Effects of aqueous hop (Humulus Lupulus L.) extract on vascular reactivity in rats: mechanisms and influence of gender and hormonal status.

Introduction

Phytoestrogens are naturally occurring plant compounds, members of polyphenol classes, with a chemical structure comparable to that of estradiol (Anderson et al., 1999). They structurally and functionally mimic mammalian estrogens and therefore are considered to play an important role in the prevention of age-related diseases (Ososki and Kennelly, 2003). Soy is the prevalent source of isoflavonoid phytoestrogens but they are present in other human foodstuffs including beans, sprouts, cabbage, spinach, grains and hop (Humfrey, 1998). Indeed, a number of polyphenols have been identified in hop, a dioecious plant of the Cannabaceae family, which is grown in most of the temperate climate zones of the world (De Keukeleire et al., 1999).

Phytoestrogenic molecules have received a great deal of attention over the last few years, particularly because of their potentially preventive roles against cardiovascular diseases (CVD), the leading cause of death in postmenopausal women. Indeed, the Anderson's meta-analysis in humans, on the effects of soy protein supplement on serum lipids, found decreases in total cholesterol, LDL cholesterol and in triglycerides serum concentrations (Anderson et al., 1995). Moreover, in both female and male monkeys, isoflavones in soy protein have been found to improve CVD risk factors (Anthony et al., 1996), in particular serum lipids. Another possibility of phytoestrogens action is their possible effects on arterial walls through an effect on vascular reactivity (van der Schouw et al., 2002). Animal in vitro studies have suggested that genistein (one of the major dietary isoflavones) leads to relaxation of male rat arteries by a nitric oxide (NO) dependent mechanism (Mishra et al., 2000) and enhances the dilator response to acetylcholine (ACh) of atherosclerotic female macaque arteries (Honore et al., 1997). Furthermore, genistein supplementation has been shown to improve endothelial dysfunction induced by ovariectomy in female rats (Squadrito et al., 2000).

The female flowers ("cones") of hop (Humulus Lupulus L.) have been used primarily as a preservative and a flavouring agent in beer. Beer is the alcoholic beverage of choice in many parts of the world and may represent a vehicle for increasing the consumption of natural products with antioxidant and other health-promoting properties (Stevens and Page, 2004). Indeed, it has been suggested that hop have a powerful estrogenic activity, and that beer would have also such an activity (Milligan et al., 1999). The potential estrogenic activity of hop was attributed to the presence of several flavonoid phytoestrogens: isoxanthohumol, 6-prenylnaringenin, 8-prenylnaringenin and geranylated flavonoids (Milligan et al., 2000; Gerhauser et al., 2002). On the other hand, antiestrogenic activity in vitro (Milligan et al., 2000) and in vivo (Gerhauser et al., 2002) was found for the major flavonoid component of the hop cone, xanthohumol.

As epidemiological studies have indicated that regular consumption of natural polyphenols, particularly red wine and green tea, is associated with a reduced risk of coronary heart diseases (Renaud and De Lorgeril, 1992; Rimm et al., 1999; Nakachi et al., 2000; Sasazuki et al., 2000), it is possible that hop may similarly contribute to the reported health-beneficial effects of moderate beer consumption (Klatsky et al., 1997). Moreover, polyphenols contained in red wine and in various grape products have been shown to facilitate the release of NO from the vascular endothelial cells to exert vasodilation (Fitzpatrick et al., 1993; Wallerath et al., 2003). However, there is no information regarding the effects of hop on vascular tissue. So, the first aim of this study was to assess the effects of an aqueous Humulus Lupulus L. extract on rat isolated thoracic aorta. The second aim of the study was to elucidate the possible mechanisms involved in aqueous hop extract-induced vascular reactivity modifications. Current evidences suggest an involvement of several pathways in plant derived estrogen-induced vasodilation such as endothelial NO (Mishra et al., 2000; Vera et al., 2005), vasodilator prostaglandins (Hermenegildo et al., 2005), or calcium-sensitive potassium ([K.sub.Ca]) channels pathways (Nevala et al., 2001). Moreover, calcium regulation via ATP-dependent [Ca.sup.2+] pumps of the sarcoplasmic reticulum or endoplasmic reticulum (SERCA) plays an important role in vessel tone. Thus, NO synthase blocker, cyclooxygenase blocker, [K.sub.Ca] channel inhibitors and SERCA blocker were used in the present study. Finally, we attempted to determine whether hop effects on vascular tissue were gender- and/or hormonal status-dependent. In this way, we used thoracic arterial rings from male rats, sham-ovariectomized and ovariectomized female rats.

