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Comparison of antioxidative capacities and inhibitory effects on cholesterol biosynthesis of quercetin and potential metabolites.

Summary

The flavonol quercetin is known to be rapidly metabolized after ingestion by enterocytes and bacteria in the intestinal tract which may influence the biological, e.g. antioxidative potency of this compound. Therefore, quercetin and several of its possible metabolites were compared with regard to their antioxidant activity and their capacity to inhibit hepatocellular cholesterol biosynthesis. Using the 2,2,-diphenylpicrylhydrazyl radical scavenger assay, all compounds with an ortho diphenolic structure acted as strong antioxidants. In contrast, in a cellular assay focusing on lipid peroxidation in cultured rat hepatocytes challenged with tert.-butylhydroperoxide only the lipophilic compounds quercetin and 3,4-dihydroxytoluene were active. Concerning the inhibition of cholesterol biosynthesis, 3,4-dihydroxytoluene surprisingly mimicked the effect of quercetin in primary rat hepatocytes, but much less so in HepG2 cells. All other metabolites were almost ineffective in both cell types. These results suggest that some of the biological functions of flavonoids detectable by in vitro assays may persist in vivo as long as comparably potent metabolites are systemically present.

Key words: flavonoids, quercetin metabolites, antioxidative activity, cholesterol biosynthesis, hepatocyte cultures, HepG2 cells

* Introduction

Flavonoids comprise a large group of naturally occuring compounds widely distributed in the plant kingdom. Some of these compounds have been reported to exert potent activities including antioxidative (Bors et al., 1990; Diplock et al., 1998), tissue-protective (Ames et al., 1993) and tumoristatic (Wang et al., 1998) effects as well as inhibition of hepatic cholesterol biosynthesis (Gebhardt, 1998). These functions were most convincingly demonstrated using appropriate in vitro assays (Gebhardt, 2000) that allow insight into the underlying molecular mechanisms. For example, some specific flavonoids, quercetin and luteolin, were recently reported to act as heavy metal chelators (Brown et al., 1998), to scavenge superoxide radicals (Yuting et al., 1990) and hydroxyl radicals (Chimi et al., 1991), reduce lipid peroxyl radicals (Torel et al., 1986) and inhibit lipid peroxidation (Avanas'ev et al., 1989; Brown and Rice-Evans, 1998; Gebhardt, 1997; Gebhardt and Fausel, 1997).

However, if the potential benefit of these activities for human and animal health is discussed, it has to be considered that flavonoids are rarely applied topically, but rather consumed as ingredients of natural food or in form of food additives, herbal medicinal products and phytomedicines. Since many flavonoids are rapidly metabolized in the intestinal tract by enterocytes and the intestinal flora (Wiseman, 1999; Hollman and Katan, 1999; Graefe et al., 1999; Gee et al., 2000), it is doubtful whether flavonoids may function per se or in form of their metabolites. Although the metabolic fate may vary considerably among the various flavonoids, there is no doubt that considerable concentrations of metabolites may appear in blood (Manach et al., 1995). Again, quercetin and rutin are among the most studied examples in animals (Booth et al., 1956; Spencer et al., 1999; Ader et al., 2000) as well as in man (Sawai et al., 1987; Manach et al., 1998; Graefe and Veit, 1999).

The present study was performed in order to compare the relative activities of quercetin and some possible metabolites (Booth et al., 1956) with respect to antioxidative effects and inhibition of hepatocellular cholesterol biosynthesis. For that purpose, the DPPH radical scavenger assay, the production of malondialdehyde by cultured rat hepatocytes exposed to t-butylhydroperoxide, and the incorporation of radiolabelled acetate into cholesterol were used. It was found that the different effects of the parent flavonol quercetin were mimicked to a variable degree by some of the metabolites depending on the presence or absence of distinct structural features.

