Phloroglucinols with Immunosuppressive Activities from the Fruits of

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Phloroglucinols with Immunosuppressive Activities from the Fruits of Eucalyptus globulus Thi-Anh Pham,†,∥,# Xiao-Long Hu,†,# Xiao-Jun Huang,‡ Ming-Xi Ma,§ Jia-Hao Feng,† Jun-Yan Li,† Ji-Qin Hou,† Pei-Lin Zhang,† Van-Hung Nguyen,∥ Manh-Tuyen Nguyen,⊥ Fei Xiong,§ Chun-Lin Fan,‡ Xiao-Qi Zhang,‡ Wen-Cai Ye,‡ and Hao Wang*,†

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†

State Key Laboratory of Natural Medicines, Department of TCM Pharmaceuticals, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 210009, People’s Republic of China ‡ Institute of Traditional Chinese Medicine and Natural Products, Jinan University, Guangzhou 510632, People’s Republic of China § State Key Laboratory of Bioelectronics, Jiangsu Laboratory for Biomaterials and Devices, Southeast University, Nanjing 210009, People’s Republic of China ∥ Department of Traditional Medicine, School of Pharmacy, Haiphong University of Medicine and Pharmacy, 72 A Nguyen Binh Khiem, Haiphong City, Vietnam ⊥ Department of Traditional Medicine, Hanoi University of Pharmacy, 13-15 Le Thanh Tong, Hoan Kiem, Hanoi, Vietnam S Supporting Information *

ABSTRACT: Five new phloroglucinol derivatives, eucalyptins C−G (1−5), together with 13 known analogues (6−18) were isolated from the fruits of Eucalyptus globulus. The structures and absolute configurations of 1−5 were established by means of spectroscopic data analysis, computational calculation methods, and single-crystal X-ray diffraction. Compounds 1−18 were investigated for their immunosuppressive effects in vitro, and 1, 2, 6, and 7 displayed moderate inhibitory activities with IC50 values of 11.8, 10.2, 18.2, and 19.1 μM, respectively. The stimulation index (SI) of 1 was 64.2 and was compared to that of cyclosporine A (SI = 149.57). Further study demonstrated that 1 exhibited an immunosuppressive effect through inducing apoptosis and inhibiting cytokine secretion. fractionation led to the isolation of five new phloroglucinolterpene adducts (1−5), and 13 known phloroglucinol derivatives (6−18). The structures and absolute configurations of 1−5 were determined by spectrometric and spectroscopic data analysis, electronic circular dichroism (ECD) spectra, quantum chemical calculations of the 13C NMR data, as well as single-crystal X-ray diffraction. Furthermore, all isolated compounds (1−18) were evaluated for their immunosuppressive effects in vitro. As a result, 1, 2, 6, and 7 revealed moderate inhibitory activities with IC50 values ranging from 10.2 μM to 19.1 μM. Notably, these compounds displayed almost no cytotoxicity on splenocytes, so that they gave a good stimulation index value, ranging from 4.6 to 64.2. Further investigation demonstrated that 1 exhibits an immunosuppressive effect through inducing apoptosis and inhibiting cytokine secretion. Herein, the isolation, structural elucidation, and biological activity of these compounds are described.

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hloroglucinol-terpene adducts are a class of secondary metabolites with structures unique to plants in the family Myrtaceae, especially Eucalyptus species.1−3 Eucalyptus globulus Labill., a tall timber tree, grows mainly in southern and southwestern mainland China, especially in Guangxi and Yunnan Provinces. The fruits of E. globulus, having the traditional Chinese name “Yi-Kou-Zhong,” have been used in herbal medications for treatment of inflammation, eczema, influenza, and rheumatoid arthritis, which might be related to immune regulation. From the leaves and fruits of E. globulus, flavonoids, terpenes, and phloroglucinol derivatives have been isolated.4,5 The major characteristic compounds, phloroglucinol derivatives, are reported to have antiviral,7 cytotoxic,6 antibacterial, as well as HIV-RTase inhibitory effects.8 Our previous study reported that phloroglucinol derivatives from E. globulus showed potential anticancer activities through inducing the apoptosis of cancer cells.9 In a continuing investigation for phloroglucinol derivatives with diversified bioactivities from Myrtaceae plants, an ethyl acetate of the fruits of E. globulus was found to possess moderate immunosuppressive activity against activated lymph node cells with an IC50 value of 12.2 μg/mL. Bioassay-guided © XXXX American Chemical Society and American Society of Pharmacognosy

Received: November 2, 2018

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DOI: 10.1021/acs.jnatprod.8b00920 J. Nat. Prod. XXXX, XXX, XXX−XXX

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RESULTS AND DISCUSSION The air-dried fruits of E. globulus were percolated using 95% aqueous EtOH at room temperature to obtain a crude extract, which was successively partitioned with petroleum ether, ethyl acetate, and n-BuOH. The EtOAc portion was fractionated using a series of chromatographic methods, to obtain compounds 1−18. Of these, 13 known phloroglucinol derivatives were subsequently identified as eucalyptin A (6),10 eucalyptin B (7),9 macrocarpal A (8),11 macrocarpal B (9),11 macrocarpal C (10),12 macrocarpal D (11),4 macrocarpal E (12),5 macrocarpal Q (13),5 eucarobustol E (14),13 euglobal-V (15),14 euglobal-III (16),15 1-(2,6-dihydroxy-4methoxy-3,5-dimethylphenyl)-2-methylbutan-1-one (17),16 and 1-(2,4-dihydroxy-6-methoxy-3,5-dimethylphenyl)-3-methylbutan-1-one (18).16 Among them, the (1R, 4S, 5S, 6R, 7R, 10R, 9′R) absolute configuration of eucarobustol E (14) was elucidated for the first time by single-crystal X-ray diffraction with Cu Kα radiation, for which the structure was determined previously by means of NMR spectroscopic analysis.13 Eucalyptin C (1), as colorless crystal, was established to have a molecular formula of C23H30O6 on the basis of its HRESIMS data (m/z 401.1963 [M − H]−, calcd for C23H29O6, 401.1964). The IR spectrum exhibited the presence of hydroxy (3423 cm−1) and carbonyl groups (1639 cm−1). The 1H NMR data of 1 showed signals for two formyl groups [δH 10.05 (1H,

