Evaluation of DiarylheptanoidâTerpene Adduct Enantiomers from

Article Cite This: J. Nat. Prod. 2018, 81, 162−170

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Evaluation of Diarylheptanoid−Terpene Adduct Enantiomers from Alpinia of f icinarum for Neuroprotective Activities Hui Liu,†,‡,§,⊥ Zhen-Long Wu,†,‡,§,⊥ Xiao-Jun Huang,†,‡,§ Yinghui Peng,‡,§ Xiaojie Huang,‡,§ Lei Shi,‡,§ Ying Wang,*,†,‡,§ and Wen-Cai Ye*,†,‡,§ †

Institute of Traditional Chinese Medicine and Natural Products, Jinan University, Guangzhou 510632, People’s Republic of China JNU-HKUST Joint Laboratory for Neuroscience and Innovative Drug Research, Jinan University, Guangzhou 510632, People’s Republic of China § Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan University, Guangzhou 510632, People’s Republic of China ‡

S Supporting Information *

ABSTRACT: Two pairs of new diarylheptanoid−monoterpene adduct enantiomers, (±)-alpininoids A and B [(±)-1 and (±)-2], as well as three pairs of new diarylheptanoid−sesquiterpene adduct enantiomers, (±)-alpininoids C−E [(±)-3−(±)-5], together with four known diarylheptanoids (6−9) were isolated from the rhizomes of Alpinia of f icinarum. Their structures with absolute configurations were elucidated on the basis of comprehensive spectroscopic analyses and computational calculation methods. The skeletons of these cyclohexenecontaining hybrid natural products were hypothesized to be generated via a crucial Diels−Alder cycloaddition between the diarylheptanoids (7 and 8) and terpenes, of which 1 represents a new carbon skeleton. All isolated compounds were evaluated for their neuroprotective effects against MPP+ (1-methyl-4-phenylpyridinium)-induced cortical neuron injury. At a concentration of 16 μM, (+)-1 significantly increased cell viability when compared with MPP+ treatment alone. flavonoids,10,19 and terpenes9 and in the purification of an essential oil.23 Recently, both a monoterpene-based (officinaruminane B) and a sesquiterpene-based (alpinisin A) diarylheptanoid−terpene adduct with novel carbon skeletons were reported from the rhizomes of A. off icinarum. However, officinaruminane B was reported only as a planar structure, and alpinisin A was assigned to be an optically pure compound possessing negative specific rotation, with the relative configuration of the latter substance deduced based on a NOESY experiment.5,24 During work in searching for structurally unique and biologically interesting constituents from medicinal plants distributed in southern regions of the People’s Republic of China, two known diarylheptanoids, 7-(4-hydroxyphenyl)-1phenyl-4E-hepten-3-one and 7-(4-hydroxy-3-methoxyphenyl)1-phenyl-4E-hepten-3-one, were isolated from a chloroformsoluble fraction of the rhizomes of A. of f icinarum. These two diarylheptanoids showed potent activities in neuronal differentiation and neurite outgrowth, and the latter compound also exhibited protective effects against Aβ-induced damage in PC12 cells and hippocampal neurons through the PI3K-mTOR pathway.16,25 In continuing work, the less polar fraction of the

D

iarylheptanoids are a group of naturally occurring plant phenols.1 Based on their characteristic 1,7-diphenylheptane core structure, diarylheptanoids exhibit an enormous structural diversity, as a result of intramolecular cyclization,2,3 polymerization,4−6 coupling with different functional groups, and hybridization.5,7 These compounds are of particular interest for drug research and development due to their diverse and significant biological effects, such as anti-inflammatory,8,9 antioxidant,10 antitumor,11,12 antiviral,13 and hepatoprotective and neuroprotective activities.14−16 Curcumin, the best known diarylheptanoid initially obtained from turmeric (Curcuma longa L., Zingiberaceae), has reached phase II clinical trials for the treatment of pancreatic cancer.17 Recent pharmacological studies have demonstrated that curcumin also shows therapeutic potential in preventing or treating a number of neurological diseases.18−20 The rhizomes of Alpinia of f icinarum Hance (Zingiberaceae), commonly known as lesser galangal, is a well-known traditional Chinese medicine and has been widely used for the remedy of gastrointestinal diseases such as stomachache, dyspepsia, and gastrofrigid vomiting.5,21 Due to its pungent, spicy taste and aromatic odor, the rhizomes of A. of f icinarum have also been used as a popular culinary spice exhibiting aromatic smell and providing peppery flavor in stir-fry, curry, and other meat dishes in Southeast Asia.22 Previous phytochemical studies on A. off icinarum had resulted in the isolation of diarylheptanoids,5,6 © 2018 American Chemical Society and American Society of Pharmacognosy

