HEALTH and WELLNESS
Ultraviolet Radiation (UVR)
Green Tea Catechins (GTCs)
Background: Safe systemic protection from the health hazards of ultraviolet radiation (UVR) in sunlight is desirable. Green tea is consumed globally and is reported to have anti-inflammatory properties, which may be mediated through the impact on cyclooxygenase and lipoxygenase pathways. Recent data suggest that green tea catechins (GTCs) reduce acute UVR effects, but human trials examining their photoprotective potential are scarce.
Objective: We performed a double-blind, randomized, placebo-controlled trial to examine whether GTCs protect against clinical, histologic, and biochemical indicators of UVR-induced inflammation.
Design: Healthy adults (aged 18–65 y, phototypes I–II) were randomly allocated to 1350 mg encapsulated green tea extract (540 mg GTC) with 50 mg vitamin C or placebo twice daily for 3 mo. Impact on skin erythema, dermal leukocytic infiltration, and concentrations of proinflammatory eicosanoids was assessed after solar-simulated UVR challenge, and subject compliance was determined through assay of urinary GTC metabolite epigallocatechin glucuronide.
Results: Volunteers were assigned to the active (n = 25) or the placebo (n = 25) group. After supplementation, median (IQR) sunburn threshold (minimal erythema dose) was 28 (20–28) and 20 (20–28) mJ/cm2 in the active and placebo groups, respectively (nonsignificant), with no difference in AUC analysis for measured erythema index after a geometric series of 10 UVR doses. Skin immunohistochemistry showed increased neutrophil and CD3+ T-lymphocyte numbers post-UVR in both groups (P < 0.01) with no statistically significant differences between groups after supplementation. Cyclooxygenase and lipoxygenase metabolites prostaglandin E2 (vasodilator) and 12-hydroxyeicosatetraenoicacid (chemoattractant), respectively, increased after UVR (P 2 cups of tea/d, or currently pregnant or breastfeeding. Ethical approval was obtained from the North Manchester Research Ethics Committee (reference 08/H1006/79). Written informed consent was obtained from the participants and the study adhered to Declaration of Helsinki principles. The study was conducted in the Photobiology Unit, Dermatology Centre, Salford Royal Hospital, Manchester, United Kingdom, between November 2010 and August 2011. Subjects were randomly assigned to receive green tea extract plus vitamin C or placebo maltodextrin (1:1; block randomization with random block size between 4 and 8; StatsDirect v2.7.8, StatsDirect Ltd.). Containers containing active and placebo supplement were sequentially numbered. Subjects and investigators were blinded to the intervention, and the randomization code was held securely until completion of the study.
Green tea supplements were gelatin capsules each containing 450 mg green tea extract (180 mg GTC) (27) and a further set of capsules each containing 25 mg vitamin C. Actively supplemented subjects took 3 green tea and 2 vitamin C capsules twice daily (with breakfast and evening meal; total daily dose 1080 mg GTC, 100 mg vitamin C; Table 1) for 12 wk. Low-dose vitamin C stabilizes the green tea extract in the gut lumen and has been shown to have no impact itself on UVR erythema (28, 29). Placebo subjects took supplements comprising maltodextrin in gelatin capsules of identical appearance to those containing green tea and vitamin C. The primary outcome was change in the minimal erythema dose (MED) of UVR at 12 wk. Secondary outcomes were change in UVR-induced neutrophils and CD3+ T lymphocytes in skin biopsy sections, as well as PGE2 and 12-HETE in suction blister fluid at 12 wk. All supplements were provided by Nestec Ltd. and packaged in bottles with identical appearance by Laboratoire LPH. Compliance was assessed by counting residual capsules in dispensed containers returned by volunteers and measurement of the green tea urinary marker epigallocatechin glucuronide as described below.
Total daily amount of green tea extract constituents consumed by active group subjects
Total daily amount of green tea extract constituents consumed by active group subjects
UVR exposure and assessment of erythema response
Exposures were performed by using a solar simulator with emission of UVB and UVA mimicking that of sunlight (emission 290–400 nm; 5% UVB, 95% UVA; Newport Spectra-Physics Ltd). Irradiance was measured 10 cm from the source before each irradiation by using a radiometer (model IL 730A; International Light) calibrated for use with the light source to ensure consistency of doses applied.
The MED of UVR (i.e., the sunburn threshold) for each subject was assessed at baseline and postsupplementation, after application of a geometric series of 10 doses of solar-simulated UVR (erythemally weighted, 7–80 mJ/cm2) to upper buttock skin (1-cm-diameter circular sites). Irradiated sites were examined visually after 24 h, with the MED defined as the lowest dose producing visually discernible erythema.
