Coumarinolignoids and Taraxerane Triterpenoids from Sapium

Article Cite This: J. Nat. Prod. 2018, 81, 2251−2258

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Coumarinolignoids and Taraxerane Triterpenoids from Sapium discolor and Their Inhibitory Potential on Microglial Nitric Oxide Production Gui-Jie Zhang,†,# Qi-Ming Pan,‡,# Yong-Li Zhang,† Hai-Bing Liao,‡ Yan-Qiu Yang,§ Yue Hou,*,§ and Dong Liang*,‡ †

College of Pharmacy, Guilin Medical University, Guilin 541004, People’s Republic of China State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmaceutical Sciences, Guangxi Normal University, Guilin 541004, People’s Republic of China § College of Life and Health Sciences, Northeastern University, Shenyang 110819, People’s Republic of China

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‡

S Supporting Information *

ABSTRACT: Seventeen compounds, including three new pairs of coumarinolignoid enantiomers, (7′S,8′S)-sapiumins A−C (1a−3a) and (7′R,8′R)sapiumins A−C (1b−3b), six new taraxerane triterpenoids, sapiumic acids A−F (4−9), and five known taraxerane triterpenoids (10−14), were isolated from an ethanol extract prepared from the stems and leaves of Sapium discolor. The structures of 1−9 and their relative configurations were determined by spectroscopic data analysis, and the absolute configurations of the coumarinolignoids 1a/1b−3a/3b and triterpenoids 6−9 were assigned using experimental and calculated ECD data. Compounds 1a/1b−3a/3b are the first coumarinolignoids to be reported from the genus Sapium. Among all the isolates, compounds 1b, 2a/2b, 3a/3b, and 6−9 inhibited nitric oxide production in lipopolysaccharide-stimulated BV-2 microglial cells, with IC50 values of 1.7−15.3 μM.

C

report of the isolation of coumarinolignoids from the genus Sapium. Herein, we report the isolation, structural elucidation, and NO inhibitory effects of the new compounds.

oumarinolignoids are a class of natural products in which a phenylpropanoid unit is linked to a coumarin moiety through a 1,4-dioxane bridge.1 These substances are relatively rare and exhibit a wide variety of biological properties, such as anti-inflammatory, antioxidant, and hepatoprotective effects.2−4 Taraxeranes, a type of triterpenoids derived biosynthetically from oleananes,5 display cytotoxic and allelopathic activities.6−8 Both coumarinolignoids and taraxerane triterpenoids occur in plants in the family Euphorbiaceae.9−12 Sapium discolor (Champ. ex Benth.) Muell. Arg., which is distributed widely in southern mainland China, is a tree or shrub belonging to the family Euphorbiaceae.13 The root bark and leaves of this plant have long been used in folk medicine to treat snakebites, allergic dermatitis, and eczema.13,14 Previous phytochemical research has indicated the presence of four diterpenoids and other constituents in this species.15,16 In an ongoing search for antineuroinflammatory agents from folk medicines,17,18 the EtOAc-soluble fraction of a 95% aqueous EtOH extract of S. discolor was shown to inhibit the production of nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. Thus, a bioassay-guided phytochemical study of the active fractions led to the isolation of three new pairs of coumarinolignoid enantiomers, (7′S,8′S)-sapiumins A−C (1a−3a) and (7′R,8′R)-sapiumins A−C (1b−3b), and six new taraxerane triterpenoids, sapiumic acids A−F (4−9), along with five known taraxeranes (10−14). This is the first © 2018 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION A 95% aqueous EtOH extract from the stems and leaves of S. discolor was suspended in H2O and partitioned successively with petroleum ether, EtOAc, and n-BuOH. The EtOAc-soluble portion was subjected to various column chromatographic (CC) steps followed by semipreparative reversed-phase (RP)-HPLC to afford three new pairs of coumarinolignoid enantiomers (1a/1b−3a/3b), as well as six new (4−9) and five known taraxerane triterpenoids. Sapiumin A (1) was purified as an amorphous powder with a molecular formula of C18H14O7, as indicated by the negative-ion HRESIMS data (m/z 341.0658 [M − H]−, calcd 341.0667) in conjunction with the 1H and 13C NMR data of this compound. The UV spectrum of sapiumin A (1) showed absorptions characteristic of a coumarin derivative (λmax 230, 261, 289, and 344 nm).4 The presence of hydroxy (3449 cm−1), carbonyl (1725 cm−1), and aromatic ring (1628, 1563, 1440 cm−1) functionalities was determined from its IR absorption bands. In the low-field region of the 1H NMR spectrum of compound 1, Received: July 18, 2018 Published: October 16, 2018 2251

DOI: 10.1021/acs.jnatprod.8b00585 J. Nat. Prod. 2018, 81, 2251−2258

Journal of Natural Products

Article

Chart 1

The mixture showed opposite electronic circular dichroism (ECD) curves and specific rotation values ([α]20D −42 for 1a and [α]20D +41 for 1b). A comparison of the calculated and experimental ECD data (Figure 2) supported the (7′S,8′S) and (7′R,8′R) absolute configurations for 1a and 1b, respectively.

a pair of doublets characteristic of the H-3 and H-4 of a coumarin moiety [δH 7.91 and 6.30 (each 1H, d, J = 9.5 Hz)] were present, along with resonances at δH 6.81 (1H, d, J = 2.0 Hz), 6.75 (1H, d, J = 8.0 Hz), and 6.71 (1H, dd, J = 8.0, 2.0 Hz) for the protons of a 1,2,4-trisubstituted aromatic unit and two aromatic singlets at δH 7.26 and 7.00 (each 1H, s). The 13C NMR and HSQC spectra showed 18 carbon resonances comprising 14 aromatic or olefinic carbons in the δC 103.9−148.9 region, a carbonyl carbon (δC 160.4), a methylene (δC 59.9), and two methines (δC 78.9 and 75.5). The 1D NMR data in the aromatic region, two sp3 methines (δC 78.9, δH 4.20 and δC 75.5, δH 4.87), and one sp3 methylene (δC 59.9, δH 3.56, 3.34) represented the typical characteristics of a phenylpropanoid unit,4 which was verified by a 2D NMR analysis. The deshielded doublet at δH 4.87 (H-7′), typical of a benzylic methine with an oxygen substituent, along with the multiplet at δH 4.20 (H-8′) implied the presence of a 1,4-dioxane ring between the coumarin moiety and the phenylpropanoid unit, and this structure corresponds to that of a coumarinolignoid skeleton.4,19 Coumarinolignoids occur naturally as regioisomeric pairs,3,4 so the linkage of coumarin and the phenylpropanoid unit was confirmed with an HMBC experiment. After optimizing the 2,3J(C,H) value for the long-range correlation to 4.0 Hz,4 HMBC correlations from H-5 (δH 7.26) to C-4/C-6/C-9, from H-8 (δH 7.00) to C-6/C-7/C-9/C-10, and from H-7′ to C-6 were observed (Figure 1). In addition, the large coupling constant of 7.9 Hz

