Anti-inflammatory neolignans from the roots of Magnolia officinalis

Anti-inflammatory neolignans from the roots of Magnolia officinalis

Bioorganic & Medicinal Chemistry 24 (2016) 1439–1445 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: ww...

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Bioorganic & Medicinal Chemistry 24 (2016) 1439–1445

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc

Anti-inflammatory neolignans from the roots of Magnolia officinalis Hung-Cheng Shih a,y, Ping-Chung Kuo b,y, Shwu-Jen Wu c,y, Tsong-Long Hwang d,e, Hsin-Yi Hung a, De-Yang Shen a, Po-Chuen Shieh f, Yu-Ren Liao a, E-Jian Lee g,h, Qiong Gu i, Kuo-Hsiung Lee i,j,⇑, Tian-Shung Wu a,f,⇑ a

School of Pharmacy, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan, ROC Department of Biotechnology, National Formosa University, Yunlin 632, Taiwan, ROC Department of Medical Technology, Chung Hua University of Medical Technology, Tainan 717, Taiwan, ROC d Graduate Institute of Natural Products, Chang Gung University, Taoyuan 333, Taiwan, ROC e Chinese Herbal Medicine Research Team, Healthy Aging Research Center, Chang Gung University, Taoyuan 333, Taiwan, ROC f Department of Pharmacy, Tajen University, Pintung 907, Taiwan, ROC g Department of Surgery, and Institute of Biomedical Engineering, National Cheng Kung University, Medical Center and Medical School, Tainan 701, Taiwan, ROC h Department of Anesthesiology, and Institute of Biomedical Engineering, National Cheng Kung University, Medical Center and Medical School, Tainan 701, Taiwan, ROC i Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA j Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung 401, Taiwan, ROC b c

a r t i c l e

i n f o

Article history: Received 6 November 2015 Revised 15 January 2016 Accepted 25 January 2016 Available online 27 January 2016 Keywords: Neolignans Anti-inflammatory activity Magnolia officinalis Houpulins E–M

a b s t r a c t Nine neolignan derivatives (1–9) were characterized from the roots of Magnolia officinalis, and their structures were elucidated based on spectroscopic and physicochemical analyses. Among them, houpulins E (1) and M (9) possess novel homo- and trinor-neolignan skeletons. In addition, 15 known compounds (10–24) were identified by comparison of their spectroscopic and physical data with those reported in the literature. Some of the purified constituents were examined for anti-inflammatory activity and, among the tested compounds, houpulins G (3), I (5), J (6), and 2,20 -dihydroxy-3-methoxy-5,50 -di-(2propenylbiphenyl) (19) significantly inhibited superoxide anion generation and elastase release with IC50 values ranging from 3.54 to 5.48 lM and 2.16 to 3.39 lM, respectively. Therefore, these neolignan derivatives have tremendous potential to be explored as anti-inflammatory agents. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction A growing body of evidence has suggested that several human diseases, including rheumatoid arthritis, ischemia, reperfusion injury, chronic obstructive pulmonary disease, and asthma, are linked to neutrophil overexpression.1–5 In response to diverse stimuli, activated neutrophils secrete a series of cytotoxins, such as superoxide anions and elastase.6,7 Therefore, under physiological conditions, reducing the concentrations of superoxide anions and elastase in infected tissues and organs is critical. Inhibition of neutrophil activation by natural compounds is considered to be an effective approach to treat inflammatory disorders. However, currently, there are only a few clinical drugs that directly modulate neutrophil activation. It is well known that tyrosine kinases are significant messengers in regulating neutrophil functions.8 Hence,

⇑ Corresponding authors. Tel.: +1 919 962 0066; fax: +1 919 966 3893 (K.-H.L.); tel.: +886 6 2757575x65333; fax: +886 6 2740552 (T.-S.W.). E-mail addresses: [email protected] (K.-H. Lee), [email protected] (T.-S. Wu). y These authors contributed equally to this work. http://dx.doi.org/10.1016/j.bmc.2016.01.049 0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.

the tyrosine kinase inhibitor sorafenib could be used as a positive control.9 As a part of our continuing program aimed at discovering novel anti-inflammatory drugs, Magnolia officinalis was selected as a target. M. officinalis Rehd. et Wils. (Magnoliaceae) is termed Hou-pu in Chinese and is one of the rare and endangered species listed under national protection as a Class II species. In traditional Chinese medicines, the roots, stems, and branches of M. officinalis have been used extensively for treating various diseases and symptoms, including depression,10 cough, asthma, liver disease, shoulder pain, urinary problems, and diarrhea.11 Previous chemical studies of Magnolia genus have reported various neolignans, sesquiterpenes, sesquiterpene–neolignans,12 phenylpropanoids,13 and alkaloids.14 In various studies, these constituents exhibited antimicrobial,15,16 antioxidant,17 antitumor,12 antibacterial,18 cytotoxic,19 and antiinflammatory bioactivities.18,20,21 Among the identified oligomeric neolignans, linkages through the aromatic ring (C6) include ortho, ortho (o,o)-linked dimers, (o,o)-linked trimers, dimers and trimers involving an o,O-linkage, and o,o-/o,p-linked trimers.22 Certain oligomeric neolignans have displayed significant potential as novel

