Chinese Chemical Letters  2020, Vol. 31 Issue (5): 1254-1258   PDF    
New norlignan enantiomers from the fruit of Crataegus pinnatifida with neuroprotective activities
Rui Guoa, Tianming Lva, Fengying Hana, Zilin Houa, Guodong Yaoa, Bin Linb, Xiaobo Wangc, Xiaoxiao Huanga,c,*, Shaojiang Songa,*     
a Key Laboratory of Computational Chemistry-Based Natural Antitumor Drug Research&Development, Liaoning Province, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China;
b School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, China;
c Chinese People's Liberation Army Logistics Support Force No. 967 Hospital, Dalian 116021, China
Abstract: (±)-Crataegusnorin A (1a/1b) and B (2a/2b), two pairs of rare 8, 9'-epoxy-type norlignan enantiomers featuring a γ-butyrolactone ring, were isolated from the fruit of Crataegus pinnatifida. Their structures were determined via extensive spectroscopic analyses. Gauge-independent atomic orbital (GIAO) NMR chemical shift calculations, combined with the advanced statistical method DP4+ were employed to establish the relative configurations of four compounds. Next, chiral separation was accomplished by chiral chromatographic column and the absolute configurations of the four compounds were unambiguously assigned by comparison between their experimental electronic circular dichroism curves with the quantum-mechanically calculated curves based on time-dependent density functional theory (TDDFT). All the isolates were evaluated for their neuroprotective activities against H2O2-induced cell injury in human neuroblastoma SH-SY5Y cells. The results showed that two pairs of enantiomers 1a/1b and 2a/2b displayed diff; erent effect on neuroprotective activity. Among them, compound 2a displayed the most potent neuroprotective effect. Further flow cytometry analysis indicated that 2a could protect SH-SY5Y cells from oxidative damage through inhibiting cell apoptosis.
Keywords: Crataegus pinnatifida    Norlignan enantiomer    Neuroprotective activity    SH-SY5Y cells    Apoptosis    

Crataegus pinnatifida, a traditional Chinese medicinal herb, belongs to the Rosaceae family. It is widely distributed in Asia, Europe and North America [1]. People use it as a medicinal plant to improve digestion, promote blood circulation and resolve blood stasis both in traditional and folk medicine [2]. Modern inves-tigations have demonstrated that C. pinnatifida has various pharmacological effects on the cardiovascular, digestive, and endocrine systems as well as killing some pathogenic micro-organisms [3-5]. In the research on the chemical constituents of C. pinnatifida, a variety of secondary metabolites, such as flavonoids, triterpenoids, steroids, lignans and organic acids, have been isolated and identified [6-9].

Herein, we carried out the continued search for biologically active and structurally unique compounds, which resulted in the isolation of two rare norlignans (1 and 2) from the fruit of Crataegus pinnatifida. For compounds 1 and 2, they were assigned as new 8, 9'-epoxy-type norlignans featuring a γ-butyrolactone ring. Structurally, they possessed the same C6C3-C2C6 carbon skeleton and only differed in the substituents present on phenyl moiety (Fig. 1).

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Fig. 1. The structures of compounds 1a/1b and 2a/2b.

Due to their negligible optical activities, compounds 1 and 2 were suggested as racemic mixtures, which was further supported by the absence of Cotton effects in their ECD spectra. The following chiral separation resulted in two pairs of enantiomers (1a/1b and 2a/2b) using chiral chromatographic column. Their relative configurations were established by employing NMR chemical shifts calculations using Gauge-independent atomic orbital (GIAO) method [10], combined with the advanced statistical method DP4+ [11]. Additionally, their absolute configurations were established by comparison of experimental and calculated ECD spectra. Their neuroprotective activities against H2O2-induced oxidative injury in human neuroblastoma SH-SY5Y cells were investigated. This is the first report of the configurational assignment of 8, 9' -epoxy-type norlignans with a flexible skeleton by quantum chemical calculations.

