Octulosonic Acid Derivatives from Roman Chamomile (Chamaemelum

Article pubs.acs.org/jnp

Octulosonic Acid Derivatives from Roman Chamomile (Chamaemelum nobile) with Activities against Inflammation and Metabolic Disorder Jianping Zhao,† Shabana I. Khan,†,‡ Mei Wang,† Yelkaira Vasquez,‡ Min Hye Yang,† Bharathi Avula,† Yan-Hong Wang,† Cristina Avonto,† Troy J. Smillie,† and Ikhlas A. Khan*,†,‡,§ †

National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, University of Mississippi, University, Mississippi 38677, United States ‡ Department of Pharmacognosy, School of Pharmacy, University of Mississippi, University, Mississippi 38677, United States § Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia S Supporting Information *

ABSTRACT: Six new octulosonic acid derivatives (1−6) were isolated from the flower heads of Roman chamomile (Chamaemelum nobile). Their structures were elucidated by means of spectroscopic interpretation. The biological activity of the isolated compounds was evaluated toward multiple targets related to inflammation and metabolic disorder such as NAG-1, NF-κB, iNOS, ROS, PPARα, PPARγ, and LXR. Similar to the action of NSAIDs, all the six compounds (1−6) increased NAG-1 activity 2−3-fold. They also decreased cellular oxidative stress by inhibiting ROS generation. Compounds 3, 5, and 6 activated PPARγ 1.6−2.1-fold, while PPARα was activated 1.4-fold by compounds 5 and 6 only. None of the compounds showed significant activity against iNOS or NF-κB. This is the first report of biological activity of octulosonic acid derivatives toward multiple pathways related to inflammation and metabolic disorder. The reported anti-inflammatory, hypoglycemic, antiedemic, and antioxidant activities of Roman chamomile could be partly explained as due to the presence of these constituents.

profile the characteristic secondary metabolites in Roman chamomile, six new caffeoyl derivatives possessing a rare octulosonic acid skeleton (1−6) were isolated and identified from the flower heads of C. nobile. A multitargeted screening approach was followed to determine the pharmacological effects of these metabolites. The anti-inflammatory activity of isolated compounds was tested through a series of cellular assays targeting NAG-1 (NSAIDs-activated gene-1), NF-κB (nuclear transcription factor κB), iNOS (inducible nitric oxide synthase), and ROS (reactive oxygen species). The compounds were also tested for their effects on peroxisome proliferatoractivated receptors (PPARα and PPARγ) and liver X receptor (LXR), which are not only considered to be significant targets related to metabolic disorder (diabetes and cardiovascular disease) but also linked to inflammatory processes.9,10 Herein, the isolation and structural elucidation of these new compounds and their biological activity are reported.

Chamaemelum nobile (L.) All. (syn. Anthemis nobilis L. and Chamomilla nobilis Godr.) is a perennial herb of the Asteraceae family, with the common name Roman chamomile. The plant is native to southern Europe but is now widely cultivated in all parts of Europe, northern Africa, North America, and southwest Asia. Roman chamomile is listed by the Council of Europe as a natural source of food flavoring (category N2).1 The flowering tops of the plant are used to make teas, liquid extracts, capsules, or tablets sold as dietary supplements. Roman chamomile is listed as GRAS (generally recognized as safe) in the United States.2,3 Traditionally, the plant has been used internally (in oral dosage forms) or externally (topical application forms) as a household medicine for the treatment of a variety of health disorders, such as dyspepsia, nausea, rheumatic pain, eczema, wounds, hemorrhoids, and neuralgia. Pharmacological studies have shown that Roman chamomile possesses therapeutic properties such as anti-inflammatory, hypoglycemic, antiedemic, and antioxidant activities.2,4,5 Another herb, Chamomilla recutita L. Rauschert (syn. Matricaria recutita L.), belonging to the same family and commonly known as German chamomile, is traditionally used in a similar way to Roman chamomile. Previous studies have revealed that both Roman and German chamomiles contain flavonoids, terpenoids, coumarins, steroids, polysaccharides, and organic acids.5−7 However, differences exist between the chemical compositions of these two herbs.8 In an effort to © 2014 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The ethyl acetate fraction obtained from the methanolic extract of the flower heads of C. nobile was subjected to a series of Special Issue: Special Issue in Honor of Otto Sticher Received: September 24, 2013 Published: January 28, 2014 509