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anti-inflammatory lead drugs.21,23 However, relatively few reports have appeared on the chemical composition of the roots of Magnolia species due to the rareness of the plant materials. In our previous paper, ethanol extracts of M. officinalis roots were investigated and four novel oligomeric neolignans and a possible biogenetic pathway were presented therein.23 Herein, additional studies on the chemical composition of M. officinalis and the structural determination of isolated neolignan derivatives are reported. Moreover, the purified neolignans were examined for inhibition of superoxide anion generation and elastase release, thereby evaluating their anti-inflammatory potential.

protons at d 7.44 and 7.46, and one formyl proton at d 9.93. In addition, two allyl groups at d 3.44, 6.00, and 5.14 and at d 3.38, 5.97, and 5.08 were observed. In the NOESY spectrum of 1, NOE crosspeaks corresponding to H-7/H-2 and H-6 and H-70 /H-20 and H-60 , respectively, were observed. Based on the above spectroscopic data and the biomimetic synthesis proposed in prior studies,22,24,25 compound 1 has a magnolol carbon skeleton with a C-50 formyl group, as shown in Figure 1. This conclusion was further corroborated by 2J, 3J-HMBC correlations from H-60 to C-40 and C-100 , which indicated the presence of hydroxy and formyl groups at C-40 and C50 , respectively. The HMBC correlations from H-20 to C-40 and C-3, and from H-2 to C-30 and C-4 also supported the neolignan skeleton. Compound 1 was named houpulin E according to the previous convention.23 Although the chemical synthesis of 1 was reported previously,26 this compound was characterized from a natural source for the first time. Compounds 2–8 exhibited UV and IR spectroscopic characteristics similar to those of 1, and accordingly, were assigned as neolignan derivatives. Comparison of the spectroscopic data with those of 1 suggested the presence of one magnolol (IHD = 10) and one monocyclic monoterpenoid moieties (IHD = 1) in 2. Furthermore, the spectroscopic characteristics of the monoterpenoid moiety coincided well with those of terpineol.12,13 Therefore, the structure of 2 was proposed as shown in Figure 1. This postulate was also supported by 2J, 3J-HMBC correlations from H-6 to C-300 ; from H-700 to C-600 , C-200 and C-100 ; and from both methyl groups CH3-900 and CH3-1000 to C-400 , attesting the connectivity of the terpineol fragment at C-5. Moreover, the NOESY crosspeaks corresponding

2. Results and discussion 2.1. Compound identification Dried and powdered M. officinalis roots were refluxed in ethanol, and the filtrate was concentrated to yield a crude extract, which was further partitioned and acidified to yield a non-alkaloid fraction. The resulting fraction was subjected to further purification by a combination of conventional chromatographic techniques to yield nine neolignan derivatives 1–9 (Fig. 1). Compound 1, C19H18O3 with 11 indices of hydrogen deficiency, exhibited a characteristic absorption maximum of a neolignan chromophore at 290 nm in UV spectrum.20 The 1H NMR and 13C NMR signals were summarized in Tables 1 and 2. The 1H NMR and COSY spectrum displayed one set of ABX-type aromatic resonances at d 6.99, 7.06, and 7.14, one set of meta-coupled aromatic

7' OH

3' 3

HO 7''CH3 1''

1'

3''

H O

OH

1 7

H3C 10''

CH3 9''

7' OH

7' 7'' CH3 1' 1'' OH OH 3' 3'' 3 OH H3C CH3 1 10'' 9'' 7

1 7

H3C CH3 10'' 9''

O

H3C 10''

CH3 9''

H3C 7'' 1''

3' 3

3''

1'

OH

1 7

H3C 10''

7'

1'

OH

B O H

9''

O

7''

8''

1 7

1''

C

3''

OH

7

A

3' 3

3'''

OH D 1''' 7'''

OH

1 7

7' 1'

OH O

3

O 4'

1 7

3'' CH3 9''

1'

B O H

7'' O 1''

9'' 8''

C HO

3' 3

1'

6

5 7'

3'

OH

3 7'

HO 7'' CH3 1''

4

3

OH

1 7

7'

2

HO

A

7'' CH3 1'' 3''

3' 3

1

OH

HO

1'

3'''

OH

OCH3

3

3'' OH

D 1'''

OH 1'

1 7 9

7'''

8

Figure 1. Chemical structures of compounds 1–9 characterized from the roots of M. officinalis.

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H.-C. Shih et al. / Bioorg. Med. Chem. 24 (2016) 1439–1445 Table 1 H NMR data for compounds 1–9 [dH mult. (J, Hz)]

1

Position 1a

5a

6a 6.37 s

6.88 d (2.2)

7.04 d (2.2)

7.10 s

7.01 d (2.2)

6.93 d (2.2)

7.05 s

7

3.44 d (6.5) 3.36 br

3.34 d (7.4)

3.38 d (6.9)

3.41 d (7.1)

8 9 10 20 30 50 60

6.00 m 5.14 m

5.95 m 5.10 m

5.94 m 5.10 m

5.95 m 5.10 m

6.00 m 5.08 m

7.46 s

7.09 s

7.07 d (2.3)

7.11 d (2.2)

7.21 d (2.2)

7.44 s

6.93 d (8.5) 7.08 s

7.18 d (8.3) 6.95 d (8.6) 7.00 d (8.3) 7.12 dd (2.3, 8.3) 7.09 dd (2.2, 8.6) 7.12 dd (2.2,

