Dietary Supplements - American Chemical Society

Chapter 20

Antiinflammatory and Anticancer Activities of Garcinol 1

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Downloaded by UNIV OF NEW HAMPSHIRE on March 5, 2015 | http://pubs.acs.org Publication Date: September 1, 2008 | doi: 10.1021/bk-2008-0987.ch020

Mou-Tuan Huang , Yue Liu , Vladimir Badmaev , and Chi-Tang H o

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Department of Chemical Biology, Laboratory for Cancer Research, School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854-8020 Sabinsa Corporation, 70 Ethel Road West, Unit 6, Piscataway, NJ 08854 Department of Food Science, Rutgers, The State University of New Jersey, 65 Dudley Road, New Brunswick, NJ 08901-8520 2

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Garcinol was isolatedfromfruitrinds of Garcinia indica, and it contains one phenolic catechol group and 3 beta-diketone groups. Due to its novel chemical structure, it possesses strong antioxidant and anti-inflammatory activities. Topical application or oral administration of garcinol to CD-1. mice markedly inhibited TPA-induced ear inflammation in a dosedependent manner. Topical application of garcinol to ears of CD-1 mice also markedly inhibited TPA-induced upexpression of pro-inflammatory cytokine IL-6 protein levels in ears in dose-dependent fashion. Oral administration of garcinol inhibited UVB-induced ear inflammation, and blocked upexpression of pro-inflammatory cytokine IL-1 beta and IL-6 protein levels in ears. Topical treatment of garcinol to backs of mice strongly inhibited TPA-induced skin tumor promotion in mice previously initiated with 7,12-dimethylbenz[a]anthracene (DMBA) both the numbers of skin tumors per mouse and percent of mice bearing with skin tumors.

© 2008 American Chemical Society

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

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Introduction Garcinol that is a polyisoprenylated benzophenone derivative was isolated from the fruit rinds of Garcinia indica (1-4). Subsequently, in 1981 Krishnamurthy et al established the chemical structure of garcinol (1-2) and then revised by Bakana et al (3). The chemical structure of garcinol is shown in Figure 1. Garcinol contains one ortho-hydroxyl group (catechol group) and three beta-diketone groups. It has a conjugated double bond that exhibits strong antioxidant property (1-6). Yamaguchi et al demonstrated that garcinol had strong antioxidant property by using several in vitro assay systems (6). Electron spin resonance spectrometry (ESR) studies indicate garcinol had free radical scavenging activity. In the hypoxanthine oxidase assay system showed that garcinol was able to suppress superoxide anion as similar degree as alphatocopherol. Garcinol also was shown to suppress superoxide anion, hydroxyl radical and methyl radical in the H 0 /NaOH/DMSO system assay. In additional, Garcinol suppressed hydroxyl radical more strongly than DL-alphatocopherol in the Fenton reaction system. Taken together, garcinol is a potent free radical scavenger and is able to scavenge both hydrophilic and hydrophobic free radicals including reactive oxygen species. Garcinol competitively inhibited xanthine oxidase activity with an IC value of 52 \iM (7). Liao et al also demonstrated that garcinol inhibited LPS-induced formation NO free radical in astrocytes (7). Garcinol inhibited formation NO free radical in the mouse RAW 264.7 macrophage cells in cultures (7-9). In this report, we demonstrated that garcinol had strong anti-inflammatory activity, and inhibited TPA- or UVBinduced ear inflammation and up-expression of pro-inflammatory cytokine IL-1 beta and IL-6 protein levels in ears of mice as well as was a potent inhibitor of TPA-induced skin tumor promoting activity in DMBA-initiated mice. 2

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Garcinol Figure 1. Chemical structure of garcinol.


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Materials and Methods Materials


7,12-Dimethylbenz[a]anthracene and 12-0-tetradecanoylphorbol-13-acetate were purchased from Sigma-Aldrich (St. Louis, MO). Acetone and 10% formalin-phosphate buffer were purchased from Fisher Scientific (Springfield, NJ).

Animals Female CD-I mice (3-4 weeks old for ear inflammation experiment, or 7-8 weeks old for skin tumor experiment were purchased from Charles River Laboratories (Kingston, NY). The mice were kept in our animal facility for at least one week before use. Mice were fed a Purina Laboratory Chow 5001 diet from Ralston-Purina Co. (St Louis, MO) and water ad libitum and kept on a 12 h light/12 h dark cycle. The dorsal area of each mouse was shaved with electric clippers at least 2 days before treatment with TPA or DMBA. Only mice that did not show signs of hair regrowth were used.

