Acyclic diterpene and norsesquiterpene from the seed of Aphanamixis polystachya

Fitoterapia 142 (2020) 104518

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Acyclic diterpene and norsesquiterpene from the seed of Aphanamixis polystachya

T

Shang Xue, Panpan Zhang, Pengfei Tang, Chengcheng Wang, Lingyi Kong , Jun Luo ⁎

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Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key Laboratory of Natural Medicines, School of Traditional Chinese Pharmacy, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People's Republic of China

ARTICLE INFO

ABSTRACT

Keywords: Aphanamixis polystachya Mosher method Acyclic norsesquiterpene NO production inhibitory activity Acyclic diterpene

Aphanamoxene A-D (1–4), three new acyclic diterpene derivatives and one new acyclic norsesquiterpene were isolated from the seed of Aphanamixis polystachya. Their structures were elucidated on the basis of extensive spectroscopic methods, including 1D and 2D NMR and HRESIMS. And the absolute configuration of 1 was achieved by Mosher method. These acyclic terpenoids (1–4) showed obvious nitric oxide production inhibitory activity on lipopolysaccharide-Induced RAW264.7 macrophages with IC50 values of 17.6 ± 1.4, 9.8 ± 0.7, 16.6 ± 1.2, and 14.2 ± 0.9 μM, respectively.

1. Introduction The genus Aphanamixis (order Meliaceae, family Aphanamixis Bl.), which include approximately 25 species, were widely distributed over southern China, India, Indochina, Indonesia and Malaysia [1]. There are four species were discovered and identified as a part of genus Aphanamixis within the scope of China. Moreover, Aphanamixis polystachya was the earliest species that has been investigated [2].The A. polystachya is an evergreen timber tree, and the bark has been used as a folk medicine which is used as an astringent and hemostasis herb [3]. Meanwhile, its seed oil can treat rheumatoid joint pain and limbs numbness [4]. Previous phytochemical investigations of this species revealed its chemical compositions, including limonoids [5,6], diterpenoids [7,8], triterpenoids [9] and diterpenoids dimers [10,11]. Besides, based on many botany chemical researches, these compounds have series of bioactivities, such as insect antifeedant [12], anti-inflammatory [13], antimalarial [14] and cytotoxic [15]. The acyclic diterpenoid is a common diterpene sort which were isolated from genus Aphanamixis. And it possesses an unusual linear structure without intact carbon rings [16,17]. Our previous research in 2013 [18] discovered that the acyclic diterpenoid is regarded as a monomer of acyclic diterpenoids dimmer. Consequently, the acyclic diterpenoid became a research hotspot in recent years. In order to screen various pharmacological activities and explore structure diversities, we take efforts to search the acyclic diterpenoids from the seed of A. polystachya. As a result, three new acyclic diterpenoids and one new norsesquiterpene derivative, named aphanamoxenes A-D were isolated and

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characterized. Herein, we report the isolation, structures determinations and bioactivities of these compounds. 2. Experimental 2.1. General HRESIMS data were measured on an Agilent 6520B Q-TOF mass. NMR data were obtained rely on Bruker Avance-500 or Ascend-600 NMR in CDCl3 with TMS as internal standard. Optical rotations (in MeOH, c ln g/mL) were acquired with a JASCO P-1020 polar meter. UV data were showed on a Shimadzu UV-2450 spectrophotometer. IR spectra were detected with KBr-disc on a Bruker Tensor 27. Column chromatography (CC) were carried out with silica gel (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), MCI and ODS (40–63 μm, Fuji Silysia Chemical, Kasugai, Aichi, Japan), successively. Preparative HPLC was applied on a Shimadzu LC6 AD series with a Shim-park RPC18 column (20 × 200 mm) and a Shimadzu SPD-20A detector. Analytical HPLC was performed on an Agilent 1200 Series using a Shim-pack VP–ODS column (4.6 × 250 mm) with a DAD detector. Chiral analysis was chromatographed on Phenomenex Lux 5u Cellulose2 column (4.6 × 250 mm) with a DAD detector. 2.2. Plant The seeds of A. polystachya were collected from Xishuangbanna, Yunnan Province of China in March 2017, and were identified by

Corresponding authors. E-mail addresses: [email protected] (L. Kong), [email protected] (J. Luo).

https://doi.org/10.1016/j.fitote.2020.104518 Received 7 January 2020; Received in revised form 15 February 2020; Accepted 16 February 2020 Available online 22 February 2020 0367-326X/ © 2020 Elsevier B.V. All rights reserved.

