Inhibitory effect of Lutein on Cytochrome P450



Introduction:  Lutein from Tagetes erecta L. is extracted from
marigold oleoresin. It affects the pharmacokinetics of drugs which are
metabolised by cytochrome P450. The aim of the study is to find the inhibitory
effect of lutein on cytochrome P450.


Materials and Methods:  Cytochrome P450 enzymes functions
to metabolise potentially toxic compounds, including drugs and products of
endogenous metabolism.Lutein at
different concentrations from 5 – 100µg/ml was examined for its inhibitory
property towards Cytochrome P 450 isoform CYP3A4.


Results: All the tested concentrations of Lutein showed potent inhibition against
CYP3A4 in a dose – dependent manner. The IC50 value of lutein for
CYP3A4 inhibitory activity was found to be 35.27µg/ml.


Conclusion: The inhibitory effects of lutein indicate the possibilities of
herb-drug interaction. If these extracts are co – administered with prescribed
drugs that are metabolised by these CYPs and the necessity for further in vivo


Keywords: Lutein, cytochrome P450, inhibitory assay, marigold oleoresin, IC50.

















                        Lutein from Tagetes erecta L. is a purified extract
obtained from marigold oleoresin, which is extracted from the petals of
marigold flowers with organic solvents. The final product, after
saponification, contains, as a major component, lutein and a smaller proportion
of zeaxanthin. Lutein (3R,3’R,6’R-??-carotene-3,3’-diol) is a member of a group
of pigments known as xanthophyll and has no provitamin A activity. It is used
as a food colouring agent and nutrient supplement (food additive) in a wide
range of baked goods and baking mixes, beverages and beverage bases, breakfast
cereals, chewing gum, dairy product analogs, egg products, fats and oils,
frozen dairy desserts and mixes, gravies and sauces, soft and hard candy,
infant and toddler foods, milk products, processed fruits and fruit juices,
soups and soup mixes in levels ranging from 2 to 330 mg/kg. 1


                        Marigold flower (Tagetes erecta  L.) represents a rich source of lutein.
It  is grown for  business purposes  in Mexico, Peru, Ecuador, Spain, India or
China. Dried Marigold flowers contain 0.1–0.2% dry matter (DM) of  carotenoids, 
out  of  which 
80%  are  lutein 
diesters. By  the  extraction 
of  dried  and 
ground  flowers,  a  non
polar oleoresin extract  is acquired. 2  The recent evidence suggests that lutein
is one of the abundant carotenoids in the diet and in human blood possesses
strong antioxidant capabilities and may be useful in reduction of incidence of
cancer.3 The purification of lutein fatty acid esters from marigold
flowers was patented by Philip in 1977(U.S.Pat.No. 4,048,203).4


                         The xanthophylls, a
major group of carotenoids, primarily include astaxanthin (AS), b-cryptoxanthin
(bC), canthaxanthin (CA), lutein (LU), and zeaxanthin (ZE) (Kotake-Nara and
Nagao, 2011; Zaripheh and Erdman, 2002). Unpredicted drug interactions have led
to severe adverse effects or treatment failures. Many of these interactions
involve the inhibition or induction of drug-metabolising cytochrome P450 (CYP)
enzymes. Similarly, dietary supplements or nutrients may be inhibitors or
inducers of CYP enzymes and have an effect on the pharmacokinetics of any
co-medicated drugs. There are few reports about interactions between drug-metabolising
enzymes and AS, bC, CA, LU, and ZE. 5


                           The aim of the present
study was thus to investigate the reversible inhibitory or time-dependent
inhibitory effects of  LU affecting the
pharmacokinetics of the drug, which is metabolised by cytochrome P450.

Materials and methods:


                           Marigold  flower 
concentrates  in the forms of
granules (samples L1–L16) and powder (samples 
L17–L34)  used  for 
the  production  of 
food supplements were analysed at least three months before their
expiration  time.  The concentrates were purchased directly from
the producers (from China, India, Israel, and Mexico). These declared the
content of lutein to range from 5% to 80%. 
Standard  solution  of 
lutein  was prepared  by 
dissolution  of  accurate 
amount  of  lutein standard (0.50 ± 0.01 mg;
Extrasynthese,  Genay, France) in
acetone-methanol mixture (50 ml; 1 : 1 v/v). 
Calibration  curve was plotted  based 
on the signals  of  various 
volumes  of  standard 
lutein  solution  (1, 
2,  3,  5, 
8,  and  10 
µl)  injected  into 
the  HPLC column. Calibration was
always implemented on the day  of  analysis.2 


                       5-100 micrograms concentrations of lutein,
potassium phosphate buffer, CYP450   reagent
and substrate 7-Benzyloxy-4-trifluoromethylcoumarin (BFC) were added to a
96-well plate.The mixtures were pre – incubated for 20 min at room temperature.
The reaction was started by a mixture of reconstituted substrate and NADP+ and
incubated at room temperature for 30-60 min. The reaction was stopped by
Tris-HCl buffer, pH 10.5. The fluorescent intensities of the products were
measured by Perkin Elmer Enspire fluorescence reader using an excitation and
emission wavelength of 405 nm and 460 nm, respectively.IC50 was calculated by
plotting concentrations of lutein against the corresponding percent inhibition.