Figure 2. Experimental ECD spectra of 1a and 1b and calculated ECD spectra of 7′S,8′S-1 and 7′R,8′R-1.

The molecular formula of sapiumin B (2) was determined to be C19H16O8 based on its 13C NMR and HRESIMS data (m/z 371.0779 [M − H]−, calcd 371.0772). The NMR data of 2 (Table 1) showed many similarities to those of 1, except for the signals of a methoxy group [δC 61.0, δH 3.91 (3H, s)]. The C-8 in 2 (δC 135.1; ΔδC +31.2) was deshielded compared to the same position in 1, which along with the HMBC crosspeak from OMe (δH 3.91) to C-8 located the methoxy group at C-8 in 2. The comparison of the NMR data of 2 with those of 1 (Table 1) revealed that the signals of the two vicinal oxymethines were almost superimposable, suggesting the linkage of the coumarin core with the phenylpropane unit in 2 as being the same as that in 1. This was verified further by the HMBC cross-peaks from H-5 (δH 7.02) to C-4/C-6/C-9 and from H-7′ (δH 4.89) to C-6. The large coupling constant of 3 J7′,8′ (8.0 Hz) suggested a trans relative configuration between

Figure 1. Selected HMBC correlations of 1 and 3.

between the two vicinal oxymethines supported the trans diaxial arrangement of H-7′ and H-8′ in the 1,4-dioxane ring.3,4,20,21 Thus, the structure of sapiumin A (1) was defined as shown. Most coumarinolignans reported so far have been isolated as racemates and thus are optically inactive.1 The specific rotation of 1 was nearly zero, and subsequent chiral HPLC resolution of 1 using a Daicel Chiralpak AD-H column yielded an approximately 1:1 ratio of 1a and 1b (Figure S9, Supporting Information). 2252



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The 1H and 13C NMR spectra (Table 1) of 3 were similar to those of 1, except for the chemical shifts of the two vicinal oxymethines. The comparison of the 13C NMR data of 3 with those of 1 (Table 1) suggested that 3 is a regioisomer of 1, as deduced from the shifts in the signals for C-7′ and C-8′ from δC 75.5 and 78.9 to δC 76.4 and 78.0, respectively. The linkage of the coumarin core with the phenylpropane moiety was also confirmed by HMBC experiments. In the HMBC spectrum, cross-peaks from H-5 (δH 7.29) to C-4/C-6/C-9, from H-8 (δH 7.00) to C-6/C-7/C-9/C-10, and from H-7′ (δH 4.96) to C-7 were observed (Figure 1). The mutual coupling constant (J = 7.9 Hz) between H-7′ and H-8′ confirmed their trans relative configuration. Once again, the chiral HPLC analysis revealed that 3 was obtained as a pair of enantiomers, 3a ([α]20D −21) and 3b ([α]20D +20). The comparison of the calculated and experimental ECD spectra (Figure 4) of these compounds facilitated the

Table 1. 1H NMR (500 MHz) and 13C NMR (125 MHz) Data of Compounds 1−3 in DMSO-d6 1 position

δC

2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′

160.4 113.4 144.0 114.6 140.7 147.0 103.9 148.9 112.5 127.0 115.0 145.3 145.9 115.5 118.9

7′ 8′ 9′

75.5 78.9 59.9

δH (J in Hz) 6.30, d (9.5) 7.91, d (9.5) 7.26, s

7.00, s

6.81, d (2.0)

6.75, d (8.0) 6.71, dd (8.0, 2.0)

2 δC 159.9 113.6 144.4 108.9 141.2 140.7 135.1 141.8 112.1 127.0 115.0 145.3 145.9 115.5 118.9

4.87, d (7.9) 75.3 4.20, m 78.7 3.56, dd (12.5, 2.0) 59.9 3.34, dd (12.5, 4.0)

OMe

61.0

δH (J in Hz) 6.31, d (9.0) 7.89, d (9.0) 7.02, s

6.81, br s

6.75, d (8.0) 6.71, br d (8.0) 4.89, d (8.0) 4.20, m 3.62, br d (12.0) 3.37, dd (12.0, 4.0) 3.91, s

3 δC 160.4 113.4 144.1 114.5 140.4 147.3 104.1 148.7 112.7 126.8 115.0 145.3 146.0 115.5 118.9 76.4 78.0 60.0

δH (J in Hz) 6.31, d (9.5) 7.94, d (9.5) 7.29, s

7.00, s

6.80, d (2.0)

6.75, d (8.0) 6.71, dd (8.0, 2.0) 4.96, d (7.9) 4.11, m 3.54, br d (12.5) 3.34a

a

Signal overlapped by solvent peaks.