* Materials and Methods

Materials

3,4-dihydroxyphenylacetic acid, 3,4-dihydroxytoluene, 4-hydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, hippuric acid, caempherol and quercetin (x2[H.sub.2]O) were purchased from Fluka (Neu-Ulm, Germany). Rutin (x3[H.sub.2]O) was received from Roth (Karlsruhe, Germany). Homovanillic acid and Trolox were obtained from Sigma (Deisenhofen, Germany).

Collagenase was provided by Boehringer Mannheim GmbH (Mannheim, Germany). Williams Medium E was obtained from Bio Whittaker (Verviers, Belgium), Dulbecco's MEM (1x) from Biochrom KG (Berlin, Germany) and fetal calf serum from PAA Laboratories GmbH (Colbe, Germany). All other chemicals were from Boehringer Mannheim GmbH, Merck (Darmstadt, Germany), Roth (Karlsruhe, Germany) or Sigma (Daisenhofen, Germany). The cell culture plates with tissue culture quality were from Techno Plastic Products AG (Trasadingen, Switzerland).

Animals

Male Sprague-Dawley rats (220-290 g) were kept in a controlled 12 h light and 12 h dark cycle on an standardized diet of Alma[R] H 1003 (Botzenhardt, Kempten) and tap water ad libitum.

Isolation and cultivation of cells

Liver parenchymal cells were isolated from the rats according to the two-step collagenase perfusion technique as described (Gebhardt, 1997) and plated at 7 x 106 cells per petri dish (100 x 20 mm), at 1.25 x [10.sup.6] cells per well of six-well plates or at 0,25 x [10.sup.6] cells per well of 24-well plates in Williams medium E supplemented with 10% of fetal calf serum, 2 mM glutamine, [10.sup.-7] M dexamethasone, 50 U/ml Penicillin and 50 [mu]g/ml Streptomycin. After two hours the medium was replaced by fresh Williams medium containing the same supplements except serum. On the next day, incubations with the test substances began. The cells were always incubated at 37 [degrees]C and 90% humidity in an atmosphere containing 5% C[O.sub.2] and 95% air.

The human hepatoblastoma cell line HepG2 was maintained in Dulbecco's MEM (1x) medium supplemented with 10% fetal calf serum, 2 mM glutamine, [10.sup.-7] M dexamethasone, 50 U/ml Penicillin and 50 [mu]g/ml Streptomycin as described (Fahrner et al., 1993; Gebhardt et al., 1994).

Determination of antioxidative activity: DPPH assay

A stock solution (0.1 mM) of DPPH was prepared in ethanol. This solution was mixed with an equal volume of a solution of the test compounds in water. Compounds with low solubility in water were first dissolved in DMSO and then diluted with water such that the final concentration of DMSO in the reaction mixture did not exceed 1%. After mixing, the reaction was allowed to complete in the dark. Measurements were then carried out according to Wang et al. (1999).

MDA assay

Hepatocytes were incubated simultaneously with t-BHP (final concentration 1.5 mM) and different concentrations of the test compounds for 40 min. After this time, the cells were washed with 0.9% NaCl and resuspended in one ml 50 mM potassium phosphate buffer (pH 7.4) and homogenized by ultrasound for 10 s (15% of the maximum power, Sonopuls HD 2200, Bandelin electronic, Berlin, Germany). MDA in cell homogenates was then determined by the thiobarbituric acid (TBA) assay (Esterbauer and Cheeseman, 1990; Gebhardt, 1997).