Received: September 21, 2017 Published: January 11, 2018 162

DOI: 10.1021/acs.jnatprod.7b00803 J. Nat. Prod. 2018, 81, 162−170

Journal of Natural Products

Article

Chart 1

(4-hydroxyphenyl)-4E-en-3-heptanone (8), and 1,7-diphenyl4E-en-3-heptanone (9).5,9 Alpininoid A (1) was isolated as a yellow oil. The molecular formula of 1 was deduced as C30H38O3 based on the deprotonated molecular ion [M − H]− at m/z 445.2744 (calcd for C30H37O3, 445.2743) in its HRESIMS. The UV spectrum of 1 exhibited absorption maxima at 204 and 282 nm. The IR spectrum suggested the presence of hydroxy group (3425 cm−1), carbonyl (1702 cm−1), and aromatic ring (1648 and 1519 cm−1) absorbances. The 1H and 13C NMR spectra of 1 exhibited signals due to a ketone carbonyl (δC 214.0), a monosubstituted benzene ring [δH 7.15−7.25 (5H, overlapped); δC 141.3, 128.6 × 2, 128.5 × 2, and 126.2)], a 1,3,4-trisubstituted benzene ring [δH 6.81 (1H, d, J = 8.0 Hz), 6.63 (1H, d, J = 1.9 Hz), and 6.61 (1H, dd, J = 8.0, 1.9 Hz); δC 146.5, 143.8, 134.2, 121.0, 114.3, and 111.0], two trisubstituted olefinic bonds [δH 5.36 (1H, br s) and 5.07 (1H, m); δC 135.8, 131.7, 124.2, and 119.7], a methoxy group [δH 3.86 (3H, s); δC 56.0], two methines [δH 2.51 (1H, m) and 1.89 (1H, overlapped); δC 52.9 and 34.6], eight methylenes, and two methyls [δH 1.67 (3H, s) and 1.59 (3H, s); δC 25.8 and 17.9]. With the aid of 1 H−1 H COSY, HSQC, and HMBC experiments, all proton and carbon signals of 1 were assigned as shown in Table 1. The 1H−1H COSY spectrum of 1 revealed the presence of five spin-coupling systems shown in bold (Figure 1). In the HMBC spectrum, the correlations between H2-1 and C-3/C2′/C-6′, between H2-2 and C-1′, between H-4 and C-3, between H2-7 and C-2″/C-6″, between OCH3-3″ and C-3″, and between OH-4″ and C-3″/C-5″ led to the establishment of a diarylheptanoid moiety (1a) with a methoxy group and a hydroxy group at the C-3″ and C-4″ positions, respectively.

ethanol extract of the same plant species was investigated. As a result, five pairs of new diarylheptanoid−terpene adduct enantiomers, (±)-alpininoids A−E [(±)-1−(±)-5], along with four known biosynthetically related diarylheptanoids (6− 9), were isolated using chiral HPLC columns. Structurally, the skeletons of 1−5 featuring a cyclohexene motif probably arise from a diarylheptanoid moiety incorporating a mono- or sesquiterpenoid unit via a key Diels−Alder cycloaddition. All molecular structures of the isolated compounds with absolute configuration were elucidated based on comprehensive spectroscopic analysis and quantum chemical calculations of 13 C NMR data and electronic circular dichroism (ECD) curves. Notably, 1 was found to be a novel diarylheptanoid−terpene adduct possessing a new carbon skeleton. Herein are reported the isolation, structure elucidation, and a plausible biogenetic pathway of these new diarylheptanoid−terpene adducts. In addition, all the isolated compounds were evaluated for their in vitro neuroprotective activities against 1-methyl-4-phenylpyridinium (MPP+)-induced damage in cortical neurons.

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RESULTS AND DISCUSSION The air-dried rhizomes of A. of ficinarum were extracted with 95% EtOH at room temperature to give a crude extract, which was suspended in water and successively partitioned with petroleum ether, chloroform, and n-BuOH. The petroleum ether-soluble fraction was separated using a series of chromatographic procedures, to result in the isolation of five pairs of new diarylheptanoid−terpene adduct enantiomers, (±)-1−(±)-5 (Chart 1), along with four known diarylheptanoids, (5R)-5hydroxy-1,7-diphenyl-3-heptanone (6),5 1-phenyl-7-(4-hydroxy-3-methoxyphenyl)-4E-en-3-heptanone (7),8 1-phenyl-7163



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Table 1. 1H and 13C NMR Spectroscopic Data for 1 and 2 (in CDCl3)a 1 δC

type

1 2 3 4 5 6

29.7 44.3 214.0 52.9 34.6 36.3

CH2 CH2 C CH CH CH2

7

32.8

CH2

1′ 2′ 6′ 3′ 5′ 4′ 1″ 2″

141.3 128.5 128.6 126.2 134.2 111.0

C CH CH CH C CH

3″ 4″ 5″

146.5 143.8 114.3

C C CH

6″

121.0

CH

1‴

30.3

CH2

2‴ 3‴ 4‴

119.7 135.8 31.2

CH C CH2

5‴ 6‴ 7‴ 8‴ 9‴ 10‴ OCH3-3″ OH-4″

37.5 26.5 124.2 131.7 25.8 17.9 56.0

CH2 CH2 CH C CH3 CH3

position

H-7‴ and C-9‴/C-10‴ supported the presence of a linear monoterpenoid unit (1b). In addition, the HMBC correlations between H-4 and C-1‴/C-3‴, as well as H-2‴ and C-5, indicated the two fragments 1a and 1b to be connected via C4−C-4‴ and C-5−C-1‴ bonds to form a cyclohexene ring (Figure 1). This spectroscopic analysis allowed the determination of the planar structure of compound 1, which was elucidated as a novel diarylheptanoid−terpene adduct with a new carbon skeleton. Unfortunately, the NOESY experiment did not afford any useful NOE correlations for the elucidation of the configuration of the two adjacent chiral centers (C-4 and C-5) in the flexible cyclohexene ring. In order to establish the relative configuration of 1, the theoretical calculations of 13C NMR data of the two possible isomers (4R*,5R*)-1 and (4R*,5S*)-1 were predicted using the GIAO method with the Gaussian 09 software at the B3LYP/6-31G(d,p) level.26,27 Comparison of the experimental and calculated 13C NMR data allowed the determination of the relative configuration of 1 as 4R*,5R* with a DP4 probability of approximately 97% (Figure 2). Although there were two asymmetric carbons in the molecule, the optical rotation value of 1 was very close to zero, suggesting that 1 was isolated as a racemic mixture. Subsequently, 1 was resolved into two enantiomers, (+)-1 and (−)-1, at a ratio of 1:1 on a chiral HPLC column (Figure S52, Supporting Information). The ECD spectra of (+)-1 and (−)-1 displayed similar signal intensities but opposite Cotton effects, confirming their enantiomeric relationship. The absolute configurations of the two enantiomers of 1 were determined further by comparison of their experimental ECD spectra with those predicted by time-dependent density functional theory (TDDFT) calculation at the B3LYP/6-31G(d) level.27 As a result, the calculated ECD curves of (4S,5S)-1 and (4R,5R)-1 displayed good agreement with the experimental values of (+)-1 and (−)-1, respectively (Figure 4). Therefore, the absolute configurations of (+)-1 and (−)-1 were elucidated as 4S,5S and 4R,5R (Chart 1), respectively. Alpininoid B (2) showed the same molecular formula, C30H38O3, as 1 by HRESIMS at m/z 445.2744 [M − H]− (calcd for C30H37O3, 445.2743). The UV and IR spectra of 2 also exhibited typical hydroxy group, carbonyl, and aromatic ring absorptions. Similar to 1, the 1H and 13C NMR spectra of 2 showed characteristic signals due to diarylheptanoid and monoterpenoid moieties, suggesting that 2 is also a diarylheptanoid−monoterpene adduct. A comprehensive analysis of the 1H−1H COSY, HSQC, and HMBC NMR spectroscopic