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Professor Shun, Cheng Zhang, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, People's Republic of China. A voucher specimen (No. AP201703) was deposited in the Department of Natural Medicinal Chemistry, China Pharmaceutical University.

2.3.3. Aphanamoxene C (3) Yellowish gum; UV (MeOH) λmax (log ɛ) 209 (2.01) nm; IR (KBr) νmax 3435, 2936, 1719, 1438, 1383, 1227, 1152 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 357.2037 [M + Na] + (calcd. For C20H31O4,m/z 357.2036).

2.3. Extraction and isolation

2.3.4. Aphanamoxene D (4) Light yellowish oil; UV (MeOH) λmax (log ɛ) 205 (2.18) nm; IR (KBr) νmax 3436, 2918, 1721, 1641, 1440, 1153, 1116 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 273.1463 [M + Na] + (calcd. For C15H23O3, 273.1461).

The air-dried seeds (4.0 kg) of A. polystachya were powdered and refluxed with 95% EtOH three times. The concentrated exact (1.1 kg) was suspended in H2O and extracted with petroleum ether and CH2Cl2 successively. The petroleum ether extract (471 g) was further sectioned between petroleum ether and 95% MeOH-H2O, and the petroleum ether residue (287 g) was subjected to a silica gel column eluted with petroleum ether and EtOAc mixtures (100:1–100:2–100:3–100:5–10:1–5:1) to give five fractions F1-F5. Fraction F5 (30 g) was chromatographed on a silica gel column eluting with petroleum ether and EtOAc mixtures (15:1–10:1–5:1–2:1–1:1) to give five fractions F5A-F5E. The 2:1 petroleum ether-EtOAc fraction F5D (6.7 g) was applied to MCI gel column eluted with MeOH-H2O (60:40–90:10) to yield three fractions F5D1–3. F5D1 was chromatographed on ODS column with step gradient of CH3CN-H2O (45:55–75:25), yielded four fraction F5D1–1-F5D1–4, F5D1–1 further separated by preparative HPLC with MeOH-H2O (65:35, 10 mL/min) to obtained 1 (75 mg). F5D1–2 was purified on preparative HPLC with MeOH-H2O (70:30,10 mL/min) to obtained 2 (18 mg) and 3 (10 mg), F5D1–3 was chromatographed on preparative HPLC with MeOH-H2O (73:27, 10 mL/min) to obtained 4 (8 mg).

2.4. Inhibition of nitric oxide production assay Inhibition of NO production was carried on LPS-induced RAW 264.7 macrophage cell lines. RAW264.7 cells were incubated with the compounds in serum-free medium. After 24 h of incubation, MTT (20 μL, 5 mg/mL) was added to each well and incubated with cells for 4 h. Then, carefully removing the culture medium and adding 150 μL dimethyl sulfoxide (DMSO) to dissolve the crystals by shaking for 10 min. The absorbance was measured at 490 nm. DMSO is also considered a blank control. All experiments were repeated three times [19]. The RAW264.7 cells seeded on 96-well plates were incubated with the compound for 3 h, and then 1 μg/mL LPS was added for 24 h. The Griess reagent was used to measure the NO content at 540 nm, and the IC50 value of the compound was determined based on the inhibition rate (%) compared with the blank control and the model control [20].

2.3.1. Aphanamoxene A (1) Yellowish gum; [α] 25 -8.4 (c 0.1, MeOH); UV (MeOH) λmax (log ɛ) D 213 (2.23) nm; IR (KBr) νmax 3436, 2932, 2858, 1720, 1438, 1226, −1 1 13 1150 cm ; H and C NMR data, see Table 1; HRESIMS m/z 375.2507 [M + Na] + (calcd. For C21H36O4Na, m/z 375.2506).

3. Results and discussion Aphanamoxene A (1) was obtained as a yellowish oil, and defined the molecular formula of C21H36O4 based on HRESIMS ion peak 375.2507 [M + Na] + (calcd. For C21H36O4Na, m/z 375.2506), accounting for four degrees of unsaturation. The 1H NMR data of 1 (Table 1) exhibited signals were assigned to five methyl groups [two tertiary at δH 1.14, 1.18 and three vinylic at δH 1.59, 1.60, 2.15 (each s, 3H)] and a methoxy at δH 3.67 (s, 3H), three olefin protons at δH 5.07 (s, br, 1H), δH 5.15 (t, 1H), δH 5.66 (s, 1H). while the 13C NMR (Table 1) data showed 21 signals including one ester carbon at δC 167.5, three