H-7′ and H-8′. Accordingly, the structure of sapiumin B (2) was established as shown. Compound 2 was also obtained as a racemate, as evident from the lack of any specific rotation. Chiral HPLC separation of 2 using a Daicel Chiralpak ID column (Figure S16, Supporting Information) afforded the pair of enantiomers 2a ([α]20D −45) and 2b ([α]20D +39). By comparing their calculated and experimental ECD spectra, the absolute configurations of 2a and 2b were deduced to be (7′S,8′S) and (7′R,8′R), respectively (Figure 3).


assignment of the (7′S,8′S) and (7′R,8′R) absolute configurations for 3a and 3b, respectively. Sapiumic acid A (4) was acquired as a white, amorphous powder. The molecular formula of 4 was determined to be C32H50O5 based on its deprotonated-ion HRESIMS data, m/z 513.3590 [M − H]− (calcd 513.3585), and 13C NMR data. The IR spectrum showed absorption peaks indicative of hydroxy (3444 cm−1), carbonyl (1714 and 1692 cm−1), and olefinic (1634 cm−1) groups. The 1H NMR spectrum exhibited signals at δH 0.92 (H3-23), 0.93 (H3-24), 0.95 (H3-25 and H3-29), 1.06 (H3-30), 1.16 (H3-27), and 1.21 (H3-26) for seven tertiary methyl groups. The 13C NMR data together with the HSQC data for compound 4 revealed 32 carbon signals, attributable to eight methyl carbons, nine methylenes, five sp3 methines, six sp3 quaternary carbons, one double bond (δC117.0 and 161.1), and two carbonyl carbons (δC 170.6 and 180.3). The 1H NMR signal of one olefinic proton at δH 5.82 (dd, J = 8.0, 3.0 Hz) together with the 13C NMR chemical shifts of C-14 (δC 161.1) and C-15 (δC 117.0) suggested that 4 is a taraxer-14-ene derivative.22,23 A detailed analysis of its NMR data indicated 4 to be related structurally to aleuritolic acid (10),24 differing only by the presence of signals from an additional oxygenated methine (δH 3.79, δC 70.9) and an acetyl group [δH 2.03 (3H, s); δC 170.6 and 21.2]. The key HMBC correlations from H-1 (δH 3.79) to C-3/C-5 and from H3-25 (δH 0.95) to C-1 (δC 70.9) were used to locate a hydroxy group at C-1, and the cross-peak from H-3 (δH 5.57) to the carbonyl carbon


Sapiumin C(3), obtained as an amorphous powder, was found to possess the same molecular formula as that of 1, C18H14O7, based on the negative-ion HRESIMS (m/z 341.0668 [M − H]−, calcd 341.0667) and 13C NMR data of this compound. 2253



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Figure 5. Selected HMBC and NOESY correlations of 4.

[δH 7.22 (1H, d, J = 8.0 Hz), 7.30 (1H, br d, J = 8.0 Hz), and 7.41 (1H, br s)] and a methoxy group [δH 3.79 (3H, s)]. In the HMBC spectrum, the correlations from OMe (δH 3.79) to C-6′ (δC 149.1) and from H-3 (δH 5.08) to C-1′ (δC 166.6) indicated the trans-p-coumaroyl group at C-3 in 6 to be replaced by a trans-feruloyl group in 7. The configuration of 7 was consistent with that of 6, according to their NOESY interactions and ECD data (Figure 6). Hence, the structure of 7, sapiumic acid D, was defined as shown. Sapiumic acid E (8) yielded a molecular formula of C39H50O6, as determined by the deprotonated HRESIMS ion at m/z 613.3542 [M − H]− (calcd 613.3535). An extensive analysis of the 1D and 2D NMR data of 8 suggested that this compound is also a taraxerane triterpenoid bearing a trans-pcoumaroyl group and that 8 is structurally related to 5 and 6. In the 1D NMR spectra of 8, signals of one ketocarbonyl carbon (δC 204.2) and one 1,2-disubstituted double bond (δC 125.3 and 158.6; δH 5.95 and 7.03) established an α, β-unsaturated carbonyl moiety (C-3−C-2−C-1), which was assigned by the 1H−1H COSY correlation of H-1 (δH 7.03)/ H-2 (δH 5.95) and the HMBCs from H-1 to C-3/C-5/C-9/C10. The trans-p-coumaroyl group was deduced to be attached at C-29 based on the key HMBCs from H2-29 (δH 4.22, 4.18) to C-19/C-20/C-21/C-30/C-1′, along with the NOESY crosspeak of H-18 (δH 2.93)/H3-30 (δH 1.26) (Figure 7). The large coupling constant of 3J2′,3′ (16.0 Hz) confirmed the presence of a 2′,3′-trans double bond. The ECD spectrum of 8 exhibited Cotton effects (CEs) at 242 (Δε +27.80) and 347 (Δε −5.51) nm (Figure 8), corresponding to the π → π* and n → π* transitions of the α,β-unsaturated cyclohexanone chromophore,25−27 indicating that 8 possesses a 10R configuration. The structure of 8, sapiumic acid E, was thus established as shown. The deprotonated HRESIMS ion at m/z 613.3538 [M − H]− (calcd 613.3535) revealed that sapiumic acid F (9) has the same molecular formula, C39H50O6, as that of 8. Analysis of the NMR data of 9 (Tables 2 and 3) showed the structure of 9 to be closely related to that of 8, except for the configuration of the p-coumaroyl group. The 1H NMR signals of olefinic protons at δH 6.07 (H-2′) and 7.00 (H-3′) with a distinctly smaller coupling constant (J = 12.5 Hz) indicated the presence of a 2′,3′-cis olefinic unit, which was verified by the NOESY crosspeak of H-2′/H-3′. The HMBCs from H2-29 (δH 4.14, 4.11) to C-19/C-20/C-21/C-30/C-1′, combined with the NOESY cross-peak of H-18 (δH 2.89)/H3-30 (δH 1.20), suggested that the cis-p-coumaroyl group is also located at C-29. A strong positive CE at 240 nm and a weak negative CE at 346 nm in the ECD spectrum (Figure 8) suggested that 9 has the same 10R configuration as seen in 8. Thus, the structure of sapiumic acid F (9) was defined as shown. The known taraxerane triterpenoids isolated in this study were identified as aleuritolic acid (10),24 3-acetylaleuritolic

