Sesquiterpenoids from Chloranthus anhuiensis with Neuroprotective

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Sesquiterpenoids from Chloranthus anhuiensis with Neuroprotective Effects in PC12 Cells Jian Xu,† Hui-Lin Zhu,† Jie Zhang,† Tao Du,† Er-Yan Guo,† Wen-Yuan Liu,‡ Jian-Guang Luo,† Feng Ye,† Feng Feng,*,†,⊥,§ and Wei Qu*,†,⊥ †

Department of Natural Medicinal Chemistry, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 210009, People’s Republic of China ‡ Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, China Pharmaceutical University, Nanjing 210009, People’s Republic of China ⊥ Key Laboratory of Biomedical Functional Materials, China Pharmaceutical University, Nanjing 211198, People’s Republic of China § Jiangsu Food & Pharmaceutical Science College, Huaian 223003, People’s Republic of China S Supporting Information *

ABSTRACT: Glutamate-induced excitotoxicity plays a vital role in neurodegenerative diseases. Neuroprotection against excitotoxicity has been considered as an effective experimental approach for preventing and/or treating excitotoxicity-mediated diseases. In the present study, six new sesquiterpenoids (1−6) and 26 known compounds of this type (7−32) were isolated and characterized from the whole plants of Chloranthus anhuiensis. Chlorantolide A (1) is the first example of a 5,6-seco-germacrane-type sesquiterpenoid, while phacadinane E (2) is a rare 4,5-seco-cadinane-type sesquiterpenoid. The structures of the new compounds were determined by spectroscopic analysis and by calculations of electronic circular dichroism (ECD) spectra. Their neuroprotective effects in mediating glutamate-induced PC12 cell apoptosis were evaluated. Compound 26 exhibited potent neuroprotective activity with an EC50 value of 3.3 ± 0.9 μM. Using Hoechst 33258 staining, a caspase-3 activity assay, and Western blot analysis it was demonstrated that this compound reduces the apoptosis of PC12 cells through inhibition of caspase-3 activity, while activating the Akt signaling pathway.

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Chloranthus anhuiensis K.F. Wu (Chloranthaceae) is an endemic species from Anhui Province in the People’s Republic of China. To date, a number of sesquiterpene lactones and diterpenes have been reported from this plant.8 However, the biological effects of this species and its chemical constituents have not been investigated previously. In this paper, we describe 32 sesquiterpenes from the whole plants of C. anhuiensis, including a unique 5,6-seco-germacrane-type sesquiterpenoid (1), a rare 4,5-seco-cadinane-type sesquiterpenoid (2), and four further new sesquiterpenoids (3−6) (see Chart 1). Also isolated were 26 known sesquiterpenoids, 8,12-epoxy1α-hydroxy-4αH,5αH-eudesma-7,11-diene-6,9-dione (7),9 chlorantene C (8),10 4α-hydroxy-8,12-epoxyeudesma-7,11diene-1,6-dione (9),10 1α-hydroxy-8,12-epoxyeudesma-4,7,11triene-6,9-dione (10),11 zedoarofuran (11),12 curcolonol (12),13 9α-hydroxycurcolone (13),14 lasianthuslactone A (14),15 atractylenolide I (15),16 atractylenolide III (16),17 6αhydroxyeudesma-4(15),7(11),8(9)-trien-12,8-olide (17),18 4β,8β-dihydroxy-5αH-eudesm-7(11)-en-8,12-olide (18),19 codonolactone (19),15 shizukafuranol (20),20 shizukolidol (21),20 ent-(3R)-3-hydroxyatractylenolide III (22),21 (7S,10S)-

lutamate, the major excitatory neurotransmitter in the brain, has been implicated in rapid synaptic transmission, neuroplasticity, learning, and memory.1 However, excessive release of glutamate may cause massive Ca2+ influx and oxidative glutamate toxicity through overstimulation of the ionotropic glutamate receptors, which can further induce neuronal cellular death.2 This excitotoxicity plays a vital role in both chronic neurodegenerative diseases and acute conditions, such as dementia and stoke.1,3,4 Therefore, it is considered that improvement of this excitotoxicity is an effective therapeutic strategy for preventing and/or reversing these glutamate-mediated diseases.5 Recently, several neuroprotective sesquiterpenes have been reported.6,7 For example, atractylenolide III, a sesquiterpene lactone, showed neuroprotective effects against neuronal apoptosis induced by glutamate through inhibition of the caspase pathway in a cellular model.8 The impairment of learning and memory in a homocysteine-induced rat model can be significantly improved by this molecule through inhibition of ROS production and upregulation of p-PKC levels.7 These studies have suggested that certain sesquiterpenes may have value as potential neuroprotective agents in excitotoxicity-mediated neurological diseases. © XXXX American Chemical Society and American Society of Pharmacognosy