Determination of acetate incorporation into and separation of non-saponifiable neutral lipids

The incorporation of [C.sup.14] into non-saponifiable neutral lipids of hepatocytes and HepG2 cells was determined as described (Gebhardt, 1991, 1998). Briefly, the culture medium was removed and fresh medium was added containing [C.sup.14]acetate (18.5 KBq/ml (0.5 [mu]Ci/ml)) and the test compounds in the appropriate dilutions. After incubation at 37 [degrees]C for 2 h, the medium was removed and the cell-layer was washed twice with saline and scraped into 2 ml of distilled water. The cells were homogenized by sonication for 20 sec. (Gebhardt, 1993). After saponification of the homogenates with 0.5 M KOH in EtOH efficient separation of fractions was performed on Extrelut[R]-columns (large-pore kieselgur) according to Pill et al., 1985. The neutral lipophilic nonsaponifiable substances were eluted with n-heptane (Gebhardt, 1993; Aufenanger et al., 1986). The precursors such as [[C.sup.14]]acetate are retained on the column to more than 99%. For measurements of incorporation, the eluate was collected directly in scintillation vials and measured in the scintillation counter after addition of 10 ml of Ultima Gold[R] (Packard, Merident, Connecticut). The yield of the elution step (93 [+ or -] 2%) and the recovery were determined as described (Gebhardt, 1993). In previous work applying silver-ion thin-layer chromatography (SI-TLC) or HPLC, it was confirmed that the radioactivity was found to more than 93% in cholesterol and approx. 5% in lanosterol (Gebhardt, 1993).

Determination of cytotoxicity and other analytical procedures

Cytotoxicity of the extracts and isolated compounds tested was determined by means of the MTT assay as described (Gebhardt and Fausel, 1997). Protein was measured following the procedure of Lowry et al. (1951).

Statistical evaluation

The data were evaluated statistically using Student's t-test. Data are given as means [+ or -] standard deviation (SD).

2 Results

DPPH assay

Antioxidative activity of quercetin, its glycoside rutin and some of its metabolites was first assessed using the DPPH radical scavenger assay. As shown in Fig. 1, rutin, quercetin and 3,4-DHPAA exerted a concentration-dependent scavenging effect in the range of one and 30 [mu]M, whereas 4-HPAA was almost ineffective up to 500 [mu]M. According to the [EC.sub.50]-values determined from such curves for these and the other compounds (Table 1) the radical scavenging activity decreased in the order querectin [greater than or equal to] rutin > 3,4-DHPAA > 3,4-DHT > HVA >> HA = 4-HPAA = 3-HPAA. The first four compounds of this sequence leaded by quercetin with an [EC.sub.50]-value of 3.4 [mu]M were even stronger than trolox ([EC.sub.50]-value 11 [mu]M) , a soluble derivative of vitamin E which is often used as an antioxidative standard.

MDA assay

When the antioxidative activity was determined in cultured rat hepatocytes that were challenged by t-BHP, quercetin again was the most potent substance with an [EC.sub.50]-value of about 16 [mu]M. In this assay not only t-BHP-induced but also endogenous lipid peroxidation was blocked at high concentrations. As shown in Fig. 2, none of the metabolites was effective up to 70 [mu]M except 3,4-DHT which seemed equally potent to quercetin (Gebhardt et al., 1999).

Cholesterol biosynthesis

The second biological activity of quercetin studied in cultured hepatocytes and HepG2 cells concerns the reduction of cholesterol biosynthesis determined by incorporation of radiolabelled acetate into the fraction of non-saponifiable neutral lipids. As demonstrated elsewhere, this fraction consists of more than 93% cholesterol under the experimental conditions used herein (Gebhardt, 1993) and, thus, truely reflects cholesterol biosynthesis. While quercetin showed a moderate inhibition (maximally about 50%) of cholesterol biosynthesis in primary cultured rat hepatocytes, rutin was much less potent (Fig. 3). Concerning the metabolites, only 3,4-DHT was able to inhibit cholesterol biosynthesis, while all other compounds were inactive at 50 [mu]M (Fig. 3B). A detailed comparison between quercetin and 3,4-DHT revealed an almost equal inhibiting potential of both compounds in primary rat hepatocytes (Fig. 3A) with quercetin being slightly stronger at concentrations around 20 [mu]M. Interestingly, mixing of qu ercetin and 3,4-DHT at several concentrations failed to produce clear additive effects. For instance, at 50 [mu]M acetate incorporation amounted to 68.7 [+ or -] 1.5% and 72.3 [+ or -] 3.1% for quercetin and 3,4-DHT, respectively. The mixture of these two compounds at 50 [mu]M each led to an incorporation of 62.3 [+ or -] 1.7%, which was significantly different (P <0.01) from the value for 100 [mu]M quercetin amounting to 55.7 [+ or -] 1.6%. This suggests a different mode of action of both molecules, most probably at different sites in the same cascade of regulatory events.