2 δH (mult., J in Hz) 2.87 2.75 (m) 2.51 (m) 1.89 a 1.63 b 1.35 (m) a 2.61 (m) b 2.43 (m) 7.15 7.25 7.17 6.63 (d, 1.9)

6.81 (d, 8.0) 6.61 (dd, 8.0, 1.9) a 2.28 (m) b 1.74 (m) 5.37 (br s) a 2.03 b 1.95 1.93 2.05 5.07 (m) 1.67 1.59 3.86 5.47

(s) (s) (s) (s)

δC

type

29.7 44.2 214.0 52.4 35.0 36.4

CH2 CH2 C CH CH CH2

32.7

CH2

141.3 128.5 128.6 126.2 134.2 111.0

C CH CH CH C CH

146.5 143.8 114.3

C C CH

121.0

CH

28.2

CH2

118.6 136.8 33.3

CH C CH2

37.6 26.5 124.2 131.7 25.8 17.9 56.0

CH2 CH2 CH C CH3 CH3

δH (mult., J in Hz) 2.88 2.75 (m) 2.47 1.98 a 1.64 b 1.37 (m) a 2.63 (m) b 2.46 7.15 7.25 7.17 6.63 (d, 1.9)

6.82 (d, 8.0) 6.61 (dd, 8.0, 1.9) 2.10 5.37 (br s) a 2.16 b 1.76 1.96 2.09 5.09 (m) 1.69 1.61 3.86 5.50

(s) (s) (s) (s)

a

Assignments were performed by analysis of 1D and 2D NMR spectra. Overlapped signals are reported without designating multiplicity.

Moreover, the HMBC correlations between H2-1‴ and C-3‴, H2-4‴ and C-2‴, H2-5‴ and C-4‴, H2-6‴ and C-3‴/C-8‴, and

Figure 1. Key 1H−1H COSY and HMBC correlations of 1−3. 164



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to zero, compound 2 was also considered as a racemic mixture. The racemate of (±)-2 was further resolved by HPLC using a chiral column to afford a pair of enantiomers, (+)-2 and (−)-2, in a ratio of approximately 1:1 (Figure S53, Supporting Information). Similarly, the experimental ECD spectra of (+)-2 and (−)-2 clearly defined their enantiomeric relationship. Finally, the absolute configurations of (+)-2 and (−)-2 were then assigned as 4S,5S and 4R,5R (Chart 1), respectively, by comparing their experimental and calculated ECD data (Figure 4). Alpininoid C (3) was isolated as a white, amorphous powder. The molecular formula of 3 was determined to be C35H46O3 by its HRESIMS (m/z 515.3537 [M + H]+, calcd for C35H47O3, 515.3520). The UV spectrum revealed absorption bands at λmax 205 and 283 nm. The IR spectrum showed the presence of hydroxy group (3423 cm−1), carbonyl (1735 cm−1), and aromatic ring (1648 and 1550 cm−1) absorptions. The analysis of 1H and 13C NMR spectra of 3 indicated the presence of a ketone carbonyl (δC 215.3), a monosubstituted benzene ring [δH 7.13−7.23 (5H, overlapped); δC 141.4, 128.6 × 2, 128.5 × 2, and 126.1], a 1,3,4-trisubstituted benzene ring [δH 6.80 (1H, d, J = 8.0 Hz), 6.58 (1H, d, J = 1.9 Hz), and 6.56 (1H, dd, J = 8.0, 1.9 Hz); δC 146.5, 143.8, 134.1, 121.0, 114.3, and 110.9], three trisubstituted double bonds [δH 5.49 (1H, d, J = 4.7 Hz), 5.05 (1H, ddd, J = 6.8, 4.0, 1.2 Hz), and 4.91 (1H, td, J = 6.3, 1.0 Hz); δC 137.2, 135.0, 131.5, 124.4, 122.8, and 120.3], a methoxy group [δH 3.84 (3H, s); δC 56.0], three methines [δH 2.48 (1H, overlapped), 2.45 (1H, overlapped), and 1.81 (1H, m); δC 57.7, 42.0, and 36.5], eight methylenes, and four methyls [δH 1.65 (3H, s), 1.62 (3H, s), 1.57 (3H, s), and 1.53 (3H, s); δC 25.9, 21.2, 17.8, and 16.6]. However, in contrast to 1 and 2, these spectroscopic data suggested that 3 is a diarylheptanoid−sesquiterpene adduct. Detailed analysis of 1 H−1H COSY, HSQC, and HMBC spectra resulted in unambiguous assignment of all proton and carbon signals of 3 as shown in Table 2. Five spin-coupling systems were established based on the correlations observed in the 1H−1H COSY spectrum of 3 (Figure 1). The key HMBC correlations between H2-1 and C3/C-2′/C-6′, between H2-2 and C-1′, between H2-7 and C-2″/ C-6″, between OCH3-3″ and C-3″, and between OH-4″ and C3″/C-5″ established a diarylheptanoid unit (3a), which was identical to that of 1 and 2 (Figure 1). Comparison of the NMR data assigned for 3a with those of 1 and 2 confirmed this conclusion. Moreover, the HMBC spectrum showed correlations between H2-1‴ and C-3‴, H2-5‴ and C-3‴/C-7‴, H-6‴