Crataegusnorin A (1) was obtained as pale yellow oil. Its molecular formula was established as C22H26O9 from the 13C NMR data and an HRESIMS ion at m/z 457.1472 [M + Na]+ (calcd. for C22H26NaO9: 457.1469), indicating 10 indices of hydrogen deficiency. The 1H NMR data of 1 (Table 1 and Fig. S4 in Supporting information) showed two sets of aromatic protons [δH 6.31 (d, 1H, J = 2.0 Hz, H-2), 6.78 (d, 1H, J = 8.2 Hz, H-5), 6.48 (dd, 1H, J = 8.2, 2.0 Hz, H-6)] and [δH 6.44 (br s, 2H, H-2' /H-6')] arising from a 1, 3, 4-trisubstituted and a symmetrical 1, 3, 4, 5-tetrasubstituted aromatic ring system, respectively. In addition, four aliphatic proton signals were evident at δH 3.41 (d, 1H, J = 5.7 Hz, H-7), 4.02 (d, 1H, J = 8.4 Hz, H-7'), 2.69 (ddd, 1H, J = 8.4, 5.7, 2.6 Hz, H-8'), and 5.52 (d, 1H, J = 2.6 Hz, H-9'), and the protons of five methoxy groups resonated at δH 3.26 (s, 3H), 3.55 (s, 3H), 3.74 (s, 3H), and 3.81 (s, 6H). The 13C NMR data of 1 (Table 1 and Fig. S7 in Supporting information) showed 22 carbon signals comprising two benzene rings, five methoxy groups, an ester carbonyl group, and four methines (two of which were oxygenated). The HMBC correlations of H-7, H-8' and H-9' with C-8 revealed the presence of the γ-butyrolactone ring in 1 (Fig. 2 and Fig. S8 in Supporting information). The multiplet centered at δH 2.69 (ddd, 1H, J = 8.4, 5.7, 2.6 Hz, H-8') displayed correlations with C-1/C-7/C-1'/C-8, indicating the γ-butyrolactone moiety of cyclo[C7-C8-O-C9'-C8'], with C-8' connected to the C-7' methine and C-7 to the phenyl ring at C-1. According to the above observations, this structure was determined as a norlignan compound with a C6C3-C2C6 skeleton. The HMBC correlations for CH3O-3/C-3, CH3O-3'/C-3', CH3O-5'/C-5', CH3O-7'/C-7', and CH3O-9'/C-9' revealed that these methoxy groups were located at C-3, C-3', C-5', C-7', C-9', respectively. Thus, the planar structure of 1 was established and named crataegusnorin A.

Table 1
1H NMR data (400 MHz) and 13C NMR data (100 MHz) for compounds 1a/1b-2a/2b in CDCl3.

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Fig. 2. Key HMBC correlations of compounds 1 and 2.

In the NOESY experiment of 1 (Fig. 3 and Fig. S10 in Supporting information), the correlations of H-8' with H-2/6, and CH3O-9' indicated anti-configuration between H-7 and H-8', H-8' and H-9'. In addition, the lack of NOE between H-7' and CH3O-9' also confirmed an opposite spatial orientation of H-8' with H-9'. However, due to the free rotation of the exible chain (C1'-C7'), the relative configuration assignment of C-7' could not be determined when solely based upon interproton distances deduced from NOESY or J-based NMR spectroscopic analysis. The configurational establishment of these molecules has always been a challenge in the structure elucidation of natural products. We therefore turned to quantum mechanical calculations for the determination of the relative configuration.

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Fig. 3. Key NOESY correlations of compounds 1 and 2.

In recent years, quantum chemical calculations coupled with several correlative approaches, such as CP3, DP4, and DP4+, have been successfully employed in natural products elucidation. We performed such calculations here. The relative configuration at C-7' was assigned by employing calculations of shielding tensor values with support from DP4+ probability analysis (Fig. 4 and Tables S1 and S2 in Supporting information). The theoretical calculations of 1H and 13C NMR chemical shifts of the two possible isomers (7S*, 7'S*, 8'S*, 9'R*)-1 and (7S*, 7'R*, 8'S*, 9'R*)-1 were pre-dicted using the GIAO method with the Gaussian 09 software at the B3LYP/6-311+G(d, p) level utilizing the polarizable continuum model (PCM) in chloroform [12]. This assignment was confirmed by comparing the experimental and calculated NMR chemical shifts. With a DP4+ probability of approximately 100%, the relative configuration of 1 was defined as 7S*, 7'R*, 8'S*, 9'R*.