Material and methods

Animals

Twenty four rats (20-week old) were used: eight male Sprague-Dawley (SD) rats (MALE), eight sham-operated (Sham OVX) SD female rats and eight ovariectomized (OVX) SD female rats. Animals were purchased from Charles River Laboratories (L'Arbresle, France). They were kept under control environmental conditions: temperature (22-24 [degrees]C), relative humidity (40-60%) and photoperiod (12 light/dark schedule). They were allowed free access to water and standard laboratory chow (AO4--SAFE, Villemoisson--Epinay-Sur-Orge--France). Experiments were approved by the European Community for the care and the use of animals (L 358-86/609EEC).

Preparation of vessel segments

Rats were anaesthetized with an intraperitoneal injection of sodium pentobarbital (6 g for 100 ml; Ceva Sante animale--Libourne, France). The thoracic aorta was removed and placed in cold Krebs'solution at pH 7.4 containing: 118 mM NaCl, 4.7 mM KCl, 1.2 mM MgS[O.sub.4], 2.5 mM Ca[Cl.sub.2], 1.2mM K[H.sub.2]P[O.sub.4], 25 mM NaHC[O.sub.3] and 11 mM glucose. Then, aorta was cleaned of residual blood, fat and connective tissue. The aortic segment was cut into rings approximately of 2 mm length.

Bioassay of vasoreactivity

The rings were mounted between two L-shaped stainless steel hooks and suspended in a 10 ml organ bath chamber containing Krebs' solution maintained at 37 [degrees]C, bubbled with 95% [O.sub.2] and 5% C[O.sub.2] mixture at pH 7.4. During a 90-min-equilibration period, aortic rings were progressively stretched to their optimal passive tension (2g) and washed every 15min as previously described by Laurant and Berthelot (1996). The force of contraction was measured with an isometric force-displacement transducer (F 60 Myograph, Narco-Biosystems) connected to the upper hook. The presence of endothelium was confirmed by observing an over 70% relaxation response to ACh in precontracted rings with [10.sub.-7] M norepinephrine (NE).

Endothelium response was evaluated with cumulative concentrations of aqueous Humulus Lupulus L. extract ([10.sup.-9]-[10.sup.-2] g/l) in aortic rings precontracted with NE ([10.sup.-7] M).

To elucidate the possible mechanisms involved in vascular reactivity modifications after cumulative concentrations of aqueous Humulus Lupulus L. extract, cumulative response curves of aqueous hop extract were established after a 15 min-incubation with N-nitro-L-arginine methyl ester (L-NAME, [10.sup.-4] M) a NOS blocker, with indomethacin ([10.sup.-5] M) a cyclooxygenase blocker, with three calcium-activated K+ ([K.sub.Ca]) channel inhibitors: iberiotoxin ([3.10.sup.-8] M), apamin ([3.10.sup.-8] M) and tetraethylammonium (TEA, [3.10.sup.-4] M) and with thapsigargin ([10.sup.-4] M) an inhibitor of ATP-dependent [Ca.sup.2+] pumps of the sarcoplasmic reticulum or endoplasmic reticulum (SERCA).

Plant material and preparation of extract

Female flowers ("cones") of Humulus Lupulus L. were purchased from ADP Labo pharmaceutique (Reventin Vaugris, France). The aqueous hop extract was obtained by macerating 1 g of dry female flowers in 11 of boiling water, for 4h. After filtration, the extractive solution was collected. Cumulative doses of aqueous hop extract were expressed as g/1 of raw material (corresponding after evaporation to 258 mg/l).

Drugs

All compounds were obtained from Sigma (Saint Quentin Fallavier, France). Noradrenaline bitartate, acetylcholine chloride, L-NAME, iberiotoxin, apamin, thapsigargin were dissolved in water, indomethacin in ethanol and TEA in dimethylsulphoxide (DMSO, VWR International SAS--Strasbourg, France). Corresponding solvent controls were used. All solutions were prepared just before their use and protected from the light. Their concentrations (referred in the text) are the final concentrations of the chamber solutions.

Data and statistical analysis

The aqueous hop extract-induced relaxation (%) in aortic rings was calculated as the percentage of contraction to NE ([10.sup.-7] M). Maximal relaxation values (Max %) were determined for each individual response curve by linear regression analysis (Sigma-Plot[R] 8.0). In addition, sensitivity of the vessels to hop extract (pD2 value) was expressed as the negative log of the dose required to produce a half-maximal response. All data were expressed as means [+ or -] SEM. Statistical comparisons were made using 1-way ANOVA followed by Fischer's LSD test. Dose-response relationships were analysed using 2-way ANOVA followed by Fischer's LSD test. When normality test failed, a 2-way ANOVA on ranks was performed. A paired t-test permitted us to compare the force of contraction induced by NE ([10.sup.-7] M) after incubation with vehicle to that induced by NE after incubation with inhibitors. Differences were considered statistically significant at p < 0.05.

