Anticomplement Monoterpenoid Glucosides from the Root Bark of

Dec 30, 2013 - Ming-Hui Shao , Wei Dai , Si-Wen Yuan , Yan Lu , Dao-Feng Chen , Qi ... Xu Xu , Bei Wu , Yanzhi Zhan , Wenping Huang , Shilin Yang , Qu...
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Anticomplement Monoterpenoid Glucosides from the Root Bark of Paeonia suff ruticosa Wei-Hua Song, Zhi-Hong Cheng, and Dao-Feng Chen* Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai 201203, People’s Republic of China S Supporting Information *

ABSTRACT: Six new (1−6) and 19 known monoterpenoid glucosides were isolated from the root bark of Paeonia suf f ruticosa. The monoterpenoid glucosides 1, 2, 7, 10−19, and 22 exhibited anticomplement effects with CH50 and AP50 values ranging from 0.14 to 2.67 mM and 0.25 to 3.67 mM, respectively. In a mechanistic study, suffrupaeoniflorin A (1) interacted with C1q, C3, C5, and C9, while galloylpaeoniflorin (12) and galloyloxypaeoniflorin (19) acted on C1q, C3, and C5 components in the complement activation cascade.

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RESULTS AND DISCUSSION Bioassay-guided fractionation of an ethanolic extract of the root bark of P. suf f ruticosa showed that both the EtOAc-soluble and n-BuOH-soluble fractions possessed anticomplement activity with CH50 values of 0.15 ± 0.05 mg/mL and 0.08 ± 0.03 mg/ mL, respectively. The two active fractions were separated by repeated column chromatography and preparative HPLC to afford 25 monoterpenoid glucosides. Compound 1 was obtained as a white, amorphous solid. Its HRESIMS showed a quasi-molecular ion peak at m/z 785.2248 [M + Na]+, indicating a molecular formula of C36H42O18. Absorptions for hydroxy (3422 cm−1) and ester carbonyl (1707 cm−1) groups were present in the IR spectrum. The 1H NMR spectrum of 1 (Table 1) showed characteristic signals for a pinane-type monoterpenoid, e.g., paeoniflorin,6 at δH 1.66 (1H, overlap, Ha-3), 1.83 (1H, d, J = 12.5 Hz, Hb-3), 2.49 (1H, overlap, H-5), 1.69 (1H, overlap, Ha-6), 2.45 (1H, overlap, Hb6), 4.64 (2H, br s, H-8), 5.36 (1H, s, H-9), and 1.20 (3H, s, CH3-10). In addition, two anomeric protons resonated at δH 4.59 (1H, d, J = 7.8 Hz) and 4.40 (1H, d, J = 7.8 Hz). The 13C NMR spectrum (Table 2) of 1 was similar to that of paeoniflorin B (7),8 isolated from the same plant, with resonances for a pinane-type skeleton, a benzoyl group, and two glucose moieties. HMBC correlations from H-1‴ (δH 4.59) to C-1 (δC 89.3), H-1⁗ (δH 4.40) to C-4‴ (δC 81.3), and H-6‴ (δH 4.83) to C-7″ (δC 167.5) indicated that the glucosyl moieties were connected to C-1 and C-4‴ and the benzoyl

he complement system provides an important means to protect a host from foreign invasive organisms, such as bacteria, fungi, and viruses. It comprises more than 30 plasma and membrane-bound proteins and can be activated through the classical pathway (CP), the alternative pathway (AP), and the lectin pathway. 1 However, unwanted complement activation results in various inflammatory diseases, including ischemia-reperfusion injury, glomerulonephritis, systemic lupus erythematosus,2 and rheumatoid arthritis.3 Thus, complement inhibitors are in high demand as attractive therapeutic agents for these diseases. The root bark of Paeonia suf f ruticosa Andr. (Moutan Cortex) is a well-known traditional Chinese medicine with antiinflammatory, analgesic, and sedative activities. It is commonly used to treat atherosclerosis, inflammation, cardiovascular disease, and menstrual problems.4,5 Previous studies showed that this Chinese herb is rich in monoterpenoid glucosides with a “cage-like” pinane skeleton. More than 40 monoterpenoid glucosides have been isolated from this plant,6−12 some of which have shown antidiabetic,13 anti-inflammatory,7,8 and antibacterial9 activities. In our search for anticomplement agents from traditional Chinese medicines,14−16 an ethanolic extract of the root bark of P. suf f ruticosa exhibited anticomplement activity. Bioactivity-guided fractionation of this extract led to the isolation of six new (1−6) and 19 known (7−25) monoterpenoid glucosides. Herein, we report the isolation and structure elucidation of the new compounds and the anticomplement activities of the 25 compounds. © 2013 American Chemical Society and American Society of Pharmacognosy

