This clears the cell of molecular rubbish, thereby preventing molecular damage, and ageing.2 According to the literature, citrus fruits rich in bioflavonoids such as naringin and hesperidin3 can help to protect the body against toxin overload when taken orally.4 The use of such natural actives is continuously increasing as the understanding of their ability to slow the ageing process improves. One of the most powerful detoxification mechanisms is centred on the phase I and phase II enzymes. Phase I activation of lipophilic compounds is carried out by enzymes of the CYP450 family.5 This phase I biotransformation creates an activated intermediate that is either directly eliminated from the body, or more commonly becomes a substrate for one of the phase II conjugation enzymes prior to elimination from the cell. Phase II enzymes, such as quinone reductase, perform a broad variety of detoxification reactions. Quinone reductase is able to detoxify a broad range of quinones produced by oxidative metabolism and is known to be expressed in human keratinocytes. Here we summarise two in vitro studies which show that a standardised grapefruit extract rich in naringin and hesperidin3 significantly induces the activity of both phase I and phase II enzymes in primary human keratinocytes.
Citrus fruits are commonly used in commercial juice preparations for human consumption. Fruits from citrus species are widely used in traditional medicine, reflecting the particularly high abundance of bioactive compounds in the peel of the fruit. Many active components of medicinal value have been isolated from citrus species, including anti-allergic, antioxidant, anti-tumor and immunomodulating agents.6 Citrus fruits contain a wide variety of phytochemicals, including flavonoids, such as hesperetin, hesperidin, naringin and narirutin (Fig. 1). The biological action of these flavonoids is possibly linked to their interactions with key regulatory enzymes involved in cell activation and receptor binding.6 In addition, these flavonoids function as antioxidants. Polyphenols (such as the flavonoids) may be regarded as xenobiotics by animal cells, and are known to interact with the phase I and phase II enzyme systems. It has also been shown that flavonoids modulate the expression of glutathione, an important enzyme in both cellular antioxidant defences and detoxification of xenobiotics.4 One important task for cellular glutathione is to scavenge free radicals and peroxides produced during normal cellular respiration, which would otherwise oxidise proteins, lipids, and nucleic acids. One mechanism operating to counteract oxidative damage involves transactivation of genes encoding enzymes that participate in glutathione metabolism and synthesis. Typically, these enzymes belong to the phase I and II families of detoxification genes.4 Through both routine metabolism and environmental exposure, the skin continuously accumulates a variety of endogenous and exogenous metabolites. Without prompt detoxification, these compounds can accelerate the ageing process (Fig. 2).7 The natural defence mechanisms of the skin cells are centered on detoxifying phase I and phase II enzymes that transform the waste molecules prior to excretion from the cell.1 Phase I and phase II enzymes may be either constitutively expressed or inducible. Importantly, certain environmental and nutritional agents have been found to influence the induction and activities of specific phase I and phase II enzymes. In these studies, phase I and phase II enzymes were evaluated in the presence and absence of a standardised grapefruit extract (GE).
Materials and methods
The standardised grapefruit extract (Cayoma Grapefruit, Qenax AG, Zug, Switzerland) with an active content of >10% flavonoids (measured as naringin) was used in all studies. The grapefruit extract is an aqueous alcoholic extract of the Citrus paradisi fruit and peel, standardised to >10% naringin. Both studies were performed with human primary epidermal keratinocytes (HPEK) grown in CnT-07 culture medium (both CELLnTEC Advanced Cell Systems AG, Bern, Switzerland). All other chemicals were purchased from Sigma Aldrich, Buchs(SG), Switzerland, except as follows: Fluorescamine (Applichem, Darmstadt, Germany), Oltipraz (Alexis Biochemicals, San Diego, USA). Fluorometer: Wallac Victor2 1420 Multilabel Counter (Perkin Elmer, Schwerzenbach, Switzerland) and Wallac Workout software, (Perkin Elmer, Schwerzenbach, Switzerland).
Phase I enzymes
The cytochrome P450 family of enzymes plays a critical role in the biotransformation of drugs, carcinogens, steroid hormones, and environmental toxicants.5 In this study we evaluated the induction of cytochrome P450 (CYP1A1) activity in human primary epidermal keratinocytes using the EROD (ethoxyresorufin-O-deethylation)8 induction method. To quantify the effect of the grapefruit extract on the activity of this enzyme, a method using the known inducer BAP (benzo-?-pyrene) and known inhibitor (?-naphthoflavone) followed by fluorescence quantification was used. HPEK (passage 4) were seeded into 96 well plates in CnT-07 (100 ?L volume). Twenty-four hours later, 100 ?L of 2x concentration grapefruit extract solution (in CnT-07, for final concentration of 0.0065% or 0.013%), benzo-?-pyrene (final concentration 100 nM), or CnT-07 (Control) were added to the wells. Three days later, cells were washed with phenolred free reaction medium. The EROD reaction was initiated with 100 ?L of reaction buffer (8 ?M ethoxyresorufin, 9 ?M dicoumarol in reaction medium). As an inhibition control, ?-naphtoflavone was added to a final concentration of 1 ?M in some wells. Base reaction medium without any ER/dicoumarol was used as a blank. Twenty minutes after initiation, a first round of five measurements was done with the fluorometer at 544 nm excitation and 590 nm emission. Another round of five measurements was performed 24-26 hours later. Mean values of the five measurements were taken. The results in fluorescent units could then be converted to resorufin concentrations using a resorufin standard curve. Protein measurements were done using fluorescamine (using BSA for standard curve). The EROD results are presented as percentage inductions relative to the control. To confirm that an EROD increase would result from a direct induction as opposed to a cytotoxic affect, an additional cytotoxicity comparison of BAP and GE was conducted.
