Beneficial actions of citrus polymethoxy flavonoids in treating acne.

Introduction

Acne vulgaris is an inflammatory disease in sebaceous glands and pilosebaceous units in the skin, and is characterised by excess sebum production, hypercornification of the follicular wall epidermis, local infection with Propionibacterium acnes, and local inflammatory events such as the augmentation of proinflammatory cytokine production and the formation of papules and pustules [1]. Treatments for acne include: benzoyl peroxide, retinoic acids such as tretinoin and isotretinoin, and adapalene; antibiotics are also widely prescribed [2-4]. In addition, herbal medicines are well known to be effective for the prevention and/or remission of various diseases including acne [5-10]. Flavonoids from medicinal plants have also been reported to possess various pharmacological effects including anti-inflammatory and anti-ageing effects against skin disorders [11-13]. We recently found a citrus polymethoxy flavonoid, nobiletin (5,6,7,8,3',4'-hexamethoxyflavone), that exhibits multiple pharmacological actions in both in vivo and in vitro models of rheumatoid arthritis, tumorigenesis, tumour invasion and metastasis, and photoageing [14-21]. This review describes the novel effects of nobiletin and related citrus polymethoxy flavonoids on sebaceous lipogenesis and sebum excretion [22], and discusses their application as acne therapies.

Acne pathology

The pathogenesis of acne is characteristic of: (1) excess sebum production and hyperplasia of sebaceous glands; (2) formation of microcomedones closely associated with hyperkeratinisation of follicular walls and infundibulum; and (3) induction of inflammatory reactions such as the enhancement of cytokine production and arachidonic acid metabolism in keratinocytes, sebocytes, and invading inflammatory cells [1]. Androgens such as testosterone and 5 [alpha]-dihydrotestosterone (5 [alpha] -DHT), insulin, prostaglandin J2, and peroxisome-proliferation stimulating factors have been reported to augment lipogenesis in sebaceous glands [23-28]. P. acnes, a Gram-positive anaerobe microbial species, contributes to the development of acne vulgaris in sebaceous glands and pilosebaceous units in the skin [29]. P. acnes lipase degrades triacylglycerols, a major component of sebum and a nutritional element of P. acnes, to produce free fatty acids, which are in turn associated with the hyperkeratinisation of follicular walls and infundibulum [1]. In addition, P. acnes-derived factor(s) have been reported to induce inflammatory reactions such as the augmentation of inflammatory cytokine production and extracellular matrix remodelling [30-33]. Furthermore, recent studies suggest a possibility that the skin flora of Gram-negative bacteria take part in the appearance and increase in deterioration of acne by locally enhancing inflammatory events in concert with P. acnes [29,34,35].

Application of nobiletin for acne therapy

Antilipogenic activity

Flavonoids have many pharmacological actions in vivo and in vitro. They are antioxidants, free radical scavengers and have anti-inflammatory, antitumourigenic and antitumour metastatic activities [16,17,36,37]. In the regulation of lipid synthesis, a common dietary flavonoid, quercetin, has been reported to exhibit the suppressive action of de novo triacylglycerol synthesis in human intestinal cells [38]. In addition, a citrus flavonoid, hesperetin, has been shown to inhibit triacylglycerol synthesis in rat hepatocytes [39]. Although the formulation of herbal medicines has been effective in improving the constitution of acne patients [10], there is little evidence on the regulation of sebaceous lipogenesis by herbal medicines. In this context, wogonin, a flavonoid derived from Japanese-Chinese traditional herbal medicines, has been reported to inhibit lipogenesis in hamster sebaceous glands [5]. We recently found that nobiletin (Figure 1), a major component in the juice of Citrus depressa Hayata (Rutaceae), inhibits sebum production by decreasing de novo triacylglycerol synthesis in vivo and in vitro [22]. Figure 2 shows a build-up of sebum in sebaceous glands and ducts (Panel A), and the decrease of the accumulation of sebum, and its level on the skin surface, in nobiletin-treated hamsters (Panel B).

[FIGURE 2 OMITTED]

Retinoic acids such as tretinoin (all-trans retinoic acid; ATRA) and isotretinoin (13-cis-retinoic acid; 13-cis-RA) have been topically and/or systemically used for acne therapy [2-4]. Both ATRA and 13-cis-RA have been shown to decrease sebum production and sebaceous gland enlargement by inhibiting lipogenesis and cell proliferation in humans, rats and hamsters [22,40-43]. We have previously demonstrated that ATRA and 13-cis-RA significantly diminish triacylglycerol production in hamster sebocytes in vivo and in vitro, and further that topical application of ATRA for 2 weeks causes hyperkeratinisation of the epidermis and infundibulum [22]. Therefore, these results suggest that the formulation of nobiletin may be clinically superior to at least ATRA in the decrease and/or avoidance of adverse effects such as scaling although the inhibitory activity on sebum production in sebaceous glands is similar.