dx.doi.org/10.1021/np400780n | J. Nat. Prod. 2014, 77, 509−515

Journal of Natural Products

Article

of a 2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]octane-5-carboxylate moiety. The presence of a bicyclo-ketal group in this octulosonic acid moiety was supported by the downfield shift of the signals of two oxygenated methinic carbons at δC 78.2 (C-1) and 80.6 (C-7).11 The downfield-shifted signals (δH 5.00 and 4.72) of the methylene group (C-9) and their HMBC correlations to the carbon of the carboxylic group in the caffeoyl moiety indicated that the caffeoyl moiety is attached to the octulosonic acid moiety at C-9 through an ester linkage. zCOSY experiments were conducted to determine the vicinal proton−proton coupling constants (3JH,H) of the compound.12 The coupling constant values between H-4a (axial H at C-4) and H-3, H-4e (equatorial H at C-4) and H-3, H-3 and H-2, and H-2 and H-1 were 5.0, 1.2, 5.1, and 4.8 Hz, respectively, indicating the equatorial orientations of both H-1 and H-3 and the axial orientation of H-2 on the pyran ring. The observation of a NOE correlation between H-4a (δH 2.31) and H-2 (δH 4.06) supported the assignment of the axial orientation of H-2 (Figure 1). To clarify the relative configuration at C-7, 2D excitation-sculptured indirect-detection (EXSIDE) experiments were performed to measure the three-bond carbon−proton coupling constants (3JC,H).13 The results showed the 3JC,H values for the couplings of C-2/H-7 and C-7/H-2 to be 7.6 and 6.8 Hz, respectively. The large 3JC,H values of these carbon−proton couplings indicated that the spatial orientations of these carbon/proton pairs are anti.14 Thus, the structure of compound 1 was determined as rel-(1S,2R,3R,5S,7R)-7-[(E)caffeoyloxymethyl]-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]octane-5-carboxylic acid. Compound 2 was isolated as a light yellow, amorphous solid. Its UV absorption pattern (λmax 327, 244, 218 nm) was similar to that of compound 1. The HRESIMS analysis, which showed a [M + H]+ ion at m/z 545.1306, revealed a molecular formula of C26H24O13 for 2. In comparison to 1, compound 2 was found

chromatographic separation steps to afford six new compounds (1−6). Compound 1 was obtained as an amorphous solid. Its molecular formula was determined as C17H18O10 based on the [M + H]+ ion peak at m/z 383.0992 (calcd 383.0978) in the HRESIMS. Its UV spectrum showed strong maximum absorptions at 327 and 245 nm, which implied the presence of an unsaturated carbonyl conjugated with an aromatic ring. The 13C and DEPT NMR spectra of 1 showed the presence of 17 carbons, consisting of two carbonyl groups, five olefinic, four oxygenated methine, two methylene, and four quaternary carbons. Its 1H NMR spectrum showed signals at δH 7.05 (1H, d, J = 2.1 Hz), 6.90 (1H, dd, J = 8.2, 2.1 Hz), and 6.78 (1H, d, J = 8.2 Hz) for a 1,2,4-trisubstituted benzene ring and signals at δH 7.54 (1H, d, J = 15.9 Hz) and 6.25 (1H, d, J = 15.9 Hz) for a pair of trans-coupled olefinic protons. They were assigned to a caffeoyl moiety on the basis of their 1H and 13C NMR chemical shifts, coupling constants, and 2D HSQC and HMBC correlation information (Tables 1 and 2). The COSY and HSQC spectra established connections of the protonated carbons from C-4 to C-9 (Figure 1). The HMBC correlations between H-4e/H-4a (δH 2.31/2.26) and C-10 (δC 175.2), between H-3 (δH 4.16) and C-5 (δC 107.0), and between H-1 (δH 4.44) and C-5 (δC 107.0) further led to the establishment

Table 1. 1H NMR Data (δH in ppm, J in Hz) of Compounds 1−6 (400 MHz, MeOD) position 1 2 3 4 7 9 2′ 3′ 5′ 8′ 9′ 2″ 3″ 5″ 8″ 9″ 2‴ 3‴ 5‴ 8‴ 9‴ Me

1

2

3

4

5

6

4.44 (dd, 4.8, 4.6) 4.06 (dd, 5.1, 4.8) 4.16 (ddd, 5.1, 5.0, 1.2) 2.31 (dd, 15.1, 1.2) 2.26 (dd, 15.1, 5.0) 4.39 (ddd, 7.9, 4.6, 3.2) 5.00 (dd, 12.2, 7.9) 4.72 (dd, 12.2, 3.2) 6.25 (d, 15.9) 7.54 (d, 15.9) 7.05 (d, 2.1) 6.78 (d, 8.2) 6.90 (dd, 8.2, 2.1)

4.55 (dd, 4.8, 3.8) 4.27 (dd, 5.2, 4.8) 5.46 (ddd, 5.0, 5.2, 1.3) 2.38 (dd, 15.4, 5.0) 2.34 (dd, 15.4, 1.3) 4.49 (ddd, 8.6, 3.3, 3.8) 5.29 (dd, 12.1, 8.6) 4.63 (dd, 12.1, 3.3) 6.43 (d,15.9) 7.59 (d, 15.9) 7.08 (d, 2.1) 6.74 (d, 8.2) 6.97 (dd, 8.2, 2.1) 6.32 (d, 15.9) 7.63 (d, 15.9) 7.14 (d, 2.1) 6.79 (d, 8.1) 6.96 (dd, 8.1, 2.1)

4.73 (dd, 5.3, 5.1) 5.47 (dd, 5.6, 5.3) 5.64 (ddd, 5.6, 5.4, 1.0) 2.54 (dd, 15.4, 5.4) 2.43 (dd, 15.4, 1.0) 4.60 (ddd, 7.9, 5.1, 5.0) 5.04 (dd, 11.6, 7.9) 4.81 (dd, 11.6, 5.0) 6.43 (d, 15.9) 7.60 (d, 15.9) 7.00 (d, 2.1) 6.76 (d, 8.0) 6.95 (dd, 8.0, 2.0) 6.10 (d, 15.9) 7.46 (d, 15.9) 6.95 (d, 2.0) 6.61 (d, 8.0) 6.78 (d, 8.0, 2.0) 6.25 (d, 15.9) 7.52 (d, 15.9) 7.14 (d, 2.0) 6.76 (d, 8.0) 6.85 (dd, 8.0, 2.0)