Erythema intensity at each of the 10 UVR-exposed sites was quantified by using a reflectance instrument (Diastron). Readings were taken in triplicate from each exposed site and from adjacent unexposed skin and erythema expressed as the difference between these readings (ΔE). Dose-response modeling was performed by using a dedicated data analysis package (Regional Medical Physics Department, Gateshead & Tyneside Health Authority) to calculate each subject’s D30, the UVR dose producing a ΔE of 30 arbitrary units, which is a threshold value approximating an individual’s visual MED.
Skin biopsy and suction blister fluid sampling
At 24 h before skin tissue and blister fluid sampling, doses of UVR of 3× the individual’s presupplementation MED were given to sites on one buttock to provoke an inflammatory response sufficient to significantly elevate cutaneous eicosanoid concentrations (9). UVR-exposed and UVR-protected areas of upper buttock skin were sampled at baseline and postsupplementation; UVR exposures were limited to one buttock, and the other buttock provided the unexposed skin and blister fluid samples. Skin punch biopsy specimens (5 mm diameter) were taken after intradermal injection of lignocaine as described (9), snap frozen, and stored at −80°C. Suction blisters were raised by using suctions cups with a central aperture diameter of 1 cm and vacuum of 250 mm Hg as described previously (9). Skin blister fluid was aspirated with a 23-gauge needle, snap frozen in liquid nitrogen, and stored at −80°C until analysis.
Immunohistochemical staining and assessment
Monoclonal anti–neutrophil elastase (clone NP57) and polyclonal anti-CD3 antibodies were obtained from Dako UK Ltd. Briefly, frozen biopsy specimens were sectioned (5 μm) and endogenous peroxidase activity blocked by incubation in 0.6% (vol:vol) hydrogen peroxide in methanol. Sections were incubated overnight at 4°C with primary antibody and then visualized by using the ImmPRESS system or Vectastain Elite ABC kit (Vector Laboratories Ltd.) for neutrophil elastase and CD3, respectively. All sections were blinded and randomized before assessment. For each biopsy sample, 3 high-power fields (hpfs) were assessed microscopically from each of 3 biopsy sections. Cell number per hpf was determined for neutrophils and CD3+ T lymphocytes.
Analysis of skin blister fluid
Eicosanoids in skin blister fluid were quantified by liquid chromatography coupled to electrospray ionization tandem mass spectrometry as described previously (30–32). Briefly, skin fluid samples (typically 50–200 μL) were diluted with methanol-water (15% w/w) up to 3 mL. Internal standards (40 ng PGB2-d4 and 80 ng 12-HETE-d8; Cayman Chemicals) were then added and resultant solutions acidified to pH 3.0, followed by solid-phase extraction (C18-E cartridges; Phenomenex) to reduce matrix effects and semi-purify the lipid mediators. Eicosanoids were analyzed on a C18 column (Luna 5 μm; Phenomenex) by using a Waters Alliance 2695 HPLC pump coupled to a triple-quadrupole mass spectrometer equipped with an electrospray ionization probe (Quattro Ultima; Waters). Instrument control and data acquisition were performed by using MassLynx 4.0 software (Waters). Multiple-reaction monitoring transitions used were as follows: PGE2, m/z 351 > 271; PGE1, m/z 353 > 317; PGE3, m/z 349 > 269; PGJ2, m/z 333 > 271; PGD1, m/z 353 > 317; PGD2, m/z 351 > 271; PGF2α, m/z 353 > 193; 13,14-dihydro-15-keto PGE2, m/z 351 > 333; 13,14-dihydro-15-keto-PGE1, m/z 353 > 335; 12-HETE, m/z 319 > 179; 11-HETE, m/z 319 > 167; 15-HETE, m/z 319 > 175; 15-hydroxyeicosatrienoic acid, m/z 321 > 221; 9-hydroxyoctadecadienoic acid, m/z 295 > 171; and 13-hydroxyoctadecadienoic acid, m/z 295 > 195.
Urinary analysis of GTC metabolites
The urinary GTC metabolite epigallocatechin glucuronide was assayed by liquid chromatography–tandem mass spectrometry as previously described (33) to establish compliance with supplementation. Volunteers provided 24-h urine samples before supplementation and after 1 d, 6 wk, and 12 wk of supplementation.
Based on previous oral flavonoid photoprotection studies (34, 35), a sample size of at least 22 subjects per group was estimated to be required to detect a 25% difference in the MED and UVR erythema dose-response between groups, at a 5% significance level with 90% power. Differences in UVR-induced inflammatory and eicosanoid responses between active and placebo groups postsupplementation were compared by ANCOVA of ln-transformed data with baseline data as the covariate. Intent to treat was the primary analysis for comparisons of outcomes between treatment groups with multiple imputation of missing data (n = 50). A per-protocol analysis was also performed (n = 45) to assess effectiveness of the supplementation. Wilcoxon’s Signed Rank test was used to compare unexposed and UVR-exposed skin within groups. Analyses were performed by using SPSS 20 (SPSS, Inc.). Erythemal dose-response curves were analyzed by using GraphPad Prism (v5.01; GraphPad Software). Statistical significance was accepted at P < 0.05.