Five novel triterpenoid saponins from Hovenia dulcis and their Nrf2 inhibitory activities

1 Introduction

Hovenia dulcis Thunb. belongs to the genus of Hovenia, family of Rhamnaceae, widely grown in East Asia conutries, such as China, India and Japan, Korea (Sinicae Agendae Academiae Sinicae Edita, 1982). The seeds of H. dulcis, named as "Zhijuzi", had been used to relieve the thirst, damp heat and drunk in China with a long history (Hyun et al., 2010; Nanjing University of Chinese Medicine, 2006). Modern pharmacology research showed the crude extracts and pure compounds from Hovenia showed hepatoprotective, anti-viral, anti-hyperuricemia, anti-alcoholism and anti-tumor activities (Hase et al., 1997; Yu and Xu, 2019; Xu et al., 2020; Song et al., 2016; Zhou et al., 2013). Phytochemical works indicated that triterpenoid saponins, flavonoids, polysaccharides and phenylpropanoids were the main types constituents in this plant (Zhou et al., 2013; Xu et al., 2012; Wang et al.,2012; Kang et al.,2017; Xu et al., 2011; Zhang et al., 2012).

The nuclear factor-erythroid 2-related factor (Nrf2) is the transcription factor with redox-sensitive property, that participates the cellular oxidative stress response. The Nrf2 expressed in low levels in normal tissues but in high levels in tumors (Chen et al., 2009). The activated Nrf2 in tumor tissues not only enhances tumor survival but also increases the resistance to anticancer drugs. The natural chemical Nrf2 inhibitors have been reported the anticancer effects through suppressing cancer cells proliferation or sensitizing the cells to chemo- and radiotherapy (Lin et al., 2020). As a part of our ongoing program to discover more good biological antitumor drugs, five novel triterpenoid saponins hovenidulciosides C−G (1–5), together with one known analogue hovenidulcioside A₁ (6) were obtained from the seeds of H. dulcis. All structures were elucudated by the comprehensive spectroscopic data analysis and the Nrf2 inhibitory activities were tested by luciferase reporter gene assay and western blotting analysis.

2 Materials and methods

3 Results and discussion

The seeds of H. dulcis were extracted with 65% EtOH and then separated by D101 macroporous resin column. The 50% ethanol– H₂O fraction was chromatographed on various column chromatographic separations and afforded 6 triterpenoid saponins including five new ones (1–5) (seeFig. 1).

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Fig. 1

Structures of compounds 1–6.

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Compound 1 was white amorphous powder with formula C₅₁H₈₂O₂₁ by the negative HR-ESI-MS peaks [M + CH₂O₂-H]⁻ at m/z 1075.5319 (C₅₂H₈₃O₂₃, calcd for 1075.5320) and [M−H]⁻ at m/z 1029.5261 (C₅₁H₈₁O₂₁, calcd for 1029.5265). The IR spectrum displayed the presence of the OH (3358 cm⁻¹) and C⚌C (1635 cm⁻¹). The ¹H NMR spectrum showed signals for seven methyl groups [δ_H 0.85 (3H, s, H₃-21), 0.88 (3H, s, H₃-19), 1.04 (3H, s, H₃-28), 1.14 (6H, s, H₃-18, 21), 1.69 (3H, s, H₃-27), 1.72 (3H, s, H₃-26)], two oxygenated methylene protons [(4.02 (1H, d, J = 7.8 Hz), 3.93 (1H, m), H-30], two oxygenated methine protons [δ_H 3.05 (1H, dd, J = 11.4, 3.6 Hz, H-3), 4.68 (1H, d, J = 7.2 Hz, H-23) ], one olefinic proton δ_H 5.16 (1H, d, J = 7.8 Hz, H-24) and four anomeric protons [δ_H 4.33 (1H, d, J = 7.8 Hz, H-1′ in Ara), 4.85 (1H, m, overlapped, H-1″ in Glc), 4.84 (1H, d, J = 8.4 Hz, H-1″′ in Ara), 4.68 (1H, d, J = 7.8 Hz, H-1″″ in Xyl)]. There were 51 carbon signals, including 7 methyls, 13 methylenes, 23 methines, 6 quaternary carbons and 2 olefinic carbon signals according to ¹³C NMR and DEPT spectra. The hydrogen signals were assigned to the relevant carbon atoms though the HSQC data. All the above data indicated that 1 had a triterpenoid skeleton with four sugar residues. Comparison of the NMR spectral data of 1 with those of the known compounds isolated from this species, revealed that the sapogenin of 1 was jujubogenin (Kimura et al., 1981). ROESY correlations between H-3 (δ_H 3.05) and H-1′ (δ_H 4.33), H-5 (δ_H 0.72), between H-23 (δ_H 4.68) and H₃-18 (δ_H 1.14), indicated the β configuration at C-3 and C-23 (Fig. 2). Acid hydrolysis of 1 by 2 N HCl and gas chromatography analysis of the silyl derivatives of sugars obtained D-glucose, L-arabose and L-xyloside. The sugar chain and glycosylation position could be deduced by HMBC correlations between δ_H 4.68 (H-1″″ in Xyl) and δ_C 84.3 (C-3″′ in Ara), between δ_H 4.85 (H-1″ in Glc) and δ_C 77.0 (C-2′ in Ara), between δ_H 4.84 (H-1″′ in Ara) and δ_C 82.0 (C-3′ in Ara), and between δ_H 4.33 (H-1′ in Ara) and δ_C 91.0 (C-3) of aglycone (Fig. 2). The assignment of proton signals in the spin system of each sugar residue was determined by the correlations in TOCSY spectrum. Therefore, 1 was elucidated as 3-O-{β-D-xylopyranosyl (1 → 3)- α-L-arabinopyranosyl (1 → 3)- [β-D-glucopyranosyl (1 → 2)]-α-L-arabinopyranosyl}- jujubogenin, and named as hovenidulcioside C.