s, H-8), 10.14 (1H, s, H-9)], two olefinic protons [δH 5.74 (1H, d, J = 10.0 Hz, H-3′) and 5.90 (1H, dd, J = 1.6, 10.0 Hz, H-2′)], two methylene protons [δH 1.60, 1.73 (each 1H, m, H10a, 10b), 1.47 (1H, m, H-5′b), and 1.88 (1H, dd, J = 3.2, 13.2 Hz, H-5′a)], four methine protons [δH 1.82 (2H, overlapped, H-11, 8′), 2.24 (1H, dd, J = 2.0, 13.2 Hz, H-6′), and 2.86 (1H, dd, J = 3.6, 9.2 Hz, H-7)], and five methyl groups [δH 0.99 (3H, d, J = 7.2 Hz, H3-9′), 1.00 (3H, d, J = 6.4 Hz, H3-13), 1.02 (3H, d, J = 6.4 Hz, H3-12), 1.04 (3H, d, J = 7.2 Hz, H310′), and 1.57 (3H, s, H3-7′)]. The 13C NMR data (Table 1) revealed the presence of 23 carbon signals, including one 3,5diformyl phloroglucinol, two quaternary carbons, five methyls, two methylenes, and six methines, indicating that 1 could be a phloroglucinol-sesquiterpenoid adduct.17 These data were closely comparable with those of euglobal-Ia1, isolated from the same plant,17 except for the signals due to the oxygenated carbon C-4′ (δ 73.6). Further HMBC correlations from H3-9′ to C-4′ (δ 73.6), C-8′ (δ 36.7), and C-10′ (δ 17.6) and from H3-10′ to C-4′ (δ 73.6), C-8′ (δ 36.7), and C-9′ (17.2) confirmed that the hydroxy group is located at C-4′ (Figure 1). Moreover, the monoterpenoid moiety was elaborated by observed HMBC correlations from H3-7′ to C-1′ (δ 78.5) and C-2′ (δ 138.7) and H-5′ to C-1′ (δ 78.5), C-3′ (δ 132.0), and C-4′ (δ 73.6). The relative configuration of 1 was established via a NOESY experiment (Figure 2). The NOESY B

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Table 1. 1H NMR and 13C NMR Data of Compounds 1 and 2 1a position 1 2 3 4 5 6 7 8 9 10 11 12 13 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′

δH (J in Hz)

2.86 dd (9.2, 3.6) 10.05 s 10.14 s a 1.73 m b 1.60 m 1.82 overlapped 1.02 d (6.4) 1.00 d (6.4) 5.90 dd (10.0, 1.6) 5.74 d (10.0) a 1.88 dd (13.2, 3.2) b 1.47 m 2.24 dd (2.0, 13.2) 1.57 s 1.82 overlapped 0.99 d (7.2) 1.04 d (6.8)