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Fig. 4. (A) Calculated 13C NMR spectroscopic data of two pairs of C-7' epimers of (7S*, 7'S*, 8'S*, 9'R*)-1 and (7S*, 7'R*, 8'S*, 9'R*)-1, (7S*, 7'S*, 8'S*, 9'R*)-2 and (7S*, 7'R*, 8'S*, 9'R*)-2 at B3LYP/6-311+G(d, p) level in CDCl3. (B) DP4+ probability of 1H and 13C NMR chemical shifts of 1 and 2.

With an optical rotation value close to zero and no Cotton effect in its ECD spectrum, compound 1 was considered as a racemic mixture. Subsequent chiral resolution of 1 afforded the anticipated enantiomers 1a and 1b, which showed mirror image-like ECD curves and specific rotations (1a: 25.0, c 0.10, MeOH; 1b:+27.0, c 0.10, MeOH). The absolute configuration of the enantiomers were determined by comparison of its experimental and quantum chemical ECD spectra calculated at the B3LYP/6-311++G(2d, p) level in MeOH. The calculated ECD of 7S, 7'R, 8'S, 9'R-1 is consistent with the measured ECD of 1a but opposite to that of 1b (Fig. 5). Therefore, the structure of (-)-1, namely, (-)-crataegusnorin A, was determined to be (7S, 7'R, 8'S, 9'R), and (+)-1, namely, (+)-crataegusnorin A, was determined to be (7R, 7'S, 8'R, 9'S), respectively.

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Fig. 5. Experimental and calculated ECD spectra for 1a/1b and 2a/2b in MeOH.

The molecular formula of compound 2 was determined to be C23H28O10 in agreement with its HRESIMS data (m/z 487.1572 [M+Na]+, calcd. for C23H28NaO10: 487.1575). The 1H and 13C NMR spectroscopy data (Table 1, Figs. S15 and S18 in Supporting information), along with the HSQC experiment, showed resonances for two benzene rings, six methoxy groups, an ester carbonyl group, and four methines. The NMR spectra of 2 are similar to those of 1, except for the presence of the C-5 methoxy group in 2, which were confirmed by HMBC correlations from the methoxy protons (δH 3.75) to C-5. A comparative analysis of the remaining data in the two-dimensional NMR spectra (HSQC and HMBC) allowed us to assign all the resonances of 2 (Fig. 2, Figs. S19 and S20 in Supporting information). Similar to 1, the 7, 8' -anti and 8', 9'-anti configuration were determined by NOESY experiment in which correlations of H-8'/H-2 and H-8' /CH3O-9' were observed (Fig. 3 and Fig. S21 in Supporting information). The unobserved correlation of H-7'/CH3O-9' also confirmed the orientation of H-8' and H-9'. In addition, the DP4+ protocol was again applied to the simulated 1H and 13C NMR chemical shifts of the two possible epimers (Fig. 4 and Tables S3 and S4 in Supporting information). The statistical results indicated that the epimer possessing the R configuration at C-7', was the correct structure for 2, with 100% probability. The optical rotation value of 2 was very close to zero and there was no Cotton effect in its ECD spectrum, suggesting that it was a racemic mixture. Subsequently, the chiral HPLC purifica-tion of 2 afforded the enantiomers 2a and 2b with opposite specific rotation (2a: 27.0, c 0.10, MeOH); 2b: +28.0, c 0.10, MeOH) and mirror imaged Cotton effects in their ECD spectrum. To determine the absolute configurations of 2a and 2b, the ECD spectra of 2a and 2b were measured in MeOH and compared with the calculated ECD spectra of the enantiomers (Fig. 5). On the basis of the matching of the experimental and computed ECD spectra, the absolute configurations of (-)-2 and (+)-2 were determined as (7S, 7'R, 8'S, 9'R)-2 and (7R, 7'S, 8'R, 9'S)-2, and were assigned the names (-)-crataegusnorin B and (+)-crataegusnorin B, respectively.

