Dietary Supplements - American Chemical Society

Chapter 12

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Antiinflammatory Constituents in Noni (Morinda citrifolia) Fruits 1

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Haiqing Yu , Shiming Li , Mou-Tuan Huang , and Chi-Tang Ho

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Department of Food Science, Rutgers, The State University of New Jersey, 65 Dudley Road, New Brunswick, NJ 08901-8520 Susan Lehman Cullman Laboratory of Cancer Research, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 164 Frelinghuysen Road, Piscataway, NJ 08854-8020 2

Morinda citrifolia (Noni) is a tropical plant whose leaves, barks, roots and fruits have been used as traditional remedy for various diseases. Although inflammation was considered as one of the indications for noni fruits, and fruit extract was demonstrated to have immunomodulatory activity in vitro, no principle has been identified. In our study, we attempted to identify constituents that may be responsible for anti inflammatory activity, and to elucidate the possible roles on anti-inflammatory mediators for interested compounds. Solvent extraction and column chromatography were major techniques used for isolation of compounds, while structures were elucidated by integration of data from UV, IR, MS and NMR analysis. Anti-inflammatory activity was assessed by mouse ear edema model. Subsequent Elisa analysis was carried out for their effects on anti-inflammatory mediators such as IL-Iβ, IL-6, PGE and myeloperoxidase. Scopoletin, quercetin, ursolic acid were identified as major anti inflammatory constituents. Since ursolic acid was known to have anti-inflammatory activity, we characterized the mode of action of scopoletin and quercetin in mouse ear inflammation model. The most potent inhibition occurred at 0.5 μmol level 2

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In Dietary Supplements; Ho, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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180 for both quercetin (62.3%) and scopoletin (50.4%). Scopoletin mainly inhibited production of myeloperoxidase and PGE while quercetin had significant suppressing effect on IL-6 production. Both compounds showed moderate inhibition on IL-ip production.


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Morinda citrifolia L. (Rubiaceae), common name noni, is a tropical evergreen shrub mainly distributed in French Polynesia, Hawaii, India and Southeast Asia. The leaves, fruits, barks and roots have been used traditionally for treatment of cancer, hypertension, diabetes, etc. (7). Exploration of chemical constituents of noni flower started as early as in 1960s, however, information on other plant tissues continues to accumulate. Noni leave extract significantly upregulated LDL receptor in vitro, although it doesn't have a significant antioxidant activity (2). This result suggests that the cardiovascular protective effect was not due to prevention of LDL oxidation, but due to enhanced activity of LDL receptor. Noni leaves were also reported as containing antitubercular constituents (E-phytol, cycloartenol, stigmasterol, betasitosterol, campesta-5,7,22-trien-3p-ol and the ketosteroids stigmasta-4-en-3-one and stigmasta-4,22-dien-3-one) in the nonpolarfraction(3). Sang et al isolated citrifolinin B, citrifolinoside A, citrifolinoside 1 and five flavonol (quercetin or kaempferol) glycosides. Among those compounds, citrifolinin B is a new iridoid glycoside with weak DPPH scavenging activity, while citrifolinoside 1, significantly inhibited UVB induced Activator Protein-1 (AP-1) activity in vitro (4-6). Anthraquinones such as damnacanthal, 7-hydroxy-8-methoxy-2ethylanthraquinone, morenone 1, morenone 2 (7,5), and anthraquinone glycosides (Physcion, molindone, Physcion-8-O-alpha-L-arabinopyranosyl ( l - » 3 ) P-D-galactopyranosyl (l->6)-P-D-galactopyranoside) were isolated from the Noni root (9). Among those, damnacanthal was proved to have induction effect on normal phenotypes in ras-transformed cells (10). In the mean time, damnacanthal has potent inhibitory activity towards tyrosine kinases, phosphorylation was enhanced for both extracellular signal regulated kinases (ERK) and stress-activated protein kinases (SAPK) pretreated with damnacanthal followed by UV treatment in vitro (11). Short Chain fatty acids such as hexanoic acid and octanoic acid were found to be abundant in ripefruitextract. Specifically, octanoic acid was tested as toxic to Drosophila species (72). These short chain fatty acids are the major source of the soapy foul flavor of nonifruit.Interestingly, hexanoic acid and octanoic acid are mainly present as esters of di- or tri-saccharides in thefruitextract (73,74). Among them, 6-0-(P-D-glucopyranosyl)-l-0-octanoyl-P-D-glucopyranose was found to inhibit AP-1 transduction in vitro, thus affecting cell transformation in the cancer proliferation stage (75). Along with the above compounds, 3