Received: July 14, 2013 Published: December 30, 2013 42

dx.doi.org/10.1021/np400571x | J. Nat. Prod. 2014, 77, 42−48

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Chart 1

group was connected to C-6‴ (Figure 1). Comparison of the ESIMS and NMR data of 1 and 7 suggested the presence of a phydroxybenzoyl unit in 1, confirmed by 1H NMR resonances (Table 1) at δH 7.89 (H-2′, 6′) and 6.82 (H-3′, 5′) as well as corresponding 13C NMR resonances at δC 122.0 (C-1′), 133.0 (C-2′, 6′), 116.2 (C-3′, 5′), and 163.7 (C-4′). HMBC correlations of H-8 (δH 4.64) to C-7′ (δC 167.9), H-2′ (δH 7.89) to C-7′, and H-2′ to C-4′ (δC 163.7) revealed that the phydroxybenzoyl unit was attached to C-8 of the aglycone core (Figure 1). The anomeric protons showed large coupling constants, consistent with β-glucosidic linkages. Both sugars were assigned D-configuration on the basis of GC-MS analysis of the per (trimethyl) silylated sugars in the hydrolysate of 1. The specific rotation of 1 {[α]20D −22 (c 0.1, MeOH)} was similar to that of 7 {[α]20D −21 (c 0.1, MeOH)}, suggesting that 1 and 7 had the same stereochemistry. ROESY correlations between CH3-10 and H-3 and between H-8 and H-9 confirmed that the monoterpenoid aglycones of 1 (Figure 1) and 7 had the same relative configuration. Consequently, the structure of 1, named suffrupaeoniflorin A, was established unambiguously as shown.

Compound 2, a white, amorphous solid, was assigned a molecular formula of C31H34O13 from HRESIMS data ([M + Na]+, m/z 637.1888). The 1H and 13C NMR spectra of 2 were strikingly similar to those of mudanpioside A (13),6 isolated from the same plant. Compounds 2 and 13 differed only in reversed positions of their p-methoxybenzoyl and benzoyl groups. In 2, these two groups are connected to C-8 and C-6‴, respectively, as indicated by HMBC correlations from H-8 (δH 4.67) to C-7′ (δC 167.8), H-2′ (δH 7.98) to C-4′ (δC 165.3) and C-7′, and H-6‴ (δH 4.49) to C-7″ (δC 167.6). The structure of 2, named suffrupaeoniflorin B, was thus fully established. Compound 3 was obtained as a white, amorphous solid. A quasi-molecular ion peak at m/z 651.2065 ([M + Na]+) in the HRESIMS indicated a molecular formula of C32H36O13, one CH2 larger than 2. Compounds 2 and 3 had comparable 1H and 13C NMR spectra (Tables 1 and 2), except for signals of an additional O-methyl group (δH 3.27, s, 3H) in 3. On the basis of an HMBC correlation between methoxy protons (δH 3.27) and C-4 (δC 109.3), the additional O-methyl group in 3 was 43

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Table 1. 1H NMR Data for 1−6 (400 MHz) in Methanol-d4a 1b 3 5 6 8 9 10 2′ 3′ 4′ 5′ 6′ 2″ 3″ 4″ 5″ 6″ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ CH3O-4 CH3O-9 CH3O-4′ CH3O-4″

c

2

3

4

1.66 1.83 d (12.5) 2.49c 1.69c 2.45c 4.64 s

1.68c 1.83 d (12.5) 2.50c 1.69c 2.47c 4.67 s

1.67 1.78 2.63 1.61 2.43 4.66

d (12.5) d (12.5) d (7.0) d (11.0) dd (11.0, 7.0) s

5.36 1.20 7.89 6.82

s s d (8.6) d (8.6)

5.37 1.24 7.98 6.97

s s d (8.6) d (8.6)

5.38 1.24 7.96 6.98

s s d (9.0) d (9.0)

6.82 7.89 8.04 7.48 7.61 7.48 8.04 4.59 3.29 3.53 3.57 3.75 4.60 4.83

d (8.6) d (8.6) d (7.4) d (7.4) d (7.4) d (7.4) d (7.4) d (7.8) m m m m d (11.3) d (11.3)

6.97 7.98 8.05 7.49 7.61 7.49 8.05 4.57 3.25 3.38 3.35 3.60 4.49 4.70

d (8.6) d (8.6) d (7.8) t (7.8) t (7.8) t (7.8) d (7.8) d (7.4) t (7.8) m m t (7.4) dd (11.7, 7.4) m

6.98 7.96 8.04 7.48 7.61 7.48 8.04 4.55 3.23 3.35 3.31 3.59 4.51 4.61 3.27

d (9.0) d (9.0) d (7.4) t (7.4) t (7.4) t (7.4) d (7.4) d (7.4) m m m m d (11.7) d (11.7) s

2.29 2.67 2.94 2.14 3.02 4.59 4.88 4.77 1.35 7.94 7.47 7.59 7.47 7.94 8.02 7.43 7.55 7.43 8.02 4.62 3.30 3.43 3.40 3.64 4.54 4.66

d (16.8) d (16.8) d (7.0) d (10.9) dd (10.9, 7.0) d (11.7) d (11.7) s s d (7.8) d (7.8) d (7.8) d (7.8) d (7.8) d (7.4) d (7.4) d (7.4) d (7.4) d (7.4) d (7.8) m m m m dd (12.1, 6.6) dd (12.1, 1.5)

2.37 d 2.49 d 2.85c 1.85 d 2.89c 4.65 d 4.74 d 5.03 s 1.30 s 7.95 d 6.98 d 6.98 7.95 8.02 7.46 7.60 7.46 8.02 4.60 3.28 3.39 3.36 3.58 4.55 4.63

3.34 s 3.86 s

6 2.36 d (18.0) 2.45 d (18.0) 2.86c 1.83 d (10.1) 2.90c 4.66 d (12.1) 4.78 d (12.1) 5.04 s 1.29 s 7.98 d (7.8) 7.46 t (7.8) 7.60 t (7.8) 7.46 t (7.8) 7.98 d (7.8) 7.95 d (8.6) 6.93 d (8.6)