(δC 170.6) indicated the linkage of the acetyl group to C-3 (Figure 5). The β-orientation of H-1 and the α-orientation of H-3 were assigned by the cross-peaks of H-1/H3-25, H-3/H-5 (δH 1.69), and H-3/H3-23 (δH 0.92) observed in the NOESY spectrum (Figure 5). Hence, the structure of 4 was established as shown, and this compound was named sapiumic acid A. The molecular formula C39H54O6 was assigned to sapiumic acid B (5) on the basis of its HRESIMS data (m/z 617.3848 [M − H]−, calcd 617.3848) with the aid of its 13C NMR data. In the 13 C NMR spectrum of 5, in addition to 30 signals assignable to a triterpenoid moiety, nine additional signals were present, including eight sp2 carbons [δC 116.0, 116.9 (2C), 126.3, 130.6 (2C), 144.8, and 161.4] and one ester carbon (δC 167.3). The 1H NMR spectrum of 5 showed resonances characteristic of a trans double bond [δH 6.69, 8.00 (each 1 H, d, J = 16.0 Hz)] and a 1,4-disubstituted aromatic moiety [δH 7.17, 7.63 (each 2H, d, J = 8.5 Hz)]. According to the above data, together with the 2D NMR analysis, the structure of 5 was similar to that of 4, except for the presence of a trans-p-coumaroyl unit at C-3 in 5, which replaced the acetyl group in 4. These structural features were confirmed by the key HMBC cross-peak from H-3 (δH 5.78) to C-1′ (δC 167.3). On the basis the NMR data (Tables 2 and 3) and the NOESY interactions, the relative configuration with regard to the triterpenoid moiety in 5 was found to be identical to that in 4. The coupling constant (J = 16.0 Hz) between H-2′ and H-3′ confirmed an E configuration of the double bond. The structure of sapiumic acid B (5) was thus established as shown. Sapiumic acid C (6) was determined to have the molecular formula C39H52O6 from its 13C NMR data and (−)-HRESIMS ion at m/z 615.3697 [M − H]− (calcd 615.3691). The 1D NMR data of 6 (Tables 2 and 3) closely resembled those of 5. However, an apparent difference in the 13C NMR spectra of these two compounds, namely, signals at δC 71.0 for 5 and δC 210.7 for 6, indicated that the C-1 hydroxy group in 5 was replaced by a ketocarbonyl group in 6. The key HMBC correlations from H2-2 (δH 3.23, 2.74), H-3 (δH 5.07), and H3-25 (δH 1.30) to C-1 (δC 210.7) verified the above conclusion. The relative configuration of C-3 and the geometry of the 2′,3′-trans double bond in 6 were the same as those in 5 based on the NOESY data and coupling constants. Furthermore, by comparing its calculated and experimental ECD spectra (Figure 6), the absolute configuration of 6 was established. Therefore, the structure of sapiumic acid C (6) was established as shown. The molecular formula of sapiumic acid D (7) was defined as C40H54O7 based on its HRESIMS ion at m/z 645.3804 [M − H]− (calcd 645.3797) with the aid of its 13C NMR data. The 1D NMR data (Tables 2 and 3) of 7 showed a high similarity to those of 6, except for the presence of 1H NMR signals characteristic of a 1,2,4-trisubstituted aromatic moiety 2254



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Table 2. 1H NMR (500 MHz) Data of Compounds 4−9 in Pyridine-d5 position 1 2a 2b 3 5 6a 6b 7a 7b 9 11a 11b 12a 12b 15 16a 16b 18 19a 19b 21a 21b 22a 22b 23 24 25 26 27 29a 29b 30 2′ 3′ 5′ 6′ 8′ 9′ OMe

4

5

3.79, t (3.0) 2.08, m

3.84, br s 2.21, m

5.57, 1.69, 1.52, 1.43, 1.95, 1.40, 2.57, 2.16, 1.63, 2.05, 1.76, 5.82, 2.77, 2.11, 2.81, 1.35, 1.19, 1.38, 1.24, 2.04, 1.55, 0.92, 0.93, 0.95, 1.21, 1.16, 0.95,

5.78, dd (9.5, 7.0) 1.77, dd (10.5, 3.0) 1.55, m 1.48, m 1.98, m 1.43, m 2.62, dd (10.5, 9.0) 2.18, m 1.67, m 2.07, dd (13.5, 4.0) 1.77, m 5.84, dd (8.0, 3.0) 2.78, dd (14.0, 8.0) 2.13, dd (14.0, 3.0) 2.83, dd (14.0, 4.0) 1.38, t (14.0) 1.22, dd (14.0, 4.0) 1.40, m 1.25, m 2.04, m 1.57, m 1.003, s 1.05, s 1.000, s 1.24, s 1.20, s 0.97, s

dd (9.5, 7.5) dd (12.0, 3.0) m m m m dd (10.5, 9.0) m m overlapped dd (13.5, 8.5) dd (8.0, 3.0) dd (14.0, 8.0) dd (14.0, 3.0) dd (13.5, 3.5) t (13.5) dd (13.5, 3.5) m m overlapped m s s s s s s

1.06, s 2.03, s

1.07, 6.69, 8.00, 7.63, 7.17, 7.17, 7.63,

s d d d d d d

(16.0) (16.0) (8.5) (8.5) (8.5) (8.5)

6

7

8

3.23, 2.74, 5.07, 1.07, 1.50,

t (11.5) dd (11.5, 4.5) dd (11.5, 4.5) overlapped m

3.25, 2.76, 5.08, 1.07, 1.52,

t (11.5) dd (11.5, 4.5) dd (11.5, 4.5) m m

1.88, 1.23, 2.19, 2.63, 1.53, 1.98, 1.75, 5.77, 2.80, 2.13, 2.81, 1.39, 1.21, 1.39, 1.27, 2.05, 1.57, 0.93, 1.12, 1.30, 1.17, 1.17, 0.99,

m m t (9.5) m m m dd (13.5, 9.0) dd (8.5, 2.5) dd (13.5, 8.5) dd (13.5, 2.5) dd (13.5, 3.5) t (13.5) dd (13.5, 3.5) t (13.5) m m m s s s s s s

1.89, 1.24, 2.20, 2.64, 1.54, 1.99, 1.75, 5.78, 2.80, 2.14, 2.81, 1.40, 1.22, 1.40, 1.28, 2.05, 1.58, 0.94, 1.11, 1.30, 1.18, 1.18, 0.99,

m m dd (10.0, 9.0) m m m dd (13.5, 9.0) dd (8.0, 3.0) dd (14.0, 8.0) dd (14.0, 3.0) dd (13.5, 4.0) t (13.5) dd (13.5, 4.0) t (13.5) m m m s s s s s s

