Abietane-Type Diterpenoids from the Arils of Torreya grandis

Abstract

A chemical investigation of the arils of Torreya grandis led to the isolation of seven abietane-type diterpenoids (compounds 1–7) including three previously undescribed compounds, one unreported natural product, and three known analogs. The structures of these compounds were determined by means of spectroscopy, single-crystal X-ray diffraction, and ECD spectra. An antibacterial activity assay showed that compounds 5 and 6 had significant inhibitory effects on methicillin-resistant Staphylococcus aureus, with MIC values of 100 μM. Moreover, compounds 1, 3, 4, and 7 exhibited anti-neuroinflammatory activity in LPS-stimulated BV-2 microglia cells, with the IC₅₀ values ranging from 38.4 to 67.9 μM.

1. Introduction

Taxaceae plants are the economically and medicinally important coniferous evergreen trees that comprise the well-known genera Taxus, Pseudotaxus and Austrotaxus, whereas the genera Torreya and Amentotaxus attributed to this family have been controversial for a long time due to their close resemblance to the family of Cephalotaxaceae [1]. Among them, Torreya is a primitive member of the gymnospermous yew family (Taxaceae), which consists of seven species and is distributed in the Northern Hemisphere, including North America (T. taxifolia and californica), Japan (T. nucifera), and China (T. fargesii, T. grandis, T. jackii, and T. yunnanensis) [2]. However, species of the genus Torreya are similar in morphology, and the relationship within this genus is still vague [3].

Torreya grandis Fort. ex Lindl cv. Merrillii Hu (Taxaceae), a native Chinese species naturally distributed across subtropical areas in China, has high nutritional and medicinal value [4,5]. Its leaves and fruits are used in traditional Chinese medicine to cure cough, rheumatism, and malnutrition [4]. Recent chemical investigations of T. grandis have led to the discovery of structurally diverse phytochemicals, including diterpenoids [6,7], glycosides [5], flavonoids [8], phenols [9], and fatty acids [10], which exhibit various bioactivities such as antibacterial [6], antioxidant [8,9], and antinociceptive and anti-inflammatory effects [11]; attenuation of cognitive impairment [12]; and inhibitory effects against DGAT1/2 [5]. Nevertheless, chemical constituents of the arils of T. grandis have rarely been reported [13,14]. As part of our continuing search for novel and/or bioactive metabolites from Chinese medicinal plants [15,16,17], a phytochemical investigation of T. grandis arils led to the discovery of seven abietane-type diterpenoids (compounds 1–7), of which compounds 1–3 were previously undescribed, compound 4 was an unreported natural product, and compounds 5–7 were known analogs (Figure 1). Herein, the isolation, structural analysis, and bioactivities of these products are reported.

2. Results and Discussion

Torregrandin A (1) was obtained as a yellow oil, and its molecular formula was determined as C₁₇H₂₂O₂ by a molecular negative-ion peak at m/z 257.1548 [M − H]⁻ (calcd. for C₁₇H₂₁O₂, 257.1547) in its HR-ESI-MS spectrum, requiring 7 degrees of unsaturation. The ¹H NMR data (

Table 1. ¹H NMR (400 MHz) and ¹³C NMR (100 MHz) spectroscopic data for compounds 1 and 2.

No.	1 a		2 b
	δC Type	δH (J in Hz)	δC Type	δH (J in Hz)
1	38.1	2.24 (m, 1H); 1.77 (m, 1H)	39.5	2.19 (m, 1H); 1.36 (m, 1H)
2	17.8	1.71 (m, 2H)	21.5	1.70 (m, 1H); 1.66 (m, 1H)
3	32.2	1.40 (m, 1H); 1.27 (m, 1H)	43.4	1.80 (m, 1H); 1.43 (m, 1H)
4	49.9		73.2
5	43.0	1.79 (m, 1H)	53.4	1.55–1.58 (m, 1H)
6	21.4	1.75 (m, 1H); 1.26(m, 1H)	19.3	2.09 (m, 1H); 1.58–1.67 (m, 1H)
7	29.8	2.69 (m, 2H)	30.8	2.78 (m, 2H)
8	136.5		126.8
9	141.5		148.3
10	36.2		39.3
11	125.7	7.05 (d, 8.6, 1H)	111.8	6.63 (s, 1H)
12	113.3	6.56 (dd, 8.6, 3.0, 1H)	153.4
13	153.4		133.5
14	115.2	6.43 (d, 3.0, 1H)	127.4	6.75 (s, 1H)
15			27.7	3.17 (m, 1H)
16			23.2	1.16 (d, 7.0, 3H)
17			23.2	1.17 (d, 7.0, 3H)
18	206.6	9.20 (s, 1H)
19	14.1	1.09 (s, 3H)	22.9	1.19 (s, 3H)
20	25.4	1.13 (s, 3H)	24.9	1.13 (s, 3H)
18-OMe

^a Recorded in CDCl₃. ^b Recorded in CD₃OD. Chemical shifts (δ) are expressed in ppm, and J values are expressed in Hz.