181 glycoside (3-methylbut-3-enyl 6-Op-D-glucopyranosyl, asperulosidic acid, rutin) were also isolated (13,14). It has been known that elevated level of mediators including pro inflammatory cytokines (IL-lp, TNFct, IL6) and arachidonic acid metabolites (prostaglandins and leukotriens) are correlated with activated inflammation process (16-22). Although traditionally, inflammation was considered at one of the indications for noni fruits, evidence has been discrete. Li et al reported only moderate anti-inflammatory activity of Noni fruit extract compared to 23 other herbs, with IC of 163 pg/ml on inhibition of COX-1 (23). While Hirazumi reported its effectiveness in stimulating the release of several anti-inflammatory mediators, including TNF-a, IL-lp, IL-10, IL-12p70, IFN-y, and nitric oxide (NO). It has been suggested that the polysaccharide constituents in noni are responsible for its immunomodulatory activity (24). However no principle has been isolated and elucidated. More interestingly, immunomodulation was suggested to be responsible for anticancer activity of Noni (25,26). So it would be of interest to confirm and explore the anti-inflammatory principles in Noni. The present work attempts to identify new constituents that may be responsible for anti-inflammatory effects. Further studies will be carried out on preliminary elucidation of anti-inflammatory mechanism of interested compounds. 50

Materials and Method Plant Material Tahiti nonifruitswere collected and water was partially removed by drum drying to yield a paste.

Solvents and Reagents Solvents including methanol, water, acetonitrile, chloroform, ethyl acetate, hexane, n-butanol and acetone were of HPLC grade, and purchasedfromFisher Scientific (Springfield, NJ). TPA (12-O-tetradecanoyl phorbol 13-acetate), deuterated solvents including (CD ) SO, CDC1 , and CD OD were obtained from Sigma (St. Louis, MO). Silica gel (60A, 32-63 pm) for normal phase chromatography was purchased from Sorbent Technology Inc. (Atlanta, GA). Octadecyl (Ci ) derivatized silica gel (60A) for reversed phase chromatography, and Sephadex LH-20 were purchased from Sigma (St. Louis, MO). Thin Layer Chromatography (TLC) plates were purchased from Fisher Scientific (Springfield, NJ). ELISA assay kits were purchased from BioSource International, Inc (Camarillo, CA). 3

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182 Instruments NMR spectra were recorded on a Varian 300 Spectrometer (Varian Inc., Palo Alto, CA). *H-NMR and C-NMR was recorded at 300MHz with TMS served as internal standard. APCI-MS spectra were obtained on a VG-platform II (Micromass, Beverly, MA). ESI-MS spectra were obtained on a Quattro II triple quadrupole mass analyzer (Micromass, Beverly, MA). HPLC systemfromL C MS was composed of a Shimadzu (Piscataway, NJ) SCL-10A system controller, 2 LC-10AS liquid chromatograph pumps, a Supelco Discovery CI8 4.6mm X 25cm column, a variable wavelength Varian 2050 UV detector and a Waters 717 autosampler. Analytical HPLC system was composed of a Varian Vista 5500 system controller, with a variable UV wavelength Varian 9065 polychrom detector (Walnut Creek, CA). Preparative HPLC system was composed of a Water Model 590 pump, a Zorbax Rx-C18 9.4mm x 25cm column, a Milton Roy spectromonitor 3100 UV detector, and Peaksimple data system.


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Extraction and Isolation The noni paste was extracted with 95% ethanol at room temperature for one week. The extract was filtered and the residue was re-extracted for anther 2 times. The filtrates were combined and evaporated under reduced pressure to yield brown syrup-like residue. The residue was then suspended in 500 mL water and transferred to a separately funnel, where 500 mL of hexane (3 times), 500 mL of ethyl acetate (3 times), and 500 mL of a-butanol (3 times) were added successively for liquid-liquid partition. Eachfractionwas collected and dried under reduced pressure. The ethyl acetatefractionwas loaded on silica gel column, eluted with a gradient of ethyl acetate: methanol: acetic acid 50:1:0.01, 20:1:0.01, 10:1:0.01, 5:1:0.01, 1:1:0.01 consecutively. Tenfractionswere collected. Each fraction were dried under reduced pressure, and further purified on repeated silica gel column, RP-18 column and Sephadex LH-20 column to yield pure compounds. Scopoletin and quercetin was purified from 50:1:0.01 fraction, while ursolic acid, p-sitosterol and campesterol were isolatedfrom20:1:0.01 fraction.