(10.5) (12.1) (12.1)

(8.6) (8.6)

d (8.6) d (8.6) d (7.4) d (7.4) t (7.4) d (7.4) d (7.4) d (7.8) m m m m dd (12.0, 7.0) m

3.25 s 3.86 s

3.85 s

5 (18.0) (18.0)

6.93 7.95 4.59 3.30 3.41 3.36 3.61 4.51 4.55

d (8.6) d (8.6) d (7.4) m m m t (7.4) d (11.7) d (11.7)

3.23 s 3.83 s

The coupling constants (J) are in parentheses and reported in Hz; chemical shifts are given in ppm. Data for Glu-4‴: δH 4.40 d (7.8 Hz, H-1⁗), 3.25 (m, H-2⁗), 3.33 (m, H-3⁗), 3.28 (m, H-4⁗), 3.34 (m, H-5⁗), 3.64 dd (11.7 and 5.8 Hz, H-6⁗), 3.87 (m, H-6⁗). cIndicates overlapping signals. a

b

Suffrupaeonidanin E (5) was assigned the molecular formula C32H36O13 on the basis of the quasi-molecular ion at m/z 651.2039 [M + Na]+. The 1H and 13C NMR spectra of 5 were similar to those of 25,18 except that the C-8 benzoyl moiety in 25 was replaced with a p-methoxybenzoyl moiety in 5. Furthermore, 1H NMR (Table 1) resonances for a pmethoxybenzoyl group were present at δH 7.95 (H-2′, 6′), 6.98 (H-3′, 5′), and 3.86 (4′-OMe). The HMBC correlation from H-8 (δH 4.74) to C-7′ (δC 167.7) confirmed the location of the p-methoxybenzoyl moiety. The 9S configuration of 5 was determined by comparison of its 13C NMR data (C-9 of 5 at δC 107.6 compared with 107.6 in paeonidanin),17 together with the ROESY correlations between CH3-10 and H-3 and between H-3 and the C-9 methoxy protons. On the basis of 1H, 13C, and 2D NMR (COSY, HSQC, HMBC, and ROESY) data, the structure of compound 5 was defined as shown. The same molecular formula C32H36O13 as that of 5 was assigned to 6 by HRESIMS (m/z 651.2069 [M + Na]+). The 1 H and 13C NMR and DEPT spectra of 6 closely resembled those of 5. However, unlike 5, 6 has a p-methoxybenzoyl moiety at C-6‴ rather than at C-8. The HMBC correlations from H-6‴ (δH 4.55) to C-7″ (δC 167.4), H-2″ (δH 7.95) to C7″ and C-4″ (δC 165.3), and methoxy protons (δH 3.83) to C4″ confirmed the assigned position. The assignments of all proton and carbon signals were determined by extensive

located at C-4. Compound 3 was named 4-O-methylsuffrupaeoniflorin B. Compound 4 was obtained as a white, amorphous solid and was assigned a molecular formula of C31H34O12 based on the HRESIMS [M + Na]+ ion at m/z 621.1947. The 1H NMR spectrum of 4 indicated the presence of a methyl group at δH 1.35 (3H, s), a methoxy group at δH 3.34 (3H, s), an acetal proton at δH 4.77 (1H, s), an anomeric resonance at δH 4.62 (1H, d, J = 7.8 Hz), and two benzoyl moieties (Table 1). The 13 C NMR spectrum displayed 31 carbons, including an acetal carbon at δC 105.8 and a carbonyl carbon at δC 210.9. Its spectroscopic data suggested that 4 is a paeonidanin-type glucoside with an additional benzoyl moiety,17 more specifically, the C-9 epimer of paeonidanin A (25).18 An HMBC experiment established the connectivity from H-1‴ (δH 4.62) to C-1 (δC 88.1), H-8 (δH 4.88) to C-7′ (δC 167.7), and H-6‴ (δH 4.66) to C-7″ (δC 167.6) (Figure 1). The 9R configuration of 4 was defined by its 13C NMR data (C-9 at δC 105.8 in 4 and 106.0 in 9-epi-paeonidanin) as well as the ROESY correlations between H-9 and H-3, H-9 and H-5, and H-3 and CH3-10 (Figure 1).10,17 Moreover, the specific rotation of 4 {[α]20D −84 (c 0.8, MeOH)} was close to that of 9-epi-paeonidanin {[α]20D −98 (c 0.8, MeOH)},17 suggesting these two compounds have the same configuration at C-9. Thus, the structure of 4, named suffrupaeonidanin D, was fully established as shown. 44

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Table 2. 13C NMR Data for 1−6 (100 MHz) in Methanol-d4a 1 2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 7′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ CH3O-4 CH3O-9 CH3O-4′ CH3O-4″

1b

2

3

4

5

6

89.3 87.0 44.4 106.2 43.8 23.0 72.0 61.1 102.2 19.5 122.0 133.0 116.2 163.7 116.2 133.0 167.9 131.3 130.6 129.7 134.5 129.7 130.6 167.5 99.8 74.6 76.3 81.3 73.9 64.6

89.2 87.0 44.4 106.2 43.8 23.0 72.0 61.2 102.2 19.5 123.3 132.8 114.8 165.3 114.8 132.8 167.8 131.3 130.6 129.7 134.5 129.7 130.6 167.6 100.0 74.9 77.8 71.9 75.2 65.1