4.49 (dd, 4.8, 5.0) 4.03 (dd, 5.0, 4.8) 4.17 (ddd, 4.9, 5.0, 1.3) 2.30 (15.1, 1.3) 2.25 (15.1, 4.9) 4.41 (ddd, 8.4, 5.0, 3.0) 4.98 (dd, 12.2, 8.4) 4.75 (dd, 12.2, 3.0) 6.28 (d, 15.9) 7.56 (d, 15.9) 7.04 (d, 2.1) 6.77 (d, 8.2) 6.93 (dd, 8.2, 2.1)

4.57 (m) 4.26 (dd, 5.0, 5.3) 5.47 (ddd, 5.5, 5.3, 1.2) 2.42 (dd, 15.5, 5.5) 2.29 (dd, 15.5, 1.2) 4.56 (m) 5.32 (dd, 12.2, 8.5) 4.60 (dd, 12.2, 3.0) 6.45 (d, 15.8) 7.59 (d, 15.8) 7.09 (d, 2.1) 6.74 (d, 8.1) 6.98 (dd, 8.1, 2.1) 6.35 (d, 15.9) 7.65 (d, 15.9) 7.16 (d, 2.1) 6.79 (d, 8.1) 6.97 (dd, 8.1, 2.1)

3.78 (s)

3.79 (s)

4.80 (dd, 5.3, 5.1) 5.46 (dd, 5.4, 5.3) 5.66 (ddd, 5.6, 5.4, 1.0) 2.56 (dd, 15.5, 5.6) 2.38 (dd, 15.5, 1.0) 4.61 (ddd, 7.9, 5.0, 5.1) 5.08 (dd, 11.5, 7.9) 4.78 (dd, 11.5, 5.0) 6.42 (d, 15.9) 7.59 (d, 15.9) 6.99 (d, 2.0) 6.75 (d, 8.0) 6.95 (dd, 8.0, 2.0) 6.09 (d, 15.9) 7.46 (d, 15.9) 6.94 (d, 2.0) 6.61 (d, 8.0) 6.75 (d, 8.0, 2.0) 6.24 (d, 15.9) 7.51 (d, 15.9) 7.13 (d, 2.0) 6.76 (d, 8.0) 6.84 (dd, 8.0, 2.0) 3.82 (s)

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Table 2. 13C NMR Data (δC in ppm) of Compounds 1−6 (100 MHz, MeOD) position

1

2

3

4

5

6

1 2 3 4 5 7 9 10 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″ 1″ 2‴ 3‴ 4‴ 5‴ 6‴ 7‴ 8‴ 9‴ Me

78.2 70.0 66.6 41.1 107.0 80.6 64.9 175.2 169.5 115.2 147.3 127.9 115.4 146.8 149.6 116.7 123.2

78.2 68.6 68.7 39.7 106.5 80.2 64.8 174.9 169.1 115.2 147.4 128.0 115.3 146.9 149.6 116.7 123.2 169.5 115.9 147.1 127.9 115.3 146.9 149.7 116.5 123.5

76.1 68.9 64.3 39.7 106.6 79.3 64.3 174.3 168.5 115.0 147.5 127.8 115.4 146.7 149.8 116.6 123.8 167.2 113.8 148.3 127.4 115.0 146.7 149.6 116.5 124.2 169.3 114.6 148.1 127.8 115.4 146.8 149.8 116.6 123.3

78.4 69.6 65.9 40.4 104.9 81.2 64.6 169.7 169.2 115.1 147.2 127.8 115.3 146.8 149.6 116.6 123.1

78.5 67.9 68.0 38.9 104.6 81.2 64.3 169.4 168.9 114.9 147.6 127.9 115.3 146.9 149.8 116.6 123.3 169.3 115.3 147.3 127.8 115.3 146.9 149.8 116.5 123.6

53.4

53.6

76.4 68.4 65.5 39.0 104.8 80.3 63.8 169.0 168.2 114.8 147.7 127.8 115.3 146.8 150.0 116.6 123.8 167.0 113.7 148.4 127.4 115.0 146.8 149.7 116.5 124.1 169.1 114.6 148.3 127.8 115.5 146.9 149.9 116.6 123.3 53.7

Figure 1. Key 2D NMR COSY, NOE, and correlations for compound 1.