Figure 2. 13C NMR calculation results of two plausible stereoisomers at the B3LYP/6-31G(d,p) level. (a) Linear correlation plots of calculated vs experimental 13C NMR chemical shift values for each potential configuration of 1. (b) Relative errors between the calculated 13 C NMR chemical shifts of two potential structures and recorded 13C NMR data of 1. (c) DP4 probability of 13C NMR chemical shifts of 1.

data allowed the full assignment of all proton and carbon resonances of 2 (Table 1). In the 1H−1H COSY spectrum of 2, correlations between H5 and H2-4‴, as well as between H2-1‴ and H-4, were observed, indicating that the diarylheptanoid and monoterpenoid units are connected through C-4−C-1‴ and C-5−C-4‴ bonds in 2 instead of the C-4−C-4‴ and C-5−C-1‴ linkages in 1. Moreover, the HMBC correlations between H-2‴ and C-4, as well as between H-5 and C-3‴, further defined the coupling pattern of the two fragments in 2. Thus, the planar structure of 2 was established as shown in Figure 1. For the same reason as mentioned for compound 1, the relative configuration of 2 also could not be determined by a NOE experiment. Using a similar method to that described for 1, the comparison of experimental and calculated 13C NMR data allowed the determination of the relative configuration of 2 as 4R*,5R* with a DP4 probability of 98% (Figure S57, Supporting Information). With an optical rotation value close

Figure 3. Geometry of the lowest-energy conformers and key NOESY correlations of 3−5. 165



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Figure 4. Calculated and experimental ECD spectra of (+)/(−)-1−(+)/(−)-5.

ring, suggesting that 4 is a stereoisomer of 3 (Table 2). Further analysis of the 1H−1H COSY, HSQC, and HMBC NMR data of 4 indicated the two compounds possess identical planar structures. However, different from 3, the NOESY spectrum of 4 showed correlations between H-6a and H-4/H-4‴, indicating the presence of a cis-relationship between H-4 and H-4‴ in 4 (Figure 3). Thus, the above data suggested that 4 is a C-4‴ isomer of 3. This assignment was confirmed by comparing the experimental and calculated 13C NMR data of 4. With a DP4 probability of 98%, the relative configuration of 4 was defined as 4R*,5S*,4‴R*, which was consistent with the result of a NOE experiment (Figure S59, Supporting Information). The molecular formula of 5 was established as C34H44O2 on the basis of the [M − H]− ion at m/z 483.3260 (calcd for C34H43O2, 483.3263) in its HRESIMS. Similar to 3 and 4, the 1 H and 13C NMR spectra of 5 also revealed the presence of diarylheptanoid and sesquiterpenoid moieties. With the aid of 1 H−1H COSY, HSQC, and HMBC experiments, all proton and carbon resonances in 5 were assigned as shown in Table 2. The sesquiterpenoid unit of 5 was determined as being identical to 3 and 4 by comparison of their 1H and 13C NMR data. In contrast to 3 and 4, the NMR spectra of 5 showed signals due to a para-disubstituted benzene ring [δH 7.01 (2H, d, J = 8.4 Hz) and 6.74 (2H, d, J = 8.4 Hz); δC 153.6, 134.7, 129.5 × 2, and 115.8 × 2] instead of signals corresponding to a 1,3,4trisubstituted benzene ring in 3 and 4. In addition, comparison of the NMR data assigned to the diarylheptanoid unit of 5 with those of 3 and 4 revealed the absence of proton and carbon signals for a methoxy group in 5. Therefore, the planar structure of 5 was established as being a 3″-demethoxy derivative of 3 and 4. In the NOESY spectrum of 5, NOE correlations between H6a and H-4/H-4‴ were observed, which suggested that H-4 and H-4‴ are cofacial in 5 (Figure 3). Additionally, the relative configurations of the three chiral centers (C-4, C-5, and C-4‴)

and C-14‴, H2-9‴ and C-7‴/C-11‴, H-10‴ and C-12‴/C-13‴, and CH3-15‴ and C-2‴/C-4‴, which suggested the presence of a linear sesquiterpenoid moiety (3b) instead of the monoterpenoid unit in 1 and 2. In addition, the HMBC correlations between H-4 and C-5‴, H-2‴ and C-5, and H-4‴ and C-3/C-5 indicated that the two substructures 3a and 3b are connected via C-4−C-4‴ and C-5−C-1‴ bonds to form a cyclohexene ring (Figure 1). The relative configuration of 3 could be determined by the NOESY spectrum and theoretical calculations of the 13C NMR data. In the NOESY spectrum, correlations between H-6‴ and H2-8‴ indicated the presence of the E form for the Δ6‴(7‴) double bond. The NOE correlations between H-5 and H-4‴ revealed that these two protons are located in the same orientation of the cyclohexane ring. Moreover, the NOE correlations between H-4 and H-6b/H2-5‴ suggested that these protons are cofacial. Taken together, the aforementioned NOE correlations suggested a trans-relationship between H-4 and H5 as well as between H-4 and H-4‴ (Figure 3). However, due to the flexibility of the cyclohexene ring and the steric bulk of the moieties attached to the three consecutive stereogenic centers (C-5, C-4, and C-4‴), a DP4 statistical procedure based on the theoretical 13C NMR calculations was performed to verify the relative configuration of 3. According to the calculation result, the relative configuration of 3 could be established unambiguously as 4R*,5S*,4‴S*, with a probability of almost 100% (Figure S58, Supporting Information). Alpininoid D (4) exhibited an identical molecular formula to 3 from its HRESIMS data. The similarity of characteristic absorptions in the UV and IR spectra of 4 and 3, combined with the molecular formula information, indicated that 4 was also a diarylheptanoid−sesquiterpene adduct. The 1H and 13C NMR spectroscopic data of 4 were found to be very similar to those of 3, except for minor differences of resonances assigned to protons and carbons located in the connecting cyclohexene 166



