Nortriterpenoids from the fruit stalk of Schisandra chinensis (Turcz.) Baill.

1 Introduction

The fruit of Schisandra chinensis (Turcz.) Baill. (S. chinensis), as a genuine traditional chinese drugs in Northeast China, is mainly distributed in cold regions such as Heilongjiang, Jilin, and Liaoning. The 2020 edition of Chinese Pharmacopoeia recorded that it has the effect of astringing and controlling, supplementing qi while nourishing fluid, tonifying kidney, and calming heart. Its root, stem, leaf, fruit stalk and other non-medicinal parts were often removed as impurities. However, it was found that these non-medicinal parts also contained bioactive components similar to the fruit of S. chinensis^[1–2]. In particular, a series of nortriterpenoids with novel and complex skeletons as well as broad biological activities (anti-tumor, anti-HIV and anti-HBV activities) were found from the stem and leaf of Schisandraceae plants which has attracted extensive attention from scholars^[3]. Through the literature retrieving, more than 200 nortriterpenoids have been identified in Schisandraceae, including over 20 different skeleton types. Meanwhile, the chemical synthesis of nortriterpenoids has also been exploited^[4]. To date, there have not been any reports on nortriterpenoids and their biological activities in the fruit stalk of S. chinensis. Thus, we carried out the present study on the constituents of nortriterpenoids in the fruit stalk of S. chinensis. Two new nortriterpenoids, schilancitrilactone M and 25-hydroxyl schindilactone D (1 and 2), together with ten known nortriterpenoids (3–12) (Fig. 1) were isolated and characterized. The cytotoxicity of these compounds against human gastric cancer cell line MGC-803 cells was tested, and the results showed that compounds 6–8 possessed significant anti-gastric cancer activities with IC₅₀ values of 9.01, 11.77, and 2.74 μmol/L, respectively.

Fig. 1

Structures of compounds 1–12 from Schisandra chinensis

2 Materials and Methods

3 Results

Compound 1 was isolated as white amorphous powder and an ion peak of [M+H]⁺ was observed by HR-ESI-MS at m/z 545.2380 (Calcd. for C₂₉H₃₇O₁₀, 545.2387), indicating the molecular formula of C₂₉H₃₆O₁₀.

In the ¹H-NMR (400 MHz, C₅D₅N) spectrum (Fig. S1) of compound 1, a typical AB coupled signal δ_H 2.82 (1H, ABd, J=15.6 Hz) and 2.37 (1H, ABd, J=15.6 Hz) was observed. ¹³C-NMR (Fig. S2) and DEPT 135 spectrums (Fig. S3) revealed that compound 1 is composed of 29 carbons, including 5 primary carbons, 5 secondary carbons, 9 tertiary carbons and 10 quaternary carbons (3 ester carbonyl ones). HSQC spectrum (Fig. S4) analysis assigned all protons to their respective carbons unambiguously. The NMR data indicated that 1 is a highly oxygenated nortriterpenoid.

Meanwhile, the ¹H-NMR and ¹³C-NMR spectrums revealed that 1 is highly similar to schilancitrilactone A^[5] with main differences in ring A and ring B. The typical ABX coupling system formed by H-1 and H-2 of schilancitrilactone A was replaced by the signals δ_H (4.42 1H, s) and 4.72 (1H, s) in the low field region, indicating that there was a hydroxy that was further positioned to C-2. The supportive evidence came from the HMBC (Fig. S6) correlations from H-2 to C-1, C-10 and from H-1 to C-2, C-3, C-10, C-19, together with the ¹H-¹H COSY (Fig. S5) correlations of H-1/H-2 (Fig. 2). In addition, both ¹H-NMR signals of H-1 and H-2 were unimodal, indicating that the dihedral angle between H-1 and H-2 was close to 90°, which further indicated that 2-OH should be β-oriented, since H-1 was biogenetically assigned as β^[6].