When HepG2 cells were used, quercetin was much stronger in inhibiting cholesterol biosynthesis compared to rat hepatocytes (Fig. 4). In HepG2 cells, the inhibiting potency of quercetin also differed from that of 3,4-DHT. Roughly, the [EC.sub.50]-value of quercetin was about 5 [mu]M, while that of 3,4-DHT was more than 10-fold higher (Fig. 4). The other compounds were even less active than 3,4-DHT (not shown).

* Discussion

After oral administration of quercetin or rutin to animals and man considerable metabolism seems to occur, since neither free quercetin nor rutin were detected in serum and urine (Hollman and Katan, 1999; Graefe et al., 1999; Graefe and Veit, 1999). In the small intestine evidence for extensive deglycosylation and formation of glucuronides by enterocytes was obtained (Day et al., 1998; Walle et al., 2000; Gee et al., 2000), while the intestinal microflora seems to produce a number of small metabolites and degradation products (Baba et al., 1981; Sawai et al., 1987) that are also absorbed and may be further metabolized in the liver. Both, conjugates and degradation products of quercetin were transiently found in the serum (Sawai et al., 1987; Manach et al., 1998; Graefe and Veit, 1999; Boyle et al., 2000). Since intestinal metabolism may proceed quite rapidly, the question arises whether biological functions attributed to the original flavonoids by in vitro assays are relevant for the in vivo situation.

In the present study, we have used two distinct functions of quercetin, namely the antioxidative activity and the inhibitory effect on cholesterol biosynthesis, in order to compare the potency of the parent compound with that of some of its possible degradation products in the same bioassays. Concerning the DPPH radical-scavenging assay we could show that the presence of a diphenol configuration (in ortho position) was sufficient to exert a scavenging activity surmounting even that of Trolox, a soluble derivative of vitamin E serving as a standard. This was not surprising in view of many published data emphasizing the strong antioxidative action of di- and polyphenols (Osawa, 1999; Prior and Cao, 1999; Vinson, 1998; Rice-Evans, 1995). More interesting, however, was the result that only 3,4-DHT, but not the diphenols 3,4-DHAA and homovanillic acid, was effective in protecting hepatocytes against lipid peroxidation induced by t-BHP. This indicates (1) that protection does not occur by neutralizing the peroxid radical in the culture medium, since otherwise also the more water-soluble diphenols should react. This is consistent with the fact that preincubation of the cells with the compounds followed by incubation with t-BHP alone leads to similar results (not shown; see also discussion by Gebhardt, 1997, for preincubation with other antioxidants). (2) It indicates that solubility within biological membranes may be a prerequisite for antioxidants to reduce lipid peroxidation in cultured hepatocytes. These results further demonstrate that different in vitro assays including cellular test systems need to be used in studies designed to adequately assess the antioxidative potential of a given compound in biological systems (Gebhardt, 2000).

