, and C-28, along with 1H−1H COSY correlation of H-22/H2-21 confirmed the position of the hydroxyl group at C-22. The relative stereochemistry of 10 was elucidated through comparison of its 1D NMR data and NOE correlations with those of 11. Notably, the β-orientation of H-22 was verified by the observed NOE correlations of H-18/H3-30 and H3-30/H-22.

To ascertain the absolute configurations of the glycosyl moiety in 1−10, HPLC sugar analysis following acid hydrolysis and thiazolidine thiocarbamoyl derivatization was conducted [22]. The results demonstrated the occurrence of a D-xylosyl group in 9 and a D-glucuronosyl group in 1−8 and 10.

The known compounds were identified as 19α-hydroxy oleanolic acid 3-O-β-D-glucuronopyranoside-6′-O-methyl ester (11) [21], ilexoside A (12) [23], 3β-hydroxy-20-epi-ursa-12,18-dien-28-oic acid 3-O-β-D-glucuronopyranoside-6′-O-methyl ester (13) [20], ilexasoside A (14) [13], ilexoside B (15) [24], 3β-hydroxyursa-12,18-dien-28-oic acid 3-O-β-D-glucuronopyranoside-6′-O-methyl ester (16) [25], and ilexasprellanoside A (17) [14], through comparison of their NMR data with reported values.

Compounds 1−17 exhibited similar structures, especially the triterpenoid aglycone moiety with diverse functional groups. Based on the well-known triterpenoids, oleanolic acid and ursolic acid, the proposed biosynthetic pathways and structural relationships of 1−17 are shown in Additional file 1: Fig S1.

The cytotoxic activities of 1−17 against H1975 and HCC827 cells were evaluated using the CCK-8 method, with gefitinib serving as the positive control. Apart from four compounds (7, 12, 15, and 17) showing moderate cytotoxicity with IC50 values ranging from 10.85 to 48.41 μM, the other compounds did not display obvious activity (IC50 > 50 μM). Among these, compound 12 was identified as the most potent, exhibiting cytotoxicity against H1975 cells (IC50 = 10.85 ± 0.34 μM).

Experimental section

General experimental procedures

Optical rotations were determined using a Rudolph Autopol I automatic polarimeter, and X-ray crystal crystallographic data were collected on an Agilent Xcalibur Nova X-ray diffractometer. Melting points were measured on an X-4 melting instrument. UV and IR spectra were recorded using a Shimadzu UV-2600i spectrophotometer and a Bruker Tensor 37 infrared spectrophotometer, respectively. 1D and 2D NMR spectra acquired on a Bruker AM-500 spectrometer at 25 °C. HR-ESI–MS was performed using a Waters Micromass Q-TOF spectrometer. Semipreparative HPLC was performed on a Shimadzu LC-40B XR liquid chromatograph equipped with a YMC-Pack ODS-A column (250 mm × 10 mm, 5 μm). Silica gel (200 − 300 mesh, Qingdao Marine Chemical, Inc, Qingdao, China), Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Sweden), and reversed-phase C18 (RP-C18) silica gel (50 μm, Quebec City Canada) were employed for column chromatography (CC).

Plant material

The roots of I. asprella were collected in April 2023 from Hezhou City, Guangxi Zhuang Autonomous Region, China. The plants were authenticated by one of the authors (W. Li) and a voucher specimen (No. GM202304) has been deposited in the Zhongshan Institute for Drug Discovery.

Extraction and isolation

The dried roots of I. asprella (20 kg) were crushed and subjected to reflux extraction using 65% EtOH for three times (3 × 100 L, 2 h each), producing a crude extract (1.6 kg). The extract was suspended in hot water (2.0 L) and successively partitioned with EtOAc (3 × 3.0 L) and n-BuOH (3 × 3.0 L), yielding EtOAc (105 g) and n-BuOH (303 g) fractions.