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Table 2. 1H and 13C NMR Spectroscopic Data for 3−5 (in CDCl3)a 3 δC

type

1

29.3

CH2

2

46.3

CH2

3 4 5 6

215.3 57.7 36.5 36.3

C CH CH CH2

7

32.6

CH2

1′ 2′, 6′ 3′, 5′ 4′ 1″ 2″ 3″ 4″ 5″ 6″ 1‴

141.4 128.6 128.5 126.1 134.1 110.9 146.5 143.8 114.3 121.0 30.9

C CH CH CH C CH C C CH CH CH2

2‴ 3‴ 4‴ 5‴

122.8 135.0 42.0 28.2

CH C CH CH2

6‴ 7‴ 8‴ 9‴ 10‴ 11‴ 12‴ 13‴ 14‴ 15‴ OCH3-3″ OH-4″

120.3 137.2 39.9 26.6 124.4 131.5 17.8 25.9 16.6 21.2 56.0

CH C CH2 CH2 CH C CH3 CH3 CH3 CH3

position

a

4 δH (mult., J in Hz)

δC

type

2.86 (m)

29.8

CH2

2.68 (m)

44.9

CH2

211.3 55.5 29.6 36.5

C CH CH CH2

32.9

CH2

141.4 128.5 128.6 126.1 134.6 111.1 146.5 143.7 114.3 120.7 30.1

C CH CH CH C CH C C CH CH CH2

120.7 136.3 41.1 28.7

CH C CH CH2

123.8 135.9 40.1 26.8 124.5 131.5 17.8 25.8 16.3 22.9 56.1


2.45 1.81 (m) a 1.48 (m) b 1.33 (m) a 2.62 (m) b 2.36 (m) 7.15 7.23 7.13 6.58 (d, 1.9)

6.80 (d, 8.0) 6.56 (dd, 8.0, 1.9) a 2.24 (m) b 1.68 5.49 (d, 4.7) 2.48 a 2.17 (m) b 1.89 (m) 4.91 (td, 6.3, 1.0) 1.99 (m) 2.05 (m) 5.05 (ddd, 6.8, 4.0, 1.2) 1.57 1.65 1.53 1.62 3.84 5.45

(s) (s) (s) (s) (s) (br s)

5 δH (mult., J in Hz)

2.87 (m) 2.79 (m) 2.75 (m) 2.65 2.66 2.06 a 1.78 (m) b 1.24 a 2.56 (m) b 2.47 (m) 7.16 7.23 7.16 6.68 (d, 1.9)

6.81 (d, 8.0) 6.64 (dd, 8.0, 1.9) a 2.21 b 1.69 5.33 (br s) 2.22 a 2.06 b 2.00 4.97 (td, 7.0, 1.0) 1.95 (m) 2.04 5.07 (ddd, 6.8, 4.0, 1.2) 1.58 1.67 1.53 1.72 3.88 5.33

(s) (s) (s) (s) (s) (br s)

δC

type

29.8

CH2

44.9

CH2

211.6 55.4 29.7 36.5

C CH CH CH2

32.3

CH2

141.4 128.5 128.6 126.1 134.7 129.5 115.8 153.6 115.8 129.5 30.2

C CH CH CH C CH CH C CH CH CH2

120.7 136.2 41.1 28.7

CH C CH CH2

123.7 135.9 40.1 26.8 124.3 131.4 17.8 25.8 16.3 22.9


δH (mult., J in Hz) 2.88 (m) 2.79 (m) 2.77 2.65 2.66 2.06 a 1.76 (m) b 1.22 a 2.54 (m) b 2.48 (m) 7.16 7.23 7.17 7.01 (d, 8.4) 6.74 (d, 8.4) 6.74 (d, 8.4) 7.01 (d, 8.4) a 2.21 b 1.71 5.33 (br s) 2.22 a 2.08 b 2.00 4.97 (td, 7.0, 1.0) 1.95 (m) 2.05 5.08 (ddd, 6.8, 4.0, 1.2) 1.58 1.67 1.53 1.72

(s) (s) (s) (s)

5.33 (br s)

Assignments were performed by analysis of 1D and 2D NMR spectra. Overlapped signals are reported without designating multiplicity.

Compounds 1−5 are five pairs of new racemic diarylheptanoid−terpene adducts with a characteristic cyclohexene motif, the skeletons of which presumably arise from the conjugation of a diarylheptanoid unit and a mono- or sesquiterpenoid moiety via a crucial [4+2] cycloaddition. The plausible biogenetic pathways of 1−5 could be proposed as outlined in Scheme S1 (Supporting Information). The coisolated diarylheptanoids 7 and 8 could be employed as dienophiles to couple with dienes (α-farnesene or β-myrcene, which have been identified previously from the essential oil of A. off icinarum23) via a nonstereoselective Diels−Alder cycloaddition to form racemates (+)-1/(−)-1−(+)-5/(−)-5. Interestingly, when β-myrcene functions as a diene, the two possible regioisomeric products, 1 (“meta” product) and 2 (“para” product), were both obtained. In contrast, only “ortho” products (3−5) would be isolated when α-farnesene is presumed as a diene. The petroleum ether-soluble fraction of

in 5 could be deduced by comparison of their 1H and 13C NMR spectroscopic data with those of 4. Accordingly, the relative configuration of 5 was confirmed as 4R*,5S*,4‴R* by a theoretical 13C NMR calculation followed by a DP4 probability analysis (Figure S60, Supporting Information). Due to their barely measurable optical rotations, this suggested that 3−5 are also racemic mixtures, as further supported by the absence of Cotton effects in their ECD spectra. Subsequently, the chiral HPLC separation of 3−5 afforded three pairs of enantiomers, (+)-3 and (−)-3, (+)-4 and (−)-4, and (+)-5 and (−)-5, in a ratio of 1:1 (Figures S54−S56, Supporting Information), respectively. Similarly, the absolute configurations of the three pairs of enantiomers were determined as 4S,5R,4‴R and 4R,5S,4‴S, 4S,5R,4‴S and 4R,5S,4‴R, and 4S,5R,4‴S and 4R,5S,4‴R (Chart 1), respectively, by comparing their experimental and calculated ECD spectra (Figure 4). 167