2.3.2. Aphanamoxene B (2) Yellowish gum; UV (MeOH) λmax (log ɛ) 205 (2.22) nm; IR (KBr) νmax 3430, 2923, 2853, 1720, 1649, 1435, 1384, 1226, 1149 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 371.2193 [M + Na] + (calcd. For C21H33O4, m/z 371.2193). Table 1 1 H and 13C NMR spectroscopic data of 1–4 in CDCl3. Position

1

No. 1 2 3 4 5 6 7 8 9 10 11 12α 12β 13α 13β 14 15 16 17 18 19 20 1-OCH3

δH – 5.66 – 2.17 2.16 5.07 – 1.99 2.07 5.15 – 2.23 2.04 1.57 1.40 3.33 – 1.18 1.14 1.59 1.60 2.15 3.67

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2

(s) (m⁎) (m⁎) (m⁎) (q, 7.4) (m⁎) (t, 6.4) (m⁎) (m⁎) (m⁎) (m⁎) (dd, 10.5, 1.6) (3H, s) (3H, s) (3H,s) (3H, s) (3H, s) (3H, s)

δC 167.4 115.4 160.3 41.0 26.0 123.1 136.1 39.7 26.5 125.0 135.1 36.9 29.9 78.4 73.1 26.5 23.5 16.1 16.0 19.0 50.9

δH – 5.65 – 2.17 2.17 5.11 – 2.13 2.35 6.61 – – – 6.86 – 6.85 – 1.37 1.37 1.82 1.62 2.14 3.66

3

(s) (m⁎) (m⁎) (m⁎) (m⁎) (q, 7.4) (t, 7.1)

δC 167.5 115.4 160.1 40.8 25.9 124.0 135.1 38.3 27.7 143.0 138.2 192.3

(d, 15.0)

120.8

(d, 15.0)

152.6 71.3 29.6 29.6 11.9 16.2 19.0 51.0

(3H, s) (3H, s) (3H,s) (3H, s) (3H, s) (3H, s)

Overlap signals. 2

δH – 5.67 – 2.20 2.20 5.10 – 2.15 2.36 6.62 – – – 6.86 – 6.85 – 1.38 1.38 1.83 1.63 2.15

4

(s) (m⁎) (m⁎) (m⁎) (m⁎) (m⁎) (t, 7.0)

δC 170.4 115.2 162.5 41.0 25.8 123.8 135.3 38.3 27.7 143.1 138.2 192.4

(d, 15.0)

120.8

(d, 15.0)

152.5 71.5 29.6 29.6 11.9 16.2 19.2

(3H, s) (3H, s) (3H,s) (3H, s) (3H, s)

δH – 5.66 – 2.19 2.19 5.16 – 2.86 6.74 6.05 – 2.25 – 1.61 – 2.16

(3H,s)

δC 167.3 115.6 159.8 40.7 26.1 126.1 132.7 42.6 146.3 132.3 198.8 27.0

(3H,s)

16.5

(3H,s)

18.9

(s) (m⁎) (m⁎) (m) (d, 7.0) (dt, 15.9, 7.0) (d, 15.9)

3.68 (3H, s)