The n-BuOH extract was chromatographed on a silica gel column using a gradient of petroleum ether (PE)/EtOAc/MeOH solvent system (5:1:0 → 0:0:1), yielding six fractions (Frs. A − F). Fr. C (24.8 g) was processed on an ODS gel column (MeOH/H2O, 3:7 → 1:0) to afford four sub-fractions (Frs. C1 − C4). Fr. C2 (2.8 g) was separated on a Sephadex LH-20 column (MeOH) and further purified by semipreparative RP-HPLC with a YMC-park ODS-A column (CH3CN/H2O, 45:55, 3 mL/min) to obtain compounds 15 (17.5 mg, tR 38 min), 9 (16.6 mg, tR 41 min), 13 (2.7 mg, tR 43 min), and 17 (7.5 mg, tR 33 min). Fr. D (33.5 g) was subjected to repeated silica gel column chromatography with CH2Cl2/MeOH (100:1 → 0:1), affording five fractions (Frs. D1–D5). Fr. D4 (778.2 mg) was further separated using a Sephadex LH-20 column (MeOH), resulting in five subfractions (Frs. D4a–Fr. D4e). Fr. D4b (94.1 mg) was purified via HPLC (MeOH/H2O, 77:23, 2 mL/min) to yield 5 (1.9 mg, tR 28 min), 11 (7.2 mg, tR 34 min), and 12 (10.7 mg, tR 35 min). Similarly, Fr. D4c (39.1 mg) was processed using HPLC (CH3CN/H2O, 43:57, 2 mL/min) to yield 1 (19.7 mg, tR 50 min). Fr. D4e (88.5 mg) was also purified using HPLC (CH3CN/H2O, 52:48, 2 mL/min) to afford 3 (4.1 mg, tR 12 min), 14 (15.5 mg, tR 15 min), and 16 (15.7 mg, tR 17 min). Chromatography of Fr. D5 on an ODS gel column (MeOH/H2O, 1:1 → 1:0) produced six fractions (Fr. D5a–D5f). Fr. D5b (17.5 mg) was further refined using HPLC (CH3CN/H2O, 30:70, 2 mL/min) to isolate 7 (5.7 mg, tR 64 min) and 10 (9.6 mg, tR 74min). Compounds 8 (8.0 mg, tR 18 min), 4 (5.6 mg, tR 21 min), and 6 (12.7 mg, tR 22 min) were purified from Fr. D5c (50.5 mg) by HPLC (CH3CN/H2O, 45:55, 2 mL/min), and 2 (11.7 mg, tR 55 min) was obtained from Fr. D5f (24.2 mg) by HPLC (CH3CN/H2O, 38:62, 2 mL/min).

Spectroscopic data of compounds

Asprecoside A (1)

Colorless needles; [α]20D −16 (c 0.1, CH3CN); IR (KBr) νmax 3417, 2950, 1738, 1444, 1374, 1238, 1092, 1045, 1027, 982 cm−1; 1H and 13C NMR data (Tables 1 and 2); HRESIMS m/z 659.3803 [M − H]− (calcd for C37H55O10−, 659.3801).

Asprecoside B (2)

Colorless needles; [α]20D −12 (c 0.1, CH3CN); IR (KBr) νmax 3424, 2925, 1738, 1458, 1376, 1168, 1051, 1025, 910 cm−1; 1H and 13C NMR data (Tables 1 and 2); HRESIMS m/z 659.3799 [M − H]− (calcd for C37H55O10−, 659.3801).

Asprecoside C (3)

White amorphous powder; [α]20D −21 (c 0.1, MeOH); IR (KBr) νmax 3434, 2948, 1737, 1452, 1387, 1227, 1171, 1046 cm−1; 1H and 13C NMR data (Tables 1 and 2); HRESIMS m/z 659.3816 [M − H]− (calcd for C37H55O10−, 659.3801).

Asprecoside D (4)

White amorphous powder; [α]20D −11 (c 0.1, MeOH); IR (KBr) νmax 3424, 2943, 1747, 1456, 1373, 1167, 1053 cm−1; 1H and 13C NMR data (Tables 1 and 2); HRESIMS m/z 689.3907 [M + HCOO]− (calcd for C38H57O11−, 689.3906).

Asprecoside E (5)

White amorphous powder; [α]20D −14 (c 0.1, MeOH); IR (KBr) νmax 3441, 2925, 1743, 1456, 1376, 1049 cm−1; 1H and 13C NMR data (Tables 1 and 2); HRESIMS m/z 705.3857 [M + HCOO]− (calcd for C38H57O12− 705.3856).

Asprecoside F (6)

White amorphous powder; [α]20D −25 (c 0.1, CH3CN); IR (KBr) νmax 3423, 2925, 1739, 1441,1374, 1244, 1164, 1046, 1025, 914 cm−1; 1H and 13C NMR data (Tables 2 and 3); HRESIMS m/z 659.3805 [M − H]− (calcd for C37H55O10−, 659.3801).

Asprecoside G (7)

White amorphous powder; [α]20D −69 (c 0.1, CH3CN); IR (KBr) νmax 3423, 2925, 1738, 1443, 1375, 1249, 1166, 1048, 1026, 982 cm−1; 1H and 13C NMR data (Tables 2 and 3); HRESIMS m/z 677.3905 [M − H]− (calcd for C37H57O11−, 677.3906).

