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Chapter 21

Inhibition of Inflammation, Expression of Pro-inflammatory Cytokines, Formation of Leukotriene B and Tumor Promotion in Mouse Skin by Boswellia serrata Extracts

Downloaded by UCSF LIB CKM RSCS MGMT on November 21, 2014 | http://pubs.acs.org Publication Date: September 1, 2008 | doi: 10.1021/bk-2008-0987.ch021

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Mou-Tuan Huang , Yue Liu , Vladimir Badmaev , and Chi-Tang H o 3

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Department of Chemical Biology, Susan Lehman Cullman Laboratory for Cancer Research, School of Pharmacy, Rutgers University, Piscataway, NJ 08854-8020 Sabinsa Corporation, Piscataway, NJ 08854 Department of Food Science, Rutgers University, New Brunswick, NJ 08901 2

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The Boswellia serrata extract (BE) that contains β-boswellic acids and related compounds has been used as an herbal medicine (Ayurvedic system) for the treatment of arthritis and other inflammation related diseases in India and China. In this manuscript, we report that a preparation of Boswellia extract (BE) that contained about 35-40% β-boswellic acid and its derivatives inhibited 12-0-tetradecanoylphorbol-13-acetate (TPA)-induced increases in inflammation, up-expression of pro-inflammatory cytokine protein levels, and decreased formation of arachidonic acid metabolite, leukotriene B in mouse ears as well as inhibited TPA-induced increases in epidermal proliferation and tumor promotion in mouse epidermis previously initiated with 7,12-dimethylbenz[a]anthrance (DMBA) in CD-1 mice. A single topical application of TPA to ears of CD-1 mice induced a time and dose-dependent increases in acute inflammation (edema). A single topical application of 0.06 - 0,24 mg of BE 20 min prior to 1.0 nmol of TPA treatment inhibited TPA-induced acute inflammation by 40-84% in a dose-dependent manner. 4

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© 2008 American Chemical Society

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


305 Application of BE to mouse ears 20 min prior to each TPA application once a day for 4 days inhibited TPA-induced persistent inflammation as well as inhibited TPA-induced increases in up-expression of IL-1 P and 11-6 protein levels. BE also inhibited TPA-induced increases in formation of arachidonic acid metabolite. Topical application of 1.2-3.6 mg of BE 10 min prior to each TPA (5 nmol) treatment once a day for 2 days strongly inhibited TPA-induced increases in incorporation of bromodeoxyuridine (BrdUr) into epidermal DNA by 71-81%. Topical application of 1.2-3.6 mg of BE 10 min prior to each TPA (nmol) twice a week for 18 weeks treatment to initiated with DMBA mice inhibited the number of skin tumors per mouse by 82-98% and skin tumor incidence was inhibited by 48-90%. The ability of BE to inhibit TPAinduced increases in persistent inflammation, up-expression of pro-inflammatory cytokine proteins as well as to inhibit formation of leukotriene (LTB ) in mouse skin that may play a role on its inhibitory effect on TPA-induced tumor promotion. 4

Introduction The gum resin of plant Boswellia serrata grows in the dry part of China and India. The gum resin exudates of B serrata is collected from the stem of the tree B. serrata and is used as a commercial source for the herbal medicine (7). The anti-inflammatory activity of Boswellic acids is due to their ability to inhibit 5lipoxygenase activity (7-5). P-Boswellic acids, the pentacyclic triterpenic acids (Figure 1) are the main constituents in the gum resin of the plant (5). The gum resin exudates of B serrata has also been reported to have anti-hyperlipidemic and anti-atherosclerotic activities (6,7), and anti-colorectal carcinogenic effects on mouse model (8,9). P-Bosweelic acid and its derivatives also have been reported to inhibit the growth of several cancer cells in cultures (10-13). At present study, we report that topical application of BE to ears of CD-I mice inhibited TPA-induced acute and persistent inflammation and inhibited TPAinduced increases in expression of pro-inflammatory cytokine IL-lp and IL-6 protein levels in ears of mice, and decreased formation of arachidonic acid metabolite, leukotriene B in mouse ears as well as inhibited TPA-induced increases in epidermal proliferation and tumor promotion in mouse epidermis previously initiated with DMBA in CD-I mice. 4


306 CH,


R ^

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R = Ac, R = H R = H, R = O R = Ac, R = O 1

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P-boswellic acid 3-O-acetyl-P-boswellic acid 11 -keto-p-boswellic acid 3-O-acetyl-11 -keto-p-boswellic acid

Figure I. Chemical structures of P-boswellic acid and its derivatives.