1.07, 6.63, 7.98, 7.64, 7.18, 7.18, 7.64,

s d d d d d d

1.08, 6.71, 8.01, 7.41,

s d (16.0) d (16.0) br s

(15.5) (15.5) (8.5) (8.5) (8.5) (8.5)

7.22, d (8.0) 7.30, br d (8.0) 3.79, s

9

7.03, d (10.0) 5.95, d (10.0)

7.03, d (10.0) 5.94, d (10.0)

1.60, m 1.45, m

1.59, m 1.44, m

1.92, 1.29, 1.74, 1.95, 1.68, 1.99, 1.80, 5.77, 2.85, 2.16, 2.93, 1.64, 1.36, 1.65, 1.39, 2.12, 1.68, 1.19, 1.07, 1.06, 1.16, 1.07, 4.22, 4.18, 1.26, 6.76, 8.05, 7.64, 7.16, 7.16, 7.64,

1.91, 1.29, 1.72, 1.93, 1.66, 1.96, 1.77, 5.76, 2.82, 2.12, 2.89, 1.61, 1.30, 1.56, 1.36, 2.07, 1.63, 1.19, 1.07, 1.05, 1.15, 1.04, 4.14, 4.11, 1.20, 6.07, 7.00, 8.06, 7.19, 7.19, 8.06,

m m dd (10.5, 8.0) m m m m dd (8.0, 3.0) dd (14.5, 8.0) dd (14.5, 3.0) dd (13.5, 3.0) t (13.5) dd (13.5, 3.0) m m m m s s s s s d (11.0) d (11.0) s d (16.0) d (16.0) d (8.5) d (8.5) d (8.5) d (8.5)

m m m m m m m dd (8.0, 3.5) dd (14.5, 8.0) dd (14.5, 3.5) dd (13.5, 3.5) t (13.5) dd (13.5, 3.5) m m m m s s s s s d (10.5) d (10.5) s d (12.5) d (12.5) d (8.5) d (8.5) d (8.5) d (8.5)

Table 3. 13C NMR (125 MHz) Data of Compounds 4−9 in Pyridine-d5

acid (11),28 1-oxo-aleuritolic acid (12),29 sebiferone (13),30 and aleuritolonic acid (14)24 by comparing their NMR data with values in the literature. Coumarinolignoids are rather widely distributed and can be found in plants belonging to approximately 19 plant families.1 This study represents the first report of coumarinolignoids isolated from the genus Sapium. Most coumarinolignoids are angularly fused, as depicted in the literature, with only four linearly fused coumarinolignoids having been isolated so far.1 The three pairs of coumarinolignoid enantiomers reported in this study are all linearly fused. Taraxerane triterpenoids are relatively rare in Nature, with only four taraxeranes having been isolated from this genus,16 and this is the first report of triterpenoids of this type from S. discolor. The EtOAc extract of S. discolor inhibited NO production in overactivated BV-2 cells, with an IC50 value of 30.2 μg/mL. Thus, all the coumarinolignoids and taraxeranes isolated were assessed for their antineuroinflammatory activities by an NO inhibition assay in LPS-induced BV-2 cells using the Griess reaction.17,18 As shown in Table 4 and Figure 9, compounds

position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 2255

4

5

6

7

8

9

70.9 31.8 77.0 38.2 48.3 18.8 41.1 39.1 40.8 42.1 17.0 34.1 37.9 161.1 117.0 32.5 51.2 42.2

71.0 32.1 76.8 38.5 48.5 18.8 41.2 39.2 40.9 42.2 17.0 34.1 38.0 161.2 117.0 32.5 51.2 42.3

210.7 41.3 79.0 38.7 54.2 18.3 40.2 39.2 42.3 54.0 19.1 34.2 37.9 160.2 117.2 32.7 51.1 42.4

210.7 41.3 79.0 38.6 54.2 18.3 40.1 39.1 42.2 54.0 19.0 34.1 37.8 160.2 117.1 32.6 51.1 42.3

158.6 125.3 204.2 44.6 53.7 19.3 40.3 39.6 44.1 41.0 17.7 33.5 37.7 160.0 117.1 32.8 51.1 41.4

158.6 125.3 204.2 44.6 53.8 19.4 40.4 39.6 44.1 41.0 17.7 33.5 37.7 160.0 117.1 32.8 51.1 41.4



Article

Table 3. continued position

4

5

6

7

8

9

19 20 21 22 23 24 25 26 27 28 29 30 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ OMe

36.0 29.6 34.3 31.6 28.2 16.7 16.6 26.3 22.5 180.3 32.3 29.1 170.6 21.2

36.0 29.6 34.4 31.7 28.4 16.9 16.7 26.3 22.6 180.3 32.4 29.2 167.3 116.0 144.8 126.3 130.6 116.9 161.4 116.9 130.6

36.0 29.6 34.4 31.7 28.4 17.4 15.1 26.1 22.8 180.4 32.4 29.3 166.6 114.9 145.8 126.1 130.9 116.9 161.7 116.9 130.9

35.9 29.5 34.4 31.6 28.3 17.4 15.1 26.1 22.8 180.3 32.4 29.3 166.6 115.0 146.2 126.4 111.6 149.1 151.4 116.9 123.8 55.9

30.7 33.5 29.7 31.6 27.4 21.8 18.9 26.1 22.1 180.1 73.6 24.8 167.9 115.3 145.3 126.2 130.8 116.9 161.5 116.9 130.8

30.7 33.4 29.8 31.6 27.5 21.8 18.9 26.1 22.1 180.2 73.7 24.8 167.3 116.4 143.9 126.7 133.5 116.0 160.6 116.0 133.5

Figure 8. Experimental ECD spectra of 8 and 9.