determined by X-ray diffraction analysis using Cu Kα radiation with the Flack parameter [0.06(8)]. Eucalyptin D (2) was isolated as an amorphous powder with the same molecular formula of C23H30O6 as 1 based on its HRESIMS data. The 1H and 13C NMR data (Table 1) of 2 showed a close similarity to those of 1. After inspection of the NMR data of 2, its planar moiety was determined as being the same as that of 1, with significantly different signals in the vicinity of C-7, particularly with the C-1, C-10, C-7, C-5′, and C-7′ signals of 2 being upfield shifted (ΔδC −2.7, −8.6, −5.8, −9.6, and −5.1, respectively), whereas C-2 of 2 was shifted downfield (ΔδC + 0.4), leading to the assumption that 2 is a C7 epimer of 1. The NOESY spectrum of 2 showed similar correlations to those of 1 (Figure 2), allowing the relative configurations to be established at C-1′ and C-6′. In addition, the strong NOESY correlations of H-7/H-6′/H3-7′ as well as H-7/H-5′a indicated that H-7 is β-oriented. On the basis of the determined relative configurations of compound 1 and 2, one pair of stereoisomers (7R, 1′R, 4′S, 6′R and 7S, 1′R, 4′S, 6′R) was selected arbitrarily for the theoretical calculations of 13 C NMR data, respectively, using the GIAO method in Gaussian 09 software at the MPW1PW91/6-311+G(d,p) level (Figure 4). According to the calculation conducted, the absolute configuration of 2 was confirmed unambiguously as 7S*, 1′R*, 4′S*, 6′R* with the DP4 probability being almost 100% (Figure 4B). Additionally, quantum-chemical electronic circular dichroism (ECD) calculations using the time-dependent theory (TD-DFT) at the B3LYP/6-311+g(d,p) level in methanol (Figure 5) also revealed that the experimental ECD data of 2 were similar to the calculated data of the (7S, 1′R, 4′S, 6′R) diastereomer, while the Cotton effects of its enantiomer were opposite the experimental data (2). Therefore, the structure of eucalyptin D (2) was defined as shown. Eucalyptin E (3) was obtained as colorless crystals with a molecular formula of C29H42O6 as established by HRESIMS and its 13C NMR spectroscopic data. The 1H NMR data of 3 showed resonances of one methoxy group [δH 3.02 (3H, s, OCH3-10)], two formyl groups [δH 10.06 (1H, s, H-7′), 10.07 (1H, s, H-8′)], six methyls [δH 1.08 (3H, s, H3-13), 1.06 (3H, s, H3-15), 1.04 (3H, s, H3-12), 0.98 (3H, s, H3-14), 0.82 (3H, d, J = 6.5 Hz, H3-12′), 0.74 (3H, d, J = 6.5 Hz, H3-13′)], six methines [δH 3.23 (1H, dd, J = 4.5, 12.5 Hz, H-9′), 2.12 (1H, m, H-1), 1.18 (1H, t, J = 9.5 Hz, H-5), 1.16 (1H, m, H-11′), 0.59 (1H, m, H-6), 0.55 (1H, m, H-7)], as well as five methylene units. The 13C NMR data of 3 exhibited 29 carbon signals, categorized into six aromatic carbons, three quaternary carbons, five methylenes, six methyls, six methines, two carbonyl carbons, and one methoxy group. Analysis of the 1 H and 13C NMR data suggested that the structure of 3 is closely related with that of a phloroglucinol moiety coupled to an aromadendrane-type skeleton, eucarobustol E,13 except for C-6′ (ΔδC +1.1), C-9′ (ΔδC +2.9), and C-10′ (ΔδC −1.1), suggesting that 3 might be the C-9′ epimer of eucarobustol E.13 This observation was validated by analysis of the 2D NMR data, especially the NOESY spectrum, in which the correlations of H-9′/H-5, H3-14/H-5, and H-5/H3-13 showed that H3-14 and H-9′ are β-oriented. Using X-ray diffraction, the complete structure and absolute configuration of 3 (Figure 3) were confirmed to be 1R, 4S, 5S, 6R, 7R, 10R, 9′S on the basis of the Flack parameter 0.01(5). Thus, the structure of eucalyptin E (3) was established as shown. Eucalyptin F (4) was established to have a molecular formula of C28H40O6, based on its HRESIMS data. The 1H and

2a δC 105.9 163.4 105 168.5 105.3 170.2 34.9 193.5 192.8 44.7 27.5 23.8 22.0 78.5 138.7 132.0 73.6 39.2 37.5 28.2 36.7 17.2 17.6

δH (J in Hz)

3.25 m 10.03 s 10.14 s a 2.55 td (4.0, 10.4) b 1.40 m 1.76 m 1.05 d (6.4) 1.02 d (6.4) 5.95 dd (10.0, 1.2) 5.87 d (10.0) a 2.07 dd (13.0, 2.4) b 1.32 m 2.17 dd (2.8, 13.6) 1.43 s 1.83 m 0.99 d (7.2) 1.02 d (7.2)

δC 103.2 163.8 104.2 167.5 103.8 170.2 29.1 192.5 191.7 36.1 24.9 23.0 20.0 77.1 138.0 129.8 73.0 29.6 35.9 23.1 35.0 16.0 16.4

a

Data were measured in CD3OD at 400 MHz. Coupling constants (in parentheses) are in Hz. Assignments were done by HSQC, HMBC, COSY, and NOESY experiments.

Figure 1. Key 1H−1H COSY and HMBC correlations of compounds 1−5.

spectrum showed diagnostic correlations between H-6′ and H8′/H3-10′/H3-13, as well as H-6′ and H3-7′, while no NOE correlation peaks were observed between H-7 and H3-7′/H-6′, indicating H-7 is α-oriented, while H-6′, H3-7′, and the isopropyl functionality at C-4′ are β-oriented. The (7R, 1′R, 4′S, 6′R) absolute configuration of 1 (Figure 3) was C

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Figure 2. Key NOE correlations of compounds 1−5 13

due to the oxygenated quaternary carbon C-4 (δ 70.7) instead of signals corresponding to the double bond in the sesquiterpenoid portion of this molecule. Furthermore, the 1 H−1H COSY spectrum was used to establish two spin systems (C-5 to C-9 and C-7′ to C-3), indicating the presence of the partial structures shown in bold lines, as in Figure 1. The observed HMBC correlations from H3-14 to C-1/C-5/C-9, from H3-12 to C-6/C-7/C-11, and from H3-15 to C-3/C-4/C5 revealed a maaliane skeleton.18 In addition, the HMBC correlations between H-7′a,7′b and C-1,C-6′ indicated the linkage of the sesquiterpene to formyl-phloroglucinol moieties. The relative configuration of 5 was elucidated by means of the NOESY experiment (Figure 2) and by comparison with chemical shifts of eucalrobusone A.18 The NOESY correlations of H-1/H-9β, H-1/H-5, H-9β/H3-13, H3-14/H3-15, H3-14/H7′a, and H-6/H3-15 revealed α-orientations of H-6, H-7, H314, and H3-15, and β-orientations of H-1 and H-5. The experimental ECD spectrum of 5 showed a positive Cotton effect at 215.4 (Δε +1.76) nm and negative Cotton effects at 272 (Δε −2.1) nm, which were similar to the calculated spectrum for the (1R, 4S, 5S, 6R, 7R, 10S) diastereomer. The structure of eucalyptin G (5) was thus elucidated as shown. Concanavalin A (ConA) is a type of lectin that has been used widely for several years to activate T cells. The precise mechanism of action of ConA is unknown, but it is likely that ConA activates T cells by indirect cross-linking with the T cell receptor.19 Thus, in this study, a model of ConA-induced T cell proliferation was used to evaluate the immunosuppressive effects of all the isolates and cyclosporin A (CsA), the positive drug. Moreover, most immunosuppressive agents have cytotoxic effects in normal cells and may lead to liver and cardiovascular toxicity.20 In view of these facts, the cytotoxic effects of the isolates (1−18) were evaluated, and the stimulation index (SI), the value of CC50/IC50, was used as a

