Relationship between rate and extent of ... - Wiley Online Library

Vol. 46, No. 5, December 1998

BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL Pages895-903

R E L A T I O N S H I P B E T W E E N RATE AND E X T E N T OF C A T E C H I N A B S O R P T I O N AND P L A S M A A N T I O X I D A N T STATUS Piergiorgio Pietta l, Paolo Simonetti 2, Claudio Gardana 2, Antonella Brusamolino 2, Paolo Morazzoni 3 and Ezio Bombardelli 3 ITBA-CNR, Milan, Italy 2 diSTAM, Section of Human Nutrition, University of Milan, Italy 3 INDENA S.p.A., Milan, Italy. Received August 3, 1998

Summary Flavonoids are described to exert a large array of biological activities, which are mostly ascribed to their radical-scavenging, metal chelating and enzyme modulation ability. Most of these evidences have been obtained by in vitro studies on individual compounds and at doses largely exceeding those dietary. Little is known about a possible relationship between rate and extent of the absorption and modifications of plasma antioxidants. To elucidate this aspect, human volunteers were supplemented with single doses of green tea catechins in fi'ee (Greenselect TM ) or phosphotipid complex form (Gl'eenselectTM Phytosome~) equivalent to 400 mg epigallocatechingallate (EGCg). EGCg was chosen as biomarker for green tea catechin absorption, and its time course plasma concentration was correlated to the subsequent percent variations of plasma ascorbate, total gtutathione, (z-tocopherol, ~3-carotene and Total Radical Antioxidant Parameter (TRAP). Green tea catechins were absorbed more extensively when administered as phospholipid complex rather than as free catechins. Single dose intake of both forms of carechins produced a transient decrease (10-20%) of plasma ascorbate and total glutathione and an increase of plasma TRAP (16-19%). These variations were consistent with the plasmatic levels of EGCg, ascorbate and total glutathione. Key Words: Green tea catechins, phytosome, kinetics, epigallocatechingallate, ascorbate, glutathione, Total Radical Antioxidant Parameter (TRAP)

Introduction Ftavonoids are polyphenolic compounds widely distributed in nature. Some flavonoid classes, including anthocyanins, catechins, flavonols, flavones and flavanones, are daily ingested with vegetables, Duits and beverages, such as red wine and green tea. Due to their antioxidant and enzyme-inhibiting properties, these components of our diet have gained increasing interest (1). However, most positive results have been reached by performing in vitro studies on single compounds. Little is known about the antioxidant potential displayed in vivo by dietary flavonoids, and this may be due to thc scarce knowledge on their kinetics. In recent years (2-4), it has been

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proved that flavonoids m'e absorbed as aglycones or their conjugated forms (glucuronides and sulfates) at very low extent, which may account fbr about 2-3% of the ingested dose. On the other hand, dietary flavonoids are largely degraded by the intestinal microflora to yield hydroxylated phenylalkanoic acids, such as 3,4-dihydroxybenzoic, 3,4-dihydroxyphenylacetic, 3methoxy-4-hydroxybenzoic acids, and theft" conjugates with glycine. These metabolites may account for up to 30% of the ingested flavonoids, as indicated by their total concentration in the 20-72 h urine after intake (2,4). Some of these metabolites, for their catechol structure, have been recently proved to possess in vitro radical-scavenging ability comparable to that of theft" precursor or ascorbic acid and vitamin C, using either chemiluminescence (5) or Trolox Equivalent Antioxidant Capacity (6) assays. Thus, it may be assumed that dietary flavonoids play their first role in the digestive tract by inhibiting free radical formation and scavenging free radicals. Once absorbed, flavonoids and their metabolites continue to play this antioxidant role, although other interactions are possible (with enzymes not involved in rcdox processes, like adenosine deaminase (7) or with specific receptors, such as A3 adenosine or benzodiazepine receptors) (8,9). Concerning the free radical-scavenging activity in vivo, it was of interest to investigate whether this activity is exerted directly or through a cascade involving interaction with and between physiologic antioxidants. The latter chance is possible, since flavonoids and their mctabolites have one-electron reduction potentials lower than those of highly oxidising reactive oxygen species (ROS) (10,11), and are capable to reduce them. In turn, some of the resulting less aggressive aroxyl radicals (ArO') for theft" hydrophilic character may oxidise hydrophilic antioxidants, like ascorbate and urate (12). On the contrary, the oxidation of vitamin E (E7 = 5(i)0 mV) by ArO" with redox potential higher than 500 mV is unlikely, since these phenoxyl radicals are confined in the aqueous compartment. This possibility has been investigated in a previous study (13), which evidenced that intake of green tea infusions (100 mg total catechins daily) for 4 consecutive weeks provided antioxidant protection through a cascade involving endogenous antioxidants, which interact differently according to theh" redox potential and polarity. More specifically, vitamin E and ~-carotene are spared at expenses of ascorbate, urate and glutathione, resulting in a protection of RBC membrane PUFAs against oxidation. Extending this study, we investigated a possible correlation between the rate and the extent of green tea catechin absorption and the subsequent modification of selected parameters, which are significant lbr the plasma antioxidant status. To this purpose human volunteers were supplemented with single doses of green tea extracts in free (Greenselect TM) or phospholipid complex Greenselect TM Phytosome ~) form. Epigallocatechingallate (EGCg) was chosen as a plasma