400 500

3.38 d (6.6) 3.34 br 5.97 m 6.00 m 5.08 m 5.06 m 9.93 1.82 d (13.4) 1.43 d 3.35 (overlap)

700

1.52 1.55 1.54 1.74 1.50 1.19

800

600

c

4a

7.06 br s 6.90 d (2.0) 6.99 d (7.9) 7.14 d (7.9) 6.97 d (2.0)

300

a

3a

2 5 6

70 80 90 100 200

b

2a

m m m d (10.4) d s

7b

7.26 7.24 7.14 (8.4, 3.32 dd (5.6, 15.9) 3.34 3.16 dd (5.6, 15.9) 5.90 m 6.03 4.99 m 5.14 7.39 7.11 d (8.4) 6.93 d (8.4) 6.93 d (8.4) 8.3) 7.11 d (8.4) 6.90

3.36 d (7.4) 5.98 m 5.06 m

3.33 d (6.9) 5.99 m 5.05 m

3.39 d (7.6) 6.01 m 5.11 m

3.35 d (6.7) 5.97 m 5.05 m

1.84 m 1.52 m 3.12 m

3.83 d (2.0)

1.64 m 1.8 m 1.2 m 1.78 m 1.76 m 1.34 s

3.17 dd (12.5, 2.0) 2.31 m 1.46 m 1.43 m 1.86 m 1.64 m 1.26 s

4.30 dd (5.6, 1.3) 1.96 1.60 3.07 dd 3.25 (11.4, 5.5) 1.09 m 1.25 1.46 m 1.53 1.43 m 1.51 1.71 m 1.78 1.67 1.63 1.46 s 1.41

8c

9a

d (2.2) d (8.4) dd 2.2) d (6.9)

6.81 6.69 6.80 (8.0, 3.02

d (2.2) d (8.0) dd 2.2) d (6.5)

m m d (2.0)

5.77 m 4.90 m 6.90 d (2.2)

7.03 d (2.3) 6.91 d (8.3) 7.06 dd (8.3,2.3) 3.35 d (6.7) 5.97 m 5.07 m 6.93 d (1.8)

d (2.2)

6.94 d (2.2)

3.34 d (6.9) 5.99 m 5.01 m

3.34 d (6.2) 5.98 m 5.09 m

7.39 d (3.1)

7.26 d (2.0)

7.20 d (8.5)

6.87 dd (8.0)

7.02 d (8.0) 6.95 dd (1.8, 8.0)

3.92 m m m m m m m m s

1.58 m

1.55 m

1.26 m

1.98 m

1.86 m

00

9

0.84 d (6.9)

0.85 d (6.7)

0.87 d (6.9)

0.96 d (6.7)

1.11 d (6.5)

1000 2000 5000 6000 7000 8000 9000

0.72 d (6.9)

0.72 d (6.7)

0.64 d (6.9)

0.87 d (7.0)

0.98 d (6.5)

7.29 dd (8.5, 3.1) 7.12 dd (8.3, 2.0) 5.86 (2.8, 2.14 1.90 3.19 2.65

dd 2.6) m m m m

6.00 m

7.40 7.25 7.17 3.40 6.07 5.06

d (3.0) 6.96 d (2.2) d (8.4) 6.86 d (8.3) dd (8.4, 3.0) 6.98 dd (8.3, 2.2) d (6.7) 3.32 d (5.7) m 5.95 m m 5.00 m

2.37 2.15 3.26 2.78

m m m m

d (ppm); 400 MHz for 1H; CDCl3; J values (Hz) in parentheses. d (ppm); 400 MHz for 1H; pyridine-d5; J values (Hz) in parentheses. d (ppm); 500 MHz for 1H; acetone-d6; J values (Hz) in parentheses.

to H-400 /H-200 , H-800 /H-200 and H-600 , and H-300 /H-200 and H-900 confirmed the relative configuration of 2. The stereochemical structure of 2 (houpulin F) was determined conclusively as displayed in Figure 1. Compound 3 was recognized as being an isomer of 2, as the same molecular formula was assigned by HRESIMS. The UV, IR, 1 H, and 13C NMR spectroscopic characteristics of 3 were similar to those of 2. However, in the NOESY spectrum, the crosspeaks between H-7 and H-2/H-6, between H-70 and H-20 /H-60 , between H-300 and H-700 /H-500 , and between H-400 and H-600 , established the stereochemistry of the terpineol fragment and the orientation of H-300 . The complete assignments of the 1H and 13C signals of 3 were made based on the 2D NMR spectra. The structure of compound 3 (houpulin G) is shown in in Figure 1. From comparing the molecular formulas of 4 and 3, one more oxygen atom was observed in compound 4. In addition, 1H, 13C, and DEPT NMR spectra of 4 were very similar to those of 3, with the only exception being one more oxygenated methine signal at d 3.83 (1H, d, J = 2.0 Hz, H-200 ) found in the 1H NMR spectrum, which indicated that one more hydroxyl group was on the terpineol fragment. The substitution pattern (OH at C-200 ) and stereochemistry of the monoterpene moiety were determined from the NOESY correlations from H-7 to H-2 and H-6, from H-70 to H-200 and H-60 , from H-600 to H-400 , from H-3000 to H-500 , and from H-300 to H-6, respectively. These 2D spectroscopic data completed the