) showed characteristic signals of a 1,2,4-trisubstituted benzene ring at δ_H 7.05 (d, J = 8.6 Hz, 1H), 6.56 (dd, J = 8.6, 3.0 Hz, 1H), and 6.43 (d, J = 3.0 Hz, 1H), which were supported by the chemical shifts at δ_C 153.4, 141.5, 136.5, 125.7, 115.2, and 113.3 in the ¹³C NMR spectrum (Table 1). Furthermore, an aldehyde group was indicated by the ¹H NMR signals at δ_H 9.20 (s, 1H), corresponding to the carbon signal at δ_C 206.6 (Table 1). The subsequent interpretation of its 2D NMR spectra (HSQC, COSY, and HMBC) unequivocally underpinned that compound 1 was an abietane-type diterpenoid derivative. Comparing the ¹H and ¹³C NMR data of compound 1 to that of the known 13-hydroxy-8,11,13-podocarpatrien-18-oic acid [18] revealed that the carboxylic acid group located at C-18 was substituted by the aldehyde group in compound 1. This was confirmed by the HMBC correlation of H-18 with C-19 and of H₃-19 with C-18 (Figure 2). Moreover, the HMBC correlations of H₃-20 with C-1, C-5, and C-9; of H₃-19 with C-5, C-4, and C-3; of H-14 with C-7, C-9, and C-12; of H-12 with C-9, C-13, and C-14; of H-11 with C-10, C-8, and C-13; and of H-7 with C-5, combined with the ¹H−¹H COSY correlations of H₂-1/H₂-2/H₂-3, of H-5/H₂-6/H₂-7, and of H-11/H-12 unambiguously established the structure of compound 1 (Figure 2). The NOESY correlations of H-3α (δ_H 1.27) with H-5 (δ_H 1.79), of H-3β (δ_H 1.40) with H₃-19 (δ_H 1.09), and of H₃-19 (δ_H 1.09) with H₃-20 (δ_H 1.13) established the relative configuration of compound 1, as shown (Figure 3). The similar optical rotations between compound 1 ([α]²⁵_D +52.0) and 18-oxoferruginol ([α]²⁵_D +69.6) [19] revealed identical configurations of these two compounds. This finding was reinforced by comparing the experimental and calculated ECD spectra of compound 1 (Figure 4), underscoring its (4R,5R,10S)-configuration.

Compound 2 was shown to have a molecular formula of C₁₉H₂₇O₂ based on its molecular negative-ion peak at m/z 287.2018 [M − H]⁻ (calcd. for C₁₉H₂₇O₂, 287.2017) in its HR-ESI-MS spectrum. The ¹H and ¹³C NMR data (Table 1) of compound 2 were very similar to those of torregrandol A [20]. Fortunately, compound 2 was crystallized from a methanol solution, which led to the confirmation of its structure and the assignment of its absolute configuration as 4R,5R,10S, based on its single-crystal X-ray diffraction analysis (Cu Kα) (Figure 5).

Torregrandin B (compound 3) shared an identical molecular formula (C₁₉H₂₇O₂) with compound 2, according to its molecular negative-ion peak at m/z 287.2021 [M − H]⁻ (calcd. for C₁₉H₂₇O₂, 287.2017) in its HR-ESI-MS spectrum. The ¹H and ¹³C NMR data (

Table 2. ¹H NMR (400 MHz) and ¹³C NMR (100 MHz) spectroscopic data for compounds 3 and 4.