C NMR spectroscopic data (Table 2) of 4 and eucarobustol F13 were highly similar, indicating they might be stereoisomers. On comparing the 13C NMR data of 4 with those of eucarobustol F, the C-3, C-5, C-6′, and C-9′ were shifted downfield (ΔδC +3.8, +4.6, +2.2, and +4.1, respectively), whereas C-2, C-15, and C-1′ were shifted upfield (ΔδC −0.9, −4.4, and −2.5, respectively), indicating that compound 4 is the C-9′ epimer of eucarobustol F.13 This observation was confirmed by means of the NOESY experiment. The strong NOESY correlations of H3-15/H-1, H3-14/H-1, and H-6/H315 showed that H-1, H3-14, and H3-15 could have an αorientation (Figure 2). In contrast, the H-9′ proton was assigned as β-oriented by the NOESY correlations of H-9′/H5, H3-13/H-9β, H-5/H-9β, and H-5/H3-13. The absolute configuration of 4 was established by comparing its experimental ECD curve with those of the quantum chemical TDDFT calculated results (Figure 5). The experimental ECD spectrum of 4 showed a positive Cotton effect at 198 (Δε +1.21) nm and negative Cotton effects at 270 (Δε −2.05) nm and 345 (Δε −0.02) nm, similar to the calculated spectrum for (9′S, 1R, 4S, 5S, 6R, 7R, 10S) (Figure 5). Thus, the structure of eucalyptin F (4) was defined as shown. Eucalyptin G (5), isolated as an amorphous powder, gave the molecular formula C28H40O6 according to the HRESIMS at m/z 471.2749 [M − H]−. The 1H NMR spectrum of 5 showed a formyl proton signal [δH 10.69 (1H, s, H-8′)], six methyl signals [δH 1.08 (3H, s, H3-12), 1.16 (3H, s, H3-13), 1.04 (3H, s, H3-14), 1.50 (3H, s, H3-15), 1.08 (3H, d, J = 6.5 Hz, H3-12′), and 1.07 (3H, d, J = 6.5 Hz, H3-13′)], and five methine signals, as well as six methylene signals. A comparison of the 1H and 13C NMR spectroscopic data of 5 with those of eucalrobusone A18 revealed that 5 is a phloroglucinolsesquiterpene adduct with the sesquiterpenoid moiety identical to that of eucalrobusone A.18 However, a difference noted was D

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Figure 3. X-ray ORTEP drawings of 1, 3, and 14.

diagnostic index.21 These compounds showed moderate activities, with IC50 values ranging from 10.2 to 132.8 μM, and also showed very low cytotoxic effects on splenocytes, with CC50 values >500 μM (Table 3). Compounds 1−18 are mainly sesquiterpene-based phloroglucinol-terpene adducts. Of these, 1 and 2, phloroglucinol-sesquiterpene adducts with chroman rings, exhibited the most immunosuppressive effects, with IC50 values of 11.8 and 10.2 μM, respectively. However, 15 and 16, phloroglucinol-sesquiterpene adducts with a fused oxygen heterocyclic ring, were found to lack inhibitory activities, indicating the beneficial influence for immunosuppressive activity being associated with the presence of a smaller terpenoidal fragment attached to the phloroglucinol moiety. In addition, preliminary structure−activity relationship (SAR) information was determined for the groups of the phloroglucinol moiety coupled to an aromadendrane-type skeleton and to a phloroglucinol-coupled eudesmane-type skeleton. Of these, 6 and 7, both possessing an isovaleryl group, showed the most potent effects, with IC50 values of 18.3 and 19.1 μM, respectively, when compared with the other diformylphloroglucinols. It is likely that isovaleryl substitution will affect the resultant activity, which could provide useful information for future structural modification. Although the immunosuppressive effects of these compounds were inferior to CsA (IC50 = 1.3 μM), their cytotoxic effects were less evident when compared with CsA (CC50 = 200.4 μM). Therefore, the SI values of some of the isolated

compounds were promising, especially 1 (SI = 64.2; Table 3), which could be selected as a lead compound for further study. Apoptosis plays a vital role in eradicating activated T cells in the process of halting excess immune responses and keeping an appropriate immune homeostasis. However, the lack of apoptosis of the activated T cell relates to a number of immune disorders.22 Therefore, whether 1 can lead to T cell injury was confirmed using MTT and LDH assays. Results showed that 1 can decrease cell viability (Figure 6A) and increase LDH release (Figure 6B). Annexin V−PI double staining showed that 1 (1, 10, 20 μM) increased the apoptosis rate of T cells to 35.23%, 56.34%, and 78.12%, respectively (Figures 6C and 7D), demonstrating that the mechanism of action of 1 is involved in the apoptosis induction of T cells. Furthermore, Bcl-2 and Bax are apoptotic inhibitors or inducers.23 The increased ratio of Bax/Bcl-2 triggered the activation of caspase-3 and caspase-9, leading to cell death.23 The results showed that compound 1 significantly increased the ratio of Bax/Bcl-2 (Figure 7A and C) and induced the release of the cytochrome c from mitochondria to cytoplasm (Figure 7B and D). Furthermore, Figure 7E and F demonstrate that 1 activated caspase-3 and caspase-9. All these findings suggested that 1 induces apoptosis of T cells through a mitochondrial apoptotic pathway. Th1 and Th2 cells are known as subclasses of T-helper cells. IFN-γ and interleukin-4, respectively, are the trait cytokines of Th1 and Th2 cells. Th1 cells secrete pro-inflammatory E

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Figure 4. 13C NMR calculation results of two plausible stereoisomers of 1 (A) and 2 (B) at the MPW1PW91/6-311+G (d, p) level. (a) Linear correlation plots of calculated vs experimental 13C NMR chemical shift values for each potential configuration. (b) Relative errors between the calculated 13C NMR chemical shifts of two potential structures and recorded 13C NMR data. (c) The DP4+ probability of 13C NMR chemical shifts.