Asprecoside H (8)

White amorphous powder; [α]20D +18 (c 0.1, CH3CN); UV (CH3CN) λmax (log ε) 225 (3.97) nm; IR (KBr) νmax 3417, 2924, 1738, 1442, 1372, 1238, 1166, 1084, 1044 cm−1; 1H and 13C NMR data (Tables 2 and 3); HRESIMS m/z 659.3817 [M − H]− (calcd for C37H55O10−, 659.3801).

Asprecoside I (9)

White amorphous powder; [α]20D +14 (c 0.1, CH3CN); UV (CH3CN) λmax (log ε) 226 (3.99) nm; IR (KBr) νmax 3414, 2937, 2873, 1696, 1460, 1372, 1239, 1163, 1043 cm−1; 1H and 13C NMR data (Tables 2 and 3); HRESIMS m/z 585.3799 [M − H]− (calcd for C35H53O7−, 585.3797).

Asprecoside J (10)

White amorphous powder; [α]20D −13 (c 0.1, CH3CN); IR (KBr) νmax 3418, 2924, 1729, 1443, 1375, 1251, 1168, 1045, 1023, 983 cm−1; 1H and 13C NMR data (Tables 2 and 3); HRESIMS m/z 677.3898 [M − H]− (calcd for C37H57O11−, 677.3906).

X-ray crystallographic data of 1 and 2

The X-ray crystallographic data of asprecosides A (1) and B (2) have been deposited at the Cambridge Crystallographic Data Centre, with the following CCDC deposition numbers: 2413613 (1) and 2,413,615 (2).

Crystallographic data of asprecoside A (1)

C37H56O10⋅4(H2O) (M = 732.88 g/mol): monoclinic, space group P21 (no. 4), a = 13.2729(3) Å, b = 7.0751(2) Å, c = 20.4063(5) Å, β = 96.8560(10)°, V = 1902.59(8) Å3, Z = 2, T = 170.00 K, μ(CuKα) = 0.801 mm−1, Dcalc = 1.279 g/cm3, 22,369 reflections measured (6.708° ≤ 2Θ ≤ 133.79°), 6424 unique (Rint = 0.0391, Rsigma = 0.0367) which were used in all calculations. The final R1 was 0.0387 (I > 2σ(I)) and wR2 was 0.1039 (all data). Flack parameter =  − 0.02(3).

Crystallographic data of asprecoside B (2)

C37H56O10 (M = 660.81 g/mol): monoclinic, space group P21 (no. 4), a = 7.0183(2) Å, b = 26.3891(6) Å, c = 23.1192(5) Å, β = 98.73°, V = 4232.22(18) Å3, Z = 4, T = 170.15 K, μ(CuKα) = 0.605 mm−1, Dcalc = 1.037 g/cm3, 12,902 reflections measured (5.116° ≤ 2Θ ≤ 133.192°), 12,902 unique (Rsigma = 0.0806) which were used in all calculations. The final R1 was 0.0631 (I > 2σ(I)) and wR2 was 0.1789 (all data). Flack parameter = 0.09(4).

Acid hydrolysis of asprecosides A − J (1−10)

Each compound (1 mg) was heated under refluxed in 1.5 mL of 2 M HCl (dioxane/H2O, 1:1) at 90 °C for 4 h. Following hydrolysis, 5 mL of water was added, followed by extraction with EtOAc. The aqueous layer was evaporated under vacuum and the residue was dissolved in anhydrous pyridine (400 μL), followed by the addition of 2 mg of L-cysteine methyl ester hydrochloride. After stirring the mixture at 60 °C for 1 h, 50 μL of o-tolyl isothiocyanate was added, and the reaction continued at the same temperature for an additional hour. The authentic samples D-xylose and D-glucuronic acid underwent identical treatment and were analyzed by reversed-phase HPLC. D-xylose (2 mL/min, MeCN/H2O, 25:75, tR = 51.7 min) was identified in 9, whereas D-glucuronic acid (2 mL/min, MeCN/H2O, 50:50, tR = 25.2 min) was observed in 1−8 and 10.

Cell culture

H1975 and HCC827 cells, sourced from the American Type Culture Collection, were cultured in RPMI 1640 medium (Gibco BRL, USA) containing 10% fetal bovine serum (FBS). These cells were grown under conditions of 5% CO2 and 37 °C.