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performed on an Agilent 1260 liquid chromatograph equipped with a 1260 pump and a UV/vis-1260 detector with a Cosmosil 5C18-MS-II reversed-phase column (20 × 250 mm, i.d. 5 μm, Nacalai Tesque Inc.). Column chromatography was performed on silica gel (200−300 mesh, Qingdao Marine Chemical Co. Ltd., Qingdao, People’s Republic of China), octadecyl silica (ODS) (40−64 μm, Merck, Darmstadt, Germany), and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden). Plant Material. The rhizomes of Alpinia of ficinarum were collected from Longtang Town, Xuwen County, Guangdong Province, People’s Republic of China, in December 2013, and were authenticated by Prof. Guang-Xiong Zhou (Institute of Traditional Chinese Medicine and Natural Products, Jinan University). A voucher specimen (No. 20131211) was deposited in the Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University. Extraction and Isolation. The air-dried and powdered rhizomes of A. of f icinarum (20 kg) were extracted with 95% EtOH(aq) three times at room temperature (3 × 150 L, 12 h each time). The EtOH extract was concentrated under vacuum to yield a crude extract (3.7 kg), which was suspended in water and then partitioned successively with petroleum ether, chloroform, and n-BuOH. The petroleum ethersoluble fraction (500 g) was subjected to passage over a silica gel column using gradient mixtures of petroleum ether−EtOAc (1:0 → 2:1, v/v) as eluents, to afford 12 major fractions (Fr.1−Fr.12). Fr.5 (87 g) was chromatographed on a silica gel column eluted with cyclohexane−EtOAc mixtures (1:0 → 5:1, v/v) to obtain six subfractions (Fr.5A−Fr.5F). Fr.5B (12 g) was separated using a Sephadex LH-20 column (CHCl 3 −MeOH, 50:50, v/v) and subsequently purified by reversed-phase preparative HPLC, using MeOH−H2O (80:20, 6 mL/min) as mobile phase, to yield 5 (5.2 mg). Fr.5C (16 g) was subjected to a Sephadex LH-20 column (CHCl3− MeOH, 50:50, v/v) to afford six subfractions (Fr.5C-1−Fr.5C-6). Fr.5C-2 (0.8 g) was further purified by reversed-phase preparative HPLC using CH3CN−H2O (65:35, 6 mL/min) as mobile phase to yield 4 (4.3 mg). Fr.7 (65 g) was subjected to a silica gel column eluted with gradient mixtures of petroleum ether−EtOAc (100:5 → 2:1, v/v) to afford four subfractions (Fr.7A−Fr.7D). Fr.7B (8 g) was then separated using an ODS column, with mixtures of MeOH−H2O (60:40 → 80:20) as eluents, to yield five subfractions (Fr.7B-1−Fr.7B5). Compound 2 (2.7 mg) was afforded from Fr.7B-2 (0.8 g) by preparative HPLC using CH3CN−H2O (60:40, 6 mL/min) as the mobile phase. Fr.7B-3 (1.2 g) was then purified by preparative HPLC on a reversed-phase C18 column using CH3CN−H2O (65:35, 6 mL/ min) as mobile phase to yield 1 (4 mg). Fr.8 (56 g) was separated using a Sephadex LH-20 column with CHCl3−MeOH (50:50) as eluents to give four subfractions (Fr.8A−Fr.8D). Fr.8C (4.6 g) was further chromatographed on an ODS column eluted with gradient mixtures of MeOH−H2O (50:50 → 80:20) to afford five subfractions (Fr.8C-1−Fr.8C-5). Fr.8C-2 (1.2 g) was subsequently purified by reversed-phase preparative HPLC, using CH3CN−H2O (70:30, 6 mL/ min) as mobile phase, to afford 3 (2.6 mg) and 6 (16 mg). Fr.10 (76 g) was subjected to separation over a silica gel column eluted with gradient mixtures of petroleum ether−EtOAc (10:1 → 2:1) to give six subfractions (Fr.10A−Fr.10F). Fr.10C (7 g) was separated over an ODS column (MeOH−H2O, 20:80 → 80:20) and further purified by reversed-phase preparative HPLC (MeOH−H2O, 60:40, 6 mL/min) to yield 7 (13 mg) and 9 (25 mg). Purification of Fr.10E (6 g) using an ODS column (CH3CN−H2O, 40:60 → 100:0) and reversed-phase preparative HPLC (CH3CN−H2O, 65:35, 6 mL/min) gave 8 (18 mg). Chiral Separation of Enantiomers. Enantioseparations of (±)-1−(±)-5 were carried out on an Agilent 1260 liquid chromatograph system equipped with a DAD detector. Compounds (+)-1 (tR 13.2 min, 1.2 mg) and (−)-1 (tR 13.8 min, 1.3 mg) were obtained by using a Phenomenex Cellulose-2 column (4.6 × 250 mm, i.d. 5 μm, Phenomenex, CA, USA) and an elution mixture of MeOH−H2O (90:10) at a flow rate of 1 mL/min. In turn, compounds (+)-2 (tR 11.2 min, 0.5 mg) and (−)-2 (tR 12.7 min, 0.6 mg) were separated using a Phenomenex Cellulose-4 column (4.6 × 250 mm, i.d. 5 μm) by elution with CH3CN−H2O (65:35) at a flow rate of 1 mL/min. Also, (+)-3

the methanol extract of A. off icinarum fresh rhizomes was analyzed by UPLC-QTOF/MS. As a result, compounds 1−5 could be clearly detected guided by the characteristic UV profiles and ion peaks from the above crude fraction and were further identified by comparing with reference compounds (Figures S65−S73, Supporting Information). However, the theoretical “meta” products of α-farnesene and diarylheptanoids (7 and 8) did not occur in the same sample. Compounds (+)-1, (−)-1, 2, 3, (+)-4, (−)-4, (+)-5, (−)-5, 6, 7, 8, and 9 were tested for their potential neuroprotective effects against MPP+-induced damage in cortical neurons. As shown in Figure 5, the cell viability of cortical neurons