assignment of all proton and carbon signals and established the stereochemical structure of 4 (houpulin H, Fig. 1). The HRESIMS of compound 5 exhibited a sodiated pseudomolecular ion peak at m/z 441.2404 ([M+Na]+), corresponding to a molecular formula of C28H34O3, with 12 IHD, in which one water molecule was eliminated from the molecular formula of 4. Compound 5 exhibited 1H, 13C and DEPT NMR spectroscopic characteristics very similar to those of 4 (Tables 1 and 2). The major spectroscopic differences between 5 and 4 were downfield shifts of H-200 and C-200 in 5, which suggested that the hydroxyl groups at C-4 and C-200 were combined in an ether linkage between the aromatic ring and the terpineol, as shown in Figure 1. This connectivity was further demonstrated by the 2J,3J-HMBC correlations of H-200 to C-3; and H-200 to C-100 , C-300 , C-400 , C-600 , and C-700 . In addition, the NOESY crosspeaks of H-7/H-2 and H-6, H-700 /H-200 and H-600 , H300 /H-500 , H-400 /H-600 , and H-6/H-300 established the complete proton and carbon signal assignments of the neolignan moieties, the conformation of the monoterpene, and the substitution position of the monoterpene moiety, respectively. Consequently, the chemical structure of 5 (houpulin I) was unambiguously constructed as shown in Figure 1. Compound 6, C28H34O3 with 12 IHD, showed characteristic signals for one set of A2B2-type aromatic protons, one aromatic singlet, two sets of allyl groups, and one terpineol fragment in the 1 H NMR and COSY spectra. These spectroscopic signals indicated

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Table 2 C NMR data for compounds 1–9

13

a b c

Position

1a

2a

3a

4a

5a

6b

7b

8c

9a

1 2 3 4 5 6 7 8 9 10 20 30 40 50 60 70 80 90 100 100 200 300 400 500 600 700 800 900 1000 1000 2000 3000 4000 5000 6000 7000 8000 9000

132.8 130.9 124.6 152.3 118.4 130.0 38.9 136.4 116.8 132.8 140.0 127.9 155.8 120.7 133.2 39.4 137.7 115.6 197.0

132.7 128.6 125.3 148.7 128.6 127.3 39.6 137.7 115.6 132.7 129.5 125.3 151.5 116.8 131.3 39. 4 137.6 115.8

132.5 128 123.3 149.2 132.6 128.5 39.5 137.6 115.7 133.1 131.1 123.1 151.3 116.4 130.1 39.3 137.5 115.8

132.9 131.2 127.3 149.8 128.6 132.7 39.3 137.6 115.7 132.5 131.2 127.2 152.1 118.1 129.1 39.5 137.9 115.5

133.8 129.7 120.9 152.1 134.2 124.7 39.8 137.6 115.6 132.7 130.0 124.9 152.2 118.7 129.4 39.4 137.7 115.7

127.3 112.4 144.8 138.9 140.5 121.7 35.2 137.4 115.4 133.7 129.4 116.8 156.3 116.8 129.4 39.4 137.6 115.5

132.4 132.9 127.0 155.5 117.0 129.4 40.1 139.3 115.8 130.7 131.0 127.7 148.8 123.4 129.2 40.1 138.8 115.7

131.6 132.3 126.3 153.5 116.8 129.2 39.9 139.4 115.2 133.0 130.8 127.9 148.4 123.7 129.4 40.1 139.0 115.5

132.2 130.1 127.9 150.8 115.6 128.9 39.4 137.8 115.6 128.9 111.7 147.1 145.5 115.1 121.9

70.6 38.8 34.1 46.4 20.1 47.4 31.3 27.5 21.5 15.8

71.5 40.3 50.9 46.5 22.3 48.7 25.7 27.5 21.7 15.7

71.7 80.9 48.8 36.7 19.9 33.4 27.6 27.8 21.4 14.8

69.2 90.6 42.1 44.7 17.0 34.8 28.1 26.1 21.5 15.5

75.3 30.7 30.2 44.9 20.1 34.9 29.1 26.2 21.1 21.9

151.5 122.2 128.6 151.6 118.1 118.6 97.7 27.3 21.3

147.5 119.7 145.4 129.1 117.0 125.3 98.3 27.2 21.1

131.9 132.9 128.4 154.5 117.8 129.7 40.1 139.1 116.0

132.1 128.8 131.2 153.2 116.4 131.4 40.1 139.4 115.7

d (ppm); 100 MHz for d (ppm); 100 MHz for d (ppm); 125 MHz for

56.1

13

C; CDCl3. C; pyridine-d5. 13 C; acetone-d6.