1

spectra, all proton and carbon NMR signals of 2 were assigned as shown in Tables 1 and 2. The connections of the two caffeoyl moieties to the octulosonic acid moiety were at C-3 and C-9 through ester linkages according to the observations that these protons showed HMBC correlations to the carbonyl carbons at δC 169.5 and δC 169.1 of the caffeoyl moieties. The downfield-shifted chemical shifts of the proton signals (δH 5.46, 5.29, and 4.63) at C-3 and C-9 further supported such assignments. The relative configurations of the chiral centers in compound 2 were determined in a similar manner to that described for compound 1. The vicinal proton−proton coupling constants, NOEs, and the three-bond carbon−proton coupling constants were measured, and the results showed that compound 2 has the same relative configuration as compound 1. Therefore, the structure of compound 2 was identified to be rel-(1S,2R,3R,5S,7R)-7-[(E)-caffeoyloxymethyl]-3-[(E)-caffeoyloxy]-2-hydroxy-6,8-dioxabicyclo[3.2.1]octane-5-carboxylic acid. Erigoster B, an isomer of compound 2, was reported from Erigeron breviscapus.11 The structural difference between them is that the hydroxy group at C-2 is equatorially oriented in compound 2 and axial in erigoster B. Compound 3 was isolated as a light yellow, amorphous solid. The UV absorption pattern (λmax 329, 245, 217 nm) of 3 was similar to those of compounds 1 and 2. The HRESIMS data showed a [M + H]+ ion at m/z 707.1623, ascribable to the molecular formula of C35H30O16. The 1H and 13C NMR spectra of 3 revealed three sets of signals for a caffeoyl moiety and signals of an octulosonic acid moiety. Thus, compound 3 was assigned as a tricaffeoyl ester derivative. All the 1H and 13C NMR signals of 3 were assigned as shown in Tables 1 and 2 on the basis of the 1D and 2D NMR spectroscopic data. The HMBC spectrum exhibited correlations of the protons at δH 5.47 (H-2), 5.64 (H-3), 5.04 (H-9a), and 4.81 (H-9b) to the carbonyl carbons of caffeoyl moieties at δC 167.2, 169.3, and 168.5, respectively, indicating the three caffeoyl groups connected at C-2, C-3, and C-9 through ester linkages. The downfield-shifted chemical shifts of H-2, H-3, and H-9 also supported the assignments made. zCOSY, ROESY, and EXSIDE NMR experiments were conducted to verify the relative configuration of the structure of compound 3, and the results demonstrated that all the substituent groups on the octulosonic acid skeleton were spatially oriented in the same manner as those of compound 1. Accordingly, the structure of compound 3 was deduced to be rel-(1S,2R,3R,5S,7R)-7-[(E)caffeoyloxymethyl]-2,3-di[(E)-caffeoyloxy]-6,8-dioxabicyclo[3.2.1]octane-5-carboxylic acid. Compounds 4−6 were isolated from the same major chromatographic fraction. The 1H and 13C NMR spectra of compound 4 showed close similarities to those of compound 1, except that there were additional signals at δH 3.78 (singlet) and δC 53.4, which were attributed to a methoxy group. The protons of this methoxy group had a correlation to the carboxylic carbon (δC 169.7) of the octulosonic acid moiety in the HMBC spectrum of 4, suggesting that compound 4 is the methyl ester of compound 1. The HRESIMS of 4, which showed a [M + H]+ ion peak at m/z 397.1145, corresponding to its molecular formula of C18H20O10, confirmed the assignment. The NMR spectra of compounds 5 and 6 also showed similarities in spectroscopic features to those of compounds 2 and 3, respectively, except for the observation of an additional methoxy group in each of 5 and 6. Furthermore, the HRSEIMS data and the observations of HMBC correlations between the methoxy proton and

H−13C HMBC

to possess an additional C9H6O3 fragment and have an additional six degrees of unsaturation. Furthermore, in the 1H and 13C spectra of 2, there were two sets of signals of caffeoyl moiety and signals for an octulosonic acid moiety, indicating that 2 is a dicaffeoyl derivative. With the aid of 2D NMR 511



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Table 3. Potential Anti-inflammatory Activity of Compounds 1−6a

a

inhibition of NF-κB

inhibition of iNOS

inhibition of ROS generation

activation of NAG-1

compound

IC50 (μM)

IC50 (μM)

IC50 (μM)

fold induction at 50 μM

1 2 3 4 5 6 parthenolide quercetin diclofenac (20 μM)

NA NA NA NA NA 42.0 ± 5.6 1.63 ± 0.55

NA NA NA NA NA NA 1.05 ± 0.20

>100 79.0 59.5 86.0 34.5 26.0

± ± ± ± ±

2.9 3.5 9.8 3.5 1.4

3.20 2.34 2.63 3.10 2.30 2.12

± ± ± ± ± ±

0.12 0.05 0.01 0.48 0.69 0.49

11.0 ± 1.4 2.65 ± 0.12

Each value is expressed as a mean ± standard deviation of at least two independent experiments.