51.0

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pairs of olefinic carbons at δC 160.3, 136.1, 135.1, 125.0, 123.1, 115.4, two oxygenated carbons at δC 73.1, 78.4, one methoxy group at δC 50.9 with aided by HSQC. Deducting four indices of hydrogen deficiency accounted for one carbonyls and three double bonds, indicating an acyclic system in 1. These characteristic NMR data illustrated that 1 was a typical acyclic diterpene with terminal methyl ester moiety similar to melidianolic acid A [21]. The difference was the existence of a methoxy group in 1, which could be demonstrated by HMBC correlation of 1-OMe (δH 3.67) to C-1 (δC 167.4). The planar structure of aphanamoxene A was determined by HSQC, HMBC and 1He1H COSY spectra (Fig. 2). The observed HMBC cross peaks, from Me-20 (δH 2.15) to C-4 (δC 41.0), C-2 (δC 115.5), C-3(δC 160.2); from Me-19 (δH 1.60) to C-8 (δC 39.7), C-6 (δC 123.1), C-7 (δC 136.1), Me-18 (δH 1.59) with C-12 (δC 36.9), C-10 (δC 125.0), C-11(δC 135.1), verified these three methyl groups attached to double-bond Δ2(3), Δ6(7), Δ10(11) respectively. HMBC signals exhibited significant correlation between Me-16 (δH 1.18), Me-17 (δH 1.14) with C-14 (δC 78.4), C-15 (δC 73.1). The result illustrates two tertiary methyl attached to the oxygenated quaternary carbon C-15 (δC 73.1). Then, obvious 1 He1H COSY correlations of H2–4/H2–5/H-6, H2–7/H2–8/H2–9, and H2–12/H2–13/H-14 also indicted 1 was an adipose-like chemical component. Based on obvious ROESY correlation of H-6/H2–8, H10/H2–12 and H-2/Me-20, the configuration of double bonds Δ 6(7), Δ 10(11) were assigned to E-type, while the double bonds Δ 2(3) was assigned to Z-type, respectively. Therefore, the planer structure was identified as a chained diterpenoid hitherto. Chirality HPLC showed 1 was an optically pure compound, thus, the absolute configuration of the secondary alcohol moiety in 1 was elucidated by Mosher method [22]. The Mosher esters of aphanamoxene A prepared by conventional way [23]. (S)-Mosher esters 1a was made of (R)-(−)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride (MTPACl) likewise (R)-Mosher ester 1b was made of (S)-(+)-α-methoxy-α(trifluoromethyl)phenylacetyl chloride. The 1H NMR signals of the two MTPA esters were assigned unambiguously based on 1H − 1H COSY spectra, and the ΔδH (S-R) values were then calculated (Fig. 2). The configuration of chiral secondary alcohol in 1 was defined to 14R based on regular pattern. Therefore, the structure of 1 was established as shown in Fig. 1. Aphanamoxene B (2) was obtained as a yellowish gum, and defined the molecular formula of C21H32O4 based on HRESIMS, determined by ion peak m/z 371.2193 [M + Na] + (calcd. For C21H33O4, m/z 371.2193), and calculating six indices of hydrogen deficiency. The 1H

Fig. 2. Selected key HMBC and 1He1H COSY correction and key Values of ΔδH (S-R) for the MTPA Esters of 1(in CDCl3).

Fig. 3. Key HMBC and 1He1H COSY (bold) Correction of compounds 2, 4.

and 13C (Table 1) NMR data suggesting 2 was an analog of aphanamoxene A (1). The obvious difference compared to compound 1 was the presence of a α-β unsaturated ketone moiety (δC 152.6, 120.8, 192.3) which could be demonstrated by 13C NMR signals and HMBC correlations of H-13 (δH 6.86), H-14 (δH 6.85) with C-12 (δC 192.3), C-15 (δC 71.3) in Fig. 3. The key HMBC correlations from H-10 (δH 6.61) and Me18 (δH 1.82) to C-12 (δC 192.3), Me-16/17 (δH 1.38) to C-14 (δC 152.6) and C-15 (δC 71.3) indicated that the α-β unsaturated ketone located at C-12/13/14. (Fig. 3). The ROESY correlations of H-2/Me-20, H-6/H-8, Me-18/H-10 indicated an (Z)-(E)-(E)-geometry of the Δ 2(3), Δ 6(7) and Δ 10(11) double bond, the coupling constant of H-13/H-14 (J = 15.0HZ) confirmed an E-geometry of Δ 13 (14) double bond. Therefore, the structure of 2 was finally assigned and displayed in Fig. 1. Aphanamoxene C (3) was also obtained as a light yellow oil, and counted the molecular formula of C20H30O4 based on HRESIMS, suggested by ion peak HRESIMS m/z 357.2037 [M + Na] + (calcd. For C20H31O4, m/z 357.2036). The similarity and difference of the 1D NMR (Table 1) and HRESIMS data between 3 and 2 indicated that 3 was nearly the same as 2 except absence of a methoxy obviously. This speculation was also confirmed by the down-shifted chemical shift of C1 and HSQC, HMBC spectrum of 3. The (Z)-(E)-(E)-(E)-geometry of the Δ 2(3), Δ 6(7), Δ 10(11) and Δ13(14) double bonds in 3 were determined by key REOSY correlations and coupling constant of H-13/H-14 (J = 15.0HZ). Thus, the structure of compound 3 was established as shown in Fig. 1. Aphanamoxene D (4) was obtained as a yellow oil, and detected the molecular formula C15H22O3 by the aid of HR-EI-MS with m/z 273.1463 [M + Na] + (calcd. For C15H23O3, 273.1461). The 1H and 13C (Table 1) spectrum inferred compound 4 was a norsesquiterpene derivative for the existence of a methoxy group. The 1H NMR data spectrum displayed resonances for three methyl (two vinylic at δH 2.16, 1.61 and one tertiary at δH 2.25), four olefin protons at δH 6.74, 6.06, 5.66, 5.16. The 13 C NMR data showed 15 carbon signals include two carbonyl moieties,