Phase II enzymes
Quinone reductase is often chosen as a key indicator, because:
• It is widely expressed in epithelial tissues, and in skin especially appears to be one of the main players.
• It is quite sensitive, and often exhibits greater induction than some other phase II enzymes.
• It seems to respond to a wide variety of stimuli, and correlates well with other related enzymes.
These kinds of characteristics have led some people to describe it as a “global” indicator of phase II enzyme activity.9 Primary human keratinocytes (HPEK, passage 3) were seeded into 96 well plates at three standardised GE concentrations selected in an earlier proliferation test (0.003%, 0.0065%, and 0.013%). Oltipraz [5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3- thione], an anti-cancer agent known to potently induce phase II enzymes, was used at 25 ?M as a positive control. The cultures were allowed to grow for four days until the cells reached confluency. The cultures were exposed to the standardised GE concentrations, and the induction of quinone reductase evaluated using a colorimetric NADPH-quinone reductase assay.10 The results were calculated only for wells with similar cellular confluence. To calculate the final results, the average background value was first subtracted from all values. Values of treated cells were then expressed as a percentage increase above the control treatment. The experiment was repeated twice.
Results
Phase I enzymes
The bioflavonoids11 of the standardised grapefruit extract (GE) significantly induced EROD in primary epidermal keratinocytes. Both concentrations 0.013% (Fig. 3) and 0.0065% (Fig. 4) tested showed similar EROD inductions as the BAP (benzo-?-pyrene) positive control (approx. 2.4x and 1.8x respectively above untreated control cells). Cell proliferation was evaluated at a range of concentrations of both GE and BAP. Proliferation was found to be unaffected at all concentrations used in the experiment (100 nM BAP, and 0.013% GE – data not shown). In fact, proliferation was found to be normal in the presence of concentrations up to at least 800 nM BAP, or 0.025% GE and confirmed that the EROD increase did not result from a cytotoxic effect.
Phase II enzymes
All three concentrations of the standardised grapefruit extract (0.003%, 0.0065% and 0.013%) significantly induced quinone reductase (Fig. 5). The induction ranged from 24% to 39% in a concentration-dependent manner. The average induction in the positive control (treatment with the potent inducer oltipraz) was found to be 69%.
Discussion
This investigation found that treatment of primary human keratinocytes with GE significantly induced the expression of phase I detoxification enzymes as measured with the EROD assay. Both extract concentrations showed similar EROD inductions as 100 nM benzo-?- pyrene. In all cases, the addition of the EROD inhibitor aNF prevented EROD induction, and yielded resorufin levels at or slightly below those found in control (untreated) cells. A similar pattern of induction was found even after correction for protein content in the wells, indicating that cell numbers were similar between wells, and did not significantly affect the results. Normal cell proliferation in the presence of concentrations up to at least 800 nM BAP, or 0.025% GE was obtained. These results provide additional evidence that the EROD induction seen following addition of these compounds did not result from a toxic effect, but from direct induction. The second part of this investigation found a concentration-dependent induction of quinone reductase following treatment of primary keratinocytes with GE. The largest induction observed (+39%) was found at the highest extract concentration (0.013%). Oltipraz, a substituted dithiolethione, was used as the positive control. It is a synthetic anti-cancer drug, known to have a strong positive effect on the expression of various phase II enzymes. The induction of electrophilic detoxication enzymes, which results in diminished carcinogen-DNA adduct formation and reduced cytotoxicity,12 appears to be an important component of the anti-carcinogenic action of oltipraz. At a concentration of 25 ?M, oltipraz treatment resulted in a 69% induction, which was higher than the 39% induction found for the highest of the GE treatments (0.013%). However, this finding is consistent with other reports that the powerful synthetic anti-cancer drug is a stronger inducer of phase II enzymes than various natural plant extracts.13
Conclusion
The results of this study clearly show that the standardised grapefruit extract used is able to significantly induce the activity of both phase I and phase II detoxification enzymes. EROD activity, a measure of phase I enzymes, was strongly induced (2.4x) in the presence of the GE, and thus exhibited a similar action to the potent positive control (BAP). Quinone reductase activity (a phase II enzyme) was also significantly increased following GE treatment (+39%). Intra-cellular detoxification processes are an important natural defence mechanism of our body. Our constant exposure to toxic metabolites and harmful foreign chemicals from different sources requires an effective system to protect against cellular damage. These results show that fruits like the grapefruit which are rich in the flavonoids14,15 naringin and hesperidin can significantly activate the protective detoxification mechanisms naturally found in skin. In view of the well-documented links between ROS/xenobiotics and agerelated processes,3 the grapefruit extract treatments documented in this study represent a potentially highly effective method for boosting the natural defences of the skin, and thus considerably improving its ability to cope with the many significant challenges to normal homeostasis.
This article was originally presented as a poster at the IFSCC conference in Melbourne 2009 and was first published in the IFSCC Magazine Edition 1, 2010. It is reproduced in Personal Care with permission from the IFSCC.
References
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