89.1 87.0 41.9 109.3 41.1 22.9 71.6 61.2 102.4 19.6 123.2 132.7 114.9 165.3 114.9 132.7 167.7 131.3 130.6 129.7 134.5 129.7 130.6 167.5 100.0 74.9 77.8 72.1 75.1 65.1 51.5

88.1 85.3 51.1 210.9 49.1 30.0 65.0 62.4 105.8 21.0 131.3 130.6 129.7 134.5 129.7 130.6 167.7 131.1 130.5 129.7 134.4 129.7 130.5 167.6 100.1 74.9 77.7 71.9 75.3 64.7

88.5 87.3 49.5 209.1 48.3 27.3 64.9 63.5 107.6 20.7 123.2 132.8 114.9 165.4 114.9 132.8 167.7 131.3 130.6 129.7 134.6 129.7 130.6 167.6 99.9 75.0 77.8 72.0 75.3 65.1

88.5 87.3 49.5 209.1 48.3 27.2 64.8 63.8 107.5 20.7 131.1 130.6 129.6 134.4 129.6 130.6 167.8 123.3 132.6 114.9 165.3 114.9 132.6 167.4 99.8 74.9 77.8 72.1 75.3 64.7 55.7

56.0

57.2 56.0

55.8

56.0

chromatogram. Thus, these compounds are natural metabolites present in the root bark of P. suf f ruticosa. The isolated known monoterpenoid glucosides (7−25) were identified as paeoniflorin B (7),8 4-O-methyloxypaeoniflorin (8),8 paeoniflorin (9),19 oxypaeoniflorin (10),20 β-benzoyloxypaeoniflorin (11),7 galloylpaeoniflorin (12),21 mudanpiosides A (13),6 B (14),6 C (15),7 D (16),6 E (17),6 and H (18),5 galloyloxypaeoniflorin (19),22 benzoylpaeoniflorin (20),23 4-Omethybenzoylpaeoniflorin (21),18 6′-O-vanillylpaeoniflorin (22),24 paeonidanin (23),25 oxypaeonidanin (24),8 and paeonidanin A (25),17 by comparison of their NMR and MS data with reported data. The isolated compounds were evaluated for in vitro anticomplement activity, and the results are summarized in Table 3. The paeoniflorin derivatives 1, 2, 7, 10−19, and 22 exhibited anticomplement effects with CH50 and AP50 values ranging from 0.14 to 2.67 mM and 0.25 to 3.67 mM, respectively. However, paeoniflorin derivatives with a C-4 methoxy group (3, 8, and 21) and the six paeonidanin derivatives 4−6 and 23−25 showed no inhibition. These results indicate that hydroxy substitution at C-4 is required for an inhibitory effect, and a methoxy group is deleterious. Furthermore, the two most potent compounds (12 and 19) contain a galloyl moiety, which might be a determining factor for optimum anticomplement activity. In addition, compounds 1, 11, 14, and 18 with a p-hydroxybenzoyl group at C-8 were more potent than 7, 13, and 20 with an unsubstituted benzoyl group, suggesting that a hydroxy group on the benzoyl moiety enhances activity. In order to probe the anticomplement mechanism of these monoterpenoid glucosides, the new monoterpenoid diglucoside 1 without a gallic group and the two most potent monoterpenoid monoglucosides 12 and 19 with a gallic group were evaluated in the complement activation cascade using complement-depleted (C-depleted) sera. As shown in Figure 2, compound 1 did not restore hemolytic activity in C1q-, C3-, C5-, and C9-depleted sera (hemolysis percentages were less than 10%), while it did restore hemolysis significantly in C2- and C4-depleted sera (80.12 ± 3.70% and 89.29 ± 2.91%, respectively). These data indicate that 1 interacted with C1q, C3, C5, and C9. Using the same procedure, both 12 and 19 interacted with C1q, C3, C5, but not C9 components. The results indicate that these monoterpenoid glucosides can act on different targets in the complement activation cascade. It is generally accepted that the complement system is a crucial trigger for inflammation.26,27 Therefore, complement inhibition appears to arrest the process of complementdependent inflammation diseases. In the present study, monoterpenoid glucosides were identified as anticomplement

56.0

a

The assignments were based on DEPT, COSY, HSQC, and HMBC spectral. bData for Glu-4‴: δC 104.8 (C-1⁗), 74.9 (C-2⁗), 77.8 (C3⁗), 71.4 (C-4⁗), 78.3 (C-5⁗), 62.5 (C-6⁗).

analysis of COSY, HMQC, and HMBC spectra. Compound 6 was named suffrupaeonidanin F. The paeoniflorin and paeonidanin derivatives isolated from P. suf f ruticosa contain C-4 or C-9 hemiacetal functionalities, which could form artifacts during extraction and isolation.12 To verify that 3−6 were not formed by methanolysis during the isolation procedure using MeOH as solvent, an acetone extract of P. suf f ruticosa root bark was prepared and analyzed by HPLC. All four compounds were found in the HPLC

Figure 1. Key HMBC and ROESY correlations of 1 and 4. 45

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Table 3. Anticomplement Activity through the Classical Pathway (CP) and Alternative Pathway (AP)a compound 1 2 7 10 11 12 13 14