13

the presence of obovaltol10 and terpineol moieties, which accounted for 11 IHD (two benzene rings, two terminal alkenes, and one cyclic alkane ring), and implied the existence of a cyclic ether ring, as shown in Figure 1. The postulate was further corroborated by the HMBC analysis, in which 2J, 3J-correlations from H-700 to C-200 , C-600 , and C-100 ; from H-1000 to C-900 , C-800 , and C-400 ; from H900 to C-1000 , C-800 , and C-400 ; from H-7 to C-6; from H-7 to C-6; from H-300 to C-6; from H-700 to C-5; and from H-200 to C-6, were observed. These data also revealed that the monocyclic ether ring was attached to C-5 and C-6 of the neolignan skeleton. The NOESY spectrum of 6 exhibited crosspeaks of H-7/H-2, H-70 /H-20 , and H70 /H-60 , thus affording the complete proton and carbon signals assignment. In addition, based on the small coupling constant of H-300 (<3 Hz) as well as molecular model monitoring (see Supporting information), the stereochemical structure of 6 should adapt a chair/half-chair stereochemistry to achieve the most stable conformation. According to the above spectroscopic analysis, the structure of 6 (houpulin J) was elucidated as shown in Figure 1. The absolute configurations of compounds 2–6 were determined by their electronic circular dichroism (ECD) spectra using time-dependent density functional theory (TDDFT) calculations.27 The structure models of the compounds were constructed based on NOE analysis. The optimized geometries of the conformers were obtained by DFT, and the overall ECD spectra were generated according to the Boltzmann weighting of each conformer at the B3LYP/6-311G (d,p) level with the TDDFT/IEFPCM model in MeOH.28 All quantum computations were performed using

Gaussian 09 software. The calculation results (see Supporting information) confirmed the absolute configurations of 3 and 5 to be the same as indicated in Figure 1. However, the experimental ECD spectra of compounds 2, 4, and 6 showed very minor Cotton effect, so the calculated ECD spectra were not applied. The spectroscopic data of compound 7 were very close to those of the previously reported houpulin D (13).23 This assumption was further proven by the NOESY and COSY spectroscopic analyses, in which each allyl group was correlated with the corresponding proton. In the HMBC spectrum of 7, 2J, 3J-correlations from H-700 (d 5.86) to C-40 (d 148.8), C-900 (21.3), and C-100 (151.5) confirmed that the hemiacetal was located at C-700 . In addition, HMBC correlations from H-20 to C-3, C-60 , and C-40 ; from H-2 to C-30 ; from H-200 to C3000 ; and from H-2000 to C-300 , established the structure of 7 (houpulin K) as shown in Figure 1. Furthermore, the stereochemical configuration at C-700 of 7 was assigned as R based on the observation of a positive Cotton effect at 280 nm in the CD spectrum.23 The spectroscopic characteristics suggested that the structure of 8 was very similar to that of 7. This postulate was further corroborated by NOESY and COSY analyses, which provided the assignment for each subunit. In the HMBC spectrum of 8, 2J, 3J-correlations from H-700 to C-40 and C-100 ; and from H-900 to C-700 , confirmed that C-700 was a hemiacetal functionality. Moreover, HMBC correlations from H-20 to C-3, C-60 , and C-40 ; from H-2 to C-30 ; from H-200 to C-600 , C-400 , and C-300 ; and from H-2000 to C-400 , furnished the full assignments of subunits A-D. The absolute configuration at C-700 of 8 was determined as S based on the observation of a negative Cotton effect at

H.-C. Shih et al. / Bioorg. Med. Chem. 24 (2016) 1439–1445

280 nm in the CD spectrum.23 Consequently, the structure of 8 (houpulin L) was established as shown in Figure 1. Comparison of the spectroscopic data of 9 with those of 1 suggested elimination of one allyl substituent from the neolignan skeleton. The NOESY crosspeaks of H-7/H-2 and H-6, and the HMBC correlations from H-60 to C-3 and C-40 ; from OCH3-30 to C30 ; and from H-20 to C-30 and C-40 , supported the presences of a C-3/C-10 linkage between ring-A and -B and a methoxy group at C-30 . Finally, the structure of 9 (houpulin M) was confirmed conclusively as shown in Figure 1. In addition to houpulins E–M (1–9), 15 compounds were identified from the ethanol extract of M. officinalis roots, including houpulins A–D (10–13),23 magnolol (14),29 honokiol (15),13 monoterpenylmagnolol (16),12 4-methoxyhonokiol (17),30 obovatol (18),13 2,20 -dihydroxy-3-methoxy-5,50 -di-(2-propenylbiphenyl) (19),31 clovanmagnolol (20),32 4-hydroxy-3-methoxy-benzaldehyde (21),33 chavicol (22),34 and a mixture of b-sitosterol (23) and stigmasterol (24).35 The chemical structures of these known compounds were confirmed by comparison of their physical and spectroscopic data with those previously reported. 2.2. Antiinflammatory activities The purified compounds 1–9 and 16–20 were examined for inhibition of superoxide anion generation and elastase release by normal human neutrophils in response to N-formyl-L-methionylphenylalanine/cytochalasin B (FMLP/CB) (Table 3). Most of them exhibited inhibition percentages greater than 50% at the tested concentration of 30 lM and, in the examined concentration ranges, all of the compounds displayed inhibitory effects in a dose-dependent manner. Houpulins G (3), I (5), J (6) and 2,20 -dihydroxy-3methoxy-5,50 -di-(2-propenylbiphenyl) (19) displayed comparable inhibition of superoxide anion generation with IC50 values ranging from 3.54 to 4.21 lM with the reference compound sorafenib9 with IC50 values of 3.20 lM (Table 3). In addition, compounds 1, 2, 5, 6, 7, 8, 17, 18 and 19 exhibited better or similar inhibitory effects on elastase release with IC50 values ranging from 0.63 to 3.81 lM to the positive control, sorafenib, with IC50 values of 2.00 lM. The inhibitory mechanism of superoxide anion generation and elastase release in neutrophils can be controlled by modulating cellular signaling pathways and also by direct radical Table 3 Inhibitory effects of isolated compounds from M. officinalis on superoxide anion generation and elastase release by human neutrophils in response to FMLP/CB Compound