Acceleration of sebum excretion

Since sebaceous glands in acne lesions facilitate lipogenesis and thereafter accumulate abundant sebum [1], not only the inhibition of sebaceous lipogenesis but also the prompt excretion of sebum from sebaceous glands to follicular ducts or on the skin surface is likely to be efficient for the remission of acne. We have revealed a unique property of nobiletin in that it positively stimulates the excretion of triacylglycerol to culture medium of differentiated hamster sebocytes [22]. In addition, the nobiletin-mediated triacylglycerol excretion is dependent on a transient increase in intracellular cAMP level and subsequent protein kinase A (PKA) activation. However, neither 13-cis-RA nor ATRA augments triacylglycerol excretion, the intracellular cAMP level, or PKA activity in differentiated hamster sebocytes. Furthermore, although sebum secretion is generally considered to be regulated by a holocrine mechanism, which may be associated with sebaceous apoptosis [44], nobiletin-enhanced sebum excretion is independent of the apoptosis of differentiated hamster sebocytes [22]. Therefore, nobiletin is likely to stimulate sebum excretion by a holocrine-independent mechanism that is associated with a transient increase in cAMP level and its sequential PKA activation in sebocytes.

Anti-inflammatory actions

Nobiletin has been reported to show anti-inflammatory actions in various cell species in vivo and in vitro. The production of prostaglandin E2, an inflammatory mediator, is suppressed by nobiletin in ultraviolet B-irradiated human keratinocytes [19] and in interleukin-1 (IL-1)-stimulated synoviocytes from humans and rabbits [14,17]. In addition, nobiletin has been reported to inhibit the gene expression and production of IL-1, IL-6 and tumour necrosis factor- [alpha] (TNF- [alpha] ) in lipopolysaccharide-stimulated mouse macrophages [17]. Furthermore, Murakami et al. [11] reported the in vivo anti-inflammatory effect of nobiletin on the phorbol ester-induced skin inflammation model in mice. Under the inflammatory conditions of the ultraviolet B-irradiated inflammation model of hamsters [22] and mice [17], the topical treatment of nobiletin has also been shown to prevent sebum production and hyperkeratinisation in epidermis and infundibulum. Taken together, with the understanding that acne is an inflammatory skin disorder [1], we propose that the topical application of nobiletin is effective for remission and/or cure of acne vulgaris by decreasing and/or preventing inflammatory reactions.

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

Antimicrobial activity

The topical and/or oral application of antibiotics such as erythromycin, clarithromycin and clindamycin is effective in acne therapy because these antibiotics inhibit the proliferation of P. acnes in the follicles thereby decreasing the inflammatory reactions in acne lesions [2,45]. However, in recent years, antibiotic-resistant P. acnes has been isolated from the skin of acne patients prescribed antibiotics [46,47]. The presence of P. acnes with antibiotic resistance is likely to contribute to a poor prognosis in acne patients. On the other hand, flavonoids and extracts from medicinal plants have been reported to exhibit antimicrobial activity against some bacteria [48,49]. For example, the natural flavonols, kaempferol and quercetin, have been reported to exhibit antimicrobial activity against antibiotic-resistant P. acnes [50]. In addition, an extract including nobiletin and tangeretin (4',5,6,7,8-pentamethoxyflavone) has been reported to show antimicrobial activity [51]. However, we have demonstrated that nobiletin does not exhibit antimicrobial activities against Escherichia coli, Staphylococcus aureus, Bacillus subtilis or Pseudomonas aeruginosa (Ito A, Noguchi M, Sasatsu M and Sato T, unpublished data). Although this discrepancy might be due to the method of isolation or the purity of flavonoids, nobiletin is unlikely to exert direct antimicrobial activity.

It has been reported that the antibiotics tetracycline and minocycline inhibit the activity of lipase in P. acnes [10,52], which thereafter diminishes the degradation of sebum leading to the hyperkeratinisation of follicular walls and infundibulum [1]. We previously reported that nobiletin inhibits the activity of cytosolic phospholipase [A.sub.2] in human keratinocytes [19], which may contribute to the decrease of prostanoid production associated with sebum production in sebaceous glands [25,26]. However, there has been no report on the regulation of P. acnes lipase activity by isolated flavonoids and their derivatives. Herbal candidates that exert direct inhibition of P. acnes lipase may be useful for the development of novel anti-acne drugs.