Another interesting result was the finding that 3,4DHT inhibited hepatocellular cholesterol biosynthesis with almost the same potency as quercetin in primary rat hepatocytes, while it was much less effective in HepG2 cells, a human hepatoblastoma cell line. Whether this difference is due to the species difference of these cells, or due to the transformed state of the HepG2 cells remains to be established. It fits, however, to the conclusion drawn from the mixing experiments that both compounds have a different mode of action which is differently accentuated in these two cell types. Furthermore, the structural requirements seem to be more complex in this case. Obviously, again the diphenolic structure (as in the B ring of quercetin) seems necessary but not sufficient and, thus, has to be associated with other structural features. As apparent from the effect of 3,4-DHT, enhanced lipophilicity may also be a precondition for the inhibitory function at least in primary rat hepatocytes. However, since quercetin and 3,4-DHT seem to act via different mechanisms and exert different degrees of inhibition in different cells, it will be interesting to see how the real structural requirements for effective inhibition look like. Further studies designed to address this question are currently performed. In this respect, it is interesting that quercetin is able to reduce the incorporation of acetate into cholesterol only by about 40% in rat hepatocytes and by 80% in HepG2 cells, while other flavonoids such as luteolin are much more potent in both types of cells (unpublished). Thus, the presence of the additional hydroxyl group at the 3-position in quercetin seems less favorable for inhibition of cholesterol biosynthesis. Concerning the antioxidative activity no such differences are apparent between these two flavonoids (Rice-Evans et al., 1996; Gebhardt et al., 1999).

In conclusion, the present study provides evidence that certain metabolites of quercetin may exert similar biological functions as the parent flavonol. Whether a compound is active seems to depend strictly on the structural requirements of the function in question and cannot easily be predicted. These results demonstrate that studies in vitro on biological effects of flavonoids need to be complemented by studies on bioavailability and metabolism before any conclusions about their effectiveness in vivo can be drawn. As shown here for quercetin and some selected metabolites, biodegradation of the parent flavone does not automatically lead to a loss of function. It may be envisioned that in vivo such functions persists as long as adequately effective metabolites remain in the body.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]
Table 1

Comparison of the antioxidative potency of rutin, quercetin, and
potential metabolites with Trolox in the DPPH-scavenger assay and
against t-butylhydroperoxide-induced lipid peroxidation in cultured rat
hepatocytes.

 [EC.sub.50]-values (a) ([mu]M)
Compound DPPH-assay MDA-assay

Trolox 11 23
Rutin 4.0 26
Quercetin 3.4 16
4-HPAA >500 n.d. (b)
3,4-DHPAA 4.6 >150
3-HPAA >500 n.d.
Homovanillic acid 17 >100
3,4-DHT 7.3 30
Hippuric acid >500 n.d.

(a) determined from detailed plots of effect vs concentration such as
depicted in Figs. 1.

(b) n.d. - not determined


Acknowledgement

The authors would like to thank Mrs. K. Lerche for excellent technical assistance.

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Abbreviations: 3,4-DHPAA -- 3,4-dihydroxyphenylacetic acid; 3,4-DHT -- 3,4-dihydroxytoluene; DMSO -- dimethylsulfoxide; DPPH -- 2,2,-diphenylpicrylhydrazyl; HA -- hippuric acid; 3-HPAA -- 3-hydroxyphenylacetic acid; 4-HPAA -- 4-hydroxyphenylacetic acid; HVA -- homovanillic acid; MTT -- 3-[4,5-dimethylthiazol-2-y1]-2,5-diphenyl tetrazolium brornid; t-BHP -- tert-butylhydroperoxide; SD -- standard deviation.

G. GiaBer (1), E.U. Graefe (2), F. Struck (1), M. Veit (2), and R. Gebhart (1)

(1.) Institut fur Biochemie, Medizinische Fakultat, Universitat Leipzig, Leipzig, Germany

(2.) Zentralinstitut Arzneimittelforschung GmbH, Sinzig, Germany

* Address

Prof. Dr. Roif Gebhardt, Institut fur Biochemie, Liebigstra[beta]e 16, 04103 Leipzig, Germany

Tel.: ++49-34 1-9722100; Fax: ++49-34 1-9722109; e-mail: rgebhardt@medizin.uni-leipzig.de
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Author:Glaber, G.; Graefe, E.U.; Struck, F.; Veit, M.; Gebhardt, R.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:4EUGE
Date:Jan 1, 2002
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