CH50 (mM) 0.33 1.29 2.29 2.28 0.58 0.21 2.36 0.41

± ± ± ± ± ± ± ±

0.04 0.11 0.12 0.10 0.03 0.03 0.11 0.05

AP50 (mM) 0.43 1.60 NE 3.29 0.85 0.32 2.82 0.97

compound

± 0.03 ± 0.05 ± ± ± ± ±

15 16 17 18 19 22 heparinb

0.14 0.07 0.05 0.08 0.05

CH50 (mM) 0.52 2.67 0.89 0.34 0.14 2.02 0.06

± ± ± ± ± ± ±

0.07 0.03 0.11 0.06 0.03 0.10 0.02

AP50 (mM) 1.05 3.67 1.12 0.67 0.25 2.78 0.13

± ± ± ± ± ± ±

0.08 0.16 0.08 0.08 0.05 0.06 0.06

Data are expressed as mean ± SD (n = 3); NE indicates no inhibitory effect; CH50 and AP50 stand for 50% hemolytic inhibition concentration through the CP and AP, respectively. bHeparin was used as the positive control (mg/mL). a

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on an Autopol V Plus (Rudolph, Hackettstown, NJ, USA). IR spectra were recorded on a PE Spectrum RXI (PerkinElmer, Wellesley, MA, USA) using KBr pellets. ESIMS were measured on an Agilent SL G1946D single quadrupole mass spectrometer (Agilent, Foster, CA, USA), and HRESIMS were measured with a Bruker Daltonics APEXIII 7.0 TESLA FTMS (Bruker Daltonics, Billerica, MA, USA). 1H and 13C NMR spectra were recorded on a Bruker DRX 400 spectrometer (Bruker BioSpin Corporation, Billerica, MA, USA) using TMS as an internal standard. UV spectra were measured in MeOH using a Lambda 25 spectrophotometer (PerkinElmer, Wellesley, MA, USA). Preparative HPLC separation was performed on an LC3000 system with P3000 pump and UV3000 multiple wavelength detector (Beijing Chuangxintongheng Science & Technology Co. Ltd., Beijing, China) using a Thermo Hypersil Gold Aq (250 × 21.2 mm, i.d., 12 μm) column. Column chromatography was carried out over silica gel (200−300 mesh, Qingdao Haiyang Chemical Co. Ltd., Qingdao, China), Chromatorex ODS (30−50 μm, Fuji Silysia Chemical Co. Ltd., Aichi, Japan), Sephadex LH-20 (25−100 μm, Pharmacia, Germany), and Diaion HP-20 macroporous resin (Mitsubishi Chemical Corporation, Tokyo, Japan). D- and L-Glucose were obtained from Sigma-Aldrich Company Ltd., Gillingham, United Kingdom. Plant Material. The root bark of Paeonia suf f ruticosa was purchased from Bozhou, Anhui Province, China, in August 2010, and authenticated by one of the authors (D.-F.C.), at the School of Pharmacy, Fudan University, Shanghai, China, where a voucher specimen (DFC-DP-20100828) is deposited. Animals and Reagents for the Anticomplement Assay. Guinea pigs and New Zealand white rabbits were purchased from Laboratory Animals Research Institute of Fudan University (Shanghai, China). Sheep erythrocytes were collected in Alsevers’ solution. Antisheep erythrocyte antibodies were provided by Prof. Yunyi Zhang (Department of Pharmacology, Fudan University, Shanghai, China). Normal human serum (NHS) was obtained from healthy adult donors. Heparin sodium salt (≥150 IU/mg, dry basis) is a polyanionic glycosaminoglycan from porcine intestinal mucosa and was purchased from Sigma-Aldrich. Anti-C1q, human (goat); anti-C2, human (goat); anti-C5, human (rabbit); and anti-C9, human (goat) were purchased from Merck Biosciences. Anti-C3, human (goat) and anti-C4, human (goat), were purchased from Shanghai Sun Biotech Co. Ltd. Buffers: Veronal buffer saline (VBS) containing 0.1% gelatin (GVB), GVB containing 0.5 mM Mg2+ and 0.15 mM Ca2+ (GVB2+), and GVB containing 2.5 mM Mg2+ and 8 mM EGTA (GVB-Mg-EGTA) were prepared. Extraction and Isolation. Dried root bark of P. suf f ruticosa (20 kg) was powdered and extracted with 95% EtOH (40 L × 3) at room temperature. The solvent was removed under reduced pressure, leaving a dark brown residue (2.6 kg). Part of the residue (1.0 kg) was suspended in H2O (2.0 L) and partitioned successively with EtOAc (2.0 L × 5) and n-BuOH (2.0 L × 5) to afford EtOAc-soluble (390 g) and n-BuOH-soluble (260 g) fractions, respectively.