No.	3 a		4 b
	δC Type	δH (J in Hz)	δC Type	δH (J in Hz)
1	38.4	2.21 (dt, 13.0, 4.0, 1H); 1.42 (m, 1H)	36.7	2.17 (dd, 12.9, 3.5, 1H); 1.60–1.72 (m, 1H)
2	18.6	1.62 (m, 2H)	17.7	1.60–1.72 (m, 1H); 1.48 (td, 12.9, 3.8, 1H)
3	40.9	1.72 (m, 1H); 1.42 (m, 1H)	36.1	1.76 (t, 12.9, 1H); 1.60–1.72 (m, 1H)
4	72.5		46.2
5	48.8	1.42 (m, 1H)	44.0	2.45 (dd, 14.1, 3.3, 1H)
6	18.2	2.00 (m, 1H); 1.83 (m, 1H)	37.2	2.68 (dd, 17.6, 14.1, 1H); 1.98 (dd, 17.6, 3.3, 1H)
7	28.8	2.86 (m, 2H)	195.6
8	127.1		122.3
9	147.9	2.3 (m, 1H)	155.3
10	37.3		37.0
11	110.9	6.64 (s, 1H)	109.3	6.79 (s, 1H)
12	150.9		160.5
13	131.7		132.9
14	126.8	6.85 (s, 1H)	125.1	7.64 (s, 1H)
15	27.0	3.12 (m, 1H)	26.1	3.13 (m, 1H)
16	22.7	1.23 (d, 7.0, 3H)	22.2	1.13 (d, 6.4, 3H)
17	22.9	1.24 (d, 7.0, 3H)	22.4	1.14 (d, 6.4, 3H)
18	30.9	1.25 (s, 3H)	177.3
19			16.2	1.25 (s, 3H)
20	24.5	1.30 (s, 3H)	23.2	1.18 (s, 3H)
18-OMe			52.3	3.59 (s, 3H)

^a Recorded in CDCl₃. ^b Recorded in DMSO-d₆. Chemical shifts (δ) are expressed in ppm, and J values are expressed in Hz.

) of this compound were very similar with those of compound 2 except for a downfield signal at δ_C 30.9, ascribable to the 4-CH₃ in the ¹³C NMR spectrum. To the best of our knowledge, the carbon resonances of an α-oriented CH₃ group at C-4 in abietane-type diterpenoids can range from 25 to 35 ppm, whereas in a β-oriented CH₃ group, they can range from 15 to 25 ppm [6,21]. The downfield chemical shift of 4-CH₃ (δ_C 30.9) in comparison with that of compound 2 (δ_C 22.9) revealed that compound 3 was an epimer of compound 2 at C-4. Next, the NOESY correlations of H-3α (δ_H 1.72) with H-5 (δ_H 1.42) and H₃-19 (δ_H 1.25), of H-5 (δ_H 1.42) and H-1β (δ_H 2.21), and of H-1α (δ_H 1.42) with H₃-20 (δ_H 1.30) established the relative configuration of compound 3, as shown (Figure 3). The absolute configuration was determined by means of ECD calculation, which unambiguously pinpointed the (4S,5R,10S)-configuration of compound 3 (Figure 6).

Compound 4, namely methyl 12-hydroxy-7-oxodehydroabietate, was recently isolated from Torreya grandis [20], which was previously synthesized by Hamulić and co-workers [22]. However, its absolute configuration was not assigned. In the present work, the absolute configuration of compound 4 was firstly confirmed by the single-crystal X-ray diffraction analysis (Cu Kα) (Figure 7).

The remaining known diterpenoids were characterized as dehydroabietinol (5) [23], dehydroabietic acid (6) [24], and torreyagrandate (7) [25] by comparing the ¹H and ¹³C NMR data as well as mass spectrometric spectra to the published data.

All isolated compounds were tested for their antibacterial activitity against Escherichia coli, methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Salmonella. However, only compounds 5 and 6 had significant inhibitory effects on MRSA, with MIC values of 100 μM, which were comparable to those of the positive control, rifampicin (MIC = 0.625 μM). Other compounds displayed no obvious antibacterial activity at 100 μM. Moreover, the anti-neuroinflammatory activity in LPS-induced BV-2 cells was evaluated for these compounds. The results indicated that compounds 1, 3, 4, and 7 showed a weak inhibitory effect on NO production, with IC₅₀ values of 49.4, 41.9, 38.4, and 52.6 μM (

Table 3. Inhibitory effects of compounds 1, 3, 4, and 7 on NO production induced by LPS in BV-2 microglial cells.

Compound	IC50 (μM) a	Cell Viability (%) b
1	49.4 ± 0.4	81.6 ± 3.5
3	41.9 ± 1.5	85.4 ± 1.3
4	38.4 ± 0.6	87.1 ± 2.3
7	52.6 ± 2.3	96.1 ± 1.0
quercetin c	10.8 ± 1.6	99.7 ± 1.8

^a Results are presented as mean ± SD from three independent experiments. ^b Cell viability (%) of BV-2 microglial cells at 100 μM expressed as the mean ± SD from three independent experiments. ^c Positive control.

), respectively. Meanwhile, none of the remaining compounds exhibited an inhibitory effect on NO production at 100 μM. All the results are representative of three independent experiments.