Fig. 1. Structures of Aphanamoxenes A- D (1–4). 3

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Scheme 1. Biogenetic pathway proposed for compounds 1–4.

three groups of double-bond carbons at δC 159.8, 146.3, 132.7, 132.3, 126.1, 115.6. HMBC signals indicated Me-12 (δH 2.25) have obvious correlation with C-11 (δC 198.8) exclusively, suggesting Me-12 was a terminal methyl attached to a ketone moiety which similar to know compound nemoralisin D [24]. The HMBC correlations from H-9 (δH 6.74)/H-10 (δH 6.06) to C-11(δC 198.8) and C-8 (δC 42.6) indicated the presence of a Δ 9(10) double bond in 4. The E-geometries of Δ 2(3), Δ 6(7) double bonds were defined on the basis of ROESY spectrum of compound 1 because of the extremely similar spectrum characteristics. The E-geometries of Δ 9(10) double bond was verified by coupling constant H-9/H10 (J = 15.9HZ). Then, the structure of compound 4 was identified unambiguously as displayed in Fig. 1. In terms of biogenesis, the precursor of compounds 1–4 speculated to be known compounds geranylgeranoic acid and melidianolic acid A (Scheme 1). Compound 1 could yield from melidianolic acid A rely on methyl-esterification reaction. On the other route, compound 2 would obtain from melidianolic acid A after oxidation and dehydration, then esterification of 2 could yield 3. Compound 4 maybe derived from compound 3 involve oxidation and degradation steps. The acyclic diterpenes generally be reported as potential inflammatory agents, wherefore compounds 1–4 were determined for their inhibitory effects on NO production induced by LPS in a macrophage cell line RAW264.7. Cell viability was first tested by the MTT method to find whether inhibition of NO production was owing to the cytotoxicity of the tested compounds. There was no obvious cytotoxic effect (over 90% cell survival) on RAW264.7 cells treated with compounds 1–4 at concentrations of up to 40 μM. Compounds 1–4, respectively, exhibited remarkable Inhibitory activity on NO production with IC50 values of 17.6 ± 1.4, 9.8 ± 0.7, 16.6 ± 1.2, and 14.2 ± 0.9 μM. The consequent demonstrated the compound 1–4 have relative significant anti-inflammatory activity.

Declaration of Competing Interest The authors declare no conflict of interest. Acknowledgment This work was financially supported by the National Natural Science Foundation of China (31470416), the “Double First-Class” University project (CPU2018GY08, China), the 111 Project from Ministry of Education of China and the State Administration of Foreign Export Affairs of China (B18056), and the Drug Innovation Major Project (2018ZX09711-001-007). Appendix A. Supplementary data HRESIMS, NMR, UV, IR spectra of compounds 1–4 and 1a/1b are available as supporting information. Supplementary data to this article can be found online at https://doi.org/10.1016/j.fitote.2020.104518. References [1] S.-K. Chen, B.-Y. Chen, H. Li, Flora Reipublicae Popularis Sinicae, 43 Science Press, Beijing, 1997, pp. 75–80. [2] H. Peng, D.-J. Mabberley, C.-M. Pannell, J. Edmonds, B. Bartholomew, Flora of China, 11 Scientific Press, Beijing, 2008, pp. 70–75. [3] M. Gupta, S. Laskar, Surface hydrocarbons from the leaves of Amoora rohituta W&A, Biotechnol. Res. Asia 6 (2009) 379–381. [4] C.-D. Daulatabad, A.-M. Jamkhandi, A keto fatty acid from Amoora rohituka seed oil, Phytochemitry 46 (1997) 155–156. [5] Y. Zhang, J.-S. Wang, Y.-C. Gu, L.Y. Kong, Aphagranols D - H: five new Limonoids from the fruits of Aphanamixis grandifolia, Helvetica Chim. Acta 97 (2014) 1354–1364. [6] S.-P. Yang, H.-D. Chen, S.-G. Liao, B.-J. Xie, Z.-H. Miao, J.-M. Yue, Aphanamolide a, a new Limonoid from Aphanamixis polystachya, Org. Lett. 13 (2011) 150–153. [7] F.-H. Fang, W.-J. Huang, S.-Y. Zhou, Z.-Z. Han, M.-Y. Li, L.-F. Liu, X.-Z. Wu, X.J. Yao, Y. Li, C.-S. Yuan, Aphapolins A and B: Two Nemoralisin Diterpenoids Isolated from Aphanamixis polystachya (Wall.) R. Parker, Eur. J. Org. Chem. 30

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