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marker of catechin absorption, and its time course concentration was correlated to percent variations of ascorbate, total glutathione, vitamin E, [3-carotene and Total Radical Antioxidant Parameter (TRAP).

Materials and methods

Materials Green tea decaffeinated (Greenselect TM and Gmenselect TM Phytosome | INDENA S.p.A.(Milan, Italy).

extracts were fi'om

Green tea catechin administration Healthy male volunteers (n=12; aged 22+2 years) were selected with a self-administered questionnaire to assess the intake of fruits and vegetables. The study subjects had an habitual low intake of flavonoid-rich foods, were not taking antioxidants supplements, and abstained fi'om beverages containing flavonoids for 3 days before the study. The fasting subjects were randomly divided in two groups. One group (n=6) received 400 mg of fi'ee catechins (GreenselectTM), and the second group had the same amount of catechins as phospholipid complex (Greenselect TM Phytosome| Blood samples were collected fi'om each participant before and 1,2,3,4,5 and 6h after ingestion.

Epigallocatechingallate detection in plasma The time course of plasma catechins was followed by measuring EGCg for 6h after supplementation. Fasting venous blood samples were taken in vacutainer tubes containing sodium-heparin before and after 1,2,3,4,5, and 6h. Plasma was separated by centrifugation at 3000 x g. An aliquot of plasma (1 ml ) was added of 1 ml of ethylacetate, then vortexed for 30s and centrifuged for 1 rain at i0,000 x g. The supernatant was injected (20 BI) on HPLC column (Symmetry C18, 220 x 4.6 mm from Waters). The eluents were: A) 0.3% phosphoric acid, B) acetonitrile. The eluticrn was on gradient mode: 0-25%B ill 30 rain at a flow-rate of 2.0 ml/min. ECGg and ECg were detected with coulometric detector (Coulochem II, ESA,.Chelmsford, MA, USA; guard cell, 350 mV, El, -100 m V , E2, +350 mV).

Profile of human plasl~Ta a#er green tea catechin ingestion The work-up of the plasma samples was minimal, since previous trials including extraction with organic solvents and subsequent evaporation resulted in an almost complete loss of green tea catechins (unpublished results). Plasma samples showed the presence of fiee EGCg and ECg (Fig. 1). EGCg was chosen as a significant marker of green tea catechin absorption, whereas ECg and other catechin conjugates eluting with the fi'ont were not lbllowed. Experiments with EGCg spiked plasma showed that this procedure ensures 95+_10% (n=4) recovery. The detector response was linear with EGCg standards up to 2 Bg, and this allowed quantitative determination by the external standard method. The limit of detection was 2 ng.

