The Effects of Ginsenoside Re and its Metabolite, Ginsenoside Rh1, on 12-O-Tetradecanoylphorbol 13-acetate- and Oxazolone-Induced Mouse Dermatitis Models

• Abstract
• Abbreviations
• Materials and Methods
• Acknowledgements
• References

Abstract

The effects of the main constituent ginsenoside Re in ginseng and its metabolite, ginsenoside Rh1, were investigated in 12-O-tetradecanoylphorbol 13-acetate (TPA)- and oxazolone-induced mouse ear dermatitis models. Ginsenoside Rh1 potently suppressed the TPA- and oxazolone-induced swellings as well as mRNA expression levels of cyclooxygenase-2, IL-1β and TNF-α, although these were only weakly inhibited by ginsenoside Re.

Abbreviations

TPA:12-O-tetradecanoilphorbol 13-acetate

COX:cyclooxygenase

TNF-α:tumor necrosis factor-α

IL-1β:interleukin-1β

Psoriasis is a chronic and inflammatory skin disorder. Psoriasis patients have been shown to have an interferon (IFN)-γ producing Th1 bias in lesion skin and peripheral blood, although cyclooxygenase (COX)-2 is also induced [1], [2]. To evaluate the effects of antipsoriatic agents, some animal models of experimental psoriasis have been developed, using 12-O-tetradecanoylphorbol 13-acetate (TPA)-applied [3] and oxazolone-induced animals [4].

Ginseng (the root of Panax ginseng C.A. Meyer, Araliaceae) is frequently taken orally as a traditional medicine in Asian countries. The major components of ginseng are ginsenosides [5]. These ginsenosides have been reported to exhibit various biological activities, including anti-inflammatory action [6], antiallergic [7], [8] and antitumor effects [9]. The pharmacological actions of ginsenosides have been explained in terms of their biotransformations by human intestinal bacteria [10], [11]. The transformed protopanaxadiol ginsenoside, compound K, exhibited anti-passive cutaneous anaphylaxis and anti-inflammatory effects [8]. However, the antipsoriatic effects of ginsenoside Re, which is the main protopanaxatriol ginsenoside in ginseng, and its metabolite, ginsenoside Rh1 [11], [12], have not been studied. In the present study, the antipsoriatic effect of ginsenosides Re and Rh1 were investigated (Fig. [1]).

The antipsoriatic effects of ginsenosides Re and Rh1 in TPA-induced mouse ear dermatitis were also investigated (Fig. [2]). When TPA alone was applied to the mouse ear, erythema (reddening of the skin), edema and/or induration occurred. The ear thicknesses, measured as an index of skin inflammation, increased by 191 % at 48 h. The application of ginsenosides Re or Rh1 with TPA also potently suppressed ear swelling. Their suppressive rates, at a dose of 0.05 %, were 41.6 and 57.4 %, respectively, on the third day. The suppressive rate of betamethasone, used as a positive agent, at a concentration of 0.05 % was 80.7 %.

The effects of ginsenosides Re and Rh1 in oxazolone-induced mouse ear dermatitis were also evaluated (Fig. [3]). The oxazolone, applied to sensitized mice ears, caused erythema, edema and/or indurations and sometimes abrasions. The ear thickness increased about 2-fold compared to that with TPA, which reached a maximum 16 days after sensitization. The application of ginsenoside Rh1, with oxazolone, also potently suppressed the ear swelling at each time-point, although ginsenoside Re caused only weak inhibition. The suppressive rates of ginsenoside Rh1 at doses of 0.01 and 0.05 % were 27.6 and 49.2 %, respectively, at 16 days. The suppressive rate of betamethasone at a concentration of 0.05 % was 82.6 %.