important roles in carbohydrate and lipid metabolism and have been considered as significant targets in treating metabolic diseases, they have also been linked with the inflammatory process by regulating the production of inflammatory cytokines. A growing body of evidence has suggested that the activation of PPARs results in suppressing the inflammatory process.10,9 In the present study, the anti-inflammatory activity of the constituents 1−6 isolated from Roman chamomile flower heads was evaluated in terms of their effects against oxidative stress and their interaction with cellular targets related to inflammation and metabolic disorders such as NAG-1, NF-κB, iNOS, ROS, PPARα, PPARγ, and LXR, through the use of a battery of cellular assays. Five compounds (2−6) decreased cellular oxidative stress by inhibiting ROS generation. Compounds 3, 5, and 6 (IC50 59.5, 34.5, and 26.0 μM, respectively) were more effective than compounds 2 and 4 (IC50 79.0 and 86.0 μM, respectively), but they were less potent than quercetin (IC50 11.0 μM), as shown in Table 3. Compound 1 was not as effective as the other isolates tested. The effect of these compounds against oxidative stress could be related to the presence of a caffeic acid moiety in their structures, since the activity was more potent for those containing a greater number of this substituent. The results also indicated that the ROS inhibition activity was more potent for compounds when esterified (5, 6) than the ones with the same unit of caffeic acid moiety in the acid form (2, 3). All six compounds (1−6) were found to cause activation of NAG-1. As shown in Table 3, NAG-1 activity was increased 2−3-fold at a concentration of 50 μM of 1−6 in comparison to the vehicle control. Compounds 1 and 4 were more effective than others. Under the same assay conditions, a 2.7-fold increase in NAG-1 activity was observed in response to the control drug diclofenac at 20 μM. The results of the evaluation of anti-inflammatory activity indicated that the compounds resemble NSAIDs in enhancing NAG-1 activity (such as diclofenac, ibuprofen, and aspirin). Compounds 1−6 did not show any inhibition of iNOS and therefore did not affect cellular nitric oxide levels in lipopolysaccharide (LPS)-treated macrophages. The increase in transcriptional activity of NF-κB in PMA-treated cells was also not suppressed by these compounds (with the exception of compound 6, which caused a 50% inhibition of NF-κB activity at 42 μM). These results indicated that their anti-inflammatory effect is not mediated by inhibition of iNOS or NF-κB activity. The effect of octulosonic acid derivatives (1−6) on the activity of PPARα, PPARγ, and LXR is shown in Table 4. These are important regulators of lipid and carbohydrate metabolism and inflammatory signaling. At a concentration of 30 μM, compounds 2 and 4 showed an increase in LXR activity (1.59-

carboxylic carbon confirmed that compound 5 is the methyl ester of compound 2 and compound 6 that of compound 3, respectively. Other spectroscopic features of compounds 4−6, including the 3JH,H, 3JC,H, and key NOE correlations, were essentially identical to those of compounds 1−3, respectively, indicating that each compound pair has the same basic skeleton and relative configuration. Therefore, the structures of compounds 4−6 were elucidated as methyl rel(1S,2R,3R,5S,7R)-7-[(E)-caffeoyloxymethyl]-2,3-dihydroxy-6,8dioxabicyclo[3.2.1]octane-5-carboxylate, methyl rel(1S,2R,3R,5S,7R)-7-[(E)-caffeoyloxymethyl]-3-[(E)-caffeoyloxy]-2-hydroxy-6,8-dioxabicyclo[3.2.1]octane-5-carboxylate, and methyl rel-(1S,2R,3R,5S,7R)-7-[(E)-caffeoyloxymethyl]2,3-di[(E)-caffeoyloxy]-6,8-dioxabicyclo[3.2.1]octane-5-carboxylate, respectively. A compound having a similar structure to compound 5 was reported from Erigeron bonariensis,15 but its relative configuration at C-7 was not determined. However, the specific rotation value ([α] −18) of this compound showed a significant difference from that of compound 5 ([α] +150.6). Octulosonic acid derivatives are a rare class of natural products. So far, these compounds have been isolated and characterized from Erigeron breviscapus,11 E. bonariensis,15 Conyza canadensis,16 and Smallanthus sonchifolius.17 The anti-inflammatory properties of Roman chamomile flower heads are well known, and their traditional use as a medicinal tea for treating inflammation of the mouth and throat has been well documented.2 Inflammation is considered as a risk factor for several types of cancer, as well as a contributing factor in obesity and metabolic disorders. The NAG-1 gene is involved in the anti-inflammatory action of NSAIDs (nonsteroidal anti-inflammatory drugs) and is associated with the apoptotic elimination of cancer cells. The induction of NAG-1 is a COX-independent mechanism by which the NSAIDs mediate their effects.18,19 The activation of NF-κB in response to pro-inflammatory signals is associated with many diseases caused by unregulated inflammation. Since NF-κB is highly activated at the sites of inflammation in diverse diseases, the compounds that can suppress NF-κB activation have potential as anti-inflammatory agents.20 Excessive generation of nitric oxide (NO) and reactive oxygen species (ROS) also contributes significantly to the progress of inflammation and subsequent development of metabolic syndrome, characterized by obesity, diabetes, and cardiovascular disease. Inhibition of inducible nitric oxide synthase (iNOS) can reduce the intracellular NO production. NF-κB, iNOS, ROS, and NAG-1 have been considered as important targets for inflammation.21,22 Although peroxisome proliferator-activated receptors (PPARα and PPARγ) and liver X receptor (LXR) play 512



