Discovery of 2 New Dibenzocyclooctadiene Lignans Cauliflorins A and B From Schisandra cauliflora

Abstract

Sarcopenia is having a serious impact on highly aged populations. A Japanese traditional medicinal remedy containing Schisandra fruits and Astragalus roots recently showed effective treatment of sarcopenia and thus needed to be investigated further. This current study selected Schisandra cauliflora fruits for investigation of their chemical constituents and evaluation of their antisarcopenia effects. Dried S cauliflora fruits were ultrasonically extracted with methylene chloride. This extract was purified by a combination of chromatographic techniques. The isolated compounds were characterized by nuclear magnetic resonance spectroscopy, electronic circular dichroism, and high resolution electrospray ionization mass spectrometry methods. Antisarcopenia activities of the isolated compounds were evaluated by their effects on the viability and proliferation of C2C12 skeletal muscle cells. Seven dibenzocyclooctadiene lignans, including 2 that had been previously undescribed, cauliflorins A and B (1 and 2), ananolignan C (3), 6-epi-ananolignan D (4), ananolignan F (5), ananonin H (6), and ananonin J (7) were isolated from the methylene chloride extract of the fruits of S cauliflora. The C2C12 cells treated with compounds 1-7 (5 µM) exhibited proliferation in the range of 75.7% ± 7.4% to 98.2% ± 11.7%, compared to 100.0% ± 6.0% of the untreated cells. Two previously undescribed and 5 known dibenzocyclooctadiene lignans were isolated from S cauliflora fruits. All the isolated compounds were inactive on the proliferative enhancement of C2C12 skeletal muscle cells.

Keywords

cauliflorin A cauliflorin B dibenzocyclooctadiene lignan Anti-sarcopenia

Introduction

The elderly populations and age-related diseases have been receiving attention in recent decades.^1,2 Sarcopenia, a natural loss in both mass and strength of skeletal muscle, is one of the main challenges of elderly people. It is reported that the loss in mass and strength of skeletal muscle starts after the age of 40 and rapidly accelerates with increasing age. Elderly people over 80 years old lose ∼50% of their muscle mass and strength in the next decade.^2,3 Japan is known to have a highly aged population. Prevention and/or treatment of sarcopenia therefore is of much concern to the Japanese Ministry of Health. A Japanese traditional medicinal remedy, called ninjin’yoeito, recently showed positive signs in the treatment of sarcopenia.⁴ The study suggested that Schisandra fruits and Astragalus roots are active ingredients of the ninjin’yoeito formula and suggested that further investigations on these herbs were needed for the treatment of sarcopenia.⁵ In this report, we describe the discovery of 2 previously undescribed and 5 known dibenzocyclooctadiene lignans from the fruits of Schisandra cauliflora. The isolated compounds were also screened for antisarcopenia activity by evaluation of their effects on the viability and proliferation of C2C12 skeletal muscle cells.

Results and Discussion

The fruits of S cauliflora were finely ground and ultrasonically macerated in methylene chloride (MC). The MC soluble fraction was separated and purified by chromatographic techniques to yield compounds 1-7, all as yellow amorphous powders.