Analysis of the endogenous antioxidants Ascorbate, vitamin E, B-carotene, total glutathione and Total Radical Antioxidant Parameter (TRAP) were detected in plasma for 6h after supplementation.

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10

20

30

Time (min)

Figure 1: EGCg and ECg in human plasma

Extraction of the fat-soluble plasma vitamins was performed using the method of Vuilleumiel; et al. (14) and plasma ascorbic acid was determined after deproteinization and enzymatic oxidation of L-ascorbic acid to dehydro-L-ascorbic acid (15). HPLC analyses, were performed by a Waters 510 pump connected with a Perkin-Elmer LC 240 spectrofluorimeter detector or a Waters 486 spectrophotometer detector and a Waters Millenium data station. Operation conditions were the following: -d-Carotene: 4.6 mm x 250 mm column paked with 7 I.tm Lichrosorb Si60 (Merck, Darmstadt, Germany), n-hexane/dioxane (100015, v/v), isocratic flow 1.2 ml/min, spectrophotometric detection, 453 nm. -a-Tocopherol: 4.6 mm x 250 mm column paked with 7 btm Lichrosorb Si60 (Merck, Darmstadt, Germany), n-hexane/ethyl acetate (1000175, v/v), isocratic flow 2.0 ml/min, spectrofluorimetric detection, 290 nm emission and 330 nm excitation. -Ascorbic acid." 4 mm x 250 mm column packed with 5 btm Aluspher RP-select B (Merck, Darmstadt, Germany), 0.08M KH2POJmetanol (80120, v/v), pH 7.8, isocratic flow 1.0 ml/min, spectrofluorimetric detection, 304 nm emission and 425 nm excitation. Plasma total GSH was determined by HPLC according to Neuschwander-Tetri et al. (16) and TRAP was measured by a fluorimetric assay according to Ghiselli et al. (17). Results and discussion

Time course of EGCg plasma concentration after green tea ingestion The time course of EGCg plasma concentration after ingestion of Greenselect TM or Greenselect TM Phytosome | is exemplified in fig. 2. The mean peak levels of EGCg were 1.84_+0.29 ~tg/ml (4.0-2-0.6 ~M) and 0.9_-_-_+0.l~tg/ml (2.0-L-0.2 ~M) tot phospholipid complex and flee catechins, respectively. These levels were attained 2h after ingestion, and then decreased slowly to

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2,5 2,0 / [ ~ / \

_~ 1,s

--~- Greenselect Phytosome

)1,o 0,5 0,0 0

1

2

3 Time (hours)

4

5

6

Figure 2: Time course of plasma EGCg after ingestion of Greenselect TM or Greenselec( TM Phytosome |

approximately 0.4_+0.1gg/ml at 6h. Thus, catechins are absorbed in higher amounts when administered as phospholipid complex, although the percent absorption (as measuring EGCg) does not exceed 2% of the given dose. However, in addition to EGCg other catechin conjugates are present in plasma, and it is likely that the concentration of total catechins may reach higher values. Furthermore, the presence of EGCg in plasma at 6 h after ingestion could support for a possible steady state concentration of EGCg (and related compounds) following regular consumption of green tea. One cup of tea contains about 150 mg of total catechins, and its daily ingestion could assure a plasma catechin concentration in a range (0,4-0,6 ~tM) comparable to the plasma concentration of [~-carotene.

Time course of percent variation of plasma ascorbate, glutathione, vitamin E, p-carotene and TRAP alter ingestion of green tea catechins The levels of the hydrophilic ascorbate and total glutathione and the lipophilic vitamin E and 13carotene were measured at the same intervals of EGCg detection. Both ascorbate and total glutathione decreased at rate and extent different for free and phospholipid complex catechins. This decrease was transient and the initial levels of ascorbate and glutathione were recovered at 6h after ingestion. Free catechins produced a negative percent variation (104__-5%) of ascorbate around 4h after ingestion, whereas an higher decrease (21+6%) was induced at shorter time (l h) by phospholipid complex catechins (Fig. 3). Concerning total glutathione plasmatic levels, they