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acquired for measuring the long-range 1H−13C coupling constants. High-resolution mass spectra were obtained using an HRESITOFMS spectrometer with the Analyst QS software for data acquisition and processing (Agilent Series 1100 SL, ESI source model #G1969A, Agilent Technologies, Palo Alto, CA, USA). TLC was performed with silica gel 60 GF254 plates (EM Science), and a CHCl3−MeOH−H2O (7:3:0.5) solvent system was used. The isolates were visualized using short-wave UV light (254 nm), followed by spraying with 1% vanillin− H2SO4 reagent. Column chromatography was performed with flash ́ flash) and Sephadex LH-20 (Sigmasilica gel (J. T. Baker, 40 iM Aldrich). Fraction collection was carried out by means of a CF-1 fraction collector. Flash column chromatography for purifications was performed on a Biotage Isolera Four system, using SNAP or HPFC cartridges. Plant Material. The flower heads of C. nobile were obtained in October 2010 from the Medicinal Plant Garden of the University of Mississippi. The plant material was authenticated by Dr. Vijayasankar Raman, and a specimen (voucher number 9254) has been deposited in the Herbarium of the University of Mississippi. Extraction and Isolation. The dried flower heads of C. nobile (910 g) were ground to a fine powder and extracted with MeOH (2 L × 3 times) by sonication for 20 min. The filtered solvents were combined and evaporated under vacuum at a temperature of 40 °C to give a residue (172 g). The obtained extract was suspended in water (800 mL) and successively partitioned with hexane, ethyl acetate, and n-butanol, yielding 26 g, 64 g, and 32 g partition fractions, respectively, after drying. The ethyl acetate fraction (50 g) was loaded onto a silica gel column (600 g sil gel, ϕ 7 cm × 38 cm) and eluted with a MeOH− CHCl3 solvent system with increasing MeOH concentration from 5% to 100% in a total volume of 12 L. The collected fractions were analyzed by TLC, and those with similar composition were combined to give a total of nine fractions. The residue (2.2 g) obtained from the eighth fraction after drying was subjected to column chromatography over silica gel (80 g) (eluting with CHCl3−MeOH and CHCl3− MeOH−H2O of increasing polarity) and Sephadex LH-20 (60 g) (eluting with MeOH), then further purified by using a Biotage chromatography system (FLASH 12+M silica gel cartridge, eluting with MeOH−EtOAc in increasing MeOH concentration from 0% to 40%) to afford compounds 1 (22.4 mg), 2 (34.1 mg), and 3 (18.5 mg). The residue (2.8 g) obtained from the fifth fraction was treated in a manner similar to that of the eighth fraction to afford 4 (47.8 mg), 5 (69.8 mg), and 6 (59.6 mg). rel-(1S,2R,3R,5S,7R)-7-[(E)-Caffeoyloxymethyl]-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]octane-5-carboxylic acid (1): amorphous solid; [α]20 D −0.5 (c 0.48, MeOH); UV (MeOH) λmax (log ε) 328 (4.29), 243 (4.04), 218 (4.22) nm; IR (film) νmax 3385, 1694, 1601, 1524, 1443, 1273, 1181, 1118 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 383.0992 [M + H]+ (calcd for C17H19O10, 383.0978). rel-(1S,2R,3R,5S,7R)-7-[(E)-Caffeoyloxymethyl]-3-[(E)-caffeoyloxy]-2-hydroxy-6,8-dioxabicyclo[3.2.1]octane-5-carboxylic acid (2): amorphous solid; [α]20 D +107.2 (c 0.75, MeOH); UV (MeOH) λmax (log ε) 327 (4.43), 244 (4.19), 218 (4.34) nm; IR (film) νmax 3384, 1688, 1600, 1522, 1444, 1270, 1164, 1115 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 545.1306 [M + H]+ (calcd for C26H25O13, 545.1295). rel-(1S,2R,3R,5S,7R)-7-[(E)-Caffeoyloxymethyl]-2,3-di[(E)-caffeoyloxy]-6,8-dioxabicyclo[3.2.1]octane-5-carboxylic acid (3): amorphous solid; [α]20 D −180.1 (c 0.62, MeOH); UV (MeOH) λmax (log ε) 329 (4.68), 245 (4.47), 217 (4.63) nm; IR (film) νmax 3381, 1694, 1599, 1522, 1445, 1270, 1157, 1114 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 707.1623 [M + H]+ (calcd for C35H31O16, 707.1612). Methyl rel-(1S,2R,3R,5S,7R)-7-[(E)-caffeoyloxymethyl]-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]octane-5-carboxylate (4): amorphous solid; [α]20 D +6.8 (c 0.92, MeOH); UV (MeOH) λmax (log ε) 327 (4.40), 244 (4.18), 217 (4.38) nm; IR (film) νmax 3385, 1690, 1601, 1515, 1444, 1273, 1168, 1112 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 397.1145 [M + H]+ (calcd for C18H21O10, 397.1135).

Table 4. Fold Induction in the Activity of PPARα, PPARγ, and LXR by Compounds 1−6a compound (30 μM) 1 2 3 4 5 6 ciprofibrate (25 μM) ciglitazone (25 μM) 25- hydroxycholesterol (25 μM)

PPARα 1.20 1.21 1.00 1.09 1.43 1.35 4.35

± ± ± ± ± ± ±

0.11 0.22 0.09 0.08 0.15 0.21 0.55

PPARγ 1.18 1.25 1.68 1.04 1.59 2.09

± ± ± ± ± ±

0.33 0.01 0.21 0.01 0.13 0.03

LXR 1.12 1.59 1.14 1.22 1.06 1.11

± ± ± ± ± ±

0.29 0.20 0.24 0.09 0.31 0.01

3.65 ± 0.87 4.67 ± 0.37

Each value is expressed as a mean ± standard deviation of at least two independent experiments.