The molecular formula of 1, C₂₅H₃₂O₈, was deduced by a Na additive molecular ion peak, m/z 483.1996 [M + Na]⁺ (calcd. for [C₂₅H₃₂O₈+ Na]⁺, 483.1989) in the high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). The ¹H nuclear magnetic resonance (NMR) of 1 showed 2 individual olefinic protons (δ_H 6.56 and 6.61 [each, 1H, s]), 1 oxygenated methine (δ_H 5.67 [1H, s]), 3 methyl groups (δ_H 1.58 [3H, s], 1.07 [3H, d, J = 7.0 Hz], and 0.91 [3H, d, J = 7.0 Hz]), and 5 methoxy groups (δ_H 3.92, 3.90, 3.86, 3.64, and 3.50 [each 3H, s]). The ¹³C NMR of 1 contained 25 carbons, which was then sorted by Heteronuclear Single Quantum Coherence (HSQC) as 1 carbonyl, 10 nonprotonated olefinic, 1 aliphatic methylene, 2 olefinic methine, 3 aliphatic methine, 5 methoxy, and 3 methyl groups. An acetyl group was deduced by signals of carbonyl (δ_C 170.0) and methyl (δ_C 20.7/δ_H 1.58) groups. Except for the signals of acetyl and methoxy groups, a total of 18 carbons including 12 aromatic carbons of the 2 penta-substituted benzene rings and 6 sp³-hybridized carbons of an aliphatic bridge suggested 1 to be a C₁₈-dibenzocyclooctadiene lignan (Figure 1). The structural framework of 1 was then confirmed by 2-dimensional-NMR analysis (Figure 2). An aliphatic bridge was established by correlation spectroscopy (COSY) interactions of H-9 (δ_H 5.67)/H-8 (δ_H 2.02)/H-7 (δ_H 2.07)/H₂-6 (δ_H 2.64), H-8 (δ_H 2.02)/H₃-18 (δ_H 1.07), and H-7 (δ_H 2.07)/H₃-17 (δ_H 0.91). Connections between this aliphatic bridge and a biphenyl system were approved by heteronuclear multiple bond correlation (HMBC) correlations between H-9 (δ_H 5.67) and C-11 (δ_C 109.6)/C-15 (δ_C 120.7)/C-10 (δ_C 136.8), H₂-6 (δ_H 2.64) and C-16 (δ_C 123.5)/C-5 (δ_C 133.4)/C-4 (δ_C 110.4). An acetoxy group was assigned at C-9 by chemical shift values of C-9 (δ_C 82.1), H-9 (δ_H 5.67), and HMBC correlation of H-9 (δ_H 5.67)/C-1′ (δ_C 170.0) (Figure 2). The 6 de-shielded aromatic carbons (δ_C 151.6, 150.9, 150.7, 148.6, 139.8, and 138.8) and signals of 5 methoxy groups suggested the binding of 5 methoxy groups and a hydroxy group to a biaryl system. Furthermore, none of the correlations between H-11 (δ_H 6.61) and methoxy protons, but an obvious correlation between H-4 (δ_H 6.56) and methoxy protons (δ_H 3.90), were observed in the rotating frame Overhauser effect spectroscopy (ROESY) spectrum, indicating a hydroxy group at C-12 and 5 methoxy groups at the remaining positions of C-1, C-2, C-3, C-13, and C-14. The electronic circular dichroism (ECD) spectrum of 1 displayed Cotton effects with positive and negative signs at wavelengths of 212 nm (+20.9 mdeg) and 251 nm (−18.1 mdeg), respectively, which had been previously demonstrated for an S-biaryl structure.^6–8 Conformation of the cyclooctadiene ring is typically either twist-boast or twist-boast-chair⁹; however, a ROESY interaction of H-8 (δ_H 2.02)/H-11 (δ_H 6.61) indicated a twist-boast-chair conformation and axial orientation of H-8. The ROESY interactions of H₃-17 (δ_H 0.91)/H-4 (δ_H 6.56) indicated an axial orientation of methyl group C-17. Consequently, absolute configurations of 1 were established as shown in Figures 1 and 3. Compound 1 was then named cauliflorin A (Figures S1 to S11).

Figure 1.

Structure of 7 dibenzocyclooctadien lignans isolated from Schisandra cauliflora fruits.

Figure 2.

Important COSY (H─H) and HMBC (H→C) interactions for 1 and 2.

Figure 3.

Key rotating frame Overhauser effect spectroscopy (ROESY) interactions for 1 and 2.

The HR-ESI-MS of 2 displayed a sodium additive molecular ion peak at m/z 471.1996 [M + Na]⁺ (calcd. for [C₂₄H₃₂O₈+ Na]⁺, 471.1989), which indicated a molecular formula of C₂₄H₃₂O₈. ¹H-NMR analysis of 2 revealed closely similar resonant signals to those of 1, including 2 singlet aromatic methines, 2 doublet methyl groups, and 5 methoxy groups. However, additional signals of a methoxy group (δ_H 3.02) and an oxymethine group (δ_H 3.88) were observed in the ¹H NMR spectrum of 2, which replaced signals of an acetyl group and a methylene group, as shown in 1. This evidence suggested that 2 differed from 1, partly by substitutions and their locations. The Nuclear Overhauser Effect Spectroscopy (NOESY) spectrum of 2 observed interaction between H-4 (δ_H 6.54) and a methoxy group (δ_H 3.91), in contrast with the absence of ROESY interaction between H-11 (δ_H 6.43) and any methoxy group, confirming the 5 methoxy groups at C-1, C-2, C-3, C-13, and C-14; and a hydroxy group at C-12. The HMBC interactions between H-4 (δ_H 6.54)/6-OCH₃ (δ_H 3.02) and C-6 (δ_C 90.3) demonstrated the presence of an additional methoxy group at C-6, while the HMBC interaction of H-11 (δ_H 6.43)/C-9 (δ_C 80.5) and chemical shift value of H-9 (δ_H 4.62) confirmed it to be a hydroxy bearing carbon. Finally, the stereochemistry of the carbon framework of 2 was deduced in the same way as that of 1 by their sharing the same signs of Cotton effects (+10.6 mdeg at 216 nm and −7.6 mdeg at 251 nm) in the ECD, and pattern of ROESY interactions (Figure 3). Of these, an equatorial orientation of H-6 (axial orientation of 6-OCH₃) was confirmed by ROESY interactions of H₃-17 (δ_H 0.97)/H-4 (δ_H 6.54), H₃-17 (δ_H 0.97)/H-6 (δ_H 3.88), and H-4 (δ_H 6.54)/H-6 (δ_H 3.88). Therefore, the structure of compound 2 was elucidated, and named cauliflorin B (Figures S12 to S22).