Figure 5. Effects of (+)-1, (−)-1, 2, 3, (+)-4, (−)-4, (+)-5, (−)-5, 6− 9, and myrcene on cell survival in MPP+-injured primary cortical neurons. The results are expressed as mean ± SEM (n = 3) of three independent experiments. ***p < 0.001 as compared with control (DMSO); #p < 0.05 as compared with the MPP+ alone. One-way ANOVA followed by Bonferroni’s multiple comparison test was used.

decreased to 62% after exposure to 500 μM MPP+ for 20 h. Notably, (+)-1 at 16 μM significantly restored the cell viability, whereas its enantiomer (−)-1 did not show any neuroprotective effect. These data suggested the stereospecificity of this neuroprotective activity of 1. Additionally, 1-phenyl-7-(4hydroxy-3-methoxyphenyl)-4E-en-3-heptanone (7) and βmyrcene, the putative biosynthetic precursors of (+)-1/(−)-1, were also evaluated for their neuroprotective activities. However, neither of the two precursors showed discernible neuroprotective activity on the test concentrations (Figure 5), indicating that the hybridization of diarylheptanoid and monoterpene might be essential for the neuroprotective activity.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotation values were measured on a JASCO P-1020 digital polarimeter (JASCO, Tokyo, Japan). UV spectra were recorded on a JASCO V-550 UV/vis spectrophotometer. ECD spectra were obtained on a JASCO J-810 spectropolarimeter. IR spectra were conducted on a JASCO FT/IR480 plus Fourier transform infrared spectrometer. 1D and 2D NMR spectra were recorded on a Bruker AVANCE-500 NMR spectrometer with tetramethylsilane as internal reference (Bruker, Fällanden, Switzerland). HRESIMS data were obtained on a Waters Xevo G2 QTOF-MS spectrometer (Waters, Milford, MA, USA) or an Agilent 6210 ESI/TOF mass spectrometer (Agilent, Palo Alto, CA, USA). Analytical HPLC was carried on an Agilent 1260 liquid chromatograph equipped with a 1260 pump and a 1260 DAD detector with a Cosmosil 5C18-MS-II reversed-phase column (4.6 × 250 mm, i.d. 5 μm, Nacalai Tesque Inc., Tokyo, Japan). Preparative HPLC was 168



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(tR 34.5 min, 0.5 mg) and (−)-3 (tR 36.5 min, 0.5 mg) were afforded by using a Phenomenex Cellulose-2 column (4.6 × 250 mm, i.d. 5 μm), eluted with CH3CN−H2O (65:35) at a flow rate of 1 mL/min. Compounds (+)-4 (tR 10.9 min, 1.1 mg) and (−)-4 (tR 12.7 min, 1.0 mg) were obtained using a Phenomenex Cellulose-4 column (4.6 × 250 mm, i.d. 5 μm) and an eluting mixture of MeOH−H2O (90:10) at a flow rate of 1 mL/min. Finally, (+)-5 (tR 9.2 min, 1.4 mg) and (−)-5 (tR 9.7 min, 1.3 mg) were obtained using a Phenomenex Cellulose-2 column (4.6 × 250 mm, i.d. 5 μm), by elution with MeOH−H2O (65:35) at a flow rate of 1 mL/min. (±)-Alpininoid A (1): yellow oil; [α]25 D ±0 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 204 (3.00), 282 (1.89) nm; IR (KBr) νmax 3425, 2920, 1702, 1648, 1519, 1452, 1370, 1271, 1032, 697 cm−1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data, see Table 1; HRESIMS m/z 445.2744 [M − H]− (calcd for C30H37O3, 445.2743). (+)-1: yellow oil; [α]25 D +25 (c 0.1, MeOH); ECD (MeOH, Δε) λmax 228 (+6.98), 300 (−4.12) nm. (−)-1: yellow oil; [α]25 D −25 (c 0.1, MeOH); ECD (MeOH, Δε) λmax 238 (−7.68), 305 (+3.11) nm. (±)-Alpininoid B (2): yellow oil; [α]25 D ±0 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 205 (3.37), 282 (2.28) nm; IR (KBr) νmax 3448, 2916, 1704, 1607, 1514, 1453, 1373, 1269, 1231, 1149, 1032, 700 cm−1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data, see Table 1; HRESIMS m/z 445.2744 [M − H]− (calcd for C30H37O3, 445.2743). (+)-2: yellow oil; [α]25 D +21 (c 0.2, MeOH); ECD (MeOH, Δε) λmax 219 (+3.03), 236 (−2.72), 291 (+4.05) nm. (−)-2: yellow oil; [α]25 D −20 (c 0.2, MeOH); ECD (MeOH, Δε) λmax 220 (−3.32), 233 (+1.35), 291 (−5.01) nm. (±)-Alpininoid C (3): white, amorphous powder; [α]25 D ±0 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 205 (3.33), 283 (2.04) nm; IR (KBr) νmax 3423, 2926, 1735, 1672, 1648, 1550, 1519, 1455, 1380, 1073, 697 cm−1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data, see Table 2; HRESIMS m/z 515.3537 [M + H]+ (calcd for C35H47O3, 515.3520). (+)-3: white, amorphous powder; [α]25 D +26 (c 0.2, MeOH); ECD (MeOH, Δε) λmax 224 (+4.62), 296 (+2.78) nm. (−)-3: white, amorphous powder; [α]25 D −24 (c 0.2, MeOH); ECD (MeOH, Δε) λmax 229 (−3.14), 286 (−2.83) nm. (±)-Alpininoid D (4): white, amorphous powder; [α]25 D ±0 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 205 (3.42), 282 (2.25) nm; IR (KBr) νmax 3424, 2924, 1702, 1648, 1511, 1455, 1377, 1267, 1119, 1032, 697 cm−1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data, see Table 2; HRESIMS m/z 513.3381 [M − H]− (calcd for C35H45O3, 513.3369). (+)-4: white, amorphous powder; [α]25 D +19 (c 0.2, MeOH); ECD (MeOH, Δε) λmax 246 (+0.38), 296 (−4.57) nm. (−)-4: white, amorphous powder; [α]25 D −18 (c 0.2, MeOH); ECD (MeOH, Δε) λmax 237 (−2.56), 297 (+4.54) nm. (±)-Alpininoid E (5): white, amorphous powder; [α]25 D ±0 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 206 (3.39), 280 (2.33) nm; IR (KBr) νmax 3405, 2924, 1702, 1647, 1613, 1511, 1452, 1373, 1261, 1098, 1034, 697 cm−1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) data, see Table 2; HRESIMS m/z 483.3260 [M − H]− (calcd for C34H43O2, 483.3263). (+)-5: white, amorphous powder; [α]25 D +14 (c 0.2, MeOH); ECD (MeOH, Δε) λmax 244 (+5.05), 296 (−4.91) nm. (−)-5: white, amorphous powder; [α]25 D −14 (c 0.2, MeOH); ECD (MeOH, Δε) λmax 234 (−5.19), 296 (+5.16) nm. Computational Methods. Conformational analysis of the all the plausible stereoisomers of compounds 1−4 was performed by using a random search algorithm, the MMFF94s force field, and MMFF94 charge in Sybyl X 2.1 software. All the conformers obtained were screened based on the energy of optimized structures at the B3LYP/631+G(d) level in an energy window of 3 kcal/mol in the Gaussian 09 software.27 The ECD and NMR data of all the selected conformers were calculated with the TD/B3LYP/6-31+G(d) and GIAO/B3LYP/ 6-31G(d,p) methods in the gas phase, respectively. The overall simulated CD curves were weighted by Boltzmann distributions of