Anti-inflammatory Assay Eight Female CD-I mice aged 5-6 weeks were grouped into acetone + acetone (negative control) group. 6 mice were grouped into actone + TPA (0.8 nmol) (positive control) group. All other test groups were composed of 5 mice. Both ears of test mouse were treated topically with 20 pL acetone or test


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compound (contained 0.5 pmol, 1 pmol and 2 pmol respectively) in acetone, at 20 minutes before acetone or TPA (0.8 nmol) application once a day for 4 days. The mice were killed by cervical dislocation at 7.5 hours within the last dose of TPA treatment. Ear punch (6 mm in diameter) were taken and weighed. The ear sample were homogenized and kept at -80 °Cfreezeruntil ELISA analysis. Inhibition % = (ear weight of test compound) - (ear weight of negative control) (ear weight of positive control) - (ear weight of negative control)

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ELISA analysis Samples and standards, as well as analysis methods for PGE , IL-ip and IL6 assays were prepared as directedfromELISA reagent kits. Five standards were used to establish a standard curve for quantification of each protein. 2

Results and Discussion Chemistry of Potential Anti-inflammatory Constituents From the ethyl acetatefractionof noni extract, a total of 5 compounds were identified (Figure 1), which by nature are flavone, coumarins, steroid and terpenoids respectively.

Identification of Scopoletin Scopoletin was obtained as a white power. APCI MS showed a pseudomolecular ion [M-H]" at m/z 191 and [M+H] at m/z 193. Additionally, a daughter ion at m/z 176 on APCI" spectrum indicated loss of a methyl group. IR spectrum showed an intense absorption at wave number 3341.38cm" , indicating stretching vibration of O-H bond, while an strong sharp absorption at 1708.51cm" , indicating a carbonyl group. ^ - N M R (DMSO-ck, 300 MHz): 10.29 (1H, s, OH), 7.89 (1H, d, J= 9.3 Hz, H-3), 7.20 (1H, s, H-8), 6.76 (1H, s, H-5), 6.19 (1H, d, J = 9.3Hz, H-4), 3.80 (3H, s, CH ). C-NMR (DMSO-ck, 300 MHz): 160.63 (s, C-2), 151.08 (s, C-7), 149.45 (s, C-6), 145.20 (s, C-8), 144.44 (s, C-9), 111.66 (s, C-5), 110.50 (s, C-3), 109.54 (s, C-4), 102.73 (d, C-10), 55.96 (s, C-ll). +

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Scopoletin p-Sitosterol

Campesterol Ursolic Acid Figure 1. Compounds IsolatedfromEthyl Acetate Fraction

Identification of Quercetin Quercetin was obtained as a yellow power. ESI MS spectrum showed pseudo-molecular ion [M-H]~ at m/z 301 and [M+H] at m/z 303. *H-NMR (DMSO-dg, 300 MHz): 12.48 (1H, s, OH-3), 10.79 (1H, s, OH-5), 9.60 (1H, s, OH-7), 9.37 (1H, s, OH-4'), 9.30 (1H, s, OH-3'), 7.66 (1H, d, J= 2.4 Hz, H-2'), 7.52 (1H, dd, J=8.4 Hz, 2.4 Hz, H-6'), 6.86 (1H, d, J=8.4 Hz, H-5'), 6.39 (1H, d, J= 1.8 Hz, H-8), 6.17 (1H, d, J= 1.8Hz, H-6). C-NMR (DMSO-d , 300MHz, Appendix 9): 175.80, 163.85, 160.67, 156.08, 147.66, 146.74, 145.02, 135.69, 121,9, 119.94, 115.57, 115.02, 102.97, 98.16, 93.31. It has been reported that in the n-butanolfractionof nonifruitsextract, rutin (quercetin glycoside) was identified. Here we confirmed the existence of quercetin aglycone in less polar fraction. +

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Identification of Ursolic Acid Ursolic acid was obtained as a white powder. The EI MS showed molecular ion at m/z 456 (19%), with daughter ions at m/z 438 (15%), 410 (32%), 395


185 (10%), 322 (9%), 300 (27%), 287 (11%), 273 (8%), 248 (100%), 207 (32%), 203 (50%), 133 (46%). Among those, the base peak m/z 248 and the peak at 207 were products of Retro-Diels-Alder reaction. H-NMR (DMSO-d , 300 MHz, Appendix 5) and C-NMR (DMSO-do, 300 MHz) obtained agreed with literature data reported earlier (27). Ursolic acid was reported previously for its activity of stimulation of splenocytes proliferation in mice (28). Huang et al reported that ursolic acid is able to inhibit TPA-induced mouse ear inflammation (29). It was also identified from Plantago major, and demonstrated to be a selective inhibitor of COX-2 enzyme (30). Meanwhile, Suh et al reported it as a suppressing agent on iNOS enzyme in mouse macrophages (31). Most recently, Chiang et al reported ursolic acid strongly enhanced the secretion of IFN-y in human peripheral blood mononuclear cells (32). All these evidence suggest that ursolic acid is a potential anti-inflammatory agent function by modulating pro-inflammatory cytokines and inflammatory enzymes. !

