Lipid Peroxidation and Cyclooxygenase Enzyme Inhibitory

Sep 8, 2017 - The molecular formula was assigned as C20H22O5, based on the protonated molecular ion at m/z 343.1523 [M + H]+ (calcd for C20H23O5, m/z ...
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Lipid Peroxidation and Cyclooxygenase Enzyme Inhibitory Compounds from Prangos haussknechtii Amila A. Dissanayake,† Baram A. H. Ameen,‡ and Muraleedharan G. Nair*,† †

Bioactive Natural Products and Phytoceuticals Laboratory, Department of Horticulture, Michigan State University, East Lansing, Michigan 48824, United States ‡ Department of Science, Charmo University, 46023 Chamchamal-Sualimani, Kurdistan Region, Iraq S Supporting Information *

ABSTRACT: Purification of extracts from Prangos haussknechtii Bioss afforded prenylated coumarins 1 and 2, monoterpenoid 3, amino acid derivative 4, and seven known compounds. Spectroscopic methods permitted establishment of the structures and relative configuration of these compounds. The pure isolates were tested for antioxidant and anti-inflammatory activities using lipid peroxidation (LPO), 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and cyclooxygenase (COX-1 and -2) enzyme inhibitory assays. Compounds 1−4 inhibited LPO with IC50 values between 43 and 114 μM and reduced MTT to formazan blue between 48 and 128 μM. In anti-inflammatory assays using cyclooxygenase enzymes, COX-1 and -2, these compounds showed inhibition, with IC50 values ranging from 34 to 56 μM.

T

he plant genus Prangos belongs to the Umbelliferae family and consists of about 70 species. They are widely distributed in the mountainous regions of western and central Asian countries including Turkey, Iraq, Iran, Afghanistan, and Uzbekistan.1 Previous phytochemical investigations on Prangos spp. revealed the presence of terpenoids (monoterpenes and sesquiterpenes), coumarins, and furanocoumarins. The extracts and pure isolates from Prangos spp. showed antimicrobial,2 antioxidant,3 antibacterial,4 cytotoxic,5 antihelmintic,6 aphrodisiac,6 cytokine release inhibitory,2 anti-inflammatory,7 antipyretic,7 and anti-HIV activities.8 Some Prangos spp. have been used in folk medicine as a tonic for the treatment of leukoplakia, healing scars, and digestive disorders.8,9 In this study, the aerial portion of Prangos haussknechtii Bioss, an herbaceous perennial grown in the mountainous regions of Kurdistan, Iraq, has been investigated. In Kurdistan, it is one of the ingredients in the traditional medicine used as a diuretic, as a carminative, and in alleviating tooth pain. The only report available on P. haussknechtii covers the analysis of the essential oil from its air-dried seeds. It contained mono- and sesquiterpenes such as δ-3-carene, α-pinene, and β-phellandrene as major constituents.10 Therefore, the work reported herein on the isolation and characterization of major phytochemicals in the aerial portion of P. haussknechtii and the antioxidant and anti-inflammatory activities of its pure isolates for the first time.

repeated silica gel column chromatography, followed by preparative thin-layer chromatography (PTLC), afforded five coumarins including the new derivatives 1 and 2 and substituted monoterpenoid 3. Similarly, C18 Combiflash fractionation of its MeOH extract, followed by HPLC purification, afforded three known phenolic acids, malic acid, and amino acid derivative 4 (Figure 1).

■

Figure 1. Structures of compounds 1−4.

RESULTS AND DISCUSSION The aerial portion of P. haussknechtii was extracted sequentially with hexanes and MeOH. The hexanes extract, subjected to © 2017 American Chemical Society and American Society of Pharmacognosy

Received: April 13, 2017 Published: September 8, 2017 2472

DOI: 10.1021/acs.jnatprod.7b00322 J. Nat. Prod. 2017, 80, 2472−2477

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Table 1. 1D and 2D NMR Spectroscopic Data for Compounds 1 and 2 1 position 2 3 4 4a 5 6 7 8 8a 1’ 2’ 3′ 4’ 6’ 7’ 8’ 9’ 10’ 11’ 12’

δCa, type 161.2, 113.1, 143.7, 112.9, 126.5, 107.3, 160.1, 116.6, 152.9, 21.6, 126.4, 131.3, 62.4, 166.8, 116.1, 156.5, 20.2, 27.4, 21.5, 56.0,

qC CH CH qC CH CH qC qC qC CH2 CH qC CH2 qC CH qC CH3 CH3 CH3 CH3

δH (J in Hz)a

2

6.22 (d, 9.3) 7.59 (d, 9.3)

4a, 2 2, 4a, 5, 8a

7.28 (d, 8.8) 6.80 (d, 8.8)

4, 7, 8a 4a, 7, 8

3.61 (d, 7.4) 5.49 (dd, 7.3)

7, 8, 8a, 2′, 3′ 4′, 11′

4.85 (s)

1′, 2′, 3′, 6’

5.70 (2.4)