Fig. 2

Key HMBC, ¹H-¹H COSY and ROESY of compounds 1–2

Because of the γ-gauche of hydroxyl group, after the 29-CH₃ in schilancitrilactone A was oxidized, 30-CH₃ was also moved to a higher field. Therefore, both C-29 and C-30 of compound 1 were not oxidized, and they were general methyls. Meanwhile, the signals H-1/29-CH₃ in ROESY (Fig. S7) confirmed that 29-CH₃ should be β-oriented. In addition, the absolute configuration at C-20 in compound 1 was determined as S, deduced from the Cotton effects in the CD (Fig. S11) spectrum similar to those of schilancitrilactone L^[7], which showed a positive Cotton effect at 268 nm. Furthermore, the ROESY correlation of H-22 with H-24 indicated that the geometry of the C(22) = C(23) of 1 is Z. The ROESY correlations of Me-28 with H-20 and H-11α, H-8 with H-5 and H-11α suggested that Me-28, H-20, H-8 and H-5 were all positioned on the same face of the molecule and therefore assigned as α-oriented, whereas the ROESY correlations of H-19β with H-1 and H-12 suggested that H-12 was β-oriented (Fig. 2). The relative configurations of the remaining chiral centers were determined to be the same as those in schilancitrilactone A because of their similar carbon and proton chemical shifts and ROESY correlations. According to the above characteristics, the structure of compound 1 named schilancitrilactone M is shown in Fig. 1.

Compound 2 is white amorphous powder with an ion peak of [M+NH₄]⁺ detected by HR-ESI-MS at m/z 594.2576 (Calcd. for C₂₉H₄₀NO₁₂, 594.2551), indicating that the molecular formula was C₂₉H₃₆O₁₂.

Compared the 1D NMR spectrum (Fig. S12–S13) with the known compound lancifodilactone L^[8], compound 2 had the same structure segments of C, D, E, F and G, while a hemiacetal and ester structure at ring A and ring B was oxidized (δ_C 108.7 C-1, 43.5 C-2). DEPT 135 spectrum (Fig. S14) analysis indicated that C-1 is the oxidation position. The change of the chemical shift of carbon at H-19 was caused by the substitution of 1-OH of compound 2 to produce a γ-gauche, which increased the density of carbon electron cloud at position 19 and moved to the high field (δ_C 40.7). Moreover, compared with compound 4 named schindilactone D^[9], the NMR data of ring A and ring B in compound 2 were consistent with those in compound 4. Furthermore, the planar structure of compound 2 was determined by 2D NMR data (Fig. S15-S17). According to the correlations of 21-CH₃ with H-22 and H-16 and 18-CH₃ with H-22, H-16 in ROESY (Fig. S18), the same spatial orientation of 18-CH₃, 21-CH₃, H-22 and H-16 were inferred. The correlation of H-20 with H-23 and H-24 were observed, suggesting that H-23 and H-24 were in a different spatial orientation (Fig. 2). The structure of compound 2 named 25 hydroxyl schindilaractone D is depicted in Fig. 1.

Other ten compounds isolated from the preparations were already known and were identified as schindilactone C (3)^[10], schindilactone D (4) ^[9], schindilactone E (5) ^[9], schindilactone H (6)^[11], lancifodilactone E (7)^[12], lancifodilactone I (8)^[8], lancifodilactone L (9)^[8], preschisanartanin O (10)^[13], arisanlactone C (11)^[14], wuweizidilactone F (12)^[6], based the 1D NMR data from the reported compounds in the literatures.

All compounds (1–12) were tested for their possible anti-cancer activities in MGC-803 gastric cancer cells (Table 3). The results demonstrated that compounds 6–8 exhibited strong cytotoxic effects against MGC-803, as reflected by the reduced cell viability, with IC₅₀ values of 9.01, 11.77 and 2.74 μmol/L, respectively.

4 Discussion

In the present study, two new nortriterpenoids, schilancitrilactone M and 25-hydroxyl schindilactone D (1 and 2), together with ten known nortriterpenoids (3–12) were isolated and characterized which assayed for their anti-cancer activities with demonstrating significant effects of compounds 6–8. Notably, compared with 2, 6 was added a double bond at C-7 which would increase the Anti-gastric cancer activity. Compound 9 was essentially similar to compound 7, but it had weaker cytotoxicity in MGC-803 cells than the latter. It is likely because the absolute configuration of 20-bit in compound 9 was the S-configuration. Due to the first position of compound 8 was H instead of OH and the 23-bit of compound 8 was the R-configuration, the Anti-gastric cancer activity of 8 was higher than compound 3. This implied that a schisanartane nortriterpenoid with 1-H, 20S, 23R or a double bond at C-7 is essential for their cytotoxicity against MGC-803.

Supporting information

The HRESIMS, UV, IR, CD, 1D NMR and 2D NMR spectra for compounds 1 and 2 as well as the 1D NMR data for compounds 3–12 are available as supplementary materials.