Received: December 28, 2017

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DOI: 10.1021/acs.jnatprod.7b01076 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Chart 1

Table 1. 1H NMR Spectroscopic Data (δ in ppm, J in Hz) for Compounds 1−6 1a

H no. 1

5.26, t (8.8)

2

2.07, 2.53, 1.13, 1.89, 2.43,

3 4 5 6 8

m m m m dd (4.8, 6.4)

2b

3.36, dd (6.3, 8.4) 2.73, dd (6.3, 8.4)

3b 2.20, 1.56, 0.91, 1.76, 2.00, 2.36,

4b

m m m m m m

6.88, d (9.9) 6.38, d (9.9)

5c 2.07, 1.88, 2.56, 2.45,

m td (4.2,13.8) ddd (4.8, 15.0, 16.8) td (3.6, 16.8)

2.66, m 2.70, m

9

2.71, d (4.0) 2.69, d (7.6) 5.10, dd (4.0, 7.6)

7.13, d (7.5) 7.43, d (7.5)

11 12 13 14

4.67, s 2.13, s 1.73, s

1.63, s 1.63, s 2.41, s

7.39, d (1.2) 2.02, d (1.2) 1.00, s

7.18, s 2.28, d (1.2) 1.40, s

1.95, 2.06, 1.57, 2.05, 2.23, 0.99, 0.97, 1.17,

15

1.06, d (6.8)

2.22, s

4.98, s 4.78, s

2.19, s

1.76, s

β 3.03, d (16.8) α 2.94, d (16.8)

dd (3.6, 9.6) m td (3.0, 13.8) m m d (7.2) d (7.2) s

2′ a

6b 1.34, m 0.99, m 0.85, m 2.08, m

2.66, 2.78, 2.12, 5.09,

dd (2.4, 13.5) dd (3.3, 13.8) m m

2.90, dd (9.9, 12.0) 1.45, m

1.86, 4.10, 3.59, 5.06, 4.81, 2.15,

s d (12.3) d (12.3) s s s

Recorded in CDCl3 at 400 MHz. bRecorded in CDCl3 at 300 MHz. cRecorded in CDCl3 at 600 MHz.

δH 5.10 (dd, J = 4.0, 7.6 Hz, H-9); δC 77.6], one sp2 methine [δH 5.26 (t, J = 8.8 Hz, H-1); δC 124.8], three sp2 quaternary carbons [δC 159.8 (C-11), 138.8 (C-10), and 124.0 (C-7), respectively], and two carbonyl carbons (δC 178.4 and 174.8). These aforementioned data suggested that this molecule is a sesquiterpene derivative, containing two ring systems. Secondary analyses of the 1D NMR data suggested that this compound possesses the same α,β-unsaturated γ-lactone ring (unit b, shown in the Figure 1) as 4,5,11-trimethyl-8,9-seco1(10),7(11)-eremophiladien-8,12-olid-9-oic acid.31 The HMBC correlations from H3-13 (δH 2.13) to C-7, C-11, and C-12 (δC 124.0, 159.8, and 72.8, respectively), and from H-12 (δH 4.67) to C-7, C-11, and C-6 (δC 124.0, 159.8, and 174.8, respectively), supported the above suggestion. Moreover, the HMBC correlations of H3-14 (δH 1.73) with C-1, C-10, and C9 (δC 124.8, 138.8, and 77.6, respectively), of H-9 (δH 5.10)

7-hydroxyeudesma-4-en-6-one (23),22 1β-hydroxy-α-cyperone (24),23 shizukanolide C (25),17 shizukanolide H (26),24 istanbulin B (27),25 istanbulin A (28),26 isogermafurenolide (29),27 shizukaacoradienol (30),28 chlomultin C (31),29 and curcuzederone (32).30 The neuroprotective activity of these substances was investigated using PC12 cells.

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RESULTS AND DISCUSSION Chlorantolide A (1), a colorless oil, was assigned a molecular formula of C15H20O4 with six degrees of unsaturation on the basis of its HRESIMS (m/z 265.1436 [M + H]+, calcd for 265.1436). Its 1H and 13C NMR (Tables 1 and 2) and HSQC spectra showed three tertiary methyls [δH 1.06 (d, J = 6.8 Hz, H3-15), 1.73 (s, H3-14), and 2.13 (s, H3-13); δC 15.4, 22.1, and 13.0, respectively], four sp3 methylenes [one oxygenated at δH 4.67 (s, H-12); δC 72.8], two sp3 methines [one oxygenated at B



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Table 2. 13C NMR Spectroscopic Data (δ in ppm) for Compounds 1−6

a

C no.