9′, 10′

2.17 1.88 1.71 3.88

7′, 8′, 10′ 7′, 8′, 9′ 2′, 3′, 4′ 7

(d, 1.5) (d, 0.9) (d, 1.0) (s)

δ Ca

HMBCb

161.1, qC 113.1, CH 143.7, CH 112.9, qC 126.6, CH 107.3, CH 160.0, qC 116.6, qC 152.9, qC 21.6, CH2 126.7, CH 130.9, qC 63.0, CH2 173.3, qC 43.5, CH2 25.7, CH 0.95 (d, 6.9)c 1.69 (d, 0.9) 3.89 (s)

δH (J in Hz)a

HMBCb

6.22 (d, 9.3) 7.59 (d, 9.3)

4a, 2 2, 4a, 8a

7.28 (d, 8.8) 6.81 (d, 8.8)

4, 7, 8a 4a, 8

3.60 (d, 7.8) 5.49 (dd, 7.8)

7, 8, 8a, 2′, 3′ 4′, 11′

4.84 (s)

1′, 2′, 3′, 6’

2.21 (d, 6.9) 2.11 (ddd, 6.7, 6.8, 6.8) 0.95 (d, 6.9)c

8′, 9′, 10′ 7′ 6′, 7′, 8′

1.69 (d, 0.9) 3.89 (s)

2′, 3′, 4′ 7

a c

Data were measured in CDCl3 at 500 MHz (1H) and 125 MHz (13C). bHMBC correlations are from proton(s) stated to the indicated carbon. Signals were overlapped.

methylbutenoate moiety (Figure 2) (Figures S1−S10, Supporting Information). The spectroscopic data, therefore, confirmed the structure of compound 1 as shown in Figure 1.

Compound 1 was obtained as a white, amorphous powder. The molecular formula was assigned as C20H22O5, based on the protonated molecular ion at m/z 343.1523 [M + H]+ (calcd for C20H23O5, m/z 343.1545) in its positive-ion HRESITOFMS. This confirmed 10 indices of hydrogen deficiency. The UV spectrum showed characteristic absorptions for a coumarin (λmax 274 and 309 nm), and the IR spectrum showed absorption bands for carbonyl (1722 and 1608 cm−1) and aromatic ring (1652 and 1437 cm−1) functionalities.11,12 The 1 H NMR spectrum of 1 displayed two doublets at δH 6.22 and 7.59 (J = 9.4 Hz), characteristic of H-3 and H-4 of coumarins (Table 1). In addition, two proton doublets at δH 7.28 and 6.80 (J = 8.8 Hz) in its 1H NMR spectrum and 13C NMR resonances for an oxygenated aromatic carbon at δC 160.1 and an aromatic carbon at δC 116.6 indicated compound 1 as a 7,8disubstituted coumarin. The HMBC correlations observed for the −OMe signal at δH 3.88 (s, H-12′) to an oxygenated tertiary aromatic carbon at δC 160.1 in the coumarin moiety assigned its location at C-7. Similarly, HMBC correlations of H7′ (δH 5.70) to C-9′ and C-10′; H-9′ (δH 2.17) to C-7′, C-8′, C-9′, and C-10′; and H-10′ (δH 1.88) to C-7′, C-8′, and C-9′ suggested the presence of a 3-methylbutenoate moiety. The HRESITOFMS data also supported the presence of a 3methylbutenoate group, as observed by the fragment ion at m/z 243.1017 [M + H − C5H7O2]+ resulting from fragmentation of the C4′−O bond. The HMBC cross-peaks from H-1′ (δH 3.61) to C-2′ and C-3′; H-2′ (δH 5.49) to C-1′ and C-4′; H-4′ (δH 4.85) to C-1′, C-2′, C-3′, and C-11′; and H-11′ (δH 1.71) to C2′, C-3′, and C-4′ revealed the presence of an oxygenated isopentenyl group. The NOESY correlations of H-4′ (δH 4.85) to H-11′ (δH 1.71) and H-2′ (δH 5.49) supported the Econfiguration of the C-2′−C-3′ double bond. Furthermore, the connectivity of the aliphatic chain to the coumarin moieties was deduced by the HMBC correlations of H-1′ (δH 3.61) to C-8 and C-8a. The HMBC cross-peaks of H2-4′ (δH 4.85) to the C6′ carbonyl carbon (δC 166.8) suggested the presence of the 3-

Figure 2. 1H−1H COSY, key HMBC, and NOESY correlations for compounds 1 and 2.

