The Anti-Inflammatory Effect of Humulus lupulus Extract In Vivo Depends on the Galenic System of the Topical Formulation

Abstract

We demonstrated the anti-inflammatory and anti-oxidative effects of Humulus lupulus (HL) extract on solar simulator-irradiated primary human keratinocytes (PHKs) by analyzing ERK and p38 MAPK phosphorylation and production of IL-6 and IL-8. The anti-inflammatory effect of topically applied HL was further tested in vivo on human skin. To this end, we developed an oil-in-water (O/W) and a water-in-oil (W/O) cream with a lipid content of 40%. The anti-inflammatory effect of 1% HL extract incorporated in these two vehicles was assessed in a randomized, prospective, placebo controlled, double-blind UVB erythema study with 40 healthy volunteers. Hydrocortisone acetate (HCA) in the corresponding vehicle served as positive control. Surprisingly, both HL and HCA were only effective in the O/W system but not in the W/O formulation. Release studies using vertical diffusion cells (Franz cells) revealed that HCA was released in much higher amounts from the O/W cream compared to the W/O formulation. In summary, we have shown that 1% HL extract exerts anti-inflammatory effects comparable to 1% HCA, but only when incorporated in our O/W cream. Our findings confirm the critical role of the vehicle in topical anti-inflammatory systems.

Keywords: Franz diffusion cells; Humulus lupulus; UVB erythema test.

Figure 1

Effect of HL extract on the phosphorylation of p38 and ERK. PHKs were pretreated with HL extract (4 µg/mL) or HC (20 µg/mL) for 2 h and irradiated with 6 J/cm² using a solar simulator. After 20 min, immunofluorescence staining of p-p38 or p-ERK was performed. The nucleus was stained with DAPI. Representative pictures of p-p38 (a) and p-ERK staining (b) for non-irradiated PHKs (no solar simulator radiation (SSR)), after irradiation (SSR), with HL extract treatment before SSR (HL + SSR), and HC pretreatment before SSR (HC + SSR). The intensity of the staining in the nucleus was measured using the Intensity Ratio Nuclei Cytoplasm Tool (RRID:SCR_018573) in ImageJ. Results of three independent experiments are shown as mean ± SD. The statistical significances were calculated in relation to the SSR-treated sample. ** p ≤ 0.01, *** p ≤ 0.001.

Figure 2

Effect of HL extract on the secretion of IL-6 and IL-8. PHKs were pretreated with HL extract (4 µg/mL) or HC (20 µg/mL) for 2 h and irradiated with 6 J/cm² using a solar simulator. The cell culture supernatant was collected after 24 h of non-irradiated PHKs (no solar simulator radiation (SSR)), after irradiation (SSR), with HL extract treatment before SSR (HL + SSR), and HC pretreatment before SSR (HC + SSR). Then, secretion of IL-6 (a) and IL-8 (b) was measured using ELISA. Results of three independent experiments are shown as mean ± SD. The statistical significances were calculated in relation to the SSR-treated sample; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 3

Verification of the O/W and W/O formulation type using methylene blue staining. A small part of both creams was squeezed from the tube into an aqueous 0.1% methylene blue solution. After 10 min, the methylene blue solution was removed and the blue staining of the cream was verified. Blue staining of the creme indicates a O/W formulation.

Figure 4

Effect of an O/W cream (a) and a W/O cream (b) with 1% HL (Humulus lupulus) extract or 1% HCA (hydrocortisone acetate) on skin erythema of non-irradiated skin (n = 40); The statistical significances were calculated in relation to the vehicle. ns: not significant, *** p ≤ 0.001.

Figure 5

Effect of an O/W cream (a) and a W/O cream (b) containing 1% HL (Humulus lupulus) extract or 1% HCA (hydrocortisone acetate) on skin erythema of human skin irradiated with 1.5- fold MED UVB (n = 40); The statistical significances were calculated in relation to the irradiated vehicle; ns: not significant, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 6

Release profile of HCA from the O/W cream (red dots) and W/O cream (black squares). Profiles were obtained using Franz diffusion cells with polyether sulfone membranes (not rate-limiting) to separate donor cream from acceptor medium (30% ethanol). (a) The initial flux of HCA (J_ini) was determined from the first 3 h of HCA release. (b) The amount of released HCA is proportional to the square root of time, i.e., it follows the Higuchi model for drug release from non-erodible matrix systems. Bars indicate standard errors from n = 3 independent experiments.

Figure 7

HPLC fingerprint of the HL extract.

Figure 8

Microscopic images of the O/W (a) and W/O (b) cream at a magnification of 400×.

Figure 9

HPLC fingerprint of HCA from the acceptor medium.