Figure 2. Targets of compounds 1 (A), 12 (B), and 19 (C) in complement activation cascade. 1-, 12-, and 19-treated sera were mixed with various complement-depleted (C-depleted) sera, and the capacity to restore hemolytic capacity of depleted sera by the classical pathway was estimated by adding sheep antibody-sensitized erythrocytes. Cont represents the complement control group. Data expressed as mean ± SD of triplicate measurements.

constituents of P. suf f ruticosa. Among these compounds, monoterpenoid glucosides 12 and 19 showed the highest anticomplement activity and, in other studies, have also exhibited low cytotoxicity.8 Thus, compounds 12 and 19 are promising candidates for development as anticomplement agents. 46

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Acid Hydrolysis of 1 and 4. Each compound (2 mg) was dissolved in 1.0 mL of 1.0 N HCl and heated to 90 °C in a water bath for 3 h. The mixture was neutralized with excess AgCO3, and the solvent was removed under a stream of N2. The obtained residue was resuspended in H2O (2 mL) and extracted with CHCl3 (2 mL). The aqueous layer was collected and evaporated to dryness using N2. The residue was redissolved in anhydrous pyridine (0.1 mL) and mixed with 0.06 N L-cysteine methyl ester hydrochloride (0.1 mL) in pyridine. The mixture was heated to 60 °C for 2 h, followed by addition of trimethylsilylimidazole (0.1 mL), and heating was continued at 60 °C for 1 h. Water and organic solvent were removed under reduced pressure, and the residue was concentrated to dryness and partitioned between n-hexane and H2O. The organic layer was analyzed by GC-MS (Shimadzu, GCMS-QP2010 Ultra) using an Inertcap column (0.25 mm × 30 m) under the following conditions [injector temperature, 250 °C; initial temperature, 150 °C (1 min), increased at 8 °C/min to 260 °C, held for 10 min; carrier gas, He operated in the splitless mode; injection size, 0.2 μL; MS conditions: EI voltage, 70 eV; scanned-mass range, m/z 50−1000]. The hydrolysates of 1 and 4 gave peaks at 15.54 and 15.53 min, respectively. With authentic D-glucose and L-glucose, peaks were detected at 15.55 and 15.76 min, respectively. Anticomplement Assay, Classical Pathway. On the basis of methods described previously by Xu et al.,15 sensitized erythrocytes (EA) were prepared by incubation of sheep erythrocytes (4.0 × 108 cells/mL) with rabbit anti-sheep erythrocyte antibodies in GVB2+. Each compound and heparin (the positive control) were dissolved in GVB2+. Guinea pig serum (1:80) was chosen to give submaximal lysis in the absence of complement inhibitors. Various dilutions of test samples (200 μL) were preincubated with 200 μL of guinea pig serum at 37 °C for 10 min. Then, EA (200 μL) was added, and the mixture was incubated at 37 °C for 30 min. Controls [blank (200 μL of EA in 400 μL of GVB2+), 100% lysis (200 μL of EA in 400 μL of H2O), and sample control (200 μL dilution of each sample in 400 μL of GVB2+)] were incubated under the same conditions. The mixture was centrifuged (4 °C, 4000 rpm), and the optical density of the supernatant (200 μL) was measured at 405 nm (Wellscan MK3 spectrophotometer, Labsystems Dragon, Helsinki, Finland). Anticomplement activity was determined as the mean of triplicate measurements at each concentration and expressed as 50% inhibitory concentration (CH50 value). Anticomplement Assay, Alternative Pathway. The AP anticomplement assay was performed according to the method described by Xu et al.15 Briefly, various dilutions of each compound and heparin (the positive control) were prepared by adding appropriate volumes of GVB-Mg-EGTA. NHS (1:10) was chosen to give submaximal lysis in the absence of complement inhibitors. After preincubation of each sample (150 μL) with 1:10 diluted NHS (150 μL) at 37 °C for 10 min, 200 μL of rabbit erythrocytes (ERs 3.0 × 108 cells/mL) was added. Following a second incubation at 37 °C for 30 min, cell lysis was determined as described for the CP anticomplement assay. Anticomplement activity was expressed as 50% inhibitory concentration (AP50 value). Identification of Targets on the Complement Activation Cascade. According to the method of Xu et al.,15 samples were dissolved in GVB2+ and diluted to the required concentration. The concentrations of compounds 1, 12, and 19 were 1.09, 0.54, and 0.45 mM, respectively, the minimum concentrations to completely inhibit hemolysis of 1:10 diluted NHS through the CP. To determine if sample-treated serum could restore the hemolytic capacity of various depleted sera, 200 μL of EA and 200 μL of individual complementdepleted sera of C1q, C2, C3, C4, C5, and C9 were added to 200 μL sample-treated NHS, and the mixture was incubated at 37 °C for 30 min. After centrifugation, the optical density of the supernatant was measured and the percentage of hemolysis was calculated. Each individual C-depleted serum was also incubated directly with EA under the same conditions, and the hemolytic activity was calculated. Controls [blank (200 μL of EA in 400 μL of GVB2+), 100% lysis (200 μL of EA in 400 μL of water), complement control (100 μL of NHS and 200 μL of EA in 300 μL of GVB2+), and sample control (100 μL