Compound 2 was obtained as a white, amorphous powder and showed the molecular formula C20H24O5, based on the protonated molecular ion at m/z 345.1689 [M + H] + (calcd for C20H25O5, m/z 345.1702) in its positive-ion HRESITOFMS. This confirmed 9 indices of hydrogen deficiency. The UV spectrum showed characteristic absorptions for a coumarin (λmax 274 and 309 nm), and the IR spectrum showed absorption bands for carbonyl (1713 and 1607 cm−1) and aromatic ring (1652 and 1438 cm−1) functionalities.11,12 The 1 H NMR spectrum of 2 displayed two doublets at δH 6.22 and 7.59 (J = 9.3 Hz), characteristic of H-3 and H-4 of coumarins (Table 1). In addition, two proton doublets at δH 7.28 and 6.81 (J = 8.8 Hz) in its 1H NMR spectrum and 13C NMR resonances for an oxygenated aromatic carbon at δC 160 and an aromatic carbon at δC 116.6 indicated compound 2 as a 7,8disubstituted coumarin. The HMBC correlations observed for the −OMe signal at δH 3.88 (s, H-12′) to an oxygenated tertiary aromatic carbon at δC 160 in the coumarin moiety 2473

DOI: 10.1021/acs.jnatprod.7b00322 J. Nat. Prod. 2017, 80, 2472−2477

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assigned its location at C-7. Similarly, HMBC correlations of H7′ (δH 2.21) to C-8′, C-9′, and C-10′; H-8′ (δH 2.11) to C-6′, C-7′, C-9′, and C-10′; H3-9′ and H3-10′ (δH 0.95) to C-7′, C8′, and C-9′ suggested the presence of an isovaleryl moiety. The HRESITOFMS data also supported the presence of an isovaleryl group as observed by the fragment ion at m/z 243.1014 [M + H − C5H9O2]+, resulting from fragmentation of the C4′−O bond. The HMBC cross-peaks from H-1′ (δH 3.60) to C-2′ and C-3′; H-2′ (δH 5.49) to C-1′ and C-4′; H-4′ (δH 4.85) to C-2′, C-3′, and C-11′; and H-11′ (δH 1.71) to C-2′, C3′, and C-4′ revealed the presence of an oxygenated isopentenyl group. The NOESY correlations of H-4′ (δH 4.85) to H-11′ (δH 1.69) and H-2′ (δH 5.49) supported the E-configuration of the C-2′−C-3′ double bond. Furthermore, the connectivity of the aliphatic chain to the coumarin moiety was deduced by the HMBC correlations of H-1′ (δH 3.61) to C-7, C-8, and C-8a. The HMBC cross-peaks of H2-4′ (δH 4.85) to the C-6′ carbonyl carbon (δC 173.3) suggested the presence of an isovaleryl ester moiety (Figure 2) (Figures S11−S18, Supporting Information). The spectroscopic data, therefore, confirmed the structure of compound 2 as shown in Figure 1. Compound 3 was obtained as a pale yellow oil with [α]20D = −49 (c 1.0, CHCl3). The molecular formula was assigned as C15H20O5, based on the deprotonated molecular ion at m/z 279.1228 [M − H] − (calcd for C15H19O5, m/z 279.1232) in its negative-ion HRESITOFMS. This confirmed 6 indices of hydrogen deficiencies. In its 13C NMR spectrum, 15 resonances indicated two carbonyl, two quaternary sp2, an oxygenated tertiary, a methylene, five methine, and four methyl carbons. The UV and IR spectra respectively showed characteristic absorptions at λmax 248 nm and 1717 cm−1 for an α,βunsaturated carbonyl moiety in the molecule. The HMBC spectrum displayed correlations of olefinic methine H-2 (δH 5.98) to C-4 and C-15; H3-15 (δH 1.99) to C-2, C-3, and C-4; and H-4 (δH 2.76) to C-2, C-3, C-5, and C-8. The COSY spectrum also displayed strong correlations of H-2 (δH 5.98) to H3-15 (δH 1.99) and H-4 (δH 2.76) to H3-15 (δH 1.99) and suggested the presence of a β-methyl-α,β-unsaturated carbonyl moiety. In addition, HMBC correlations of H3-11 (δH 1.70) and H3-12 (δH 1.76) to C-10, C-9, and C-6 along with COSY correlations between H3-12 (δH 1.76) and olefinic H-9 (δH 5.98) provided evidence for the presence of a 2-methylpropenyl moiety. The COSY correlation between H-9 (δH 5.98) and H-7 (δH 3.54) also supported the 2-methylpropenyl moiety attached to the oxygenated tertiary C-6. The HMBC correlation from H8 (δH 2.62) to C-2 further suggested the presence of an eightmembered ring, and correlation from H-4 (δH 2.76) to C-8 and NOESY correlations from H-8 (δH 2.62) to H-5 (δH 4.84) supported an ethereal linkage between C-5 and C-8, i.e., the presence of a tetrahydrofuran moiety. An acetyl functionality was indicated based on the chemical shifts and HMBC correlations between H3-14 (δH 1.59) and carbonyl carbon C-13. The connection of the acetoxy moiety was deduced by HMBC correlations between H-7 (δH 3.54) and C-13. The 2Z configuration was confirmed by the NOESY correlation of H-2 (δH 5.98) to H3-15. Correlations from H3-14 (δH 1.59) to H4b, H-5, and H-8 were evident for an α- orientation of the acetoxy moiety, and correlations from H-7 (δH 3.54) to H3-11 and H3-12 supported a 2-methylpropene side chain in a β orientation (Figure 1) (Figures S19−S29, Supporting Information). Therefore, the spectroscopic data were in agreement with the proposed structure of compound 3 as shown in Figure 1.