Figure 5. Calculated and experimental ECD spectra of 2, 4, and 5.

cytokines like IFN-β, IFN-γ, and interleukin-2 (IL-2); their responses predominate in chronic inflammatory and autoimmune diseases. Th2 cells generate cytokines like interleukin4 (IL-4), interleukin-5 (IL-5), and interleukin-6 (IL-6), and their responses predominate in Omann’s syndrome, systemic

sclerosis, transplantation tolerance, chronic graft versus host disease, and allergic diseases.24 It has been demonstrated that those cytokines are produced through inhibiting the AKT signaling pathway.25 Our results showed that 1 (1, 10, and 20 μM) respectively decreased the level of IL-4 in the supernatant F

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Table 2. 1H NMR and 13C NMR Data of Compounds 3−5 3a δH (J in Hz)

δC

2.12 m α 1.43 m β 1.43 (m) α 0.92 ddd (4.5, 6.0, 11.5) β 1.28 overlapped

52.1 23.1

position 1 2 3 4 5 6 7 8

4b

39.0 48.6 48.1 28.5 25.2 19.5

1.18 t (9.5) 0.59 m 0.55 m α 0.87 m β 1.81 m α 1.57 brt (12.5) β 1.52 m

9 10 11 12 13 14 15 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′ OCH3

1.04 1.08 0.98 1.06

δH (J in Hz) 1.89 m α 1.59 m β 1.65 overlapped α 0.97 m β 1.42 m 1.78 m 0.59 m 0.63 m α 1.62 overlapped β 1.55 m α 1.52 brt (12.5) β 1.70 m

36.6 78.6 18.6 28.6 16.3 18.0 17.2 167.1 104.5 168.1 104.5 169.5 109.1 192.5

s s s s

10.06 10.07 3.23 dd (4.5, 13) a 2.33 td (4.0, 13.0) b 1.29 overlapped 1.13 m 0.82 d (6.5) 0.74 d (6.5) 3.02 s

1.11 1.24 1.17 1.13

s s s s

10.12 s 3.39 dd (12.5, 4.5) a 2.42 td (4.0, 13.0) b 1.45 m 1.20 m 0.83 d (6.5) 0.88 d (6.5)

26.3 24.4 21.0 47.3

δC

δH (J in Hz)

56.5 25.0

δC

1.77 overlapped α 1.92 m β 1.77 overlapped α 2.05 m β 1.77 overlapped

40.3 49.8 48.3 30.3 27.9 20.4

1.42 d (6.5) 0.93 dd (6.0, 9.0) 0.62 t (9.1) α 1.88 m β 1.65 dd, (7, 14.5) α 1.02 m β 2.10 m

43.6 74.2 21.2 25.0 17.4 31.4 18.1 170.2 106.1 170.4 106.2 169.0 111.5 193.0

10.11s

192.5 39.5 34.8

5c

1.08 1.16 1.04 1.50

s s s s

71.7 50.6 21.2 18.4 16.1 37.5

191.8 205.3 52.1

a 3.38 d (6.5) b 3.34 m 2.52 m 1.07 d (6.5) 1.08 d (6.5)

28.2 21.7 24.9

43.2

36.6 17.1 15.3 29.2 14.7 22.9 168.0 106.9 163.9 107.1 173.1 103.6 22.3

a 3.02 d (12) b 2.73 m 10.70 s

193.1 41.8 36.3

47.3 25.8

25.2 22.7 22.6

a

Data were measured in DMSO. bMeasured in CD3OD. cMeasured in C5D5N at 500 MHz. Coupling constants (in parentheses) are in Hz. Assignments were done by HSQC, HMBC, COSY, and NOESY experiments.

Table 3. In Vitro Cytotoxicity on Lymph Node Cells and Inhibitory Effects of Compounds 1−18 and Cyclosporin A on Lymph Node Cells Co-stimulated by Concanavalin A compound 1 2 3 4 5 6 7 8 9 CsA

CC50 663.5 656.3 562.5 597.2 598.3 554.3 578.2 597.3 570.2 200.4

± ± ± ± ± ± ± ± ± ±

32.1 26.3 31.2 32.2 38.3 21.2 28.3 17.2 24.3 17.9

IC50 (μM)

SIa

compound

± ± ± ± ± ± ± ± ± ±

56.3 64.2 15.7 22.7 16.9 30.5 30.2 29.2 18.4 149.6

10 11 12 13 14 15 16 17 18

11.8 10.2 35.8 26.3 35.3 18.2 19.1 20.4 31.2 1.3

4.2 3.2 5.2 5.3 4.4 4.0 3.3 3.2 4.1 0.4

CC50 541.3 596.2 602.3 544.2 578.5 588.2 600.4 623.4 670.2

± ± ± ± ± ± ± ± ±

30.0 30.1 31.6 29.3 45.4 32.0 33.0 41.0 38.2

IC50 (μM)

SIa

± ± ± ± ± ± ± ± ±

8.3 14.5 14.2 10.4 8.5 8.5 4.6 8.9 5.0

69.6 41.2 42.4 52.3 68.4 69.3 129.3 70.2 132.9

3.2 5.3 6.3 5.5 7.5 8.3 10.3 5.3 13.2

a The stimulation index (SI) was determined as the ratio of the concentration of the compound that reduced cell viability to 50% (CC50) to the concentration of the compound needed to inhibit the proliferation to 50% (IC50) of the control value.