Part of the EtOAc fraction (280 g) was subjected to column chromatography (CC) on silica gel, eluted with CH2Cl2−MeOH (30:1 to 0:1), to afford fractions Et-1−10. Et-3 (35 g) was further subjected to silica gel CC eluted with CHCl3−MeOH (30:1 to 3:1) to obtain seven subfractions (A1−A7). Further separation of subfractions A3, A6, and A7 by flash ODS with a gradient mixture of MeOH−H2O (2:8 to 8:2), followed by preparative HPLC eluted with MeCN−H2O (3:7), afforded compounds 2 (16 mg), 3 (13 mg), 4 (35 mg), 5 (10.5 mg), 6 (12 mg), 13 (28 mg), 21 (15.6 mg), 22 (30 mg), and 25 (17 mg). Fraction Et-4 (30 g) was separated with silica gel CC (CHCl3− MeOH, 20:1 to 5:1) and Sephadex LH-20 (MeOH), followed by preparative HPLC separation with MeOH−H2O (3:7), to yield compounds 11 (30.5 mg), 12 (40 mg), 14 (27 mg), 15 (50 mg), 18 (18.6 mg), and 20 (78 mg). Compounds 8 (9 mg) and 24 (10 mg) were obtained from fraction Et-6 by repeated silica gel CC (CHCl3− MeOH, 10:1 to 2:1) and Sephadex LH-20 (MeOH) purification. The n-BuOH fraction (150 g) was chromatographed on a Diaion HP-20 macroporous resin column (12 × 80 cm), using a gradient of H2O and MeOH (0:100, 25:75, 50:50, 75:25, 100:0), to give five subfractions (Bu-1−5). Fraction Bu-4 (8 g) was repeatedly subjected to silica gel CC eluted with CHCl3−MeOH (4:1), followed by successive separation on Sephadex LH-20 (MeOH) and preparative HPLC (MeCN−H2O, 1:3), to give compounds 1 (10 mg), 7 (55 mg), and 16 (25 mg). Fraction Bu-3 (40 g) was separated using flash ODS with MeOH−H2O (25:75 to 100:0), followed by Sephadex LH-20 (MeOH) and preparative HPLC (MeOH−H2O, 1:3) purification to yield compounds 9 (89 mg), 19 (19 mg), and 23 (15 mg). In a similar manner to that described for fraction Bu-3, fraction Bu-2 yielded compounds 10 (59 mg) and 17 (71 mg). Suffrupaeoniflorin A (1): white, amorphous solid; [α]20D −22 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 209 (4.14), 258 (4.03) nm; IR (KBr) νmax 3422, 2933, 1707, 1608, 1451, 1314, 1280, 1167, 1073 cm−1; 1H NMR (400 MHz, methanol-d4) and 13C NMR (100 MHz, methanol-d4) data, see Tables 1 and 2, respectively; ESIMS m/z 785 [M + Na]+; HRESIMS m/z 785.2248 [M + Na]+ (calcd for C36H42O18Na, 785.2263). Suffrupaeoniflorin B (2): white, amorphous solid; [α]20D −31 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 225 (4.19), 257 (4.25); IR (KBr) νmax 3432, 2936, 1718, 1606, 1450, 1317, 1279 cm−1; 1H NMR (400 MHz, methanol-d4) and 13C NMR (100 MHz, methanol-d4) data, see Tables 1 and 2, respectively; ESIMS m/z 632 [M + NH4]+; HRESIMS m/z 637.1888 [M + Na]+ (calcd for C31H34O13Na, 637.1891). 4-O-Methylsuffrupaeoniflorin B (3): white, amorphous solid; [α]20D −36 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 228 (4.49), 257 (4.45); IR (KBr) νmax 3432, 2938, 1718, 1606, 1451, 1317, 1278, 1170, 1077 cm−1; 1H NMR (400 MHz, methanol-d4) and 13C NMR (100 MHz, methanol-d4) data, see Tables 1 and 2, respectively; ESIMS m/z 651 [M + Na]+; HRESIMS m/z 651.2065 [M + Na]+ (calcd for C32H36O13Na, 651.2048). Suffrupaeonidanin D (4): white, amorphous solid; [α]20D −84 (c 0.8, MeOH); UV (MeOH) λmax (log ε) 231 (4.37); IR (KBr) νmax 3433, 2934, 1722, 1451, 1278, 1072 cm−1; 1H NMR (400 MHz, methanol-d4) and 13C NMR (100 MHz, methanol-d4) data, see Tables 1 and 2, respectively; ESIMS m/z 621 [M + Na]+; HRESIMS m/z 621.1947 [M + Na]+ (calcd for C31H34O12Na, 621.1942). Suffrupaeonidanin E (5): white, amorphous solid; [α]20D −29 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 228 (4.38), 257 (4.21); IR (KBr) νmax 3431, 2933, 1718, 1606, 1450, 1278, 1070 cm−1; 1H NMR (400 MHz, methanol-d4) and 13C NMR (100 MHz, methanol-d4) data, see Tables 1 and 2, respectively; ESIMS m/z 651 [M + Na]+; HRESIMS m/z 651.2039 [M + Na]+ (calcd for C32H36O13Na, 651.2048). Suffrupaeonidanin F (6): white, amorphous solid; [α]20D −33 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 229 (4.38), 258 (4.02); IR (KBr) νmax 3433, 2934, 1717, 1606, 1451, 1279, 1072 cm−1; 1H NMR (400 MHz, methanol-d4) and 13C NMR (100 MHz, methanol-d4) data, see Tables 1 and 2, respectively; ESIMS m/z 651 [M + Na]+; HRESIMS m/z 651.2069 [M + Na]+ (calcd for C32H36O13Na, 651.2048). 47

dx.doi.org/10.1021/np400571x | J. Nat. Prod. 2014, 77, 42−48

Journal of Natural Products

Article

samle in 500 μL of GVB2+)] were incubated under the same conditions.