Figure 3. 1H−1H COSY, key HMBC, and NOESY correlations for compounds 3 and 4.

Compound 4, a white, amorphous powder with [α]20D = +12 (c 1.0, H2O), gave the molecular formula C18H34N2O5 based on the protonated MeOH adduct at m/z 391.2847 [M + H + CH3OH]+ (calcd for C19H39N2O6, m/z 391.2808) in its positive-ion HRESITOFMS. This confirmed 3 indices of hydrogen deficiency. Both 1H and 13C NMR spectroscopic data also supported the molecular formula of compound 4 (Table 3). Analysis of the combined 1D and 2D NMR data established that compound 4 possessed three carbonyl carbons, six sp3 methines, three methylenes, and six methyl groups. The IR absorption at 1581 cm−1 demonstrated the presence of carbonyls, as confirmed by the presence of resonances at δC 175.5 and 174.1 in its 13C NMR spectrum. Detailed analyses of HOMODEC, HMBC, and 1H−1H COSY NMR spectroscopic data provided evidence of two substructures. For example, decoupling of the multiplet at δH 3.71 resulted in the collapse of the multiplet at δH 1.67. Similarly, selective irradiation of the multiplet at δH 1.67 resulted in collapse of the multiplet at δH 3.71 to a singlet as well as the two methyl doublets at δH 0.92 to singlets (Figures S38, S39, Supporting Information). This permitted assignment of the multiplet at δH 3.71 to H-2, δH 1.67 to H-3 and H-4, and the doublet of doublets at δH 0.92 to Me-5 and Me-6. The COSY correlations from H-2 (δH 3.71) to H-3 and H-4 (δH 1.67) and from H-3 and H-4 (δH 1.67) to H35 and H3-6 (δH 0.92) supported the presence of a 2methylpropyl fragment (Figure S45, Supporting Information). The HMBC cross-peaks of H3-5 and H3-6 (δH 0.92) to the carbonyl carbon C-1 and H-3 and H-4 (δH 1.67) to the carbonyl carbon C-1 supported the presence of a 2-amino-4methylpentanamide moiety (Figure S44, Supporting Information). The second subunit showed the presence of a six-carbon unit with two methyl, a methylene, two methane, and a carbonyl carbon. Decoupling of the doublet at δH 3.62 impacted the multiplet at δH 1.94. Similarly, irradiation of the δH 1.94 multiplet resulted in the collapse of the doublet at δH 3.62 to a singlet, the multiplets at δH 1.45 and 1.26 to two sets of ddd (7.7, 7.2, and 7.2 Hz), and the methyl doublet at δH 0.97 to a singlet. Furthermore, decoupling of the doublet at δH 0.97 changed the multiplet at δH 1.94 to a quintet (4.4, 4.4, and 4.3 Hz) (Figures S40−S43, Supporting Information). Therefore, the resonances at δH 3.62, 1.94, 1.45, 1.26, 0.91, and 0.97 were assigned to H-2′, H-3′, H-4′a, H-4′b, H3-5′, and H3-6′, respectively. The COSY correlations from H-2′ (δH 3.62) to H-3′ (δH 1.94) and from H-3′ (δH 1.94) to H-4′a (δH 1.45), H4′b (δH 1.26), H3-5′ (δH 0.91), and H3-6′ (δH 0.97) established the presence of a short-chain aliphatic moiety (Figure S45, Supporting Information). Analysis of the HMBC cross-peaks from H3-6′ (δH 0.97) to C-1′ and H3-5′ (δH 0.91) to C-1′ 2474

DOI: 10.1021/acs.jnatprod.7b00322 J. Nat. Prod. 2017, 80, 2472−2477

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Table 2. 1D and 2D NMR Spectroscopic Data for Compound 3 δC,a type

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

196.9, 127.2, 160.1, 34.9, 76.1, 78.6, 94.3, 53.5, 117.7, 132.6, 18.2, 26.0, 158.2, 27.4, 24.6,

δH (J in Hz)a

qC CH qC CH2 CH qC CH CH CH qC CH3 CH3 qC CH3 CH3

HMBCb

COSY

NOESY

5.98 (m)c

4, 15

15

15

2.76 (ddd, 5.8, 5.9, 6.3) 4.84 (dd, 5.9, 6.8)

2, 3, 5, 8 3, 6

15 4

15 4, 14

5.34 (d, 11.2) 2.62 (d, 6.8) 5.98 (m)c

6, 10, 13 1, 2, 6 12

9 11, 12

11 5, 14 12

1.70 (s) 1.76 (s)

6, 9, 10, 12 6, 9, 10, 11

9 9

7 9

1.59 (s) 1.99 (s)

13 2, 3, 4

2, 4

5, 8 2

a c

Data were measured in CDCl3 at 500 MHz (1H) and 125 MHz (13C). bHMBC correlations are from proton(s) stated to the indicated carbon. Signals were overlapped.