Superoxide anion IC50a (lM)

Elastase release IC50a (lM)

1 2 3 4 5 6 7 8 9 16 17 18 19 20 Sorafenibe

NTb 8.16 ± 3.45*** 4.21 ± 1.37*** 15.11 ± 2.07*** 4.17 ± 0.33*** 3.54 ± 1.26*** NTc 19.27 ± 2.76*** 7.89 ± 0.65*** 10.84 ± 3.61*** 14.32 ± 3.78*** NTd 5.48 ± 1.61*** >30 3.20 ± 0.42

0.63 ± 0.19*** 3.56 ± 1.06*** 2.16 ± 0.91*** 10.69 ± 1.93*** 2.59 ± 0.43*** 3.67 ± 0.15*** 2.25 ± 0.06*** 3.81 ± 1.20*** 18.25 ± 2.50*** 14.85 ± 0.38*** 8.18 ± 1.33*** 3.30 ± 0.97*** 3.39 ± 0.83*** >30*** 2.00 ± 0.13

a Concentration necessary for 50% inhibition (IC50). Results are presented as mean ± SEM (n = 3). b Alone induced superoxide generation by human neutrophils. c Induced superoxide generation in CB-priming human neutrophils. d Alone caused femicytochrome c reduction. e Sorafenib, a tyrosine kinase inhibitor, was used as a positive control. *** P <0.001 compared with the control value.

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scavenging. In our previous study,23 the oligomeric neolignan houpulin B (11) did not alter the activation of ERK, p38 MAPK, JNK, and Akt, but rather may influence calcium clearance mechanisms. Therefore, the present observations suggest that the extracts and purified compounds of the roots of M. officinalis have the potential to be developed as novel anti-inflammatory lead drugs or health foods, and further investigation of the anti-inflammatory mechanism is warranted. 3. Conclusions In this study, nine neolignan derivatives (1–9) along with 15 known compounds (10–24) were characterized from the roots of Magnolia officinalis. Among these compounds, houpulins E (1) and M (9) possess novel homoneolignan and trinorneolignan skeletons, respectively. Moreover, anti-inflammatory activities were examined and houpulins G (3), I (5), J (6), and 2,20 -dihydroxy-3methoxy-5,50 -di-(2-propenylbiphenyl) (19) displayed significant inhibition of superoxide anion generation and elastase release with IC50 values ranging from 3.54 to 5.48 lM and 2.16 to 3.39 lM, respectively, suggesting that these neolignan derivatives are highly promising for development as anti-inflammatory agents. 4. Materials and methods 4.1. General experimental procedures Optical rotations were measured using a Jasco DIP-370 digital polarimeter. UV spectra were obtained on a Hitachi UV3210 spectrophotometer. IR spectra were recorded on a Shimadzu FT-IR Prestige-21 spectrophotometer. 1H and 13C NMR, COSY, HMQC, HMBC, and NOESY spectra were recorded on Bruker Avance 300, Avance III-400, and Avance 500 NMR spectrometers using tetramethylsilane (TMS) as an internal standard. Standard pulse sequences and parameters were used for the NMR experiments, and all chemical shifts were reported in parts per million (ppm, d). Mass spectra were measured on a Bruker APEX II spectrometer with ESI ionization (positive-ion mode). All chemicals were purchased from Merck KGaA (Darmstadt, Germany), unless specifically indicated. Column chromatography was performed on silica gel (SiO2, Kieselgel 60, 70–230 mesh, Merck KGaA). Thin layer chromatography (TLC) was conducted using precoated Kieselgel 60 F 254 plates (Merck), and the compounds were visualized using UV light or by spraying with 10% (v/v) H2SO4 followed by heating at 110 °C for 10 min. 4.2. Plant material The roots of M. officinalis were provided by the Chuang SongZong Pharmaceutical Factory and were authenticated by Prof. C. S. Kuo, Department of Life Science, National Cheng Kung University. A voucher specimen (2010000013) was deposited in the Herbarium of National Cheng Kung University, Tainan, Taiwan, ROC. 4.3. Extraction and isolation The dried and powdered roots of M. officinalis (5.0 kg) were refluxed with ethanol (6  20 L) and filtered. The filtrate was concentrated to afford a crude extract (2.0 kg), which was suspended in water and partitioned with dichloromethane. The organic layers were combined and concentrated to yield a dichloromethane extract (550.0 g), which was further partitioned with 5% HCl aqueous solution to afford a dichloromethane soluble fraction (444.0 g) and a 5% HCl aqueous extract (90.0 g) The dichloromethane soluble fraction was subjected to open