The neuroprotective activities of norlignan enantiomers 1a/1b and 2a/2b against H2O2-induced oxidative injury in SH-SY5Y cells were evaluated by MTT assay, and the cell viabilities of all compounds were shown in Fig. 6. The results showed that 200 mmol/L H2O2 could significantly reduce the cell viability compared with the control group. Among them, enantiomers 2a and 2b exhibited varying degrees of neuroprotective effects, while enantiomers 1a/1b exhibited no obvious protective effects in the bioassay. Comparison of compounds 1a/1b with 2a/2b indicated that the presence of methoxy group at C-5 could increase neuroprotective effects. As shown in Fig. 6, compound 2a showed the most potent neuroprotective activity, the cell viability of which at 25 μmol/L was 79.79%, much more potent than the positive control Trolox (63.1% at 25 μmol/L) and its enantiomer 2b had a weaker protective effect at a certain concentration. These results highlighted the fact that the absolute configurations of enantiom-ers possess remarkable influences on their neuroprotective activities.

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Fig. 6. Neuroprotective effects of compounds 1a/1b and 2a/2b against H2O2-induced cell growth inhibition of SH-SY5Y cells. In the presence or absence of the tested compounds at different concentrations (12.5, 25, and 50 μmol/L), the MTT assay was used to examine the cell viability after H2O2 (200 μmol/L) treatment for 4 h. ###P < 0.001 vs. control group. *P < 0.05, ***P < 0.001 vs. the H2O2 treated group. & P < 0.05 was considered statistically significant when compared to its enantiomer.

Moreover, in order to further investigate the apoptosis-inhibiting effects of enantiomers 2a/2b in SH-SY5Y cells, Annexin V-FITC/PI staining using flow cytometry was applied to quantify the number of the apoptotic cells (Fig. 7). It can be seen that significant apoptosis occurred in model group and the apoptosis ratio reached to 20.00% compared to the control group (4.90%). Then, pretreatment of compound 2a (25 μmol/L) could decrease the apoptosis ratio to 7.10%, while 2b had no obvious effect on the apoptotic ratio in H2O2-treated cells. Consistent with the MTT results, these results indicated that 2a showed stronger neuro-protective effect compared with its enantiomer 2b. Taken together, compound 2a exerted neuroprotective effect against H2O2-induced SH-SY5Y cellular damage by inhibiting apoptosis.

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Fig. 7. Effects of enantiomers 2a and 2b on the apoptosis ratio in H2O2-treated SH-SY5Y cells. The cells were pretreated with enantiomers 2a and 2b and then incubated with H2O2 for 4 h. Flow cytometry was used to examine the apoptotic ratio after Annexin V-FITC/PI staining. The percentage of apoptotic cells was calculated in the right. ##P < 0.01 vs. the control group. **P < 0.01 vs. the H2O2-treated group. P < 0.05 was considered statistically significant when compared to its enantiomer.

In summary, two pairs of rare 8, 9' -epoxy-type norlignan enantiomers (1a/1b and 2a/2b) featuring a γ-butyrolactone ring, were isolated from the fruit of Crataegus pinnatifida. Their structures as well as the absolute configurations of four compounds were established via extensive spectroscopic analyses and quantum chemical calculations. This is the first report of the configurational assignment of 8, 9' -epoxy-type norlignans with the flexible skeleton by quantum chemical calculations. In addition, all the isolates were evaluated for their neuroprotective effects against H2O2-induced oxidative injury in SH-SY5Y cells. The results showed that compound 2a displayed the most potent neuroprotective effect and the two pairs of enantiomers 1a/1b and 2a/2b displayed diff; erent effects on neuroprotective activity. Further flow cytometry analysis indicated that 2a could protect SH-SY5Y cells from oxidative damage through inhibiting cell apoptosis. Overall, this Chinese medicine contains promising candidates for the treatment of neurodegenerative diseases.

Declaration of competing interest

The authors declare that they have no conflicts of interest to this work.

Acknowledgments

This work was supported by Career Development Support Plan for Young and Middle-aged Teachers in Shenyang Pharmaceutical University (No. ZQN2018006) and the Project of Innovation Team Foundation (No. LT2015027).

Appendix A. Supplementary data

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.cclet.2019.09.042.

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