            Lutein at different concentrations from 5 –
100µg/ml was examined for its inhibitory property towards Cytochrome P 450
isoform CYP3A4. All the tested concentrations of Lutein showed potent
inhibition against CYP3A4 in a dose – dependent manner. The IC50
value of lutein for CYP3A4 inhibitory activity was found to be 35.27µg/ml.
Figure 1 shows the inhibitory potential of different concentrations of lutein
on cytochrome P450.






Figure 1













Table 1

Effect of Lutein on Cytochrome P 450 (CYP3A4) inhibitory potential

















Values are
expressed as Mean ± SEM (n = 3)



from this study provide the information indicating the possibilities of
herb-drug interaction if lutein extracted from marigold oleoresin aqueous
extracts are co-administered with the prescribed drugs that are metabolized by
CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4. Lutein extracted from Marigold
oleoresin demonstrated inhibitory effects on CYP3A4.  Lutein extracted from marigold oleoresin,
apart from inhibitory effects, also shows potent antioxidant activities.14


                    Comparing with the other
studies, it was shown that inhibitory effects of lutein against cytochrome P450
was a potent inhibition in a dose-dependent manner. P. amarus and P. emblica
aqueous extracts on all CYP isoforms were weaker. These weak inhibitory effects
in vitro may or may not be found in vivo, however, results from these in vitro
studies suggest that further confirmatory study in vivo was recommended.6


                     In a previous study,  ECa233 demonstrated a concentration-related
inhibitory effects on the activities of CYP2B6, CYP2C19 and CYP3A4 but not or
very small effects on CYP1A2, CYP2C9, CYP2D6 and CYP2E1 at the range concentrations
of ECa233 in the reaction mixture up to 1,000 µg/ml. No inhibitory effect of
ECa233 on CYP1A2, CYP2C9, CYP2D6 and CYP2E1 rule out the potential effect of
the extract to interact with various currently used medicines that are metabolized
by these CYP isoforms.7


                      In a study by
Winitthana 16, drug – drug interactions were found out. Asiaticoside
inhibited CYP2C19 (IC50 = 412.68 ± 15.44 ?M)
and CYP3A4 (IC50 = 343.35 ± 29.35 ?M).
Madecassoside also inhibited CYP2C19 (IC50 = 539.04 ± 14.18 ?M)
and CYP3A4 (IC50 = 453.32 ± 39.33 ?M).
Asiaticoside and madecassoside had no effect on the activities of CYP1A2,
CYP2C9 and CYP2D6 and CYP2E1. Assessment of mechanism-based inhibition and the
type of inhibition were performed for asiaticoside and madecassoside with
CYP2C19 and CYP3A4. These results suggested that madecassoside is a
mechanism-based inhibitor of CYP2C19 and CYP3A4. Asiaticoside exhibited
non-competitive inhibition of CYP2C19 (Ki = 385.24 ± 8.75 ?M)
and CYP3A4 (Ki = 535.93 ± 18.99 ?M).
Madecassoside also showed non-competitive inhibition of CYP2C19 (Ki = 109.62 ± 6.14 ?M)
and CYP3A4 (Ki = 456.84 ± 16.43 ?M).

                    Similar studies
that were conducted are, aqueous extracts of Hibiscus on hepatic cytochrome
P450 and subacute toxicity in rats. The result was that there was no
modulation.8 In a study by Teresa, inhibition of cytochrome P450
metabolism by blended herbal products and vitamins was studied. The study
revealed that the extracts showed a low to moderate capacity to inhibit the
cytochrome metabolism.9  Similarly,
studies on Cocktail inhibition assays for assessing the drug-drug,
drug-botanical interactions and assessing the six major cytochrome P450 enzymes
was conducted by Guannan and Jing Jing Wan respectively.10,11

                    A study by Yu Fen Zeng,
reveals that although drug–drug interactions may be identified during drug
development and approval, food–drug interaction and supplements–drug
interaction should not be overlooked. Natural health products are being
increasingly widely used. Apart from an appraisal of product safety and
effectiveness, attention should be paid to the potential that these product
ingredients may interact with medications. In recent years, dietary
carotenoids, especially xanthophylls, have attracted significant attention
because of their activities as antioxidants and their roles in preventing
cancer and age-related macular degeneration. Although no less than 40
carotenoids are ingested through the typical diet, only a few xanthophylls have
been found in human tissues. There are a few reports of inducible effects on
CYP activities by the xanthophylls AS, bC, CA, LU, and ZE. However, their
inhibitory effects on CYPs have rarely been investigated.5



                    Lutein obtained from
marigold petals is of major commercial interest because of its use in
functional food and cosmetics, as well as in pharmaceuticals. The production
yield from each marigold plant is very important for the large-scale extraction
of lutein, in terms of cost efficiency. In conclusion, lutein extracted from
Marigold oleoresin exhibited inhibitory effect on CYP3A4. It showed a potent
inhibition against cytochrome P450. The inhibition was in a dose – dependent
manner. Results from this study provide information indicating the
possibilities of herb-drug interaction if these extracts are co-administered
with the prescribed drugs that are metabolized by CYP3A4. Further in vivo study
is needed to investigate whether these effects are clinically significant.