12.8 and 52.5 ± 10.6 pg/mL, respectively, compared to the control group (575.9 ± 8.2 pg/mL). Figure 9 shows that 1 reduces significantly the expression of p-AKT, suggesting that the potential immunosuppressive effects of 1 are related also to

fluid of cultured cells to 118.1 ± 6.1, 99.7 ± 11.5, and 68.9 ± 8.4 pg/mL, compared to a control group (142.4 ± 12.4 pg/ mL; Figure 8A). In addition, Figure 8B shows that 1 (1, 10, and 20 μM) can also inhibit the secretion of INF-γ to 347.4 ± G

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Figure 6. Effects of 1 on T cell viability and apoptosis. (A) The CCK-8 assay for ConA-induced spleen cells. (B) The LDH release in cell culture supernatant. (C) Annexin V−PI double staining. Distribution of viable (lower left, Annexin V− P−), necrotic (upper left, Annexin V− PI+), late apoptotic (upper right, Annexin V+ PI+), and early apoptotic (lower right, Annexin V+ PI−). (D) Bar graphs showed the quantitative results of AVPI. The data are represented as means ± SEM of three independent experiments. **p < 0.01 and ***p < 0.001 compared with control group. 3, 7 days each time), to obtain a dark black residue (980 g). The crude extract was partitioned sequentially with petroleum ether, EtOAc, and n-BuOH. The EtOAc extract (320 g) was subjected to silica gel column chromatography (CC) by using a stepwise elution gradient with mixtures of petroleum ether/EtOAc (20:1 to 1:1), to obtain six fractions (Frs. A−F). Fraction C (110 g) was separated on silica gel CC with petroleum ether/EtOAc (20:1 to 0:1) as the eluant to give eight subfractions (Frs. C.1−C.8). Fraction C.1 (4.5 g) was separated on silica gel CC, eluting with a gradient of CH2Cl2/MeOH (20:1 to 0:1), and purified by HPLC (CH3CN/H2O, 90:10), to yield 15 (8.7 mg) and 16 (20.3 mg). Fraction C.2 (8.5 g) was separated on an MCI gel column (MeOH/H2O, 50:50 to 100:0), yielding three subfractions (Frs. C.2.1−C.2.3). Fraction C.2.2 (212.0 mg) was separated on Sephadex LH-20 (CH2Cl2/MeOH, 50:50) and then purified via HPLC (MeOH/H2O, 85:15), to yield compounds 7 (12 mg), 17 (10.8 mg), and 18 (7.4 mg). Fraction C.3 (215 mg) was separated over ODS, eluting with a gradient of MeOH/H2O (60:40 to 100:0), and then purified via HPLC (MeOH/H2O, 90:10) to give 10 (18.3 mg). Fraction C.4 (35.0 g) was chromatographed repeatedly over MCI gel (MeOH/H2O, 60:40 to 100:0), silica gel (CH2Cl2/ MeOH, 25:1 to 0:1), and Sephadex LH-20 (CH2Cl2/MeOH, 50:50), with final purification using preparative HPLC (MeOH/H2O, 85:15) to furnish 1 (6.2 mg), 2 (5.5 mg), 4 (5.8 mg), 5 (6.7 mg), 6 (34.7 mg), 8 (107.2 mg), and 9 (75.3 mg). Fraction C.5 (12.0 g) was separated on an MCI gel column (MeOH/H2O, 60:40 to 100:0), silica gel (CH2Cl2/MeOH, 25:1 to 0:1), followed by semipreparative HPLC (MeOH-H2O, 90:10), to obtain 3 (15.5 mg), 11 (56.8 mg), 12 (6.3 mg), 13 (7.6 mg), and 14 (8.4 mg). Eucalyptin C (1). Colorless crystals; [α]D22 −42.7 (c 0.08, MeOH); UV (MeOH) λmax (log ε) 203 (4.29), 276 (4.59) nm; IR (KBr) νmax 3423, 2958, 2872, 1639, 1439, 1176, 779 cm−1; 1H and 13C NMR,

the inhibitory effects on cytokine secretion through inhibiting the phosphorylation of AKT.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were acquired using a Rudolph Autopol IV polarimeter. UV spectra were measured on a JASCO V-630 spectrophotometer. ECD data were measured on a JASCO J-810 spectropolarimeter. IR spectra (KBr disks) were measured on a Nicolet IS10 FTIR spectrometer. NMR spectra were acquired using a Bruker AV-400 or AV-500 spectrometer. An Agilent 6520 Q-TOF mass spectrometer was utilized for HRESIMS measurements. X-ray diffraction data were performed using a Gemini Ultra CCD diffractometer (λ = 1.54184 Å). Silica gel (200−300 mesh, Qingdao Marine Chemical Co., Ltd., Qingdao, People’s Republic of China), ODS-AQ (YMC Co. Ltd., Japan), MCI gel (Mitsubishi Chemical Industries Ltd., Tokyo, Japan), and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Sweden) were used for column chromatography. Thin-layer chromatography was carried out with silica gel plates (GF254, Qingdao Marine Chemical Co., Ltd.). Spots were visualized under UV light by heating silica gel plates after spraying with 10% H2SO4 reagent. Preparative HPLC was performed using a Shimadzu LC-8A liquid chromatography system equipped with a Shim-Pack Prep-ODS column (20 × 250 mm, 15 μm). Plant Material. The fruits of Eucalyptus globulus, authenticated by Prof. Min-Jian Qin, were collected at Nanning, People’s Republic of China, in August 2016. A voucher specimen (No. EG160801) has been deposited in the Department of TCM Pharmaceuticals, China Pharmaceutical University, People’s Republic of China. Extraction and Isolation. Air-dried and smashed fruits of E. globulus (12 kg) were percolated using 95% aqueous EtOH (100 L × H