3. Materials and Methods

3.1. General

A Rudolph Autopol III instrument was used to measure the optical rotations. HR-ESI-MS spectra were tested using a TripleTOF 5600+ system (AB SCIEX, Framingham, MA, USA). Infrared (IR) spectra were acquired using a Bruker TENSOR 27 spectrometer (Bruker). Ultraviolet (UV) and electronic circular dichroism (ECD) spectra were recorded by a Chirascan spectrometer (Applied Photophysics, Ltd., Leatherhead, UK). The NMR data were acquired on a Bruker Avance-400 spectrometer (Beijing Oubeire Co., Ltd., Beijing, China). Column chromatography (CC) was executed using silica (200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, China), C18 reversed-phase silica (ODS-AQ-HG GEL, AQG12S50, YMC, Co., Ltd., Kyoto, Japan), and Sephadex LH-20 gels (GE Healthcare, Inc., Uppsala, Sweden). An Agilent 1100 series system (Agilent Technologies, Inc., Agilent Technologies, Inc., CA, USA) with a Prazis absolute C18 column (5 µm, 10 mm × 250 mm) was used for the HPLC analysis and preparation. Fractions were monitored by TLC (Qingdao Marine Chemical, Ltd., Qingdao, China), with the spots visualized using the vanillin–sulfuric acid color method.

3.2. Plant Materials

The arils of Torreya grandis Fort. ex Lindl. cv. Merrillii. (Taxaceae) were collected in November of 2022 in Yuexi, Anhui Province, People’s Republic of China. The plant sample was identified by Prof. Zhen-Hai Wu at College of Life Sciences, Northwest A&F University. A voucher specimen has been deposited in our institute with the following accession number: TG-arils-2022-AH.

3.3. Extraction and Isolation

The air-dried powder of the arils of Torreya grandis (1.5 kg) was presoaked three times with 95% EtOH (3 × 2 L) at room temperature to produce an extract (338 g) which was first re-suspended in H₂O and then partitioned with petroleum ether (PE), ethyl acetate (EtOAc), and n-butanol. The EtOAc extract (75 g) was eluted with PE-EtOAc (v/v, 50:1→1:1), which was then combined under the guidance of the thin-layer chromatography (TLC) analysis to obtain eight fractions (A–H). Fraction E (11.6 g) was subjected to a silica gel.

Gel CC with PE-EtOAc (v/v, 10:1→2:1) produced five fractions (E1–E5). Fraction E2 (1.2 g) was isolated on a Sephadex LH-20 column eluted with MeOH to yield five fractions (E2a–E2e). Fraction E2b (300 mg) was separated on a reverse-phase C18 silica gel CC column eluted with MeOH-H₂O (20–100%, v/v) to yield three fractions (E2b1–E2b3). Fraction E2b2 (120 mg) was purified by semipreparative RP-HPLC eluted with CH₃OH–H₂O (60:40, v/v) to yield compounds 2 (7.8 mg, t_R 22.7 min) and 3 (5.4 mg, t_R 23.5 min). Fraction E3 (2.2 g) was separated on a silica gel CC column and eluted with PE-EtOAc (v/v, 10:1→2:1) to obtain five fractions (E3a–E3e). Fraction E3c (320 mg) was chromatographed over a Sephadex LH-20 column with MeOH to produce three fractions (E3c1–E3c3). After purification by semipreparative RP-HPLC with CH₃OH–H₂O (60:40, v/v), fraction E3c2 (90 mg) yielded compound 4 (4.8 mg, t_R 21.5 min). Fraction E4 (900 mg) was separated by a Sephadex LH-20 column with MeOH to yield three major fractions (E4a–E4c). Fraction E4b (85 mg) was purified by semipreparative RP-HPLC with CH₃OH–H₂O (62:38, v/v) to yield compound 7 (21 mg, t_R 28 min). Fraction F (2.5 g) was chromatographed over a silica gel CC column with PE-EtOAc (v/v, 10:1→1:1) to yield five fractions (F1–F5). Fraction F2 (220 mg) was subjected to a Sephadex LH-20 column with MeOH to yield three fractions (F2a–F2c). Fraction F2b (45 mg) yielded compound 5 (17 mg, t_R 25.7 min) after purification by semipreparative RP-HPLC with CH₃OH–H₂O (60:40, v/v). Fraction G (10.5 g) was subjected to a silica gel CC column with PE-EtOAc (v/v, 10:1→1:1) to yield five fractions (G1–G4). Fraction G2 (1.8 g) was subjected to a reverse-phase C18 silica gel CC column eluted with MeOH-H₂O (20–90%, v/v) to obtain three fractions (G2a–G2c). After re-purification using a Sephadex LH-20 column, compound 1 (12 mg, t_R 19.5 min) was obtained from the fraction G2b (320 mg) through a semipreparative RP-HPLC column with CH₃OH–H₂O (65:35, v/v). Fraction G2c (500 mg) was purified by a Sephadex LH-20 column eluted with MeOH to generate three fractions (G2c1–G2c3). Then, fraction G2c2 (105 mg) was separated by a semipreparative RP-HPLC column with CH₃OH–H₂O (60:40, v/v) to yield compound 6 (30 mg, t_R 31.3 min).