[FIGURE 5 OMITTED]

Other flavonoids

In addition to nobiletin, another citrus polymethoxy flavonoid, 3-hydroxy-5,6,7,8,3',4'-hexamethoxyflavone (natsudaidain) (Figure 1), has been isolated and characterised to exhibit antitumourigenic and anti-inflammatory activities [21,53]. We have demonstrated a number of characteristics. As in the case of nobiletin, natsudaidain inhibits the production of triacylglycerol (Figure 3A) by decreasing diacylglycerol acyltransferase activity in hamster sebocytes (data not shown). In addition, we found that natsudaidain augments the extracellular levels of triacylglycerol in differentiated hamster sebocytes (Figure 4A) and it also transiently increases the intracellular cAMP level in the differentiated sebocytes (1.6 [+ or -] 0.3-fold at 64 [micro]M vs. control cells). However, the structural and functional properties of natsudaidain have been shown to differ from those of nobiletin on the regulation of mitogen-activated protein/extracellular signal-regulated kinase [21].

Well-known dietary flavonoids, quercetin (Figure 1) and genistein, have been shown to exhibit various beneficial anti-inflammatory, antitumourigenic and antioxidant properties [54,55]. Quercetin inhibits intracellular triacylglycerol production in differentiated hamster sebocytes (Figure 3B), a result which is supported by Casaschi et al. [38]. In contrast to the polymethoxy flavonoids that we tested, quercetin did not increase the the excretion of triacylglycerol in the differentiated sebocytes (Figure 4B). The most likely reason for this was the finding that there was no increase in the intracellular level of cAMP (0.9 [+ or -] 0.3-fold at 64 [micro]M vs. control cells). However, it has been reported that dietary polyphenols, including quercetin, increase the intracellular cAMP level in rat pheochromocytoma PC12D cells [56] and in adipocytes from both rats [57] and mice [58]. Therefore, the regulation of cAMP production by polyphenols seems to be dependent on cell species. In fact, as shown in Figure 5, the bifunctional actions on the regulation of sebum production and excretion are likely to be specific to polymethoxy flavones such as nobiletin and natsudaidain rather than flavonols including quercetin.

Future prospects

Treatment with tretinoin, isotretinoin, antibiotics and steroid hormones results in remission and/or cure of acne [2-4]. However, there are many adverse effects associated with their use such as skin irritation and scaling and teratogenicity as well as the induction of bacterial resistance [2,46,47,59]. Novel anti-acne agents are required not only to have antilipogenic and anti-inflammatory properties, but also to have a reduced side-effect profile. Taken together with our in vivo findings of antilipogenesis in hamster sebocytes [22] and previous reports [15-21], nobiletin is a useful candidate for a novel anti-acne agent that demonstrates both anti-inflammatory and antilipogenic activity in sebaceous glands, pilosebaceous units and infiltrated inflammatory cells in acne lesions, but has few or no adverse effects or cytotoxicity. Moreover, Nagai et al. [60] have reported that hamster and rat sebocytes secrete microvesicles (termed sebosomes) containing histone H3 and squalene. Since histone peptides have been shown to exert antimicrobial activity [61], a physiological antimicrobial mechanism may be programmed in sebaceous glands. Compounds that accelerate the secretion of sebosomes from sebaceous glands may be also useful for the improvement of skin immunity, leading to a lower incidence of acne and other skin infection disorders. Finally, these studies may unexpectedly give hints on the development of novel drugs and strategies for acne therapy.

Acknowledgements

This work was supported by a grant to private universities provided by the Promotion and Mutual Aid Corporation for Private Schools of Japan, and by the Fifth TUPLS Innovative Research Promotion Grant. We also wish to thank Dr N. Akimoto for her technical assistance and helpful discussion, and Ms. A. Takahashi and Ms M. Kojima for their technical assistance.

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Correspondence to: Dr Takashi Sato, Department of Biochemistry and Molecular Biology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. (email: [e-mail]).

Takashi Sato and Akira Ito Department of Biochemistry and Molecular Biology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences

Figure 1: Chemical structures of flavonoids. Natsudaidain,
3-hydroxy-5,6,7,8,3',4'-hexamethoxyflavone.

Flavonoid           [R.sub.1]       [R.sub.2]    [R.sub.3]    [R.sub.4]

Nobiletin           H               OMe          Ome          OMe
Natsudaidain        OH              OMe          OMe          OMe
Quercetin           OH              OH           H            OH

Flavonoid           [R.sub.5]       [R.sub.6]    [R.sub.7]

Nobiletin           OMe             OMe          OMe
Natsudaidain        OMe             OMe          OMe
Quercetin           H               OH           OH