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(21) Shimizu, M.; Hayashi, T.; Moria, N.; Kiuchi, F.; Noguchi, H.; Sankawa, U. Chem. Pharm. Bull. 1983, 31, 577−583. (22) Mastuda, H.; Ohta, T.; Kawaguchi, A.; Yoshikawa, M. Chem. Pharm. Bull. 2001, 49, 69−72. (23) Braca, A.; Kiem, P. V.; Yen, P. H.; Nhiem, N. X.; Quang, T. H.; Cuong, N. X.; Minh, C. V. Fitoterapia 2008, 79, 117−120. (24) Tanaka, T.; Kataoka, M.; Tsuboi, N.; Kouno, I. Chem. Pharm. Bull. 2000, 48, 201−207. (25) Kosiova, I. N.; Simeonov, M. F.; Todorova, D. I. Phytochemistry 1998, 47, 1303−1307. (26) Chen, M.; Muchersie, E.; Luo, C.; Forrester, J. V.; Xu, H. P. Eur. J. Immunol. 2010, 40, 2870−2881. (27) Ignatius, A.; Schoengraf, P.; Kreja, L.; Liedert, A.; Rechnagel, S.; Kandert, S.; Brenner, R. E.; Schneider, M.; Lambris, J. D. J. Cell. Biochem. 2011, 112, 2594−2605.

ASSOCIATED CONTENT

S Supporting Information *

NMR spectra of the new compounds 1−6, HPLC chromatogram of acetone extract of P. suf f ruticosa, and GC-MS diagrams obtained with L-cysteine methyl ester hydrochloride derivatives of hydrolysates of 1 and 4 are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-21-51980135. Fax: +86-21-51980017. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This investigation was supported by grants from the National Natural Science Foundation of China (30925042, 81330089) and the State Key Program for Innovative Drugs from the Ministry of Science and Technology, China (2009ZX09502013). The authors are grateful to Dr. S. L. Morris-Natschke at the University of North Carolina at Chapel Hill for polishing the English.

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REFERENCES

(1) Kirschfink, M. Immunopharmacology 1997, 38, 51−62. (2) Holders, V. M. J. Clin. Invest. 2004, 114, 616−619. (3) Ballanti, E.; Perricone, C.; Muzio, G. D.; Kroegler, B.; Chimenti, M. S; Graceffa, D.; Perricone, R. Autoimmun. Rev. 2011, 10, 617−623. (4) Hong, M. H.; Kim, J. H.; Na, S. H.; Bae, H.; Shim, Y. C.; Kin, S. H.; Ko, S. G. Biosci. Biotechnol. Biochem. 2010, 74, 1152−1156. (5) Ding, H. Y.; Wu, Y. C.; Lin, H. C.; Chan, Y. Y.; Wu, P. L.; Wu, T. S. Chem. Pharm. Bull. 1999, 47, 652−655. (6) Lin, H. C.; Ding, H. Y.; Wu, T. S.; Wu, P. L. Phytochemistry 1996, 41, 237−242. (7) Ding, L. Q.; Jiang, Z. H.; Liu, Y.; Chen, L. X.; Zhao, Q.; Yao, X. S.; Zhao, F.; Qiu, F. Fitoterapia 2012, 83, 1598−1603. (8) Ding, L. Q.; Zhao, F.; Chen, L. X.; Jiang, Z. H.; Liu, Y.; Li, Z. M.; Qiu, F.; Yao, X. S. Bioorg. Med. Chem. Lett. 2012, 22, 7243−7247. (9) An, R. B.; Kim, H. C.; Lee, S. H.; Jeong, G. S.; Sohn, D. H.; Park, H.; Kwon, D. Y.; Lee, J. H.; Kim, Y. C. Arch. Pharm. Res. 2006, 29, 815−820. (10) Yang, Y.; Hu, H. Y.; Yu, N. J.; Zhang, Y.; Zhao, Y. M. Helv. Chim. Acta 2010, 93, 1622−1627. (11) Furuya, R.; Hu, H. H.; Zhang, Z. Y.; Shigemori, H. Molecules 2012, 17, 4915−4923. (12) Ha, D. T.; Ngoc, T. M.; Lee, I.; Lee, Y. M.; Kim, J. S.; Jung, H. J.; Lee, S. M.; Na, M. K.; Bae, K. H. J. Nat. Prod. 2009, 72, 1465−1470. (13) Hsu, F. L.; Lai, C. W.; Cheng, J. T. Planta Med. 1997, 63, 323− 325. (14) Zhang, T.; Chen, D. F. J. Ethnopharmacol. 2008, 117, 351−361. (15) Xu, H.; Zhang, Y. Y.; Zhang, J. W.; Chen, D. F. Int. Immunopharmacol. 2007, 7, 175−182. (16) Du, D. S.; Cheng, Z. H.; Chen, D. F. Nat. Prod. Commun. 2012, 7, 501−505. (17) Okasaka, M.; Kashiwada, Y.; Kodzhimatov, O. K.; Ashurmetov, O.; Takaishi, Y. Phytochemistry 2008, 69, 1767−1772. (18) Duan, W. J.; Yang, J. Y.; Chen, L. X.; Zhang, L. J.; Jiang, Z. H.; Cai, X. D.; Zhang, X.; Qiu, F. J. Nat. Prod. 2009, 72, 1579−1584. (19) Lemmich, J. Phytochemistry 1996, 41, 1337−1340. (20) Kaneda, M.; Iitaka, Y.; Shibata, S. Tetrahedron 1972, 28, 4309− 4011. 48

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