Materials and Methods

Preparation of Boswellin serrata Extract and Chemical Composition Oleogum resin exudates of Boswellia serrata (100 g) was extracted with methanol (200 mL x 3), the methanol evaporated in vacuum to give 45 g of dried extract. The dried extract (30 g) was dissolved in 2% KOH solution (200 mL) and extracted with ethyl acetyl and the ethyl acetatefractionwas discarded. The aqueous solution was neutralized with 2% HC1 to pH 6.0, and then extracted again with ethyl acetate (5 x 150 mL). The combined ethyl acetate solution was washed with water, and dried with anhydrous Na S0 overnight, and then evaporated to dryness to produce 18 g of residue (Boswellin, a mixture of boswellic axcid and its derivatives). Boswellin (BE) contains about 13-18% of P-boswellic acid, 11-17% 3-0-acetyl-P-boswellic acid, 6-8% 11-keto-Pboswellic acid and 4-7% 3-O-acetyl-l 1-keto-P-boswellic acid. 2

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Animals Female CD-I mice (23-25 days for ear inflammation experiments), or (6-7 weeks old for skin tumor experiments) were purchased from Charles River Laboratories (Kingston, NY). The mice were kept in the Susan Lehman Cullman Laboratory for Cancer Research animal facility for at least 1 week before use. Mice were fed with a Purina Laboratory Chow 5001 diet from Ralston-Purina Co. (St Louis, NO) and water ad libitum and kept on a 12 h light/12 h dark cycle. The dorsal region of each mouse was shaved by electric clippers at least 2 days before treatment with TPA or DMBA. Only mice that did not show signs of hair re-growth were used.

Measurement of TPA-induced Acute Inflammation in Ears of CD-I Mice Measurement of mouse ear edema was done according to the following procedure. Both ears of female CD-I mice (23-25 days old; 5-6 mice per group) weren treated topically with 20 pL acetone, 0.5 nmol TPA in acetone or Boswellin extract (BE) together with 0.5 nmol TPA in acetone. Six hours later the mice were sacrificed by cervical dislocation, and 6-mm (diameter) ear punches biopsies were taken and weighed. The increases in weight of ear punches were directly proportional to the degree of inflammation (75).

Determination of TPA-induced Persistent Inflammation, Upexpression of Cytokine IL-1 and IL-6 proteine Levels, Formationof PGE2 and LTB4 Levels in Ears of CD-I Mice For persistent inflammation, both ears of female CD-I mice were treated topically once a day for 4 days either 10 pL acetone (vehicle) or test compound in acetone 20 min prior to each application of acetone or 0.8 nmol TPA in acetone. Ears were persistent inflammation during the 4 days of TPA treatment. The mice were sacrificed 6 h after the last TPA treatment. Ear punches (6-mm in diameter) were taken and weighed. Ear samples from each group were pooled and homogenized as described in the next section (75)

Preparation of Ear Homogenate for ELISA Assays Ear tissues were homogenized in a PBS containing 0.4 M NaCl, 0.05% Tween-20, 0.5% BSA, 0.1 mM PMSF, 0.1 mM benzethonium, 10 mM EDTA, and 20 U aprotinin per ml. The supernatantfractionwas used for determination


308 of cytokine protein levels. A two-site sandwich ELISA was used to assay for cytokines (75).

Determination of Proliferation of Epidermis by Bromodeoxyuridine (BrdUr) Labeling Index


Immunohistochemical staining for BrdUr was performed according to the procedure of the commercial assay kit as described in our previous publication

(91

Tumor Studies on Mouse Skin The dorsal region of female mice CD-I mice (7-8 weeks old) was shaved with electric clippers. For studies on the inhibitory effect of topical application of BE on TPA-induce tumor promotion, the mice (30 per group) were treated topically with 200 nmol of 7,12-dimethylbenz[a]athracene (DMBA) in 100 uL acetone. After 1 week of the mice were treated topically with 200 |xl of acetone, 5 nmol of TPA, or 1.2 or 3.6 mg of BE 10 min prior to 5 nmol TPA twice weekly for 20 weeks. A control group of mice received 200 ul acetone alone. Skin tumors greater than 1 mm in diameter were counted and recorded every 2 weeks. All skin tumors were examined histopathologically (16).

Results Inhibitory Effect of BE on TPA-induced Increases in Acute Inflammation Mouse Ears The possibility that BE could inhibit TPA-induced edema (acute inflammation) was evaluated by studying the effects of BE on TPA-induced edema of mouse ears. A preparation of Boswellia extract (BE) contained about 35-40% of total P-boswellic acids (w/w) was used for our studies. Topical application of 0.06-0.24 mg of BE with 0.5 nmol of TPA to the ears of mice inhibited TPA-induced edema by 40-84% (Figure 2). The inhibitory effect of BE on TPA-induced ear edema was dose-dependent when BE was given together with TPA or 30 min before TPA. Several preparations of BE were evaluated their anti-inflammatory activity. The preparation of BE containing the higher total amount of p-boswellic acids was the higher the anti-inflammatory activity.



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Figure 2. Effect ofBE on TPA-induced acute inflammation in ears of CD-I mice. Both ears of mice were treated topically with acetone or test compound in acetone at 20 min prior to topical application of acetone or TPA in acetone. The mice were sacrificed at6h after TPA treatment. Ear punches were taken and weighed. Data are mean±SE from 10 ear samples per group. ^Statistically different fro the group 2 (P

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