Table 4. Effects of the Isolates on NO Production in LPSInduced BV-2 Microglial Cells

a

compound

IC50 (μM)

compound

IC50 (μM)

1a 1b 2a 2b 3a 3b 4 5 6

>50 5.8 ± 1.4 4.2 ± 1.5 11.8 ± 1.9 7.2 ± 2.2 13.8 ± 1.9 >50 46.5 ± 3.8 14.0 ± 2.0

7 8 9 10 11 12 13 14 minocyclinea

15.3 ± 2.7 2.7 ± 1.6 1.7 ± 1.0 >50 >50 >50 >50 >50 4.9 ± 2.1

Positive control.

were inactive (IC50 > 50 μM), which suggested that the introduction of a p-coumaroyl or feruloyl unit might be critical for the mechanism of their antineuroinflammatory activities.

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EXPERIMENTAL SECTION

General Experimental Procedures. HPLC-ECD spectra were obtained by means of chromatography on a JASCO LC-2000 instrument combined with a JASCO CD-2095 detector (from 220 to 420 nm). The other instruments and spectroscopic measurements for the purified compounds and the materials used in the isolation of the compounds were the same as those previously reported.31,32 Plant Material. The stems and leaves of Sapium discolor were collected in August 2014 in Guilin, Guangxi Province, People’s Republic of China. After specimen authentication by Professor Shao-Qing Tang (College of Life Science, Guangxi Normal University), a voucher specimen (No. SD-201408) was deposited at the State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, Guangxi Normal University. Extraction and Isolation. The air-dried stems and leaves of S. discolor (20 kg) were extracted with 95% EtOH/H2O (3 × 100 L,

Figure 6. Experimental ECD spectra of 6 and 7 and calculated ECD spectrum of 6.

1b, 2a/2b, 3a/3b, and 6−9 greatly inhibited NO production, with IC50 values in the range of 1.7−15.3 μM. Minocycline was used as a positive control (IC50 = 4.9 μM). At the same time, the cytotoxicities of these compounds on BV-2 cells were assessed by the MTT method to avoid possible effects on NO release due to reduced viability. None showed obvious cytotoxic activity at the tested concentrations (1, 10, 30, and 100 μM). For the taraxerane triterpenoids, compounds 6−9 exhibited potent inhibitory effects, while compounds 4 and 10−14

Figure 7. Selected HMBC and NOESY correlations of 8. 2256



Article

Figure 9. Effects of compounds 1a/1b−3a/3b and 5−9 on LPS-induced NO production in BV-2 microglial cells. (Data are expressed as means ± SEM (n = 3); #p < 0.05 compared with the control group, *p < 0.05 compared with LPS group; MINO = minocycline.) tR 8.6 min) and 1b (1.96 mg, tR 11.5 min) were separated from 1 by a Chiralpak AD-H column, eluting with EtOH−n-hexane (38:62). Compounds 2 and 3 were chromatographed on a Chiralpak ID column with EtOH−n-hexane (32:68 and 50:50, respectively) as a mobile phase, to yield 2a (1.95 mg, tR 9.0 min), 2b (1.95 mg, tR 12.1 min), 3a (3.14 mg, tR 6.7 min), and 3b (3.70 mg, tR 9.2 min). Sapiumin A (1): white, amorphous powder; UV (MeOH) λmax (log ε) 230 (4.36), 261 (3.79), 289 (4.01), 344 (4.06) nm; IR (KBr) νmax 3449, 1725, 1628, 1563, 1440, 1296, 1153, 1029, 832 cm−1; 1H and 13 C NMR data, see Table 1; (−) HRESIMS m/z 341.0658 [M − H]−, calcd for C18H13O7, 341.0667. (7′S,8′S)-Sapiumin A (1a): [α]20D −42 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 288 (−1.66) nm. (7′R,8′R)-Sapiumin A (1b): [α]20D +41 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 296 (+1.12) nm. Sapiumin B (2)”. white, amorphous powder; UV (MeOH) λmax (log ε) 231 (4.44), 306 (4.08) nm; IR (KBr) νmax 3430, 1698, 1615, 1570, 1490, 1415, 1310, 1093, 1052, 820 cm−1; 1H and 13 C NMR data, see Table 1; (−) HRESIMS m/z 371.0779 [M − H]−, calcd for C19H15O8, 371.0772. (7′S,8′S)-Sapiumin B (2a): [α]20D −45 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 314 (−0.55) nm. (7′R,8′R)-Sapiumin B (2b): [α]20D +39 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 314 (+0.43) nm. Sapiumin C (3): white, amorphous powder; UV (MeOH) λmax (log ε) 229 (4.31), 260 (3.73), 289 (3.97), 343 (4.04) nm; IR (KBr) νmax 3437, 1690, 1627, 1566, 1446, 1399, 1301, 1152, 824 cm−1; 1H and 13C NMR data, see Table 1; (−) HRESIMS m/z 341.0668 [M − H]−, calcd for C18H13O7, 341.0667. (7′S,8′S)-Sapiumin C (3a): [α]20D −21 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 298 (+0.24) nm. (7′R,8′R)-Sapiumin C (3b): [α]20D +20 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 284 (−0.39) nm. Sapiumic acid A (4): white, amorphous powder; [α]20D +31 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 202 (4.54) nm; IR (KBr) νmax 3444, 2941, 2869, 1714, 1692, 1634, 1466, 1381, 1261, 1209 cm−1; 1H and 13C NMR data, see Tables 2 and 3; (−) HRESIMS m/z 513.3590 [M − H]−, calcd for C32H49O5, 513.3585. Sapiumic acid B (5): white, amorphous powder; [α]20D +42 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 202 (4.58), 313 (4.30) nm; IR (KBr) νmax 3433, 2939, 2867, 1690, 1606, 1515, 1454, 1385, 1266,