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Figure 7. Effects of 1 on mitochondrial apoptosis pathway. (A) Original bands of Bax, Bcl-2, and actin. (B) Quantitative analysis of the ratio of Bax/Bcl-2. (C) Original bands of cytochrome c in cytoplasm and mitochondria. (D) Quantitative analysis of the ratio of cytochrome c (cytoplasm/ mitochondria). (E) Activity of active caspase-3. (F) The activity of active caspase-9. The data are represented as mean ± SEM of three independent experiments. **p < 0.01 and ***p < 0.001 compared with control group.

Figure 8. Effects of 1 on the secretion of IL-4 and IFN-γ. (A) The level of IL-4 in the supernatant of cells. (B) The level of IFN-γ in the supernatant of cells. The data are represented as means ± SEM of three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 compared with control group. Table 1; (−)-HRESIMS m/z 401.1963 [M − H]− (calcd for C23H29O6, 401.1964). Eucalyptin D (2). Amorphous powder; [α]D22 −30.3 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 205 (4.32), 277 (4.60) nm; IR (KBr) νmax 3445, 2951, 2869, 1635, 1178, 782 cm−1; 1H and 13C NMR, Table 1; (−)-HRESIMS 401.1959 [M − H]− (calcd for C23H29O6, 401.1964). Eucalyptin E (3). Colorless crystals; [α]D22 −52.5 (c 0.21, MeOH); UV (MeOH) λmax (log ε) 217 (4.25), 276 (4.43), 390 (3.67) nm; IR (KBr) νmax 3404, 2954, 2864, 1622, 1185, 1057, 853, 799 cm−1; 1H and 13C NMR, Table 2; (−)-HRESIMS m/z 485.2918 [M − H]− (calcd for C29H41O6, 485.2903). Eucalyptin F (4). Amorphous powder; [α]D22 −36.8 (c 0.28, MeOH); UV (MeOH) λmax (log ε) 205 (4.24), 276 (4.37) nm; IR

(KBr) νmax 3397, 2952, 2867, 1627, 1455, 1181, 1074, 845, 797 cm−1; 1 H and 13C NMR, Table 2; (−)-HRESIMS m/z 471.2745 [M − H]− (calcd for C28H39O6, 471.2747). Eucalyptin G (5). Amorphous powder; [α]D22 −59.1 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 203 (4.36), 288 nm (4.17), 3.84 (3.69) nm; IR (KBr) νmax 3397, 2931, 2866, 1620, 1439, 1188, 877 cm−1; 1H and 13C NMR, Table 2; (−)-HRESIMS: m/z 471.2749 [M − H]− (calcd for C28H39O6, 471.2747). Crystallographic Data of Eucalyptin C (1). Orthorhombic crystal, C23H30O6 (M = 402.47 g/mol), space group P212121 (no. 19), crystal size: 0.130 × 0.120 × 0.110 mm3, a = 10.16244(10) Å, b = 12.47826(13) Å, c = 33.6950(3) Å, V = 4272.84(8) Å3, Z = 8, Dcalcd = 1.251 g/cm3, μ = 0.732 mm−1, T = 100.00(10) K, 22 577 reflections measured, independent reflections: 8064 (Rint = 0.0370). The final R I