Table 3. 1D and 2D NMR Spectroscopic Data for Compound 4 position 1 2 3 4 5 6 1′ 2′ 3′ 4′a 4′b 5′ 6′

δCa, type 175.5, 53.3, 39.7, 24.0, 21.9, 20.7, 174.1, 59.4, 35.8, 24.3,

qC CH CH2 CH CH3 CH3 qC CH CH CH2

11.0, CH3 14.6, CH3

δH (J in Hz)a

HMBCb

Table 4. Antioxidant Activity of Compounds 1−4 as Determined by MTT and Lipid Peroxidation Assays COSY

IC50 (μM) compound

3.71 1.67 1.67 0.92

(m) (m)c (m)c (dd, 5.8, 5.8)c

1, 1, 2, 2, 2,

3, 2, 3, 3, 3,

4 4 5 4 4

3.62 1.94 1.45 1.26 0.91 0.97

(d, 3.9) (m) (m) (m) (dd, 7.3, 7.3) (d, 6.8)

1′, 4′ 2′, 3′, 3′, 1′,

3′, 4′, 6′ 3′, 5′, 6′ 5′, 6′ 4′ 2′, 3′, 4′

3 2, 3, 4, 4,

1 2 3 4 vitamin C TBHQ BHT BHA

4 5, 6 6 5

3′ 2′, 4′b 4′b, 5′ 5′, 4′a 4′a, 4′b 3′

MTTa,b 121.9 128.2 67.1 48.6 142.1 150.4 NDd NDd

± ± ± ±

4.9 3.7 2.7 2.4

LPOa,c 62.6 86.1 113.9 43.0 NDd 10 6.9 5.0

± ± ± ±

3.1 4.2 4.3 2.8

a Data were calculated as 50% inhibitory concentration (IC50). Values are expressed as mean ± SEM (n = 2). bVitamin C and TBHQ were used as the positive controls for the MTT assay. cTBHQ, BHT, and BHA were used as the positive controls for the LPO assay. dData not available.

a

Data were measured in D2O at 500 MHz (1H) and 125 MHz (13C). HMBC correlations are from proton(s) stated to the indicated carbon. cSignals were overlapped.

b

Arachidonic acid converts to prostaglandins, the inflammation-causing hormones, by the catalysis of cyclooxygenase (COX) enzymes.14 Therefore, inhibition of COX enzymes prevents the production of prostaglandins and other inflammation-causing intermediates. Coumarins 1 and 2 showed COX-1 enzyme inhibitory activity with IC50 values of 36.8 and 47.7 μM, respectively, when compared with the standards aspirin, ibuprofen, and naproxen, with IC50 values of 600, 72.8, and 52.2 μM, respectively (Table 5). Compound 3 at 100 μM concentration showed weak inhibition of both COX enzymes, whereas compound 4 exhibited inhibition of the COX-2 enzyme with an IC50 value of 34.6 μM when compared with the standards Celebrex and naproxen, with IC50 values of 1.3 and 52.2 μM, respectively (Table 5). It is significant to note that the COX enzyme inhibitory activity of coumarins 1 and 2 was comparable to the activity of the over-the-counter nonsteroidal anti-inflammatory drugs (NSAIDs) aspirin, naproxen, and ibuprofen. Compound 4 inhibited the COX-2 enzyme at a higher rate than the COX-1 enzyme, a trend similar to the inhibitory activity of the prescription anti-inflammatory drug Celebrex. Other isolates from P. haussknechtii were identified as isoimperatorin,15 osthol,15 oxypeucedanin,15 malic acid,16 p-

established the presence of a 2-amino-3-methylpentanamide moiety (Figure S44, Supporting Information). Therefore, the structure of compound 4 was assigned as shown in Figure 1. Potential antioxidant activity was determined for all pure isolates using the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT)13 and lipid peroxidation (LPO)14 assays. The MTT assay detects reducing compounds, and Table 4 shows the IC50 values obtained for the pure compounds 1−4. Compounds 3 and 4 exhibit strong antioxidant activity with IC50 values of 67.1 and 48.6 μM, respectively and coumarins 1 and 2 exhibit IC50 values of 121.9 and 128.2 μM, respectively, when compared with the positive controls vitamin C and TBHQ (tert-butylhydroquinone) with IC50 values of 142.1 and 150.4 μM, respectively. The LPO assay detects compounds that can scavenge free radicals. The antioxidant activity (LPO) of isolates 1−4 was determined by using the large unilamellar vesicles (LUVs) model system,14 and they exhibited weak IC50 values of 43−113.9 μM as compared to the commercial antioxidants BHT (butylated hydroxytoluene), BHA (butylated hydroxyanisole), and TBHQ, with IC50 values of 10, 6.9, and 5 μM, respectively (Table 4). 2475