each 3 h) under reflux. The residue (1.6 kg) was dispersed in H2O and sequentially partitioned with petroleum ether (PE), EtOAc, and n-BuOH. The EtOAc fraction (168.3 g) was subjected to silica gel (200− 300 mesh) column chromatography (CC), eluting with PE/acetone (10:1 to 1:1) and then CH2Cl2/MeOH (6:1 to 2:1) to yield seven fractions (A−G). Fractions A, B, and E inhibited NO production with IC50 values of 18.0, 13.1, and 4.6 μg/mL, respectively. Therefore, these active fractions were subjected to further phytochemical investigations. Fraction A (4.7 g) was separated into nine subfractions (A1−A9) via reversed-phase C18 (RP-C18) CC, eluting with MeOH/H2O (50:50 to 100:0). Subfractions A4 (60.5 mg), A5 (113.5 mg), A6 (65.0 mg), A7 (178.0 mg), and A9 (146.4 mg) were further purified by semipreparative RP-HPLC at a flow rate of 8 mL/min to obtain compounds 4 (CH3CN/H2O, 70:30, 6.0 mg, tR 43.1 min), 13 (CH3CN/H2O, 75:25, 15.0 mg, tR 43.1 min), 10 (CH3CN/H2O, 80:20, 8.7 mg, tR 43.5 min), 14 (CH3CN/H2O, 85:15, 3.0 mg, tR 49.6 min), and 11 (CH3CN/H2O, 90:10, 28.0 mg, tR 45.9 min), respectively. Fraction B (6.0 g) was separated into 15 subfractions (B1−B15) via RP-C18 CC uing a gradient of MeOH/H2O (20:80 to 100:0). Furthermore, subfractions B10 (74.0 mg), B11 (103.2 mg), B12 (137.0 mg), and B13 (90.0 mg) were purified using semipreparative RP-HPLC with an 8 mL/min flow rate to yield 12 (CH3CN/H2O, 70:30, 6.6 mg, tR 32.3 min), 9 (CH3CN/H2O, 70:30, 6.5 mg, tR 34.8 min), 8 (CH3CN/H2O, 72:28, 15.0 mg, tR 31.1 min), and 5 (CH3CN/H2O, 75:25, 12.5 mg, tR 19.8 min), respectively. Semipreparative RP-HPLC purification of subfraction B15 (418.0 mg) with CH3CN/H2O (80:20, 8 mL/min) as an isocratic solvent system yielded 6 (17.2 mg, tR 46.9 min) and 7 (6.0 mg, tR 51.5 min). Fraction E (16.7 g) was separated by an MCI column (MeOH/H2O, 30:70 to 100:0) to obtain 11 subfractions (E1−E11). Subfraction E7 (3.3 g) was further fractionated into nine subfractions (E7a−E7i) via RP-C18 CC, eluting with a mobile phase of MeOH/H2O (35:65 to 70:30). Subfraction E7e (250.0 mg) was separated by a Sephadex LH-20 column (MeOH) and then semipreparative RP-HPLC with MeOH/H2O (40:60) at 6 mL/min to yield 3 (17.2 mg, tR 50.3 min). Semipreparative RP-HPLC purification of E7f (162.0 mg) with CH3CN/H2O (25:75) at 8 mL/min afforded 1 (8.7 mg, tR 37.5 min) and 2 (11.2 mg, tR 39.8 min). The chiral HPLC separations were conducted on an Agilent 1260 system with a Daicel Chiralpak AD-H or Chiralpak ID column (250 × 4.6 mm, 5 μm) at a flow rate of 1 mL/min. Compounds 1a (1.72 mg, 2257



■

1171, 831 cm−1; 1H and 13C NMR data, see Tables 2 and 3; (−) HRESIMS m/z 617.3848 [M − H]−, calcd for C39H53O6, 617.3848. Sapiumic acid C (6): white, amorphous powder; [α]20D +38 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 202 (4.54), 224 (4.14), 314 (4.34) nm; ECD (MeOH) λmax (Δε) 218 (+3.30), 282 (−5.06), 327 (+3.61) nm; IR (KBr) νmax 3415, 2943, 2869, 1700, 1605, 1514, 1455, 1257, 1167, 830 cm−1; 1H and 13C NMR data, see Tables 2 and 3; (−) HRESIMS m/z 615.3697 [M − H]−, calcd for C39H51O6, 615.3691. Sapiumic acid D (7): white, amorphous powder; [α]20D +35 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 202 (4.40), 234 (3.95), 326 (4.15) nm; ECD (MeOH) λmax (Δε) 216 (+3.44), 287 (−4.32), 326 (+2.98) nm; IR (KBr) νmax 3443, 2943, 2869, 1704, 1633, 1516, 1461, 1256, 1164, 821 cm−1; 1H and 13C NMR data, see Tables 2 and 3; (−) HRESIMS m/z 645.3804 [M − H]−, calcd for C40H53O7, 645.3797. Sapiumic acid E (8): white, amorphous powder; [α]20D +59 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (4.94), 313 (4.51) nm; ECD (MeOH) λmax (Δε) 242 (+27.80), 347 (−5.51) nm; IR (KBr) νmax 3275, 2942, 2872, 1724, 1656, 1606, 1513, 1453, 1283, 1178, 830 cm−1; 1H and 13C NMR data, see Tables 2 and 3; (−) HRESIMS m/z 613.3542 [M − H]−, calcd for C39H49O6, 613.3535. Sapiumic acid F (9): white, amorphous powder; [α]20D +46 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 203 (4.63), 309 (4.10) nm; ECD (MeOH) λmax (Δε) 240 (+23.48), 346 (−4.55) nm; IR (KBr) νmax 3431, 2938, 2870, 1695, 1606, 1514, 1455, 1384, 1163, 831 cm−1; 1H and 13C NMR data, see Tables 2 and 3; (−) HRESIMS m/z 613.3538 [M − H]−, calcd for C39H49O6, 613.3535. ECD Calculations. The ECD calculations for compounds 1−3 and 6 were performed as those previously described,31 and the process is also described in detail in the Supporting Information. NO Production Measurements and Cell Viability Assays. As previously reported, the Griess reaction was used to measure the accumulation of nitrite, and cell viability was evaluated by the MTT assay.17,18 The BV-2 microglial cell line was kindly provided by Professor Yuanqiang Guo’s laboratory, Nankai University.