Betamethasone and FK-506 are useful for psoriasis [4], [13]; however, they exhibit side effects, such as severe nephrotoxicity and neurotoxicity. Therefore, new agents for clinical uses should be developed from Chinese traditional medicines. We screened potential antipsoriatic compounds from antiallergic natural products. Ginseng was found to show inhibitory activity in TPA- and oxazolone-induced mouse ear dermatitis models. Ginsenoside Rh1 significantly inhibited the swelling in the mouse ear dermatitis models induced by TPA and oxazolone. Ginsenoside Rh1 also inhibited the mRNA expression levels of the induced COX-2, IFN-γ, TNF-α and IL-1β in mouse ear dermatitis, as well as histopathologically improved the swelling and abrasion (data not shown).

However, the inhibitory effect of ginsenoside Re was relatively weak compared to that of ginsenoside Rh1. Nevertheless, the in vivo effect of ginsenoside Re may be similar to that of ginsenoside Rh1, as ginsenoside Re was easily metabolized to ginsenoside Rh1 when the ginseng extract was orally administered to rats [8], [11], [12]. Furthermore, when ginseng is orally administered, its protopanaxatriol ginsenoside Re can be easily metabolized to ginsenoside Rh1 [11]. Based on these findings, ginsenoside Rh1 and ginseng may improve irritant or allergic contact dermatitis, and potentially psoriasis, by regulation of the expressions of COX-2, TNF-α and IL-1β produced by macrophage cells and that of IFN-γ produced by Th1 cells.

Materials and Methods

Isolation of ginsenoside Re and its metabolite ginsenoside Rh1 from human intestinal microflora: Fresh human feces (5 g) was suspended in 100 mL of TS broth, centrifuged at 500 × g for 10 min, and the resulting supernatant centrifuged at 10 000 × g for 30 min. The resulting precipitate was suspended in 100 mL of 20mM phosphate buffer (pH 7.0), containing 150 mg of ginsenoside Re (purity, > 90 %), according to the previous method [8], incubated for 24 h at 37 °C, and extracted with BuOH. The BuOH fraction was subjected to chromatography on a silica gel column, using CHCl₃-MeOH-H₂O (10 : 3:1, lower layer) as eluent to isolate 35 mg of ginsenoside Rh1 (35 mg; purity, > 95 %) as the main metabolite.

Contact dermatitis: The ICR mice (20 - 25 g) were supplied from the Orient Experimental Animal Breeding Center (Seoul, Korea). All animals were housed in wire cages at 20 - 22 °C, with 50 ± 10 % humidity, fed standard laboratory chow (Orient Experimental Animal Breeding Center, Seoul, Korea) and allowed water ad libitum. All procedures relating to the animals and their care conformed to the international guidelines of the ‘Principles of Laboratory Animals Care’ (NIH publication no. 85 - 23, revised 1985).

TPA (Sigma Co., St Louis, MO, USA)-induced mouse ear dermatitis was measured according to the previous method of Park et al. [14]. Oxazolone (Sigma Co.)-induced dermatitis was measured according to the previous method of Fujii et al. [4]. The ear thickness, measured as an index of ear skin inflammation, was measured using a Digimatic Micrometer (Mitsutoyo Co., Tokyo, Japan) 72 h after each application of oxazolone and 3 h after the final treatment of ginsenosides. RT-PCR analysis of the ear tissues was performed by the method of Shin et al. [15].

All data were expressed as the mean ± standard deviation. The statistical significance was analyzed by a one-way ANOVA, followed by a Student-Newman-Keuls test.

Acknowledgements

This work was supported by a grant of the Korean Food Research Institute (2003).

References

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  PubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar

Prof. Dr. Dong-Hyun Kim

College of Pharmacy

Kyung-Hee University

1 Hoegi

Dongdaemun-ku

Seoul 130-701

Korea

Fax: +82-2-957-5030

Email: dhkim@khu.ac.kr

References

• 1 Nicoloff B J. The cytokine network in psoriasis.  Arch Dermatol. 1991;  127 871-84
  CrossrefPubMedSearch in Google Scholar
• 2 Hernandez G L, Volpert O V, Iniguez M A, Lorenzo E, Martinez-Martinez S, Grau R. et al . Selective inhibition of vascular endothelial growth factor-mediated angiogenesis by cyclosporin A: roles of the nuclear factor of activated T cells and cyclooxygenase 2.  J Exp Med. 2001;  193 607-20
  CrossrefPubMedSearch in Google Scholar
• 3 Reynolds N J, Voorhees J J, Fisher G H. Cyclsoporin A inhibits 12-O-tetradecanoylphorbol 13-acetate-induced cutaneous inflammation in severe combined immunodeficient mice that lack functional lymphocytes.  Br J Dermatol. 1998;  139 16-22
  CrossrefPubMedSearch in Google Scholar
• 4 Fujii Y, Takeuchi H, Tanaka K, Sakuma S, Ohkubo Y, Mutoh S. Effects of FK-506 (tacrolimus hydrate) on chronic oxazolone-induced dermatitis in rats.  Eur J Pharmacol. 2002;  456 115-21
  CrossrefPubMedSearch in Google Scholar
• 5 Shibata S, Fujita M, Itokawa H, Tanaka O, Ishii T. Panaxadiol, a sapongenin of ginseng roots (1).  Chem Pharm Bull. 1963;  11 759-64
  PubMedSearch in Google Scholar
• 6 Wu J Y, Gardner B H, Murphy C I, Seals J R, Kensil C R, Recchia J. et al . Saponin adjuvant enhancement of antigen-specific immune responses to an experimental HIV-1 vaccine.  J Immunol. 1992;  148 1519-25
  PubMedSearch in Google Scholar
• 7 Choo M K, Park E K, Han M J, Kim D H. Antiallergic activity of ginseng and its ginsenoside.  Planta Med. 2003;  69 518-22
  Thieme ConnectPubMedSearch in Google Scholar
• 8 Park E K, Choo M K, Han M J, Kim D H. Ginsenoside Rh1 possesses antiallergic and anti-inflammatory activities.  Int Arch Allergy Immunol. 2004;  133 113-20
  CrossrefPubMedSearch in Google Scholar
• 9 Wakabayashi C, Hasegawa H, Murata J, Saiki I. In vivo antimetastatic action of ginseng protopanaxadiol saponins is based on their intestinal bacterial metabolites after oral administration.  Oncol Res. 1998;  9 411-7
  PubMedSearch in Google Scholar
• 10 Akao T, Kida H, Kanaoka M, Hattori M, Kobashi K. Intestinal bacterial hydrolysis is required for the appearance of compound K in rat plasma after oral administration of ginsenoside Rb1 from Panax ginseng .  J Pharm Pharmacol. 1988;  50 1155-60
  PubMedSearch in Google Scholar
• 11 Tawab M A, Bahr U, Karas M, Wurglics M, Schubert-Zsilavecz M. Degradation of ginsenosides in humans after oral administration.  Drug Metab Dispos. 2003;  31 1065-71
  CrossrefPubMedSearch in Google Scholar
• 12 Hasegawa H, Sung J H, Matsumiya S, Uchiyama M. Main ginseng saponin metabolites formed by intestinal bacteria.  Planta Med. 1966;  62 453-7
  PubMedSearch in Google Scholar
• 13 Schafer-Korting M, Schmid M H, Korting H C. Topical glucocorticoids with improved risk-benefit ratio.  Drug Saf. 1996;  14 375-85
  CrossrefPubMedSearch in Google Scholar
• 14 Park B K, Heo M Y, Pak H, Kim H P. Inhibition of TPA-induced cyclooxygenase-2 and skin inflammation in mice by wogonin, a plant flavone from Scutellaria radix .  Eur J Pharmacol. 2001;  425 53-8
  PubMedSearch in Google Scholar
• 15 Shin Y W, Bae E A, Kim S S, Lee Y C, Kim D H. Effect of ginsenoside Rb1 and compound K in chronic oxazolone-induced mouse dermatitis.  Int Immunopharmacol. 2005;  5 1183-91
  CrossrefPubMedSearch in Google Scholar

Prof. Dr. Dong-Hyun Kim

College of Pharmacy

Kyung-Hee University

1 Hoegi

Dongdaemun-ku

Seoul 130-701

Korea

Fax: +82-2-957-5030

Email: dhkim@khu.ac.kr