a

and 1.22-fold), while 3, 5, and 6 increased PPARγ activity (1.68-, 1.59-, and 2.09-fold, respectively). PPARα was activated by compounds 5 and 6 only (1.43- and 1.35-fold at 30 μM). Compounds 1 and 2 were not as effective toward PPARs. No cytotoxicity was observed for any of these compounds against mouse macrophages (RAW 264.7) or human hepatoma cells (HepG2) up to a concentration of 50 μM (data not shown). The methanolic extract of the flower heads of C. nobile showed inhibition of intracellular oxidative stress (45% inhibition of ROS generation at 200 μg/mL) and inhibition of NF-κB activity (50% inhibition at 17 μg/mL). The extract was also found to activate NAG-1 (2.2-fold at 50 μg/mL), PPARα (1.8-fold at 25 μg/mL), and PPARγ (3.0-fold at 25 μg/ mL). The results indicated that compounds 1−6 play a contributing role in the anti-inflammatory properties of the extract of Roman chamomile. They may regulate the process of inflammation through mediating multiple targets that are involved in several biochemical and metabolic pathways. The presence of these constituents could partly explain the reported anti-inflammatory, hypoglycemic, antiedemic, and antioxidant activities of Roman chamomile. Further studies are warranted to explore their anti-inflammatory potential in more detail.

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EXPERIMENTAL SECTION

General Experimental Procedures. All chemicals and reagents were supplied by Fisher Scientific and Sigma-Aldrich unless otherwise stated. The solvents used for column chromatography were of certified ACS grade. Deuterated methanol (CD3OD, 99.8% D), chloroform (CDCl3, 99.9% D), and water (D2O, 99.9% D) were obtained from Cambridge Isotope Laboratories. Specific rotations were measured using a Rudolph Research AutoPol IV polarimeter at room temperature; UV spectra were recorded by a Hewlett-Packard 8452A UV−Vis spectrometer; IR spectra were acquired using Bruker Tensor 27 and MIRacle ATR FT-IR spectrometers (Bruker Optics). 1D NMR spectra were measured at 25 °C on a Bruker AVANCE DRX400 NMR spectrometer (Bruker BioSpin, Billerica, MA, USA) with a BBO probe at 400 MHz for 1H and 100 MHz for 13C with Bruker Topspin software (v. 1.3). 2D NMR spectra (COSY, HSQC, HMBC, ROESY, and Sel-EXSIDE) were acquired on an Agilent DD2500 NMR spectrometer (Santa Clara, CA, USA) with a OneNMR probe at 500 MHz for 1H and 125 MHz for 13C using the pulse programs provided in the Agilent Vnmrj 3.2 software. Chemical shifts were referenced to the residual solvent signal (MeOD: δH 3.31 ppm, δC 49.15 ppm). ROESY spectra were recorded with a mixing time of 300 ms. HSQC experiments were acquired by using 146 Hz as the one-bond proton−carbon coupling constant. HMBC experiments were conducted in which a multiple-bond 1H−13C coupling constant of 8 Hz was used for calculating the evolution time. Sel-EXSIDE (selective-excitation-sculptured indirect detection) experiments were 513



Article

Methyl rel-(1S,2R,3R,5S,7R)-7-[(E)-caffeoyloxymethyl]-3-[(E)caffeoyloxy]-2-hydroxy-6,8-dioxabicyclo[3.2.1]octane-5-carboxylate (5): amorphous solid; [α]20 D +150.6 (c 0.51, MeOH); UV (MeOH) λmax (log ε) 329 (4.53), 245 (4.29), 218 (4.44) nm; IR (film) νmax 3389, 1684, 1600, 1515, 1443, 1277, 1159, 1113 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 559.1437 [M + H]+ (calcd for C27H27O13, 559.1452). Methyl rel-(1S,2R,3R,5S,7R)-7-[(E)-caffeoyloxymethyl]-2,3-di[(E)-caffeoyloxy]-6,8-dioxabicyclo[3.2.1]octane-5-carboxylate (6): amorphous solid; [α]20 D −165.8 (c 0.38, MeOH); UV (MeOH) λmax (log ε) 330 (4.67), 246 (4.42), 218 (4.57) nm; IR (film) νmax 3387, 1697, 1601, 1516, 1444, 1276, 1159, 1114 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 721.1772 [M + H]+ (calcd for C35H31O16, 721.1769). Reporter Gene Assay for the Activation of NSAID-Activated Gene-1 (NAG-1). Human chondrosarcoma (SW1353) cells were cultured in DMEM/F12 medium supplemented with 10% FBS, 100 U/mL penicillin G sodium, and 100 μg/mL streptomycin at 37 °C in an atmosphere of 5% CO2 and 95% humidity. For the assay, cells were transfected with 25 μg of pcDNA3.1-NAG-1 plasmid containing a fulllength NAG-1 cDNA (a gift from Dr. Thomas E. Eling, NIH, Research Triangle Park, NC, USA) by electroporation at 160 V and one 70 ms pulse using a BTX Electro Square Porator T 820 (BTX I, San Diego, CA, USA). Transfected cells were plated in 96-well plates at 1 × 105 cells/200 μL/well in DMEM/F12 supplemented with 10% FBS. After 24 h, the cells were treated with different concentrations of test compounds for 24 h. Luciferase activity was measured using a Luciferase Assay Kit (Promega, Madison, WI, USA). Light output was detected on a SpectraMax plate reader. Fold induction in NAG-1 activity was calculated in comparison to vehicle control. Diclofenac (Sigma-Aldrich, St Louis, MO, USA) was used as a positive control.23 Reporter Gene Assay for the Inhibition of NF-κB Activity. Human chondrosarcoma (SW1353) cells were cultured as indicated in the above paragraph. The assay was performed as described earlier.24 In brief, cells transfected with NF-κB luciferase plasmid construct were plated in 96-well plates at a density of 1.25 × 105 cells/well. After 24 h, cells were treated with the test compounds, and after incubating for 30 min, phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich) (70 ng/ mL) was added and further incubated for 6−8 h. Luciferase activity was measured as described above. Percent decrease in luciferase activity was calculated relative to the vehicle control. Parthenolide (Sigma-Aldrich) was used as a positive control. Assay for the Inhibition of iNOS Activity. The assay was performed in mouse macrophages (RAW264.7) cultured in phenol red-free RPMI medium with 10% bovine calf serum, 100 U/mL penicillin G sodium, and 100 μg/mL streptomycin. Cells were seeded in 96-well plates (100 000 cells/well) and incubated for 24 h for a confluency of 75% or more. The cells were treated with the test compounds, and after 30 min, LPS (Sigma-Aldrich) (5 μg/mL) was added and further incubated for 24 h. The activity of iNOS was determined in terms of the concentration of NO by measuring the level of nitrite in the cell culture supernatant using Griess reagent (Sigma-Aldrich). Percent inhibition of nitrite production by the test compound was calculated in comparison to the vehicle control. IC50 values were obtained from dose−response curves. Parthenolide was used as the positive control.25 Reporter Gene Assay for the Activation of PPARs and LXR. Activation of PPARα, PPARγ, and LXR was determined in human hepatoma (HepG2) cells as described previously.26 In brief, HepG2 cells were cultured in DMEM supplemented with 10% FBS, 100 units/ mL penicillin G sodium, and 100 μg/mL streptomycin. For the PPARα and PPARγ activation assay, HepG2 cells were transfected with pSG5-PPARα and PPRE X3-tk-luc or pCMV-rPPARγ and pPPREaP2-tk-luc plasmid DNA (25 μg/1.5 mL cell suspension), respectively, by electroporation at 160 V for a single 70 ms pulse using a BTX Electro Square Porator T 820. For the LXR activation assay, cells were transfected with pCMX-hLXRα and LXRE-tk-luc plasmid DNA (gift from Dr. Laura Liscum, The University School of Medicine, Boston, MA, USA) in a similar manner. Transfected cells were plated at a density of 5 × 104 cells/well in 96-well tissue culture plates and