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Anti-inflammatory Activity of Scopoletin and Quercetin Among the isolated compounds, scopoletin and quercetin was selected for further bioactivity test. Scopoletin was previously reported as dose dependently inhibited iNOS enzyme (33), indicating its possible role as having effects on anti-inflammatory mediators, as iNOS induction can be mediated by different pro-inflammatory cytokines and other stimulants. Quercetin is another abundant constituent. It is well known as a strong antioxidant and modifies eicosanoid biosynthesis (34), indicating that quercetin may possess anti-inflammatory activity.

Anti-inflammatory Effects on TPA-induced Mouse Ear Edema Model Quercetin and scopoletin were tested for their in vivo anti-inflammatory using TPA-induced mouse ear edema model. TPA (12-O-tetradecanoylphorbol13-acetate), a phorbol ester, is a natural product exists in seed of Croton tiglium L. It is a potent tumor promoter and inflammation inducer that can be used to stimulate production of pro-inflammatory cytokines (35-37). It was reported that multiple topical applications of TPA to mouse ears will induce a prolonged inflammatory reaction, which results in increases in ear weight, inflammatory cell infiltration and epidermal hyperplasia (35). Therefore, in our model, the weight increase of mouse ears will be an indicator of inflammation. Three levels of each compound and a combination of them both at 0.5 pM level were evaluated. Results were shown in Figure 2. Both quercetin and scopoletin showed anti-inflammatory effects on TPA induced mouse ear edema



186 at the test levels. The most potent inhibition occurred at 0.5 pmol for both quercetin (62.3%) and scopoletin (50.4%). For quercetin, we speculate that this phenomenon may be due to its antioxidant properties, who at lower concentrations, serve as antioxidant while at higher concentrations, pro-oxidant. However, for scopoletin, it may be due to a different mechanism since it was shown to have minimal antioxidant activity (38). Combination of two compounds did not enhance each other in their anti inflammatory activities. The mixture showed an inhibition rate of 50.1%, which is weaker than quercetin alone and not significantly different from scopoletin alone according to student's T test.

Inhibitory Effects on Pro-inflammatory Mediators PGE

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In the test against prostaglandin E (PGE ) expression, both quercetin and scopoletin showed stronger inhibitory effect on production of PGE release at lower concentrations. At a concentration of 0.25 pM, scopoletin seemed to be a stronger inhibitor than quercetin, which suppressed production of PGE by 57.7%. The combination of compounds showed no synergistic effect on thenactivity, decreasing from 57.7% in the case of scopoletin and 55.4% in the case of quercetin to 44.6% (Figure 3). The inhibition effects on PGE indicate that both compounds involves in the arachidonic acid metabolism pathway. Also, as PGE is able to induce production of other pro-inflammatory cytokines, we can foresee that a wide array of inflammation cascade would be suppressed following the reduction of PGE 2

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IL-ip When tested against inhibition of IL-ip, quercetin has a significant inhibitory effect at 0.5 pmol level. As for scopoletin, the same trend seems to exist. At a low level of 0.25 pmol level, IL-ip release is reduced by 26.1 %. This trend is contrary to the case of PGE assay. This is surprising since IL-lp is reported to be able to enhance phospholipase (PLA ) activation and production of prostaglandins. In this case, other factors that act as better PGE2 enhancer than IL-lp may play a more important role. Further study on other mediators would help to explain this phenomenon. In the meantime, an examination of the effect of combination of both compounds showed no synergistic effect on the inhibition of IL-lp at tested condition (Figure 4). 2

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Figure 2. Effects of quercetin and scopoletin on TPA-induced inflammation.

Figure 3. Inhibition effects of quercetin and scopoletin on PGE

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Figure 4. Inhibition effects of quercetin and scopoletin on IL~lp.

IL-6 Figure 5 shows the effect of both compounds against IL-6. Quercetin significantly inhibited IL-6 release at both test levels, with higher inhibition at 0.5 nmol, with an inhibition rate of 51.4%. Similarly, scopoletin inhibited IL-6 protein synthesis at both test levels too, and the maximun activity occurred at 0.25 (imol with a reduction rate of 28.0%. The fact that both compounds showed higher suppression effect at lower concentration seems to agree with that of IL-ip. This is understandable since IL-ip is responsible for stimulating the production of other pro-inflammatory cytokines, including IL-6. Additionally, quercetin was shown to be a stronger inhibitor of IL-6 when applied at the same concentration with scopoletin. Again, the combination of scopoletin with quercetin did not bring any additional enhancing benefit at the test condition.

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Figure 5. Effects of quercetin and scopoletin on IL-6

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