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Fig. 2

Key ¹H–¹H COSY, HMBC and ROESY correlations in compounds 1 and 4.

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Compound 2 was obtained as amorphous powder. The quasimolecular ions [M + CH₂O₂-H]⁻ at m/z 1093.5444 (C₅₂H₈₅O₂₄, calcd for 1093.5436) and [M−H]⁻ at m/z 1047.5383 (C₅₁H₈₃O₂₂, calcd for 1047.5384) suggested the molecular formula was C₅₁H₈₃O₂₂. The NMR spectra of 2 showed the similar signal pattern to those of 1, except for one additional hydroxyl group linked to C-25 and one lost double bond between C-24 and C-25. HMBC correlations from δ_H 1.21 (s, 3H, H₃-27) and 1.26 (s, 3H, H₃-26) to δ_C 49.0 (C-24) and δ_C 71.5 (C-25) (Fig. 2) revealed that the sapogenin of 2 was ziziglaziovigenin (Correa Dos Santos et al., 2019). ROESY correlations between H-3 at δ_H 3.06 and H-1′ at δ_H 4.34, H-5 at δ_H 0.69, between H-23 at δ_H 4.22 and H-18 at δ_H 1.14, indicated the β configuration at C-3, 23 (Fig. 2). Compound 2 was identified as 3-O-{β-D-xylopyranosyl (1 → 3)-α-L- arabinopyranosyl (1 → 3)- [β-D-glucopyranosyl (1 → 2)] -α-L-arabinopyranosyl}- ziziglaziovigenin, and named as hovenidulcioside D.

Compound 3 was obtained as white powder with the formula C₄₄H₆₇O₁₇ according to the quasimolecular ions at m/z 913.4426 [M + CH₂O₂-H]⁻ (C₄₅H₆₉O₁₉, calcd for 913.4428) and m/z 867.4365 [M−H]⁻ (C₄₄H₆₇O₁₇, calcd for 867.4373). The IR spectrum displayed characteristic absorptions for hydroxyl (3356 cm⁻¹), carbonyl (1747 cm⁻¹) and olefinic (1651 cm⁻¹) groups. The NMR data indicated that 3 was a methyl-migrated 16,17-seco-dammarane triterpenoid with two sugar residues. Considering those saponins isolated from this species (Yoshikawa et al., 1996) and our previously studies (Xu et al., 2012; Xu, 2011), it was suggested that the aglycone of 3 was hovenidulcigenin A (Xu et al., 2012). Acid hydrolysis of 3 and GC analysis of the silyl derivatives of sugars afforded D-glucose. The configurations of the D-glucoses were β according to coupling constants of anomeric protons [H-1′ (d, J = 7.2 Hz) and H-1″(d, J = 7.8 Hz)] in ¹H NMR. HMBC correlations between δ_H 4.68 (H-1″ in Glc) and δ_C 81.1 (C-2′ in Glc), between δ_H 4.43 (H-1′ in Glc) and δ_C 91.1 (C-3) suggested the sequence and glycosylation position of the sugar. Therefore, 3 was elucidated as 3-O-[β-D-glucopyranosyl (1 → 2)-β-D-glucopyranosyl]- hovenidulcigenin A, and named as hovenidulcioside E.