Measurement of Mouse ear Inflammation Measurement of mouse ear inflammation was done according to the following procedure. Both ears of female CD-I mice (3-4 weeks old; 5-6 mice per group) were treated topically with 20 pL acetone, 0.5 nmol TPA in acetone or test compound in acetone 10 min prior to TPA (1 nmol) treatment. Six hours later the mice were sacrificed by cervical dislocation, and 6-mm in diameter ear punches biopsies were taken and weighed. The increase in weight of ear punches was directly proportional to the degree of inflammation.

Tumor Studies on Mouse Skin The dorsal area of female CD-I mice (8 weeks old) was shaved with electric clippers. For studies on the inhibitory effect of topical application of garcinol on TPA-induced skin tumor promotion, the mice (30 mice per group) were treated topically with 200 nmol of 7,12-dimethylbenz[a]anthracene (DMBA) in 100 pL of acetone. A control group of mice received 200 pL of acetone alone. After 1 week the mice were treated topically with 200 pL of acetone or TPA (5 nmol) in acetone. Garcinol (0.2 or 10 pmol) in acetone were treated topically on dorsal


296 area 10 min prior to TPA treatment twice weekly for 10 or 14 weeks. Skin tumors greater than 1 mm in diameter were counted and recorded every 2 weeks. All skin tumors were examined histopathologically.


Preparation of Garcinol. Garcinol was isolated and purified from the dried fruit rinds of Garcinia indica (Kokum) according to the procedure as described by Yamaguchi et al (5). The dried fruit rinds of G. indica was extracted with ethanol. The extract was fractionated by preparative ODS (Octadecyl silica) column chromatography eluted stepwise with 70-80% (v/v) ethanol monitored at UV 254 nm, and the main fraction absorption at 254 nm eluted at 80% (v/v) ethanol were concentrated and dried by the rotary evaporator under 50 °C. The dried material was resolved in hexane and solution was under 5 °C for 2 days. Yellow amorphous powder was collected from the solution and washed with cold hexane on a glass filter. After drying in a vacuum desiccator, the amorphous was solubilized in hot acetonitrile and recrystallized at room temperature. Pale yellow crystal needles (garcinol) were obtainedfromsolvent.

Results Anti-inflammatory Activity Both in vitro and in vivo tests have been used to evaluate the anti inflammatory activities of garcinol (7-9).

The In vitro Cell Culture Anti-inflammatory Assay Inhibition of lipopolysaccharide (LPS)-induced up-expression of iNOS and COX-2 in the mouse RAW264.7 macrophage cell culture has been widely used as the in vitro anti-inflammatory assays in cell culture system. Garcinol strongly inhibited LPS-induced up-expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) in astrocytic and in the mouse RAW264.7 macrophage cell cultures (7-9). Since inflammation is associated with upexpression of iNOS and COX-2 mRNA and protein levels.


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Inhibitory Effect of Topical Application of Garcinol and Curcumin on TPAinduced Edema of Mouse Ears We have tested the anti-inflammatory activity of garcinol in a mouse ear inflammation model. Garcinol had a strong anti-inflammatory activity in TPAinduced edema of mouse ear test. The results are shown in (Table I). A single topical application of 1 nmol of 12-0-tetradecanoylphorbol-13-acetate (TPA) on ears of CD-I mice resulted in increase in ear inflammation by increase an average weight of ear punches from 7.4 mg (acetone control group) to 15.1 mg (TPA-treated group) per punch. Topical application of 0.2 or 0.5 \imol of garcinol together with TPA (1.0 nmol) treatment resulted in suppressing TPAinduced ear inflammation by 39 or 78%, respectively. Topical application or 0.2 or 0.5 nmol of curcumin together with TPA (1.0 nmol) on ears of CD-I mice inhibited TPA-induced ear inflammation by 58 or 88%, respectively (Table I). Our results suggested that both garcinol and curcumin had potent anti inflammatory activity in the mouse ear inflammation model.

Table I. The Effects of Topical Application of Garcinol and Curcumin on TPA-induced Edema of Mouse Ears in CD-I Mice Treatment Acetone TPA Garcinol (0.1 nmol) + TPA Garcinol (0.5 nmol) + TPA Curcumin (0.1 nmol) + TPA Curcumin (0.5 nmol) + TPA

Average weight of ear punches (mg) 7.44±0.36* 15.05±0.82 12.14±0.81* 9.25±0.66* 10.20±0.80* 8.41±0.33*

% Inhibition —

39% 78% 58% 88%

Both ears of mice (4 weeks old; 5 mice per group) were treated topically with acetone, TPA in acetone or TPA + test compound in acetone. The mice were sacrificed 6 h after TPA treatment. Ear punches (6-mm in diameter) were taken and weighed. Data are expressed as the mean±SE. •Statistically different from the group 2 TPA alone (P

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