grown for 24 h. After 24 h, the cells were treated with the test compounds. After incubation for 24 h, the cells were lysed and the luciferase activity was measured. The fold induction of luciferase activity in treated cells was calculated in comparison to the vehicle control. Ciprofibrate, rosiglitazone (both from Cayman Chemical, Ann Arbor, MI, USA), and 25-hydroxycholesterol (Sigma-Aldrich) were used as positive controls. Assay for the Inhibition of Cellular Oxidative Stress. The cellular antioxidant activity was measured in HepG2 cells according to a method previously described.27 The method measures the ability of test compounds to prevent intracellular generation of peroxyl radicals in response to 2,2′-azobis(2-amidinopropane) dihydrochloride (ABAP, Sigma-Aldrich), a generator of peroxyl radicals. The assay is more relevant biologically than a chemical assay because it represents the complexity of a biological system and accounts for cellular uptake, bioavailability, and metabolism of the antioxidant agent being tested. For the assay, HepG2 cells were seeded in the wells of a 96-well plate at a density of 60 000 cells/well and incubated for 24 h. The medium was removed and cells were washed with PBS before treating with the test compounds diluted in serum-free medium containing 25 μM 2′,7′dichlorofluorescin diacetate (DCFH-DA, Invitrogen, Carlsbad, CA, USA) for 1 h. After removing the medium, the cells were treated with 600 μM ABAP and the plate was immediately placed on a SpectraMax plate reader for kinetic measurement every 5 min for 1 h (37 °C, emission at 538 nm and excitation at 485 nm). Quercetin (SigmaAldrich) was included as the positive control. The area under the curve (AUC) of fluorescence versus time was used to calculate cellular antioxidant activity (CAA) units from the following equation:

CAA unit = 100 − [(AUC sample/AUC control) × 100]

(1)

The IC50 value of the test samples was calculated from dose−response curves of CAA units versus test concentration. Quercetin at 11 μM caused 50% inhibition of cellular generation of peroxyl radicals in HepG2 cells. Determination of Cytotoxicity. Cytotoxicity of 1−6 toward RAW264.7 and HepG2 cells was determined using a Neutral Red assay.28

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ASSOCIATED CONTENT

S Supporting Information *

1

H and 13C NMR spectra of compounds 1−6 are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +662-915-7821. Fax: +662-915-7989. E-mail: ikhan@ olemiss.edu. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported in part by the United States Department of Agriculture, Agricultural Research Service, Specific Cooperative Agreement No. 58-6408-2-0009. The authors thank Dr. J. Parcher for helpful discussions and Dr. V. Raman for authenticating the plant material. Ms. K. Martin is acknowledged for excellent technical support in the bioassays. The authors would also like to thank Agilent Technologies for provision of the HRESITOFMS spectrometer instrumentation and software.

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DEDICATION Dedicated to Prof. Dr. Otto Sticher of ETH-Zurich, Zurich, Switzerland, for his pioneering work in pharmacognosy and phytochemistry. 514



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REFERENCES

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Octulosonic Acid Derivatives from Roman Chamomile (Chamaemelum

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