1a

2b

3b

4b

5b

6b

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

124.8 26.3 32.9 38.9 178.4 174.8 124.0 26.4 77.6 138.8 159.8 72.8 13.0 22.1 15.4

141.0 22.0 43.6 208.2 170.0 122.7 154.1 118.5 136.5 137.5 83.9 27.7 27.7 18.9 30.0

32.7 22.9 36.3 148.5 48.8 21.5 137.8 146.1 192.1 47.8 120.9 145.0 8.0 15.9 108.7

153.2 140.9 187.0 137.8 150.7 186.7 121.7 163.8 36.8 42.6 119.8 140.9 9.2 25.1 12.4

37.0 33.6 199.3 133.6 156.8 204.9 79.7 28.8 35.3 39.7 31.9 17.3 16.4 22.0 12.7

23.8 16.8 24.0 149.1 62.9 24.2 160.5 79.0 39.6 42.5 122.5 174.5 8.8 61.8 107.0 171.6 21.2

respectively], two methylenes [δH 2.73 (dd, J = 6.3, 8.4 Hz, H3) and 3.36 (dd, J = 6.3, 8.4 Hz, H-2); δC 43.6 and 22.0, respectively], one oxygenated quaternary carbon at δC 83.9 (C11), three double bonds [δH 7.43 (d, J = 7.5 Hz, H-9) and 7.13 (d, J = 7.5 Hz, H-8); δC 154.1 (C-7), 141.0 (C-1), 137.5 (C10), 136.5 (C-9), 122.7 (C-6), and 118.5 (C-8), respectively], and two carbonyl groups (δC 208.2 and 170.0). Comparison of the above data and phacadinane D35 revealed many similarities, except that the sp2 oxygenated quaternary carbon was replaced with a sp2 methine group in 2. The HMBC cross-peaks of H314 (δH 2.41) with C-1, C-9, and C-10 (δC 141.0, 136.5, and 137.5, respectively), and the 1H−1H COSY correlation of H-8/ H-9, supported the above deductions. Thus, the structure of 2 was established as shown in Figure 2 as a rare 4,5-seco-cadinanetype sesquiterpenoid derivative.

Recorded in CDCl3 at 100 MHz. bRecorded in CDCl3 at 150 MHz.

Figure 2. Key HMBC and COSY correlations for compound 2.

(5S,10S)-9-Oxo-atractylon (3) exhibited a molecular formula of C15H18O2 on the basis of the HRESIMS ion peak at m/z 231.1378 [M + H]+ (calcd for C15H19O2, 231.1387). Its 1H and 13 C NMR spectra, inclusive of the HSQC data, showed 15 carbon resonances, including two methyls [δH 1.00 (s, H3-14) and 2.02 (d, J = 1.2 Hz, H3-13); δC 15.9 and 8.0, respectively], five methylenes [one sp2 methylene at δH 4.98 (s, Ha-15) and 4.78 (s, Hb-15); δC 108.7], two methines [one olefinic at δH 7.39 (d, J = 1.2 Hz, H-12); δC 145.0], and six quaternary carbons [including four olefinic methanes at δC 120.9 (C-11), 137.8 (C-7), 146.1 (C-8), and 148.5 (C-4), and one carbonyl carbon at δC 192.1 (C-9), respectively]. These observed data were similar to those of atractylon,36 a eudesmane sesquiterpenoid lactone derivative, except that the CH2-9 carbon was replaced with a carbonyl carbon in 3. The HMBC correlations from H3-14 (δH 1.00) to C-1, C-5, C-9, and C-10 (δC 32.7, 48.8, 192.1, and 47.8, respectively) supported the above deductions (see Figure 3). The ROESY correlations of Hβ-1/Me-14 and Hα-1/H-5 in 3 indicated Me-14 and H-5 to be β- and α-oriented, respectively. Additionally, data for its experimental ECD spectra (see Figure 8) were comparable to those of the calculated ECD in the gas phase using the TDDFT method at the B3LYP/6-31+g(d,p)

Figure 1. (a) Key HMBC and COSY correlations and (b) key ROESY correlations for compound 1.

