Research paper
In vivo photoprotective and anti-inflammatory effect of hyperforin is associated with high antioxidant activity in vitro and ex vivo

https://doi.org/10.1016/j.ejpb.2012.03.002Get rights and content

Abstract

Hyperforin, a major constituent of St. John’s Wort (Hypericum perforatum, HP), provides anti-inflammatory, anti-tumor, and anti-bacterial properties. Previous studies have shown anti-oxidative properties of St. John’s Wort extracts; however, its free radical scavenging activity in skin cells or skin has not been assessed in detail so far. Therefore, the free radical scavenging activity of hyperforin was tested in the H2DCFDA-assay in vitro in HaCaT keratinocytes irradiated with solar simulated radiation. Hyperforin (EC50 0.7 μM corresponding to 0.42 μg/ml) was much more effective compared to Trolox (EC50 12 μg/ml) and N-acetylcysteine (EC50 847 μg/ml) without showing phototoxicity. The radical protection factor of a cream containing 1.5% w/w of a hyperforin-rich HP extract was determined to be 200 × 1014 radicals/mg, indicating a high radical scavenging activity. The cream was further applied ex vivo on porcine ear skin and significantly reduced radical formation after infrared irradiation. Finally, the UV-protective effect of the HP cream was tested on 20 volunteers in a randomized, double-blind, vehicle-controlled study. HP cream significantly reduced UVB-induced erythema as opposed to the vehicle. Occlusive application of HP cream on non-irradiated test sites did not cause any skin irritation. Taken together, these results demonstrate that hyperforin is a powerful free radical scavenger.

Graphical abstract

A cream containing a Hypericum perforatum extract protects against radical formation.

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Introduction

The skin is our barrier against the oxidative environment such as nitrogen oxide, cigarette smoke, and ozone [1]. The most prominent oxidative noxe is the sunlight, which induces reactive oxygen species (ROS) mainly in the UVB and UVA spectral range. However, recent investigations have shown that in the visible and infrared spectral range, ROS are also produced [2], [3]. ROS are responsible for premature skin aging and skin cancer and should be in balance with the antioxidative defense system. Therefore, the skin is equipped with an enzymatic and non-enzymatic antioxidative system. In order to strengthen the antioxidative defense system, several cosmetic formulations containing antioxidants are available for topical application. Among various plant extracts, lipophilic extract from St. John’s Wort (Hypericum perforatum, HP) is used for the treatment of atopic skin. A major constituent of such lipophilic extracts is hyperforin, which is known to provide anti-inflammatory, anti-tumor, and anti-bacterial properties [4], [5], [6]. Topical treatment of mild atopic dermatitis with a cream containing 1.5% HP extract was significantly superior to the vehicle [7]. Investigations on antioxidative properties of aqueous extracts of several Hypericum species have shown that Hypericum perforatum has the highest radical scavenging activity [8]. Hyperforin was only partly present in the aqueous extracts because of its lipophilicity. Sagratini et al. [9] have shown that more lipophilic methanol–acetone extracts contain pharmacologically important compounds that exhibited a mild to moderate antioxidant activity. Hypericum perforatum s.l. (subsp. veronense in particular) was found to be the richest in hypericin, hyperforin, and particularly in phenolic compounds [9]. Although these studies have shown antioxidative properties of HP extracts, no information about the radical scavenging activity of hyperforin in vitro on skin cells, ex vivo on excised skin, or in vivo is available.

Therefore, we investigated the antioxidative effect of hyperforin on skin cells irradiated with solar simulated radiation (SSR). The radical scavenging activity of a cream containing a hyperforin-rich HP extract was measured by determining the radical protection factor (RPF) of the formulation. Furthermore, we assessed whether HP cream application on skin biopsies can decrease subsequent infrared-induced radical formation. Finally, we tested whether skin treatment with HP cream may reduce UVB-induced skin erythema on human skin in vivo.

Section snippets

Topical formulations

For ex vivo and in vivo investigations, a verum cream containing 1.5% of a HP CO2-extract with 44.3% hyperforin (Flavex Naturextrakte GmbH, Rehlingen, Germany) and a vehicle cream were applied. The HP extract was free of hypericin.

For the determination of the radical scavenging activity of the cream (RPF determination), HP concentrations of 0.5%, 1.5%, 3.0%, and 5.0% were used.

EPR experiments

EPR spectroscopy is based on the resonant absorption of microwave radiation by paramagnetic species exposed to a

Free radical scavenging effect of hyperforin in cells after UV irradiation

Hyperforin reduced the formation of SSR-induced DCF formation in HaCaT cells from 0.625 to 10 μM, whereas the solvent had no effect (Fig. 1A). The EC50 of hyperforin (0.7 μM or 0.35 μg/ml) was more effective compared to Trolox (EC50:12 μg/ml) and N-acetylcysteine (EC50: 847 μg/ml). No phototoxic effect of hyperforin was observed (Fig. 1B).

Radical scavenging effect of the cream without irradiation

The radical scavenging activity was determined using the radical protection factor (RPF) method, which uses a semi stable radical and measures the amount of

Discussion

Hyperforin, the lipophilic main active ingredient of HP, has anti-bacterial, anti-inflammatory, and differentiation promoting properties [4], [7], [17]. In this paper, we have shown that hyperforin reduces ultraviolet-induced oxidative stress without phototoxic effects in vitro. The half maximal radical scavenging concentration lies at 0.7 μM (0.35 μg/ml), which is similar to the antitumorigenic concentrations of hyperforin that have been described earlier [18]. Compared to previously published

Acknowledgments

We would like to thank Birgit Haarhaus for her excellent technical assistance. The study was partly sponsored by Cassella-med GmbH, the Foundation of Skin Physiology, and the Federal Land of Berlin within the Program to Promote Research, Innovation and Technologies (ProFIT, Grant No. 10148423) for granting subsidies from the innovation promotion fund, with co-financing by the European Fund for Regional Development (ERDF). The competence center skintegral receives funding from the Software AG

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