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rM

Time 0 1 2 3 4 5 6

Greenselect 10.61 + 0.40 10.62 _+0.05 10.21 + 0.36 10.19 + 0.27 9.51 +0.45 9.49 + 0.38 10.19 + 0.21

Treatments GreenselectrM Phytosome| 9.78 + 0.24 7.69 -+ 0.48 9.65 + 0.30 9.87 _+0.24 9.83 +0.15 9.72 + 0.20 9.75 -+ 0.18

GreenselectPhytosome Greenselect

20 10 c O "m'

0

>

~k

~-'~~'~"~ ~ ~ 4

~

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E I1) 13.

-10

-20

-30 Figure 3: Plasma levels (means -+ SE, ~tg/ml) and percent variation of ascorbic acid after intake of Greenselect TM or Greenselect TM Phytosome|

decreased gradually reaching the maximum negative variation (18_+9%) at 2h after free catechin ingestion. On the contrary, phospholipid complex catechins determined a sharper and lower decrease (15_+4%) at 2h followed by an increase (13_+8%) at 3h (Fig. 4). These findings are well con'elated with the time course of EGCg concentration in plasma. TRAP increased steadily reaching the maximum (18+7%) at 2-3h lbr phospholipid complex catechins. This rise was in good correlation with thne course of plasma EGCg levels, ascorbate and glutathione levels (Fig. 5). In the case of free catechins, TRAP behaviour was different: after an initial low decrease, a positive variation of 17 + 5% was reached at 4h, and this increase was mostly related to the partial recovery of glutathione (Fig. 5).

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Greenselect T---g 9.77 __+0.37 8.65 + 0.32 8.02 + 0.74 8.84 + 0.41 9.28 +__0.22 9.56 + 0.17 9.97 + 0.35

Time 0 1 2 3 4 5 6

Treatments ~ree--n~~ 8.39 8.45 7.17 9.46 9.10 8.82 8.51

Phytosome | __+0.12 + 0.07 __+0.30 + 0.80 -+ 0.25 + 0.13 __+0.06

Greenselect Phytosome Greenselect 30 20 cO

10

>

0

o

Hours

-lo

12.

-20 -30

Figure 4: Plasma levels (means • SE, ~tmol/1) and percent variation of total glutathionc after intake of Greenselect TM or Greenselect TM Phytosome |

Concerning vitamin E and l-carotene plasmatic concentrations, no significant variations could be measured after intake of both lbrms of green tea catechins. It is conceivable that a single dose administration is not capable to induce modifications of these liposoluble vitamins, and a longterm consumption is requh'ed, as evidenced in a 30-day study (13). These data indicate that [1] green tea catechins are absorbed more extensively when adrninistered as phospholipid complex rather than as free catechins; [2] single dose intake of green tea catechins induce a transient decrease of plasma ascorbate and total glutathione in good conelation with rate and extent of catechin absorption; [3] TRAP, as an indicator of the plasma antioxidant status, is positively affected by green tea catechin intake in accordance to the kinetics of catechin absorption and to the changes of ascorbate and total glutathione plasma concentrations. These results represent the first evidence on the relationship between kinetic (rate and extent of absorption) and dynamic data (modification of endogenous antioxidants and plasma antioxidant

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Greenselect TM

Time 0 1 2 3 4 5 6

1 1 5 0 + 33 1167 + 27 1161 + 17

1082 + 26 1341 +71 1207 + 52 1 1 5 0 + 19

Treatments -GreenselectTM ph--~-osome~ 1359 + 24 1345 + 26 1563 + 42 1 6 1 0 + 119 1325 + 20 1345 + 14 1373 + 17

--*'- Greenselect Phytosome Greenselect 30 25 20 g t~ "r'-

C

15 10 5

O 13-

0 Hours

-5 -10 -15

Figure 5: T R A P modification (means + SE, ~tmol/1TROLOX) and percent variation after intake of Greenselect T M or Greenselect T M Phytosome |

status) data, and may contribute to understand the mechanism o f the antioxidant effect of green tea catechin consumption.