with C-5 (δC 178.4), together with the 1H−1H COSY corrections of H-1/H-2/H-3/H-4/H-15, suggested an eightmembered lactone ring (unit a, shown in the Figure 1). Additionally, 1H−1H COSY cross-peaks of H-7/H-8, and the HMBC cross-peaks of H-9 (δH 5.10) with C-8 (δC 26.4) and C7 (δC 124.0), two partial structures containing units a and b that connected to a methylene carbon (C-8), were determined. Establishing the relative configurations of this compound from the ROESY spectrum directly was not possible because of the flexibility of the eight-membered lactone ring. However, the bond angle between C(14)−C(10) and C(9)−C(10) is stable because they connect to a double bond, and the ROESY interaction between H-9 (δH 5.10) with H-14 (δH 1.73) was observed clearly, which indicated that 1 has a C-9S configuration, as supported by molecular modeling.32−34 The time-dependent density functional theory (TDDFT) method at the B3LYP/6-311g(d) level was used for calculating the electronic circular dichroism (ECD) spectra of 1a (4R,9S) and 1b (4S,9S). Comparison of these calculated data and the experimental ECD showed that the ECD spectrum of 1 matched the 1b data well (see Figure 7), which revealed that the absolute configuration of chlorantolide A (1) is 4S,9S. Phacadinane E (2), isolated as a white, amorphous powder, was assigned a molecular formula of C15H18O3 with seven degrees of unsaturation, based on its HRESIMS (m/z 269.1144 [M + Na]+, calcd for C15H18O3Na+, 269.1145). Its 1H and 13C NMR (Tables 1 and 2) and HSQC spectra displayed signals for four methyls [δH 2.41 (s, H3-14), 2.22 (s, H3-15), 1.63 (s, H312), and 1.63 (s, H3-13); δC 18.9, 30.0, 27.7, and 27.7,

Figure 3. (a) Key HMBC and COSY correlations and (b) key ROESY correlations for compound 3. C



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level, demonstrating the absolute configuration of 3 to be 5R,10R. Chlorantene J (4) displayed a molecular formula of C15H14O3 with nine degrees of unsaturation, as determined by the HRESIMS at m/z 243.1016 [M + H]+ (calcd for C15H15O3, 243.1022). Its 1H and 13C NMR spectra showed three methyls [δH 1.40 (s, H3-14), 2.19 (s, H3-15), and 2.28 (d, J = 1.2 Hz, H3-13); δC 25.1, 12.4, and 9.2, respectively], one sp3 methylene, one sp3 quaternary carbon, four olefinic bonds [δH 6.88 (d, J = 9.9 Hz, H-1), 6.38, (d, J = 9.9 Hz, H-2), and 7.18 (s, H-12); δC 119.8 (C-11), 121.7 (C-7), 137.8 (C-4), 140.9 (C-2), 140.9 (C-12), 150.7 (C-5), 153.2 (C-1), and 163.8 (C8), respectively], and two carbonyl groups (δC 187.0 and 186.7), suggesting that 4 is also a eudesmane sesquiterpenoid lactone derivative. Further analysis revealed that 4 is an analogue of chlorantene D.10 The HMBC correlations from H315 (δH 2.19) to δC C-5 (150.7), C-3 (187.0), and C-4 (137.8), from H3-14 (δH 1.40) to δC C-1 (153.2), and the 1H−1H COSY correlation of H-1/H-2 indicated a Δ1,2 double bond in 4 (see Figure 4). In the ROESY spectrum, a correlation of H3-


(1R,3S,5S,8S,10R)-14-Acetylshizukanolide (6) displayed a molecular formula of C17H20O4 from the positive molecular ion peak at m/z 289.1439 [M + H]+ (calcd for C17H21O4, 289.1441) in its HRESIMS. The 1H, 13C NMR and HSQC spectra of 6 revealed 17 carbon resonances, including two methyls [δH 1.86 (s, H3-13) and 2.15 (s, H-2′); δC 8.8 and 21.2, respectively], five methylenes [one oxygenated methylene at δH 3.59 (d, J = 12.3 Hz, Hb-14) and 4.10 (d, J = 12.3 Hz, Ha-14); δC 61.8; one sp2 methylene at δH 5.06 (s, Ha-15) and 4.81 (s, Hb-15); δC 107.0, respectively], four methines, and six quaternary carbons [three sp2 quaternary carbons at δC 122.5 (C-11), 149.1 (C-4) and 160.5 (C-7); and two carbonyl carbons at δC 174.5 (C-12) and 171.6 (C-1′), respectively]. The above NMR data suggested that 6 is structurally related to shizukanolide,37 a lindenane-type sesquiterpenoid lactone derivative, except for an additional acetyl group at C-14 in 6. The HMBC cross-peaks from δH 4.10 (Ha-14) and 3.59 (Hb14) to δC 39.6 (C-9), 42.5 (C-10), 23.8 (C-1) and 171.6 (C1′), from δH 2.15 (H-2′) to δC 171.6 (C-1′) supported the aforementioned deductions (see Figure 6). The ROESY