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column chromatography over silica gel by eluting with a mixture of n-hexane and ethyl acetate (19:1) and a stepwise gradient of ethyl acetate to obtain 10 fractions (F1–F10). Fraction 4 (F4) was further purified using silica gel column chromatography (SiO2 CC) eluting with a mixture of n-hexane and diisopropyl ether (3:1) and afforded six subfractions (F4-1–F4-4). Subfraction F41 displayed significant spots and was subjected to SiO2 CC eluting with n-hexane and ethyl acetate (9:1), to afford five minor fractions (F4-1-1–F4-1-5). F4-1-3 was further purified by SiO2 CC with n-hexane: acetone (15:1) solvent system to afford houpulin J (6) (60.6 mg). Fraction 5 (F5) was subjected to SiO2 CC eluting with a mixture of n-hexane and diisopropyl ether (1:1) to afford four subfractions (F5-1–F5-4). Subfraction F5-4 was a major fraction and displayed significant spots on thin-layer chromatography (TLC). F5-4 was subjected to SiO2 CC eluting with a mixture of n-hexane and ethyl acetate (19:1) to afford ten minor fractions (F5-4-1–F5-4-10). F5-4-3 was repeatedly purified by SiO2 CC and preparative TLC (pTLC) (eluting with n-hexane–acetone, 4:1) to afford houpulin E (1) (4.3 mg). F5-4-5 was further isolated by SiO2 CC to afford houpulin I (5) (31.4 mg). Fraction 7 (F7) was further recrystallized with chloroform–methanol to yield honokiol (15) (19.5 g), and the residual mother liquid was further purified via SiO2 CC with a mixed eluent of n-hexane and acetone (4:1) to yield 14 subfractions (F7-1–F7-14). Subfractions F7-4 and F7-5 were major fractions and displayed significant spots during TLC monitoring. F7-4 was further isolated using SiO2 CC eluting with a mixture of chloroform and diisopropyl ether (30:1) to afford eight minor fractions (F7-4-1–F74-8). F7-4-8 was repeatedly purified using SiO2 CC and pTLC (eluting with n-hexane–chloroform, 1:2) to afford houpulins F (2) (7.4 mg) and H (4) (3.8 mg). Subfraction F7-5 was subjected to SiO2 CC eluting with a step gradient of chloroform and diisopropyl ether (20:1) to afford nine subfractions (F7-5-1–F7-5-9) based on the TLC profiles. F7-5-3 was chromatographed over silica gel using a solvent system of chloroform and diisopropyl ether (40:1) and further purified by pTLC to afford houpulins K (7) (27.1 mg) and L (8) (1.6 mg). F7-5-4 was repeatedly purified by SiO2 CC and pTLC to afford houpulin M (9) (3.2 mg). F7-5-8 was repeatedly purified by SiO2 CC and pTLC to afford houpulin G (3) (2.6 mg).

see Table 2; ESIMS m/z 459 [M+Na]+; HRESIMS m/z 459.2509 [M+Na]+ (calcd for C28H36O4Na, 459.2511). 4.3.5. Houpulin I (5) Colorless syrup; [a]25 D +15 (c 1.3, MeOH); UV (MeOH) kmax (log e) 297 (3.91); IR (neat) mmax 3383, 2958, 2931, 1639, 1496, 1465, 1215, 1126 cm1; 1H NMR data, see Table 1; 13C NMR data, see Table 2; ESIMS m/z 441 [M+Na]+; HRESIMS m/z 441.2404 [M+Na]+ (calcd for C28H34O3Na, 441.2406). 4.3.6. Houpulin J (6) Colorless syrup; [a]25 D 2 (c 2.7, MeOH); UV (MeOH) kmax (log e) 291 (4.35); IR (neat) mmax 3522, 3421, 2958, 1674, 1597, 1500, 1442, 1226, 1165 cm1; 1H NMR data, see Table 1; 13C NMR data, see Table 2; ESIMS m/z 441 [M+Na]+; HRESIMS m/z 441.2404 [M +Na]+ (calcd for C28H34O3Na, 441.2406). 4.3.7. Houpulin K (7) Colorless syrup; [a]25 D 8 (c 1.3, MeOH); UV (MeOH) kmax (log e) 290 (4.04); IR (neat) mmax 3406, 2974, 1697, 1496, 1419, 1215, 1053 cm1; 1H NMR data, see Table 1; 13C NMR data, see Table 2; ESIMS m/z 569 [M+Na]+; HRESIMS m/z 569.2301 [M+Na]+ (calcd for C36H34O5Na, 569.2304). 4.3.8. Houpulin L (8) Colorless syrup; [a]25 D 72 (c 0.1, MeOH); UV (MeOH) kmax (log e) 290 (3.80); IR (neat) mmax 3414, 2924, 1642, 1600, 1496, 1219, 1126 cm1; 1H NMR data, see Table 1; 13C NMR data, see Table 2; ESIMS m/z 569 [M+Na]+; HRESIMS m/z 569.2301 [M +Na]+ (calcd for C36H34O5Na, 569.2304). 4.3.9. Houpulin M (9) Colorless syrup; UV (MeOH) kmax (log e) 295 (3.79), 256 (3.85) nm; IR (neat) mmax 3522, 2927, 1604, 1504, 1261, 1219, 1122, 1029 cm1; 1H NMR: see Table 1; 13C NMR: see Table 2; ESIMS m/z 279 [M+Na]+; HRESIMS m/z 279.0995 [M+Na]+ (calcd for C16 H16O3Na, 279.0997). 4.4. Biological assay methods

4.3.3. Houpulin G (3) Colorless syrup; [a]25 D 66 (c 0.1, MeOH); UV (MeOH) kmax (log e) 291 (3.77); IR (neat) mmax 3325, 2954, 2931, 1600, 1492, 1465, 1226, 1118 cm1; 1H NMR data, see Table 1; 13C NMR data, see Table 2; ESIMS m/z 443 [M+Na]+; HRESIMS m/z 443.2565 [M+Na]+ (calcd for C28H36O3Na, 443.2562).