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experimental procedures were approved by the Ethical Committee of the China Pharmaceutical University (No. SYXK2016-0011). Cell Preparation. The assay was performed similarly to that in a previous study with slight modifications.21 BALB/c mice were sacrificed to collect their spleens aseptically. Next, the spleens were grounded by crossing through a sterile plastic strainer with 200 mesh. After centrifugation (1000g, 5 min), erythrocytes were lysed in red blood cell lysis buffer. Then, cell pellets were washed two times with culture medium. The cell viability (95%) was detected by using the trypan-blue dye exclusion technique. Cell Proliferation Assay. Lymph node cells at a density of 1 × 105 cells/well were seeded in 96-well plates and stimulated with concanavalin A (5 μg/mL) at different concentrations of test compounds or cyclosporin A for 48 h. For the proliferation assay, CCK-8 (20 μL) was added to all wells 4 h before the end of the treatment. Finally, the absorbance intensity at 540 nm was detected with a microplate reader (Tecan, Austria). Cytotoxicity Testing. Lymph node cells at a density of 1 × 105 cells/well were seeded in 96-well plates and were treated with different concentrations of test compounds or cyclosporin A for 48 h. For the cytotoxicity assay, all wells were added with CCK-8 (20 μL) 4 h before the end of the treatment. The absorbance intensity at 540 nm was detected on a microplate reader (Tecan, Austria). Apoptosis Assay. Annexin V−PI double staining was conducted via a previous method.26 Lymph node cells (3 × 106 cells/well in sixwell plates) were stimulated with ConA (5 μg/mL) with various concentrations of 1 (1, 10, 20 μM) for 48 h. Then, cell lysates were washed twice with cold PBS. Next, the density of the cell was adjusted to 1 × 106 cells/mL using binding buffer. Finally, all samples were performed according to the specifications of the commercial kit and then analyzed using a FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA). Measurement of Cytokines. Lymph node cells at a density of 2 × 106 cells/well were seeded in 24-well plates and were stimulated with ConA (5 μg/mL) in the presence of different concentrations of 1 (1, 10, 20 μM) for 48 h. After this, the culture medium was collected to perform the ELISA assays for IL-4 and IFN-γ. The experimental procedure was conducted following the manufacturer’s instructions. Finally, the absorbance intensity at 450 nm was detected on microplate reader (Tecan, Austria). Western-Blot Analysis. Lymph node cells at a density of 2 × 106 cells/well were seeded in six-well plates and were treated with ConA (5 μg/mL) in the presence of various concentrations of 1 (1, 10, 20 μM) for 48 h. Then, cells were collected and washed two times with PBS. Protein extraction and Western blot were conducted as in our previous study.9 The images were analyzed using the ImageJ software. Active Caspase-3 and Caspase-9 Activity Assays. Lymph node cells at a density of 2 × 106 cells/well were seeded in 24-well plates and were stimulated with ConA (5 μg/mL) in the presence of different concentrations of 1 (1, 10, 20 μM) for 48 h. After the cell collection, the activities of caspase-3 and caspase-9 were determined following a previous report27 using the commercial kit. Statistics. The significances of intergroup differences were determined using one-way analysis. Results are expressed as the means ± SD of the indicated numbers of independent experiments. Statistical significance was determined for p values of 2σ (I)) and wR2 = 0.1061 (I > 2σ (I)). CCDC number: 1872379. Crystallographic Data of Eucalyptin E (3). Orthorhombic crystal, C29H42O6 (M = 518.67 g/mol), space group P212121 (no. 19), crystal size: 0.140 × 0.130 × 0.110 mm3, a = 11.02230(10) Å, b = 13.53790(10) Å, c = 18.85090(10) Å, V = 2812.91(4) Å3, Z = 4, Dcalcd = 1.225 g/cm3, μ = 0.690 mm−1, T = 100.00(10) K, 20 461 reflections measured, independent reflections: 5587 (Rint = 0.0314). The final R indices were R1 = 0.0311 (I > 2σ (I)) and wR2 = 0.0814 (I > 2σ (I)). CCDC number: 1872380. Crystallographic Data of Eucarobustol E (14). Trigonal crystal, C30H45O7.5 (M = 525.66 g/mol), space group P3221 (no. 154), crystal size: 0.12 × 0.11 × 0.1 mm3, a = 20.6895(3) Å, b = 20.6895(3) Å, c = 12.5425(2) Å, V = 4649.59(15) Å3, Z = 6, Dcalcd = 1.126 g/cm3, μ = 0.646 mm−1, T = 100.00(10) K, 22 576 reflections measured, 6078 independent reflections (Rint = 0.0347). The final R indices were R1 = 0.0515 (I > 2σ (I)) and wR2 = 0.1417 (I > 2σ (I)). CCDC number: 1872381. Biological Materials. A Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Laboratories (Japan). Fetal bovine serum and RPMI 1640 medium were from Invitrogen (San Diego, CA, USA). Cyclosporin A and concanavalin A were from Sigma Chemical Co. (St. Louis, MO, USA). A BCA protein determination kit, chemiluminescence kit, caspase-3 and caspase-9 activity determination kits, red blood cell lysis buffer, RIPA lysis buffer, and PMSF (protease inhibitor) were purchsed from Beyotime (Shanghai, People’s Republic of China). ELISA kits for mice (IL-4 and INF-γ) were from EIAAB Science Co., Ltd. (Wuhan, People’s Republic of China). Penicillin, streptomycin, Annexin V−PI double staining kit, mitochondrial protein extraction kit, and HRP-conjugated goat antirabbit (mouse) IgG were obtained from KeyGEN BioTECH (Nanjing, People’s Republic of China). Primary rabbit antibodies for Bcl-2, Bax, and cytochrome c were obtained from Cell Signaling Technology (Beverly, MA, USA). Mouse antibodies for t-AKT, pAKT, and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Animals. Male BALB/c mice (6−8 weeks old) were purchased from Nanjing University Experimental Animal Center (Nanjing, People’s Republic of China). Mice were acclimatized for 7 days before use. They were well-maintained at 22−25 °C and kept on a 12-h light−dark cycle and were allowed free access to food and water. All

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00920. Additional general experimental procedures, computational calculation methods, 1D and 2D NMR spectra and HRESIMS spectra of compounds 1−5, and ECD computational details (PDF) J

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Xiao-Jun Huang: 0000-0002-3636-4813 Wen-Cai Ye: 0000-0002-2810-1001 Hao Wang: 0000-0003-3994-9806 Author Contributions #

T.-A.P. and X.-L.H. contributed equally and are joint first authors. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was funded by the Funding of Double First-rate Discipline Innovation Team (No. CPU2018GF05), the National Natural Science Foundation of China (No. 81573309, 1803392, and 81473160), Major National Science and Technology Projects of the Chinese Thirteen Five-year Plan (No. 2017ZX09309024), the Natural Science Foundation of Jiangsu Province (No. BK20180566), the National Key R&D Program of China (No. 2017YFC1703802), the Research and Innovation Project for College Graduates of Jiangsu Province 2017 (No. KYCX17_0694), China Postdoctoral Science Foundation funded project (No. 2018M630644), the Jiangsu Province Graduate Student Training Innovation Project (No. KYLX16_1208), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the Student’s Platform for Innovation and Entrepreneurship Training Program of Jiangsu Province (No. 201810316006Y).

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