each conformer (with a half-bandwidth of 0.3 eV) and compared to the experimental ones in SpecDis 1.6 software.28 The overall theoretical NMR data were analyzed by using linear regression and DP4 probability.26 Since the planar structure of 5 is the 3″-demethoxy derivative of 3 and 4, the simulated NMR data and CD curves of all plausible stereoisomers of 3 and 4 were further utilized for the relative and absolute configuration determination of 5. Cell Culture. The use of animals was approved by the Ethics Committee on Animal Experiments at Jinan University, Guangzhou, People’s Republic of China. All experimental procedures were strictly performed according to the guidelines of the Care and Use of Laboratory Animals under the decree No. 2 of the National Science and Technology Commission of the People’s Republic of China. Cortical neurons were isolated from Sprague−Dawley rat embryos 18.5 days after gestation. Neurobasal (Gibco BRL, MD, USA) supplemented with 2% B27 (Gibco BRL) was used to culture primary cortical neurons. Neuronal cells were seeded on 96-well dishes (6 × 104 cells/well) for the MTT viability assay.29 Cell Viability Assay. The cell viability was determined using the colorimetric assay. The neurons (7 days in vitro) were incubated 12 h with compounds (4, 8, 16, and 32 μM) for 20 h and then treated with MPP+ (500 μM) for 20 h. As a known neurotoxic agent, in order to protect the laboratory workers, MPP+ was used in a biological safety cabinet and handled with gloves, face shield, and safety glasses, avoiding contact with skin, eyes, and clothing. Then, 30 μL of MTT solution (5 mg/mL; Sigma-Aldrich, St. Louis, MO, USA) was added to 100 μL of culture medium in each well. Supernatants were removed after 3 h, and 100 μL of DMSO was added to each well to dissolve the resultant formazan product. The absorbance of MTT was measured at 570 nm with a DTX880 multimode detector (Beckman Coulter, Brea, CA, USA). Each test was carried out in triplicate and repeated at least three times. Statistical Analysis. All data were expressed as means ± SEM (standard error of the mean) of at least three independent experiments. Statistical significance was determined by one-way analysis of variance (ANOVA).

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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.7b00803. The structures of the known compounds 6−9; UV, IR, HRESIMS, NMR spectral data, NMR and ECD computational details, chiral HPLC separation of compounds 1−5, and the UPLC-QTOF/MS analysis of petroleum ether-soluble fraction of the methanol extract of fresh rhizomes of A. off icinarum (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: +86 20 85223553. Fax: +86 20 85221559. *E-mail: [email protected]. ORCID

Wen-Cai Ye: 0000-0002-2810-1001 Author Contributions ⊥

H. Liu and Z.-L. Wu contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Nos. 81630095, 81622045, and U1401225) and the Science and Technology Planning Project 169



Article

Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, Revision A.02; Gaussian, Inc.: Wallingford, CT, 2009. (28) Bruhn, T.; Schaumlöffel, A.; Hemberger, Y.; Bringmann, G. SpecDis version 1.60; University of Wuerzburg: Germany, 2012. (29) Huang, X. J.; Tang, G. Y.; Liao, Y. M.; Zhuang, X. J.; Dong, X.; Liu, H.; Huang, X. J.; Ye, W. C.; Wang, Y.; Shi, L. Biol. Pharm. Bull. 2016, 39, 1961−1967.

of Guangdong Province (No. 2016B030301004). The work was also supported by the high-performance computing platform of Jinan University.

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REFERENCES

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