Cell viability assay

Cell viability was evaluated through the Cell Counting Kit-8 (CCK-8) assay. In brief, Briefly, H1975 and HCC827 cells were seeded into 96-well plates (1× 103 cells/well) and incubated for 24 h. Then, the attached cells were treated with different concentrations of compounds. Following a 3-days incubation, CCK-8 reagent (Dojindo) was added, and luminescence was measured according to the manufacturer’s protocol.

Supplementary Information

Additional file 1. (5.8MB, pdf)

Acknowledgements

This work was supported by the Qi-Huang Chief Scientist Project of the National Administration of Traditional Chinese Medicine (2020).

Author contributions

Yuwei Wu: investigation, methodology, and writing of original draft; Baihui Zhang and Wenxian Li: methodology; Lihua Peng and Welin Qiao: resources; Wei Li: data curation, revised the manuscript, and supervision; De-an Guo: supervision and project administration. All authors read and approved the final manuscript.

Availability of data and materials

All data generated and analyzed during this study are included in this published article and its supplementary information file.

Declarations

Competing interests

The authors declare that there are no conflicts of interest associated with this work.

Footnotes

Publisher's Note

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Contributor Information

Wei Li, Email: liwei1@simm.ac.cn.

De-an Guo, Email: daguo@simm.ac.cn.