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REFERENCES

(1) Begum, S. A.; Sahai, M.; Ray, A. B. Prog. Chem. Org. Nat. Prod. 2010, 93, 1−70. (2) Chen, J. J.; Wang, T. Y.; Hwang, T. L. J. Nat. Prod. 2008, 71, 212−217. (3) Yun, B. S.; Lee, I. K.; Ryoo, I. J.; Yoo, I. D. J. Nat. Prod. 2001, 64, 1238−1240. (4) Xu, J. F.; Feng, Z. M.; Liu, J.; Zhang, P. C. Chem. Biodiversity 2008, 5, 591−597. (5) Martinez, A.; Perojil, A.; Rivas, F.; Medina-O’Donnell, M.; Parra, A. Tetrahedron 2015, 71, 792−800. (6) Kuroda, M.; Aoshima, T.; Haraguchi, M.; Marx-Young, M. C.; Sakagami, H.; Mimaki, Y. J. Nat. Prod. 2006, 69, 1606−1610. (7) Hu, J.; Shi, X.; Chen, J.; Huang, H.; Zhao, C. Fitoterapia 2012, 83, 55−59. (8) Macias-Rubalcava, M. L.; Hernandez-Bautista, B. E.; JimenezEstrada, M.; Cruz-Ortega, R.; Anaya, A. L. J. Chem. Ecol. 2007, 33, 147−156. (9) Fozdar, B. I.; Khan, S. A.; Shamsuddin, T.; Shamsuddin, K. M.; Kintzinger, J. P. Phytochemistry 1989, 28, 2459−2461. (10) Cheng, X. F.; Chen, Z. L. Fitoterapia 2000, 71, 341−342. (11) Lima, E. M. C.; Medeiros, J. M. R.; Davin, L. B. Phytochemistry 2003, 63, 421−425. (12) Ahmed, Y.; Sohrab, M. H.; Al-Reza, S. M.; Tareq, F. S.; Hasan, C. M.; Sattar, M. A. Food Chem. Toxicol. 2010, 48, 549−552. (13) Ma, J. S.; Tseng, Y. C. Flora Republicae Popularis Sinicae (Zhongguo Zhiwu Zhi); Science Press: Beijing, 1997; Vol. 44, Issue 3, p 18. (14) Xie, W. Z. Quanguo Zhongcaoyao Huibian; People’s Medical Publishing House: Beijing, 1996; p 59. (15) Liu, H. B.; Zhang, H.; Yu, J. H.; Yue, J. M. J. Asian Nat. Prod. Res. 2015, 17, 1117−1128. (16) Al Muqarrabun, L. M. R.; Ahmat, N.; Aris, S. R. S. J. Ethnopharmacol. 2014, 155, 9−20. (17) Li, J.; Li, N.; Li, X.; Chen, G.; Wang, C.; Lin, B.; Hou, Y. J. Nat. Prod. 2017, 80, 3081−3092. (18) Li, N.; Ma, Z.; Li, M.; Xing, Y.; Hou, Y. J. Ethnopharmacol. 2014, 152, 508−521. (19) Yin, H. L.; Li, J. H.; Li, J.; Li, B.; Chen, L.; Tian, Y.; Liu, S. J.; Zhang, T.; Dong, J. X. Fitoterapia 2013, 84, 360−365. (20) Kumar, S.; Ray, A. B.; Konno, C.; Oshima, Y.; Hikino, H. Phytochemistry 1988, 27, 636−638. (21) Ray, A. B.; Chattopadhyay, S. K.; Kumar, S.; Konno, C.; Kiso, Y.; Hikino, H. Tetrahedron 1985, 41, 209−214. (22) Laphookhieo, S.; Karalai, C.; Ponglimanont, C.; Chantrapromma, K. J. Nat. Prod. 2004, 67, 886−888. (23) Mahato, S. B.; Kundu, A. P. Phytochemistry 1994, 37, 1517− 1575. (24) Carpenter, R. C.; Sotheeswaran, S.; Sultanbawa, M. U. S.; Ternai, B. Org. Magn. Reson. 1980, 14, 462−465. (25) Ren, Y.; Van Schoiack, A.; Chai, H. B.; Goetz, M.; Kinghorn, A. D. J. Nat. Prod. 2015, 78, 2440−2446. (26) Hao, Z. Y.; Liang, D.; Luo, H.; Liu, Y. F.; Ni, G.; Zhang, Q. J.; Li, L.; Si, Y. K.; Sun, H.; Chen, R. Y.; Yu, D. Q. J. Nat. Prod. 2012, 75, 1083−1089. (27) Snatzke, G. Tetrahedron 1965, 21, 421−438. (28) McLean, S.; Perpick-Dumont, M.; Reynolds, W. F.; Jacobs, H.; Lachmansing, S. S. Can. J. Chem. 1987, 65, 2519−2525. (29) Yang, S. M.; Liu, X. K.; Qing, C.; Wu, D. G.; Zhu, D. Y. Acta Pharm. Sin. 2007, 42, 292−296. (30) Liu, R. H.; Kong, L. Y. Chin. Chem. Lett. 2002, 13, 1079−1082. (31) Jiang, H.; Zhang, G. J.; Liu, Y. F.; Wang, H. S.; Liang, D. J. Nat. Prod. 2017, 80, 975−982. (32) Pan, Q. M.; Li, Y. H.; Hua, J.; Huang, F. P.; Wang, H. S.; Liang, D. J. Nat. Prod. 2015, 78, 1683−1688.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00585. Calculation details and ECD spectra of compounds 1−3 and 6; 1D NMR, 2D NMR, and HRESIMS of compounds 1−9; and chiral HPLC chromatograms of compounds 1−3 (PDF)

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Article

AUTHOR INFORMATION

Corresponding Authors

*E-mail (Y. Hou): [email protected]. *E-mail (D. Liang): [email protected]. ORCID

Dong Liang: 0000-0002-9765-7548 Author Contributions #

G.-J. Zhang and Q.-M. Pan contributed equally to this study.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partially supported by the National Natural Science Foundation of China (21562017, 21462006, 81473330, and U1603125), the Natural Science Foundation of Guangxi (2018GXNSFAA138051 and 2017GXNSFFA198004), and a project of the State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources (CMEMR2016-A02, CMEMR2018B01, and CMEMR2017-A06). 2258


Coumarinolignoids and Taraxerane Triterpenoids from Sapium

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