14/Hβ-9 was observed, which suggested that Me-13 is βoriented. The 10R-configuration of 4 was confirmed by comparison of its ECD data with the data calculated in methanol using the TDDFT method at the B3LYP/631+g(d,p) level (see Figure 9). The 1D NMR and HSQC spectra of (7R,10S)-7-hydroxyeudesm-4-en-3,6-dione (5), with a molecular formula of C15H22O3 from its HRESIMS (m/z 251.1645 [M + H]+, calcd for C15H23O3, 251.1647), exhibited signals for four methyls [δH 1.76 (s, H3-15), 1.17 (s, H3-14), 0.99 (d, J = 7.2 Hz, H3-12), and 0.97 (d, J = 7.2 Hz, H3-13); δC 12.7, 22.0, 17.3, and 16.4, respectively], four sp3 methylenes, one sp3 methine, two sp3 quaternary carbons (one oxygenated quaternary carbon at δC 79.7), two sp2 quaternary carbons (δC 133.6 and 156.8) and two sp2 carbonyl groups (δC 199.3 and 204.9). The NMR data of 5 were similar to those of (−)-(7S,10S)-7-hydroxyeudesm-4en-6-one,22 but the methylene group at C-3 was replaced with a carbonyl carbon in 5. These observations were confirmed by the HMBC cross-peaks of δH 1.76 (H3-15) with δC 133.6 (C4), 156.8 (C-5), and 199.3 (C-3)). The ROESY correlations of H3-14/Hβ-1 (δH 1.88), H3-14/Hβ-9 (δH 1.95), and H3-12/Hα-8 (δH 2.06) suggested that Me-14 and OH-7 are both β-oriented. Furthermore, the calculated ECD spectrum in methanol was obtained using the TDDFT method at the B3LYP/6-31+g(d,p) level. The ECD spectrum displayed Cotton effects at 205 (Δε −18.91), 233 (Δε +10.79), and 255 (Δε +34.75) nm, which matched the calculated ECD spectrum of 5a (7S,10S) well (see Figure 10). Therefore, compound 5 was defined as depicted in Figure 5.


correlations between Hβ-9 (δH 1.45) and H-14, between H14 and H-8, suggested that CH2-14 and H-8 are both βoriented, while the analogous correlations of H-14/H-2 (δH 0.85), H-5/H-3, and H-3/Hα-6 (δH 2.78) supported H-3 and H-5 as both being α-oriented. The 1R,3S,5S,8S,10R-configuration of 6 was confirmed by the comparison of its ECD data with the data calculated in the gas phase using the TDDFT method at the B3LYP/6-31+g(d,p) level (see Figure 11). Therefore, the structure of 6 was elucidated as (1R,3S,5S,8S,10R)-14-acetylshizukanolide. All compounds isolated in this investigation were evaluated for their neuroprotective potency against glutamate-induced PC12 cell death. The results showed that compounds 2−7, 9, 16, 26, 29, 31, and 32 exhibited EC50 values in the range from D



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Figure 10. Measured ECD spectrum (200−400 nm) of 5 and calculated ECD spectra of 5a (7S,10S) and the enantiomer of 5a.

Figure 7. Measured ECD spectrum (200−400 nm) of 1 and calculated ECD spectra of 1a (4R,9S) and the enantiomer of 1a.

Figure 11. Measured ECD spectrum (200−400 nm) of 6 and calculated ECD spectra of 6a (1R,3S,5S,8S,10R) and the enantiomer of 6a.