References

1.

Cook, N.C. and Samman, S. (1996) Flavonoids - Chemistry, metabolism, cardioprotective effects, and dietm-y

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Pietta, P.G., Simonetti, P., Gardana, C., Brusamolino, A., Morazzoni, P., and Bombardelli, E. (1998) Catechin

sources. J. Nutr. Biochem., 7, 66-76. metabolites after intake of green tea infusions. Biofactors, in press. 3.

Ishikawa,T., Suzukawa,M., Ito, T., Yoshida, H., Ayaori,M., Nishiwaki,M., Yonemura, A., Hara,Y., Nak~unura,H. (1997) Effect of tea flavonoid supplementation oil file susceptibility of low-density lipoprotein to oxidative modification. Am. J. Clin. Nutr., 66, 261-266.

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Pietta, P.G., Gardmm, C., Manri, P.L.(1997) Identification of Ginkgo biloba flavonol metabolites after oral administration to humans .J. Chromatogr., B, 693, 249-255.

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Merfort, I., Heilmann, J., Weiss, M., Pietta, P.G., and Gardana, C. (1996) Radical scavenger activity of three

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Pietta, P.G., Simonetti, P., Rice-Evans, C. (1998) Trolox equivalent antioxidant capacity of selected flavonol

flavonoid metabolites studied by inhibition of chelniluminescence in human PMNs. Planta Med., 62, 289-292.

and catechin metabolites, Free Rad. Res., submitted. 7.

Melzig, M.F, (t996) Inhibition of adenosine dimninase activity of aortic endothelial cells by selected flavonoids. Planta Med., 62, 20-21.

8.

Ji, X., Melman, N., Jacobson, K.A. (1996) Interactions of flavonoids and other phytochemicals with adeuosine receptors, J. Meal. Chem, 39, 781-788.

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Medina, J.h., Viola, H, Wolfman, C., Marder, M., Wasowski, C., Calvo, D., Paladini, A.C. (1997) Overview flavonoids : a new fmnily of benzodiazepine receptor ligmlds, Neurochem. Res., 22, 419-425.

10. Jovanovic, S.V., Steenken, S., Tosic, M., Marjmlovic, B., and Simic, M.G. (1994) Flavonoids as mltioxidmlts. J Am. Chem. Soc., 116, 4846-4851. 11. Buetmer, G.R. (1993) The pecking order of tree radicals and antioxid~mts: lipid peroxidation, a-tocopherol, mid ascorbate. Arch. Biochem. Biophys., 300 (2), 535-543. 12. Bors,W., Buetmer,G.R. (1997) In Vitmnin C in health and disease. Ezls L.Packer and J.Fuchs, Marcel Dekker, New York, pp. 75-94. 13. Pietta, P.G. and Simonetti,P. (1998) Dietary flavonoids and interaction with endogenous antioxidants, Biochem. Mol. Biol. Int., 44(5), 1069-1074. 14. Vuilleuanier, J.-P., Keller, H.E., Gysel, D., and Hunziker, F. (1983) Clinical chemical methods tot the routine assessment of file vitamin status in humaa populations. Iaternat. J. Vit. NuU. Res., 53, 265-272. 15. Speek, A.J., Schrijver, J,, and Schreurs, W.H.P. (1984) Fluorhnetric determination of total vitamin C in whole blood by high-performance liquid chromatography with pre-column derivatization. L Chromatogr. 305, 53-60. 16. Neuschwander-Tetri, B.A. ,'rod Roll, F.J. (1989) Glutaflfione measurement by High-Perfbrmance Liquid Chromatography separation and tluorometric detection of the glutathionc-orlhophthalaldehyde adduct. Anal. Biochem., 179, 236-241. 17. Ghiselli, A., Seraflni, M., Mmani, G., Azzmi, E., and Fen'o-Luzzi, A. (1995) A fluorescence-basod method lot measuring total plasma emtioxidant capability. Free Radic. Biol. Med., 18(1), 29-36.

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