DOI: 10.1021/acs.jnatprod.7b00322 J. Nat. Prod. 2017, 80, 2472−2477

Journal of Natural Products

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Aldrich Chemical Co., arachidonic acid was from Oxford Biomedical Research, Inc. (Oxford Biomedical Research, Inc., Oxford, MI, USA), and 1-stearoyl-2-linoleoyl-sn-glycerol-3-phosphocholine was from Avanti Polar Lipids (Alabaster, AL, USA). The fluorescent probe, 3[p-(6-phenyl)-1,3,5-hexatrienyl]phenylpropionic acid, was purchased from Molecular Probes (Eugene, OR, USA). COX-1 and -2 enzymes were prepared in our laboratory from ram seminal vesicles (Oxford Biomedical Research, Inc.) and insect cells cloned with human PGHS2 enzyme, respectively. All enzymes and reagents were stored in the Bioactive Natural Products and Phytoceuticals Laboratory at Michigan State University (East Lansing, MI, USA). Plant Material. The aerial portions of P. haussknechtii Bioss were collected from Halgard mountain (altitude 2300 m), Kurdistan, in May 2015. A voucher specimen (#6758) has been deposited at the herbarium of Salahaddin University, Erbil, Kurdistan. Extraction and Isolation. The dried aerial parts of P. haussknechtii (250 g) were sequentially extracted with hexanes (3 × 2.5 L, 8 h) and MeOH (3 × 2.5 L, 24 h) and evaporated under vacuum to afford hexanes extract PHH (15.8 g) and MeOH extract PHM (17.6 g). An aliquot of PHH (2.11 g) was fractionated by silica gel MPLC by eluting with hexanes−acetone (4:1 and 2:1, v/v), followed by acetone (100%), to yield fractions A (459 mg), B (148 mg), C (189 mg), D (264 mg), E (645 mg), F (180 mg), and G (191 mg), respectively. The major components in fractions A−C were chlorophylls and hence were kept aside. An aliquot of fraction D (41 mg) was further purified by preparative TLC using CHCl3−MeOH (50:1, v/v, developed 2×) to yield isoimperatorin15 (30 mg, Figures S48−S50, Supporting Information). Similarly, an aliquot of fraction E (37 mg) was purified by preparative TLC using hexanes−acetone (4:1, v/v, developed 2×) to yield osthol15 (25 mg, Figures S51, S52, Supporting Information). Fraction F (170 mg) was fractionated by silica gel MPLC, eluted with hexanes−acetone (4:1, 2:1, v/v), and yielded fractions H (16 mg), I (54 mg), and J (92 mg), respectively. An aliquot of fraction I (48 mg) was purified by preparative TLC using CHCl3−MeOH (100:1, v/v, developed 3×) to yield compounds 1 (15 mg, Figures S1−S10, Supporting Information), 2 (16 mg, Figures S11−S18, Supporting Information), and 3 (11 mg, Figures S19−S29, Supporting Information), respectively. Similarly, an aliquot of fraction J (38 mg) was further purified by preparative TLC using CHCl3−MeOH (100:1, v/v, developed 2×) to yield oxypeucedanin15 (25 mg, Figures S53, S54, Supporting Information). Preliminary TLC analysis of fraction G indicated that it contained primarily free fatty acids and hence was kept aside. An aliquot of PHM (1.33 g) was fractionated on a reversed-phase Combiflash purification system eluting with MeOH−H2O (1:9, 1:4, 3:7, 2:3, and 1:1, v/v) and afforded fractions L (886 mg), M (116 mg), N (89 mg), O (35 mg), P (32 mg), and Q (32 mg). Elution of the column with MeOH (100%), monitored at 210 nm and 5 mL/min flow rate, gave fraction R (91 mg). An aliquot of fraction L (80 mg) was crystallized from MeOH−H2O (4:1, v/v) to yield malic acid16 (69 mg, Figures S55, S56, Supporting Information). Purification of fractions M and N was carried out on a Waters 2010 HPLC system (Waters Corp.) equipped with a reversed-phase XTerra Preparative MS column (10 μm, 19 × 250 mm) using a flow rate of 3 mL/min. Detection was by UV, using a PDA detector, at 210 and 254 nm wavelengths. A MeOH−H2O (2:98, v/v) isocratic solvent system, used as the mobile phase for purification of fraction M (30 mg), yielded compound 4 (16 mg, 26 min, Figures S30−S46, Supporting Information). Similarly, a MeOH−H2O (85:15, v/v) isocratic solvent system, used as the mobile phase for purification of fraction N (88 mg), afforded p-coumaric acid17 (13 mg, 28 min, Figures S57, S58, Supporting Information) and 3-phenyllactic acid18 (28 mg, 33 min, Figures S59−S62, Supporting Information). Fractions O, P, and Q were complex mixtures, as indicated by HPLC profiles. The complexity of the mixture and its paucity did not permit further purification of fractions O, P, and Q. An aliquot of fraction R (30 mg) was crystallized from MeOH to yield ferulic acid14 (21 mg, Figures S63−S65, Supporting Information). Compound 1: UV (MeOH) λmax (log ε) 309.8 (3.14) and 274.5 nm (2.03); IR νmax (KBr) 2918, 1711, 1608, 1437, 1281, 1249, 1141, 1116

Table 5. Anti-inflammatory Activity of Compounds 1−4 As Determined by Cyclooxygenase Inhibitory (COX-1 and -2) Assays IC50 (μM) compound