Compound 4 was purified as white powder with the formula of C₄₆H₇₀O₁₇N by the negative HR-ESI-MS data at 954.4667 [M + CH₂O₂-H]⁻ (C₄₇H₇₂O₁₉N, calcd for 954.4693), 908.4617 [M−H]⁻ (C₄₆H₇₀O₁₇N, calcd for 908.4638). NMR data of 4 were highly consistent with those of 3, except for the existence of an additional N-acetyl amino group, which indicated by the signals at δ_H 1.99 (NHCOCH₃), δ_C 23.0 (NHCOCH₃) and 174.0 (NHCOCH₃), suggesting that 4 was an N-acetylated amino derivative of 3 (Zhang et al., 2011). HMBC correlations between δ_H 1.99 (NHCOCH₃) and δ_C 174.0 (NHCOCH₃), between δ_H 3.64 (H-2″) and δ_C 174.0 (NHCOCH₃), between δ_H 3.64 (H-2″) and δ_C 102.0 (C-1″) indicated that the N-acetamido group was attached to Glc-C-2″. The carbonal signal at δ_C 58.1 (C-2″) is the typical property for the presence of a 2-deoxy-2-acet-amidoglycosyl unit (Zhang et al., 2011). The β configurations of two sugars were deduced by coupling constants of H-1′ (d, J = 7.8 Hz) and H-1″ (d, J = 8.4 Hz) in ¹H NMR spectrum. Furthermore, the HMBC correlations between δ_H 4.89 (H-1″ in Glc-NAc) and δ_C 79.3 (C-2′ in Glc) and between δ_H 4.40 (H-1′ in Glc) and δ_C 91.4 (C-3) suggested the glycosidic chains attched to C-3 (Fig. 2). The ROESY correlations from δ_H 3.20 (H-3) to δ_H 0.80 (H-5), δ_H 0.91 (H-21) and δ_H 1.10 (H-28) indicating the β configuration of C-3. Thus, compound 4 was determined to be 3-O-[β-D-2-deoxy-2-acetylaminoglucopyranosyl (1 → 2)-β-D-glucopyranosyl]- hovenidulcigenin A, and named as hovenidulcioside F.

Compound 5 was obtained as white powder with formula of C₄₆H₇₂O₁₇N based on the quasimolecular ions at m/z 956.4828 [M + CH₂O₂-H]⁻ (C₄₇H₇₄O₁₉N, calcd for 956.4849) and m/z 910.4472 [M−H]⁻ (C₄₆H₇₂O₁₇N, calcd for 910.4795). The NMR signal pattern of 5 were very similar to those of 4, except that the vinyl group in 4 was substituted by a methylene and a methine in 5. This evidence postulated that 5 was the dihydro-analog of 4 on the Δ²⁴-olifinic moiety, which was further confirmed by the correlations between δ_H 4.59 (H-23) and δ_H 2.18/2.04 (H₂-24) and between δ_H 1.86 (H-25) and δ_H 1.23 (H₃-27) in ¹H–¹H COSY spectrum. The correlations between δ_H 4.59 (H-23) and δ_H 1.23 (H₃-27) in ROESY spectrum indicated the trans configurations of C-23 and C-25. The above data revealed that the aglycone of 5 was hovenidulcigenin B, which was previously obtained from the seeds of H. dulcis (Xu et al., 2012; Yoshikawa et al., 1996). Thus, compound 5 was determined to be 3-O-[β-D-2- deoxy-2-acetylaminoglucopyranosyl(1 → 2)-β-D- glucopyranosyl] - hovenidulcigenin B, and named as hovenidulcioside G.

In this work, compounds 1–6 were evaluated the activities on Nrf2 expression using ARE-luciferase reporter assay (Chen et al., 2016). Finally, compound 1 was found to inhibit the ARE-luciferase activity in a dose-dependent manner (Fig. 3A) while 2–6 showed neither activatory nor inhibitory activity on ARE-luciferase effect. Then the protein levels of Nrf2 in A549 cells were decreased after 1 treatment (Fig. 3B) as expected. And the cytotoxicity assay showed that the 50% inhibitory concentration of compound 1 was about 95.67 μM within 24 h. Compared with the control group, there was no significant difference to cell viability at concentrations below 40 μM. The results indicated that the compound did not cause cytotoxicity when it acted as an Nrf2 inhibitor (Fig. 3C). The results demonstrated that compound 1 could inhibit the Nrf2 pathway by reducing the Nrf2 protein level. Compounds 1 and 2 had the identical sugar moiety, the Nrf2 inhibitory effect disappeared when the double bond between C-24 and C-25 lost and the hydroxyl group linked to C-25 added in 2, suggested that importance of the double bond and the hydroxyl group between C-24 and C-25, which maybe was the active crucial group in 1.

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Fig. 3

Compound 1 selectively inhibited the Nrf2 pathway. (A) 1 inhibited ARE-dependent luciferase activity(n = 3). (B) Nrf2 expression in A549 cells, GAPDH was used as control (n = 2). (C) Cell viability of compound 1 in A549 cells (n = 3).

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4 Conclusions

In this work, five new triterpenoid saponins hovenidulcioside C–G (1–5) along with one known analogue were isolated from the seeds of H. dulcis. The inhibitory activities on Nrf2 pathway of compound 1 was observed, which had better effect at the dosage of 25 μM. As a cytoprotective transcription factor, Nrf2 plays a systemic role in various cancers and the Nrf2 inhibitor is possible became a favorable strategy for cancer therapy (Lin et al., 2020). This study suggested that the active triterpenoid saponins from Hovenia genus may serve as privileged constituents in future anti-tumor drug discovery.