The NMR and ECD spectral data of other compounds, respectively, consisted of known dibenzocycloortadiene lignans, including ananolignan C (3),¹⁰ 6-epi-ananolignan D (4),^10,11 ananolignan F (5),¹⁰ ananonin H (6),¹² and ananonin J (7).¹² 6-Epi-ananolignan D (4) was previously reported to be a product of chemical transformation from kadsulignan L,¹¹ but compound 4 has not been isolated previously from a natural source. The assignment data of 4 are reported for the first time in this study (Figures S23 to S36, Table S1). To date, over 300 dibenzocyclootadiene lignans have been discovered.¹³ Structural characteristics of such lignans are variable from substituted groups, chiral centers along an aliphatic bridge, and helicity of the biaryl system. Different substituted groups and/or absolute configurations of each derivative could result in different biological activities. Therefore, the discovery of new dibenzocyclootadiene lignans (1 and 2) not only confirm high structural diversity of such lignans, but also provide additional evidence for biological evaluation and distribution for structure–activity relationships of such lignans.

Schisandrins are well-known dibenzocyclooctadiene lignans and are major constituents of Schisandra fruits.¹³ They are believed to be responsible for their antisarcopenia activity.⁵ Thus, the 7 isolated dibenzocyclooctadiene lignans (1-7) were examined for their effects on the viability and proliferation of C2C12 skeletal muscle cells, an important factor reflecting antisarcopenia level.^14,15 Compounds 1-7 (5 µM) did not show any significant effect on cell viability, and, unfortunately, did not exhibit cell proliferation (Figure S37). In the presence of the compounds, the percentages of cell proliferation were from 75.7% ± 7.4% to 98.2% ± 11.7%, compared to 100.0% ± 6.0% of untreated cells.

Material and Methods

General

General experimental procedures and bioassay are given in the Supplementary File.

Extraction and Purification

The fruits of S cauliflora were dried overnight and then finely pulverized. Powdered fruits (1.0 kg) were extracted with MC (3 times, each 3 L of MC, 60 min in an ultrasonic bath). The extract solution was filtered through filter paper, and the solvent was evaporated to give the crude extract. This (35 g) was roughly separated by silica gel column chromatography, eluting with increasing quantities of acetone in n-hexane (0%-100% in volume) to give 8 fractions, SFD1-SFD8. Fraction SFD3 (5.2 g) was subjected to reversed phase column chromatography, eluting with methanol in water (75% methanol), to yield 4 fractions, SFD3A-SFD3D. Fraction SFD3B was equally divided into 2 portions, SFD3B1 and SFD3B2. The SFD3B1 portion was injected into a semipreHPLC column, running with CH₃CN/H₂O (60/40) to give 1 (6.5 mg, t_R 63.8 min). The SFD3B1 was injected into a semipreHPLC, running with CH₃CN/H₂O (65/35) to give 5 (5.7 mg, t_R 52.6 min). Fraction SFD3D was injected into a semipreHPLC, running with CH₃CN/H₂O (80/20) to give 7 (15.0 mg, t_R 52.8 min). Fraction SFD4 was subjected to reversed phase column chromatography and eluted with methanol in water (2.5/1) to furnish 3 fractions, SFD4A-SFD4C. Fraction SFD4A was injected into a semipreHPLC, running with CH₃CN/H₂O (55/45), to give 2 (8.3 mg, t_R 42.7 min). Fraction SFD4B was injected into a semipreHPLC, running with CH₃CN/H₂O (65/35), to give 4 (24.1 mg, t_R 31.6 min). Fraction SFD4C was also injected into a semipreHPLC, running with CH₃CN/H₂O (65/35) to give 6 (14.0 mg, t_R 59.5 min). Fraction SFD5 was first separated on a reversed phase column, eluting with methanol/water (2.5/1), and then purified by semipreHPLC, running with CH₃CN/H₂O (50/50), to obtain 3 (6.0 mg, t_R 44.1).