References

  • 1.Lacaille-Dubois MA, Wagner H. New perspectives for natural triterpene glycosides as potential adjuvants. Phytomedicine. 2017;37:49–57. [DOI] [PubMed] [Google Scholar]
  • 2.Vincken JP, Heng L, De Groot A, Gruppen H. Saponins, classification and occurrence in the plant kingdom. Phytochemistry. 2007;68:275–97. [DOI] [PubMed] [Google Scholar]
  • 3.Li Y, Wang J, Li L, Song W, Li M, Hua X, Wang Y, Yuan J, Xue Z. Natural products of pentacyclic triterpenoids: from discovery to heterologous biosynthesis. Nat Prod Rep. 2023;40:1303–53. [DOI] [PubMed] [Google Scholar]
  • 4.Dzubak P, Hajduch M, Vydra D, Hustova A, Kvasnica M, Biedermann D, Markova L, Urban M, Sarek J. Pharmacological activities of natural triterpenoids and their therapeutic implications. Nat Prod Rep. 2006;23:394–411. [DOI] [PubMed] [Google Scholar]
  • 5.Lv N, Wang L, Zeng M, Wang Y, Yu B, Zeng W, Jiang X, Suo Y. Saponins as therapeutic candidates for atherosclerosis. Phytother Res. 2024;38:1651–80. [DOI] [PubMed] [Google Scholar]
  • 6.Pham HTT, Ryu B, Cho HM, Lee BW, Yang WY, Park EJ, Tran VO, Oh WK. Oleanane hemiacetal glycosides from Gymnema latifolium and their inhibitory effects on protein tyrosine phosphatase 1B. Phytochemistry. 2020;170: 112181. [DOI] [PubMed] [Google Scholar]
  • 7.Liu XG, Sun YQ, Bian J, Han T, Yue DD, Li DQ, Gao PY. Neuroprotective effects of triterpenoid saponins from Medicago sativa L. against H2O2-induced oxidative stress in SH-SY5Y cells. Bioorg Chem. 2019;83:468–76. [DOI] [PubMed] [Google Scholar]
  • 8.Nguyen MK, Nguyen VP, Yang SY, Min BS, Kim JA. Astraoleanosides E−P, oleanane-type triterpenoid saponins from the aerial parts of Astragalus membranaceus Bunge and their β-glucuronidase inhibitory activity. Bioorg Chem. 2024;145: 107230. [DOI] [PubMed] [Google Scholar]
  • 9.Yang XW, Dai Z, Wang B, Liu YP, Zhao XD, Luo XD. Antitumor triterpenoid saponin from the fruits of Avicennia marina. Nat Prod Bioprospect. 2018;8:347–53. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Yang X, Gao X, Du B, Zhao F, Feng X, Zhang H, Zhu Z, Xing J, Han Z, Tu P, Chai X. Ilex asprella aqueous extracts exert in vivo anti-inflammatory effects by regulating the NF-κB, JAK2/STAT3, and MAPK signaling pathways. J Ethnopharmacol. 2018;225:234–43. [DOI] [PubMed] [Google Scholar]
  • 11.Bai C, Zhou X, Han L, Yu Y, Li N, Zhang M, Qu Z, Tu P. Two new 18, 19-seco triterpenoids from Ilex asprella (Hook. et Arn.) Champ. ex Benth. Fitoterapia. 2018;127:42–6. [DOI] [PubMed] [Google Scholar]
  • 12.Wen Q, Lu Y, Chao Z, Chen DF. Anticomplement triterpenoids from the roots of Ilex asprella. Bioorg Med Chem Lett. 2017;27:880–6. [DOI] [PubMed] [Google Scholar]
  • 13.Wang L, Cai Y, Zhang XQ, Fan CL, Zhang QW, Lai XP, Ye WC. New triterpenoid glycosides from the roots of Ilex asprella. Carbohydr Res. 2012;349:39–43. [DOI] [PubMed] [Google Scholar]
  • 14.Lei Y, Shi SP, Song YL, Bi D, Tu PF. Triterpene saponins from the roots of Ilex asprella. Chem Biodivers. 2014;11:767–75. [DOI] [PubMed] [Google Scholar]
  • 15.Huang XJ, Wen S, Guan XF, Wu ZL, Li MM, Fan CL, Zhang QW, Chan G, Wang Y, Ye WC. Eleven new triterpenoid glycosides from the roots of Ilex asprella. Chem Biodivers. 2019;16: e1900202. [DOI] [PubMed] [Google Scholar]
  • 16.Kashiwada Y, Zhang DC, Chen YP, Cheng CM, Chen HT, Chang HC, Chang JJ, Lee KH. Anti-tumor agents, 145. Cytotoxic asprellic acids A and C and asprellic acid B, new p-coumaroyl triterpenes, from Ilex asprella. J Nat Prod. 1993;56:2077–82. [DOI] [PubMed] [Google Scholar]
  • 17.Zhou M, Xu M, Ma XX, Zheng K, Yang K, Yang CR, Wang YF, Zhang YJ. Antiviral triterpenoid saponins from the roots of Ilex asprella. Planta Med. 2012;78:1702–5. [DOI] [PubMed] [Google Scholar]
  • 18.Li W, Gan L, Yuan FY, Sang J, Huang D, Yin AP, Pu R, Tang GH, Guo D, Yin S. Biscroyunoid A, an anti-hepatic fibrotic 19-nor-clerodane diterpenoid dimer with a C-16−C-12′ linkage from Croton yunnanensis. J Nat Prod. 2023;86:434–9. [DOI] [PubMed] [Google Scholar]
  • 19.Wang D, Mei F, Nie J, Li Z, Zhang D, Guo D, Li W. Highly oxygenated clerodane furanoditerpenoids from the leaves and twigs of Croton yunnanensis. Chin J Nat Med. 2024;22:945–54. [DOI] [PubMed] [Google Scholar]
  • 20.Li S, Zhao J, Wang W, Lu Y, Xu Q, Liu Y, Li X, Khan IA, Yang S. Seven new triterpenoids from the aerial parts of Ilex cornuta and protective effects against H2O2-induced myocardial cell injury. Phytochem Lett. 2015;14:178–84. [Google Scholar]
  • 21.Liao L, Zhou X, Liu YL, Xu QM, Li XR, Yang SL. Four new triterpenoidal saponins from Ilex cornuta and their cytotoxic activities. Phytochem Lett. 2013;6:429–34. [Google Scholar]
  • 22.Yan XL, Sang J, Zhang X, Lin Y, Long QD, Zhu QF, Liao SG. Euphorboside A, a cytotoxic meroterpenoid glycoside with an unusual humulene-phloroglucinol skeleton from Euphorbia kansuensis. Fitoterapia. 2021;153: 104966. [DOI] [PubMed] [Google Scholar]
  • 23.Inada A, Kobayashi M, Murata H, Nakanishi T. Two new triterpenoid glycosides from leaves of Ilex chinensis Sims. Chem Pharm Bull. 1987;35:841–5. [Google Scholar]
  • 24.Cai Y, Zhang Q, Li Z, Fan C, Wang L, Zhang X, Ye W. Chemical constituents from roots of Ilex asprella. Chin Tradit Herb Drugs. 2010;41:1426–9. [Google Scholar]
  • 25.Li SS, Liu YL, Xu QM, Mao CM, Yang SL. Three new triterpenoids isolated from the aerial parts of Ilex cornuta and protective effects against H2O2-induced myocardial cell injury. Chin J Nat Med. 2017;15:115–20. [DOI] [PubMed] [Google Scholar]

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Supplementary Materials

Additional file 1. (5.8MB, pdf)

Data Availability Statement

All data generated and analyzed during this study are included in this published article and its supplementary information file.

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