pretreatment of cultures with different concentrations of compound 26 (40, 20, and 10 μM, respectively). The results revealed that compound 26 can prevent cultured PC12 cells from undergoing apoptotic death induced by glutamate. Caspases, the triggers and executioners of apoptosis, play a critical role in cellular death, and especially caspase-3 is involved in the DNA fragmentation that occurs during apoptosis.39 Also, it is considered that glutmate-induced neuronal apoptosis is caspase-3-dependent.40 Moreover, Akt signaling is a main pathway involved in the survival and adaptive protection of various cell types,41 and it is also a critical regulator in the mechanism of glutamate excitotoxicity.42 Therefore, to further elucidate the neuroprotective mechanisms of compound 26, the effects of this sesquiterpenoid on caspase3 and Akt activities were assessed using a Cleavalite caspase-3 activity assay kit and Western blotting, respectively. As shown in Figures 13 and 14, the activation of caspases-3 was significantly enhanced, while a marked decrease in Akt phosphorylation was observed after treatment with 15 mM glutamate for 24 h. However, these conditions were significantly reversed when cultures were pretreated with different concentrations of compound 26 (10, 20, and 40 μM, respectively). The results revealed that compound 26 has a potential neuroprotective activity against apoptosis induced in PC12 cells by glutamate, as a result of inhibiting caspase-3, while activating Akt signaling.

Figure 8. Measured ECD spectrum (200−400 nm) of 3 and calculated ECD spectra of 3a (5S,10S) and the enantiomer of 3a.

Figure 9. Measured ECD spectrum (200−400 nm) of 4 and calculated ECD spectra of 4a (10S) and the enantiomer of 4a.

3.3 ± 0.9 to 132.0 ± 7.4 μM, with compound 26 being the most active substance (Table 3). Previous reports have revealed that both necrosis and apoptosis can be induced by glutamate in neuronal cellular death.38 To elucidate the mechanism underlying the observed neuroprotective activity of compound 26 against glutamateinduced PC12 cell death, induction of apoptosis was investigated using a Hoechst 33258 staining assay. As shown in Figure 12, 65.5% of cultured PC12 cells underwent apoptosis after exposure to 15 mM glutamate for 24 h. However, the proportion of apoptotic neurons decreased to 7.2−23.7% after

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations and ECD spectra were obtained on a PerkinElmer 341 polarimeter and an Applied Photophysics Chirascan spectrometer, respectively. A Shimadzu UV-2450 spectrophotometer and a Bruker Tensor 27 spectrometer were used to measure UV and IR spectra, respectively. A E



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Table 3. Protective Effects of Selected Compounds on PC12 Cell Injury Induced by Glutamate (EC50, μM)a,b compound

mean ± SD

compound

mean ± SD

compound

mean ± SD

2 3 4 5 6

93.1 ± 1.8 118.9 ± 3.4 105.6 ± 5.2 132.0 ± 7.4 89.5 ± 4.9

7 9 16 26 29

51.4 ± 3.6 107.1 ± 6.3 36.3 ± 2.9 3.3 ± 0.9 24.3 ± 0.8

31 32 nimodipinec

15.2 ± 0.5 70.3 ± 4.3 97.3 ± 5.4

Compounds 1, 8, 10−15, 17−25, 27, 28, and 30 were inactive. bEach value represents the mean ± SD from three independent experiments. Positive control.

a c

Figure 12. Hoechst 33258 staining showing protection by compound 26 to cultured PC12 cellular apoptotic death induced by glutamate treatment. Cultures were treated with glutamate or compound 26 and stained subsequently with fluorescent DNA-bingding dye Hoechst 23358. (A) Control culture. (B) Culture treated with 15 mM glutamate for 24 h. (C−E) Cultures treated with 15 mM glutamate + compound 26 (10, 20, and 40 μM, respectively). Columns show means, error bars, and SD. aqueous residue. The former extract was suspended in water and extracted with petroleum ether (PE) and EtOAc, respectively. The PE partition (110 g) was applied to silica gel by a stepwise gradient with mixtures of PE-EtOAc (10:0 to 0:10) to afford seven fractions, A−G. Fraction C (11 g) was chromatographed repeatedly over MCI gel (MeOH−H2O, 7:3 to 10:0), Sephadex LH-20 (PECH2Cl2−MeOH, 5:5:1), and silica gel (PE−EtOAc, 6:1), and then each of the major components was purified via semi-preparative HPLC (MeOH−H2O, 75:25, CH3CN−H2O, 85:15) to give 3 (11.2 mg), 15 (25.4 mg), 23 (20.2 mg), and 30 (17.4 mg). Fraction D was separated over ODS (MeOH−H2O, 5:5 to 10:0) and then repurified through semi-preparative HPLC (MeOH−H2O, 70:30, CH3CN−H2O, 75:25) to afford 4 (4.2 mg), 6 (8.4 mg), 7 (2.3 mg), 8 (11.2 mg), 9 (42.7 mg), 11 (13.4 mg), 21 (15.4 mg), and 29 (5.3 mg). Fraction E was chromatographed repeatedly over MCI gel (MeOH−H2O, 5:5 to 10:0), silica gel (PE−EtOAc, 30:1 to 4:1), and Sephadex LH-20 (PE− CH2Cl2−MeOH, 5:5:1), with final purification using semi-preparative HPLC (MeOH−H2O, 60:40, CH3CN−H2O, 68:32) to furnish 1 (5.6 mg), 2 (6.2 mg), 10 (4.6 mg), 12 (9.3 mg), 16 (10.4 mg), 25 (5.9 mg), and 32 (8.4 mg).