3.4. Structural Elucidation

Torregrandin A (compound 1), yellow oil; [α]_D²⁵ +52 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 200 (1.95) nm; IR (KBr) ν_max 3427, 3172, 2928, 1717, 1615, 1498, 1399, 1112 cm⁻¹; HR-ESI-MS (negative) m/z 257.1548 [M − H]⁻ (calcd. for C₁₇H₂₁O₂, 257.1547); ¹H and ¹³C NMR dataassigned and listed in Table 1.

Torregrandin B (compound 3), yellow oil; [α]_D²⁵ +59 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 200 (1.73) nm; IR (KBr) ν_max 3604, 3304, 2934, 1714, 1507, 1423, 1368, 1236, 1017 cm⁻¹; HR-ESI-MS (negative) m/z 287.2021 [M − H]⁻ (calcd. for C₁₉H₂₇O₂, 287.2017); ¹H and ¹³C NMR data assigned and listed in Table 2.

3.5. Crystal Data for Compounds 2 and 4

Compound 2, C₁₉H₂₈O₂, Mr = 288.41 block from MeOH, space group C₁2₁, a = 18.245(3) Å, b = 8.7702 (12) Å, c = 10.5065(15) Å, V = 1591.6 (4) ų, Z = 4, μ = 0.374 mm⁻¹, and F(000) = 632.0; T = 170.0; crystal dimensions: 0.08 × 0.06 × 0.04 mm³; R = 0.0393, wR = 0.0982, S = 1.090; Flack parameter = 0.17(12); crystallographic data for compound 2 has been deposited at the Cambridge Crystallographic Data Center with the accession number CCDC-2328804.

Compound 4, C₂₁H₂₈O₄, Mr = 344.43 block from MeOH, space group P2₁2₁2₁, a = 11.0818(2) Å, b = 11.2194(2) Å, c = 14.5634(2) Å, V = 1810.68(5) ų, Z = 4, μ = 0.690 mm⁻¹ and F(000) = 744.0; T = 150.0; crystal dimensions: 0.2 × 0.15× 0.1 mm³; R = 0.0713, wR = 0.1545, S = 1.135; Flack parameter = −0.05(5); crystallographic data for compound 4 has been deposited at the Cambridge Crystallographic Data Center with the accession number CCDC-2328805.

3.6. ECD Calculation

The ECD calculation was performed according to our previously reported methods [26].

3.7. Antibacterial Assay

To determine the MICs, bacterial strains of Escherichia coli, methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Salmonella were cultured in 2 mL of Mueller–Hinton broth (MHB) for 3−5 h at 37 °C, followed by a dilution of the mixture to a concentration of 1 × 10⁵ CFU/mL. Next, 100 μL of each dilution and 100 μL of bacterial suspension were successively added to 96-well plates. Subsequently, the tested compounds were added to give the final concentrations of 200, 100, 50, 25, and 12.5 μM, respectively. The positive groups were rifampicin (10, 5, 2.5, 1.25, 0.625 μM), and the negative and blank groups were 200 μL of media and bacterial solution (1 × 10⁵ CFU/mL). After incubation at 37 °C for 16–18 h, the antibacterial effects were observed by the naked eye. The lowest concentration of the tested compounds which completely inhibited the growth of bacteria was defined as the MIC value. The experiment was repeated at least three times.

3.8. Cytotoxicity and Anti-Inflammatory Assays

The bioassays for NO production and cell viability were conducted according to our previously reported methods [27,28]

4. Conclusions

In summary, seven abietane-type diterpenoids (compounds 1–7), including three previously undescribed ones and one unreported natural product as well as three known analogs, were isolated and characterized from the arils of T. grandis. Moreover, compounds 5 and 6 showed mild inhibitory effects against MRSA, and compounds 1, 3, 4, and 7 exhibited weak anti-neuroinflammatory activity in LPS-induced BV-2 microglia cells. These findings not only enrich the molecular diversity of abietane-type diterpenoids but also offer evidence for antibacterial and anti-neuroinflammatory agents that could be used against human pathogenic bacteria and neuroinflammation-related diseases.