COX-1

a,b

COX-2a,b

1 2 3 4 aspirin ibuprofen Celebrex naproxen

36.8 ± 3.1 47.7 ± 2.9 −c −c 600 72.8 −c 52.2

45.3 ± 2.8 56.4 ± 2.6 −c 34.6 ± 1.8 −c −c 1.31 52.2

a

Data were calculated as 50% inhibitory concentration (IC50). Values are expressed as mean ± SEM (n = 2). bAspirin, ibuprofen, Celebrex, and naproxen was used as the positive controls for the COX-1 and -2 assays. cNot active.

coumaric acid,17 3-phenyllactic acid,18 and ferulic acid14 by spectroscopic methods. LPO (Table 2, Supporting Information) and COX enzyme inhibitory activities (Table 2, Supporting Information) of these compounds were also significant. Bioassay results indicated that the coumarin osthol was the most active in inhibiting COX-1 and -2 enzymes, with IC50 values of 52.5 and 81.1 μM, respectively. However, the coumarins isoimperatorin, osthol, and oxypeucedanin inhibited LPO with IC50 values of 63.7, 119.7, and 74.8 μM, respectively, and malic acid and 3-phenyllactic acid with IC50 values of 97.7 and 161.4 μM, respectively. We have reported both LPO and COX enzyme inhibitory activities of p-coumaric and ferulic acids earlier.14 A mass balance on pure isolates suggested that aerial portions of P. haussknechtii contained 22 mg of coumarins per g of dry material. The in vitro bioassay results reported herein and the abundance of bioactive compounds in P. haussknechtii support the anecdotal medicinal claims such as its use in mitigating inflammatory diseases.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded on a PerkinElmer model 341 polarimeter at 20 °C. UV spectra were recorded using a PerkinElmer Lambda35 UV/vis spectrometer, and infrared (IR) spectra were acquired using a Mattson Galaxy Series FTIR 3000 spectrometer. NMR spectra were recorded on an Agilent DirectDrive2 500 MHz spectrometer (Agilent Technologies, Palo Alto, CA, USA). HRESITOFMS data were acquired on a Waters Xevo G2-S Q-TOF LC mass spectrometer (Waters Corporation, Milford, MA, USA). Merck silica gel (60 mesh size, 35−70 μm, EMD Chemicals, Inc., Gibbstown, NJ, USA) was used for MPLC. Silica gel plates (250 μm) (Analtech, Inc., Newark, DE, USA) were used for TLC. TLC plates were viewed under UV light at 254 and 366 nm in a Spectroline CX-20 ultraviolet fluorescence analysis cabinet (Spectroline Corporation, Westbury, NY, USA). COX assays were performed in a micro oxygen chamber with an oxygen electrode (Instech Laboratories, Plymouth Meeting, PA, USA) attached to a YSI model 5300 biological oxygen monitor (Yellow Springs Instrument, Inc., Yellow Springs, OH, USA). A Turner model 450 fluorometer (Barnstead Thermolyne Corporation, Dubuque, IA, USA) was used to record the resultant emission fluorescence of isolates in the LPO assay. The fluorescence in the wells of MTT assay plates was measured using a Bio-Tek Elx800 universal microplate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA). All solvents used for isolation and purification were of ACS reagent grade (SigmaAldrich Chemical Co., St. Louis, MO, USA). MTT, TBHQ, BHA, BHT, aspirin, naproxen, and ibuprofen were purchased from Sigma2476

DOI: 10.1021/acs.jnatprod.7b00322 J. Nat. Prod. 2017, 80, 2472−2477

Journal of Natural Products

Article

cm−1; 1H and 13C NMR data, see Table 1; HRESITOFMS m/z 343.1523 [M + H]+ (calcd for C20H23O5, m/z 343.1545). Compound 2: UV (MeOH) λmax (log ε) 309.4 (3.05) and 274.5 nm (2.01); IR νmax (KBr) 2959, 1713, 1608, 1283, 1250, 1118, 1092 cm−1; 1 H and 13C NMR data, see Table 1; HRESITOFMS m/z 345.1689 [M + H]+ (calcd for C20H25O5, m/z 345.1702). Compound 3: pale yellow oil; [α]20D = −49 (c 1.0, CHCl3); UV (MeOH) λmax (log ε) 248.4 nm (2.66); IR νmax (KBr) 3432, 2924, 1717, 1646, 1460, 1383 cm−1; 1H and 13C NMR data, see Table 2; HRESITOFMS m/z 279.1228 [M − H]− (calcd for C15H19O5, m/z 279.1232). Compound 4: [α]20D = +12 (c 1.0, H2O); UV (H2O) λmax (log ε) 258.8 nm (2.72); IR νmax (KBr) 3432, 2962, 1581, 1517, 1397 cm−1; 1 H and 13C NMR data, see Table 3; HRESITOFMS m/z 391.2847 [M + H + CH3OH]+ (calcd for C19H39N2O5, m/z 391.2808). MTT Antioxidant Assay. The antioxidant activity of all isolates by the MTT assay was determined according to a previous report.13 Lipid Peroxidation Inhibitory Assay. The antioxidant activity of all isolates by the LPO inhibitory assay was determined according to the reported procedure.14 Cyclooxygenase Enzymes Inhibitory Assays. The COX-1 and -2 enzyme inhibitory activity of compounds was measured according to the published procedure.14