Waters Micromass Q-TOF instrument was used to measure HRESIMS data. NMR spectra were acquired on Bruker AM-600, AM-500, and AM-300 spectrometers at 25 °C. A Shimadzu LC-20 AT instrument equipped with a SPD-M20A PDA detector and a YMCPack ODS-A column (250 × 10 mm, S-5 μm, 12 nm) were used for semi-preparative HPLC. ODS (12 nm, S-50 μm, YMC Co., Ltd.), silica gel (200−300 mesh, Qingdao Haiyang Chemical Co., Ltd.), Sephadex LH-20 gel (Amersham Biosciences), and MCI gel (CHP20P, 75−150 μm, Mitsubishi Chemical Industries Ltd.) were employed for column chromatography. Plant Material. The whole plants of Chloranthus anhuiensis were collected in June 2013 at Bozhou, Anhui Province, People’s Republic of China. The plant was authenticated by F.F. A voucher specimen (Chan-2013JX-B) is maintained at the Department of Natural Medicinal Chemistry, China Pharmaceutical University. Extraction and Isolation. The freeze-dried and powdered whole plants of Chloranthus anhuiensis (8.0 kg) were extracted three times with 95% aqueous EtOH (40 L, each 2 h) at 80 °C. The combined extracts were then evaporated in vacuo to yield 410 g of a green F



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nm; ECD (MeOH, c 3.36 mM) 218 (Δε +19.23), 252 (Δε −10.54), 305 (Δε −14.74) nm; IR (KBr) νmax 2937, 1740, 1732, 1668, 1442, 1381, 1239, 1017, 953 cm−1; for 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 231.1378 [M + H]+ (calcd for C15H19O2, 231.1387). Chlorantene J (4): colorless oil; [α]25D −40.7 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 201 (4.39), 239 (3.56), 314 (2.79) nm; ECD (MeOH, c 2.89 mM) 200 (Δε −2.96), 233 (Δε +1.79), 255 (Δε +1.45), 309 (Δε −0.97) nm; IR (KBr) νmax 2927, 1658, 1631, 1401, 1124, 1064, 835 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 243.1016 [M + H]+ (calcd for C15H15O3, 243.1022). (7S,10S)-7-Hydroxyeudesm-4-ene-3,6-dione (5): colorless oil; [α]25D +21.4 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 201 (4.11), 262 (3.62) nm; ECD (MeOH, c 3.22 mM) 205 (Δε −18.91), 233 (Δε +10.79), 255 (Δε +34.75) nm; IR (KBr) νmax 2963, 1700, 1672, 1454, 1382, 1189, 1021, 940 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 251.1645 [M + H]+ (calcd for C15H23O3, 251.1647). (1R,3S,5S,8S,10R)-14-Acetylshizukanolide (6): colorless oil; [α]25D −25.1 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 212 (3.41) nm; ECD (MeOH, c 2.16 mM) 213 (Δε +22.98) nm; IR (KBr) νmax 2928, 1745, 1683, 1385, 1239, 1032 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 289.1439 [M + H]+ (calcd for C17H21O4, 289.1441). Neuroprotection Assays. The PC12 cells were incubated in a 96well plate. In the treatment group, cells were pretreated with isolated compounds for 2 h followed by stimulation with 15 mM glutamate for 24 h. In the model group, cells were stimulated with 15 mM glutamate for 24 h, and nimodipine was used as the positive control substance. The MTT method was used to measure cell viability.43 Hoechst 33258 Staining. After test compound treatment and 4% paraformaldehyde fixation, PC12 cell nuclei were stained with 12 μg/ mL of Hoechst 3358 to evaluate chromatin condensation and/or nuclear fragmentation characteristic of apoptosis.7 Caspase-3 Activity Assay. Caspase-3 activity was evaluated by utilizing a Cleavalite caspase-3 activity assay kit (Beyotime Institute of Biotechnology, Shanghai, People’s Republic of China) following standard operating procedures. Briefly, after the compound treatment, PC12 cells were harvested and lysed by trypsinization and lysis buffer, respectively. Next, 150 μg of cell lysate protein and 100 μL of reaction buffer were incubated in a 96-well microtiter plate at 37 °C for 2 h, and then were measured at 405 nm using a plate reader.44 Western Blotting. After treatment with compound 26, PC12 cells were harvested and lysed by trypsinization and RIPA buffer, respectively. The total cellular proteins were obtained by centrifugation. A BCA protein assay kit (Beyotime Institute of Biotechnology, Shanghai, People’s Republic of China) was used to measure the concentration of proteins. Electrophoresis and immunoblot analysis were carried out as described previously.45