Cauliflorin A (1)

Yellow amorphous powder; [α]_D²⁹: +27 (c 0.1, MeOH); ECD (MeOH) θ_{(λ nm)}: +20.9₍₂₁₂₎, −18.1₍₂₅₁₎ mdeg; HR-ESI-MS: m/z 483.1996 [M + Na]⁺ (Calcd. for [C₂₅H₃₂O₈+ Na]⁺, 483.1989); ¹H NMR and ¹³C NMR data shown in Table 1.

Table 1.

¹H and ¹³C Nuclear Magnetic Resonance (NMR) Data for 1 and 2 in CDCl₃.

No.	1		2
No.	δ _C ^a	δ_H^b mult. (J in Hz)	δ _C ^a	δ_H^b mult. (J in Hz)
1	150.9	–	152.3	–
2	139.8	–	142.2	–
3	151.6	–	152.5	–
4	110.4	6.56 (s)	111.6	6.54 (s)
5	133.4	–	132.2	–
6	38.9	2.64 (m)	90.3	3.88*
7	35.0	2.07 (m)	40.9	1.93 (m)
8	42.1	2.02 (m)	40.1	2.10 (m)
9	82.1	5.67 (s)	80.5	4.62 (m)
10	136.8	–	138.8	–
11	109.6	6.61 (s)	109.0	6.43 (s)
12	148.6	–	148.7	–
13	138.8	–	138.3	–
14	150.7	–	150.9	–
15	120.7	–	119.3	–
16	123.5	–	122.4	–
17	14.7	0.91 (d, 7.0)	ND	0.97 (d, 7.0)
18	19.6	1.07 (d, 7.0)	ND	1.01 (d, 7.0)
1-OCH₃	60.2	3.64 (s)	60.4	3.64 (s)
2-OCH₃	60.7	3.86 (s)	60.9	3.91 (s)
3-OCH₃	56.0	3.90 (s)	56.1	3.91 (s)
6-OCH₃	–	–	56.2	3.02 (s)
13-OCH₃	60.9	3.92 (s)	60.5	3.90 (s)
14-OCH₃	60.1	3.50 (s)	60.0	3.76 (s)
12-OH	–	5.74 (s)	–	5.75 (s)
1′	170.0	–
2′	20.7	1.58 (s)

125 MHz.

500 MHz.

*Overlapped signals.

ND, not detected due to low intensity.

Cauliflorin B (2)

Yellow amorphous powder; [α]_D²⁹: +51 (c 0.1, MeOH); ECD (MeOH) θ_{(λ nm)}: +10.6₍₂₁₆₎, −7.6₍₂₅₁₎ mdeg; HR-ESI-MS: m/z 471.1996 [M + Na]⁺ (calcd. for [C₂₄H₃₂O₈+ Na]⁺, 471.1989); ¹H NMR and ¹³C NMR data shown in Table 1.

Conclusions

Purification of the MC extract of S cauliflora fruits resulted in the isolation of 7 dibenzocyclooctadiene lignans. Two of them (cauliflorins A and B, 1 and 2) are previously undescribed. Their structures were elucidated by a combination of spectroscopic methods, including HR-ESI-MS, NMR, and ECD. At a concentration of 5 µM, all the isolated compounds (1-7) were inactive for the enhancement of C2C12 skeletal muscle cells proliferation.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X231176406 - Supplemental material for Discovery of 2 New Dibenzocyclooctadiene Lignans Cauliflorins A and B From Schisandra cauliflora

Supplemental material, sj-docx-1-npx-10.1177_1934578X231176406 for Discovery of 2 New Dibenzocyclooctadiene Lignans Cauliflorins A and B From Schisandra cauliflora by Bui Thi Thu Trang, Nguyen Huy Hoang, Pham Hai Yen and Do Thi Trang, Nguyen Thi Cuc, Duong Thi Hai Yen, Jinyoung Kim, Junjung Choi, Seung Hyun Kim, Bui Huu Tai, Nguyen Xuan Nhiem, Phan Van Kiem in Natural Product Communications

Footnotes

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical Approval

Our institution does not require ethical approval for reporting individual cases or case series.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Vietnam Academy of Science and Technology (VAST) under grant number QTKR01.01/21-22.

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

Supplemental Material

Supplemental material for this article is available online.

ORCID iD

Nguyen Xuan Nhiem

Phan Van Kiem

Bui Thi Thu Trang

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