4.4.1. Preparation of human neutrophils The study of human neutrophils was approved by the Institutional Review Board at Chang Gung Memorial Hospital, Taoyuan, Taiwan. The written informed consent was obtained from every volunteer. Neutrophils were isolated using a standard dextran sedimentation method prior to centrifugation on a Ficoll Hypaque gradient and hypotonic lysis of erythrocytes. Blood was drawn from healthy human donors (20–30 years old) by venipuncture into heparin-coated vacutainer tubes using a protocol approved by the institutional review board at Chang Gung Memorial Hospital. Blood samples were mixed gently with an equal volume of a 3% dextran solution. Leukocyte-rich plasma was collected after sedimentation of the red cells for 30 min at room temperature. The leukocyte-rich plasma was then transferred onto 20 mL of Ficoll solution (1.077 g/mL) and centrifuged at 400g for 40 min at 20 °C. The granulocyte/erythrocyte pellets were resuspended in ice-cold 0.2% NaCl and lysed. After 30 s, the same 1.6% NaCl solution volume was added to reconstitute the isotonic condition. Purified neutrophils were pelleted and then resuspended in calcium (Ca2+)-free Hank’s balanced salt solution (HBSS) buffer at pH 7.4 and were maintained at 4 °C before use.

4.3.4. Houpulin H (4) Colorless syrup; [a]25 D 47 (c 0.2, MeOH); UV (MeOH) kmax (log e) 290 (3.82); IR (neat) mmax 3367, 2954, 2927, 1712, 1639, 1496, 1465, 1226 cm1; 1H NMR data, see Table 1; 13C NMR data,

4.4.2. Measurements of superoxide anion generation The assay for measuring superoxide anion generation was based on the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c.6 Briefly, after supplementation with 0.5 mg/

4.3.1. Houpulin E (1) Colorless syrup; UV (MeOH) kmax (log e) 345 (3.72), 283 (3.88) nm; IR (neat) mmax 3401, 2924, 2854, 2673, 1651, 1496, 1427, 1292, 1230, 1091 cm1; 1H NMR data, see Table 1; 13C NMR data, see Table 2; ESIMS m/z 317 [M+Na]+; HRESIMS m/z 317.1153 [M +Na]+ (calcd for C19H18O3Na, 317.1154). 4.3.2. Houpulin F (2) Colorless syrup; [a]25 D 3 (c 4.8, MeOH); UV (MeOH) kmax (log e) 290 (2.81); IR (neat) mmax 3325, 2958, 2927, 1639, 1500, 1415, 1230, 1072 cm1; 1H NMR data, see Table 1; 13C NMR data, see Table 2; ESIMS m/z 443 [M+Na]+; HRESIMS m/z 443.2565 [M +Na]+ (calcd for C28H36O3Na, 443.2562).

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mL ferricytochrome c and 1 mM Ca2+, neutrophils (6  105 cells/ mL) were equilibrated at 37 °C for 2 min and incubated with 100 nM FMLP during preincubation with 1 lg/mL cytochalasin B (FMLP/CB) for 3 min. Changes in the 550 nm absorbance reflecting a reduction in ferricytochrome c were continuously monitored using a double-beam, six-cell position spectrophotometer with constant stirring (Hitachi U-3010, Tokyo, Japan). Calculations were based on differences in the reactions with and without SOD (100 U/ mL) divided by the extinction coefficient for the reduction of ferricytochrome c (e = 21.1/mM/10 mm). 4.4.3. Measurements of elastase release Degranulation of azurophilic granules was determined by measuring the release of elastase as previously described.6 Experiments were performed using MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide as the elastase substrate. Briefly, after supplementation with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide (100 lM), neutrophils (6  105/mL) were equilibrated at 37 °C for 2 min and incubated with drugs or an equal volume of vehicle (0.1% DMSO, negative control) for 5 min. Cells were activated using 100 nM FMLP and 0.5 lg/mL CB, and changes in the 405 nm absorbance were continuously monitored to assay elastase release. The results are expressed as the percent of elastase release in the FMLP/ CB-activated, drug-free control system. 4.5. Statistical analysis The results are expressed as mean ± SEM. Computation of 50% inhibitory concentrations (IC50) were performed using PHARM/ PCS v.4.2 software. Statistical comparisons were made between groups using Student’s t-test. Values of P <0.05 were considered to be statistically significant. 4.6. Quantum chemical calculations The structure models of the compounds were constructed based on NOE analysis. The conformations were optimized using DFT at B3LYP-SCRF/6-31G(d) level using the integral equation formalism variant of the polarizable continuum model (IEF-PCM). The free energies and vibrational frequencies were calculated at the same level to confirm their stability and no imaginary frequencies were found. The optimized low energy conformers with energy <2 kcal mol1 were considered for ECD calculation. The TD-DFT/ B3LYP SCRF/6-311G(d,p) method was applied to calculate the excited energies, oscillator strength and rotational strength, with 60 states. All the calculations were run with Gaussian 09. The excited energies and rotational strength were used to simulate ECD spectrum of conformer by introducing the Gaussian function. The calculated ECD curve was generated using GaussSum 3.0 with r = 0.3 eV. Acknowledgements We are grateful to the National Center for High-performance Computing for computer time and facilities. This work was sup-

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