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(3) Kogure, K.; Yamauchi, I.; Tokumura, A.; Kondou, K.; Tanaka, N.; Takaishi, Y.; Fukuzawa, F. Phytomedicine 2004, 11, 645−651. (4) Iqbal, P. F.; Bhat, A. R.; Azam, A. Eur. J. Med. Chem. 2009, 44, 2252−2259. (5) Mavi, A.; Terzi, Z.; Ozgen, U.; Yildirim, A.; Coskun, M. Biol. Pharm. Bull. 2004, 27, 702−705. (6) Başer, K. H.; Demirci, B.; Demirci, F.; Bedir, E.; Weyerstahl, P.; Marschall, H.; Duman, H.; Aytaç, Z.; Hamann, M. T. Planta Med. 2000, 66, 674−677. (7) Murray, R. D. H.; Mendez, J.; Brown, S. A. In The Natural Coumarins: Occurrence, Chemistry and Biochemistry; John Wiley & Sons, 1982; Chapter 2, pp 45−55. (8) Shikishima, Y.; Takashi, Y.; Honda, G.; Ito, M.; Takeda, Y.; Kodzhimatov, O. K.; Ashurmetov, O. Chem. Pharm. Bull. 2001, 49, 877−880. (9) Razavi, S. M.; Nazemiyeh, H.; Hajiboland, R.; Kumarasamy, Y.; Delazar, A.; Nahar, L.; Sarker, S. D. Rev. Bras. Farmacogn. 2008, 18, 1− 5. (10) Sajjadi, S. E.; Mehregan, I. Daru, J. Pharm. Sci. 2003, 11, 78−81. (11) Ogawa, H.; Hasumi, K.; Sakai, K.; Murakawa, S.; Endo, A. J. J. Antibiot. 1991, 44, 762−767. (12) Hua, H. M.; Wang, S. X.; Wu, L. J.; Li, X.; Zhu, T. R. Acta Pharm. Sin. B 1992, 27, 279−282. (13) Liu, Y.; Nair, M. G. J. Nat. Prod. 2010, 77, 1193−1195. (14) Jayaprakasam, B.; Vanisree, M.; Zhang, Y.; Dewitt, D. L.; Nair, M. G. J. Agric. Food Chem. 2006, 54, 5375−5381. (15) Thanh, P. N.; Jin, W.; Song, G.; Bae, K.; Kang, S. S. Arch. Pharmacal Res. 2004, 27 (12), 1211−1215. (16) Akamatsu, F.; Hashiguchi, T.; Hisatsune, Y.; Oe, T.; Kawao, T.; Fujii, T. Food Chem. 2007, 217, 112−116. (17) Pei, K.; Ou, J.; Huang, J.; Ou, S. J. Sci. Food Agric. 2016, 96, 2952−2962. (18) Lavermicocca, P.; Valerio, F.; Visconti, A. Appl. Environ. Microbiol. 2003, 69, 634−640.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00322. 1D and 2D NMR, IR, UV, and HRESITOFMS spectra and bioassay results of compounds (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +1 517 355 0406. Fax: +1 517 353 0890. E-mail: nairm@ msu.edu. ORCID

Amila A. Dissanayake: 0000-0001-6274-6017 Muraleedharan G. Nair: 0000-0001-9604-0055 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research is a contribution from Michigan State University AgBioResearch. The authors acknowledge Mr. Al-Dalawi for collecting the plant material and Mr. Al-Makhmuri for identifying the plant material. B.A.H.A. received a Fulbright Fellowship to conduct research at MSU. The NMR spectra were recorded on instrumentation purchased in part with the funds from NIH grant no. 1-S10-RR04750, NSF grant no. CHE-88-00770, and NSF grant no. CHE-92-13241. MS data were obtained at the Michigan State University Mass Spectrometry Facility, which is supported, in part, by a grant (DRR-00480) from the Biotechnology Research Technology Program, National Center for Research Resources, National Institutes of Health.

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REFERENCES

(1) Razavi, S. M.; Nazemiyeh, H.; Delazar, A.; Hajiboland, R.; Rahman, M. M.; Gibbons, S.; Nahar, L.; Sarker, S. D. Phytochem. Lett. 2008, 1, 159−162. (2) Tada, Y.; Shikishima, Y.; Takaishi, Y.; Shibata, H.; Higuti, T.; Honda, G.; Ito, M.; Takeda, Y.; Kodzhimatov, O. K.; Ashurmetov, O.; Ohmoto, Y. Phytochemistry 2002, 59, 649−654. 2477

DOI: 10.1021/acs.jnatprod.7b00322 J. Nat. Prod. 2017, 80, 2472−2477