Figure 13. Activation of caspase-3 in cultured PC12 cells determined by utilizing Cleavalite caspase-3 activity assay kit. Cultures were treated with glutamate or compound 26 (10, 20, and 40 μM) for 24 h. (*p < 0.05, **p < 0.01, ***p < 0.005, one-way ANOVA, post hoc comparison, Tukey’s test. Columns show means, error bars, and SD. The EtOAc partition (153 g) was fractionated on a silica gel column with mixtures of PE−acetone (100:0 to 0:10) to give seven fractions, a−g. Fraction b (5.0 g) was separated by silica gel CC with mixtures of PE−EtOAc (6:1 to 2:1), and then purification over Sephadex LH-20 (PE−CH2Cl2−MeOH, 5:5:1) and ODS (MeOH−H2O, 50:50 to 90:10), to obtain seven subfractions, which was subjected to further purification by semi-preparative HPLC (MeOH−H2O, 68:32) to give 14 (3.2 mg), 20 (6.8 mg), 26 (8.1 mg), and 28 (4.1 mg). Fraction c (14.0 g) was separated by column chromatography over MCI gel (MeOH−H2O, 5:5 to 8:2), ODS (MeOH−H2O, 45:45 to 85:15), silica gel (CH2Cl2−EtOAc, 5:1), and Sephadex LH-20 (PE−CH2Cl2− MeOH, 5:5:1), and then further repurified by semi-preparative HPLC (MeOH−H2O, 60:40 to 75:25) to yield 5 (9.5 mg), 17 (10.1 mg), 18 (3.5 mg), 19 (7.8 mg), and 27 (8.9 mg). Fraction d (20.0 g) was separated on a MCI gel column (MeOH−H2O, 4:6 to 8:2), silica gel (PE−acetone, 4:1), followed by semi-preparative HPLC (MeOH− H2O, 60:40), to obtain 13 (8.4 mg), 24 (9.1 mg), 22 (11.5 mg), and 31 (6.3 mg). Chlorantolide A (1): colorless oil; [α]25D −22.7 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 206 (3.97) nm; ECD (MeOH, c 1.56 mM) 206 (Δε +10.67) nm; IR (KBr) νmax 2933, 1746, 1743, 1660, 1194, 1156, 1102, 1037 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 265.1436 [M + H]+ (calcd for C15H21O4, 265.1436). Phacadinane E (2): colorless oil; UV (MeOH) λmax (log ε) 216 (4.18), 305 (2.98) nm; IR (KBr) νmax 2921, 1750, 1703, 1455, 1369, 1307, 1263, 1228, 1085, 1046 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 269.1144 [M + Na]+ (calcd for C15H18O3Na+, 269.1145). (5R,10R)-9-Oxo-atractylon (3): colorless oil; [α]25D −11.3 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 216 (4.46), 252 (3.17), 303 (3.45)

Figure 14. Western blot analysis of p-Akt and total-Akt in cultured PC12 cells. Cultures were treated with 15 mM glutamate or compound 26 (10, 20, and 40 μM) for 24 h. (*p < 0.05, **p < 0.01, ***p < 0.005, one-way ANOVA, post hoc comparison, Tukey’s test. Columns show means, error bars, and SD. G



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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.7b01076. Additional HRESIMS, 1H NMR, 13C NMR, HSQC, HMBC, COSY, and ROESY spectra for 1−6 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel/fax: +86 25 83271038. E-mail: [email protected] (F.F.). *Tel/fax: +86 25 83271038. E-mail: [email protected] (W.Q.). ORCID

Jian Xu: 0000-0001-7033-8574 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported financially by the National Natural Science Foundation of China (Nos. 81703382 and 81573557), the China Postdoctoral Science Foundation (2017M621888), and the Priority Academic Program Development of Jiangsu Higher Educational Institutions.

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Sesquiterpenoids from Chloranthus anhuiensis with Neuroprotective

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