Results

Assessment of endothelial function integrity

Relaxation response to ACh ([10.sup.-6] M) in precontracted rings with NE ([10.sup.-7] M) was very weak in OVX group (38.93 [+ or -] 2.79%, p < 0.001) compared to MALE (89.03 [+ or -] 1.70%) and Sham OVX female rats (91 [+ or -] 1.78%).

NE-induced vasoconstriction 

As contraction level can influence the relaxation, we assessed the vasoconstrictive response induced by [10.sup.-7] M NE (expressed as % of maximal contraction induced by a single dose of 100mmol[l.sup.-1] KCl). It was significantly higher in OVX rats (97.16 [+ or -] 0.57, p < 0.001) than in MALE (86.35 [+ or -] 3.36) and Sham OVX rats (75.99 [+ or -] 1.5). In addition, contraction elicited by NE was greater in MALE compared to Sham OVX rats (p < 0.05).

Effects of aqueous Humulus Lupulus L. extract on thoracic vascular reactivity

Fig. 1 shows the dose-response curves of aqueous hop extract of the thoracic aorta in the three groups. Cumulative concentrations of aqueous hop extract induced a marked vasodilation of aortic rings from MALE and Sham OVX rats, whereas only a slight effect was observed in OVX female rats. Thus, vasodilation was significantly higher in MALE and Sham OVX than in OVX rats (p < 0.001). Moreover, relaxation elicited by aqueous hop extract was greater in Sham OVX than in MALE rats (p < 0.001). As shown in Table 1, maximal relaxation (Max %) and pD2 values in response to Humulus Lupulus L. increased significantly in MALE (p < 0.01) and Sham OVX (p < 0.001) groups in comparison with OVX group.

Because the effect of aqueous hop extract was very weak on arterial rings obtained from OVX female rats, effects of several inhibitors were evaluated only on intact arterial rings from MALE and Sham OVX rats.

[FIGURE 1 OMITTED]

Effects of L-NAME and indomethacin on aqueous hop extract-induced vasodilation

Incubation of arterial rings with L-NAME ([10.sup.-4] M) or indomethacin ([10.sup.-5] M) did not affect NE pre-contraction tone. In presence of L-NAME (Fig. 2A) and indomethacin (Fig. 2B), the aqueous hop extract-induced vasorelaxation was significantly decreased in both MALE and Sham OVX groups (p < 0.001). These results were confirmed by the values of Max % and pD2 significantly lower in both groups after L-NAME and indomethacin incubation (p < 0.05, Table 2A). L-NAME produced more effects in male rats compared to sham-operated female rats, as evidenced by the Max % values: 24.79 [+ or -] 4.67% and 83.27 [+ or -] 3.32% in MALE and Sham OVX rats respectively (Table 2A).

[FIGURE 2 OMITTED]

Effects of calcium-activated [K.sup.+] ([K.sub.Ca]) channel inhibitors on aqueous hop extract-induced vasodilation

Three different [K.sub.Ca] channel blockers including the specific antagonist of [Ca.sup.2+]-activated large conductance [K.sup.+] channels, iberiotoxin, or small conductance [K.sup.+] channels, apamin, and the non-selective [K.sup.+] channel inhibitor, TEA, were used in this study to test the hypothesis that the effect of aqueous hop extract on vasodilation changes was mediated by endothelium-derived hyperpolarizing factor (EDHF).

Incubation of arterial rings with iberiotoxin ([3.10.sup.-8] M) or apamin ([3.10.sup.-8] M) or TEA ([3.10.sup.-4] M) did not affect NE pre-contraction tone. Moreover, iberiotoxin and apamin did modify neither aqueous hop extract-induced vasorelaxation, nor Max % nor pD2 values in both MALE and Sham OVX rats (Figs. 3A, B and Table 2B). On the other hand, in the presence of TEA, aqueous hop extract-induced vasodilation was significantly attenuated in both groups (p < 0.001, Fig. 3C), as confirmed by lower Max % values (p < 0.05, Table 2B).

Effects of thapsigargin on aqueous hop extract-induced vasodilation

Incubation of arterial rings with thapsigargin ([10.sup.-4] M) did not affect NE pre-contraction tone in Sham OVX rats, whereas in MALE rats, NE-induced vasoconstriction was significantly diminished (0.289 [+ or -] 0.06 g) in comparison with NE-induced vasoconstriction after incubation with vehicle (1.114 [+ or -] 0.11 g, p < 0.001). In Sham OVX rats, treatment with SERCA inhibitor significantly decreased the aqueous hop extract-induced vasodilation (p < 0.001, Fig. 4). Max % and pD2 values were also significantly lower in comparison with values obtained in absence of thapsigargin (p < 0.05, Table 2A). On the other hand, in MALE rats, aqueous hop extract-induced vasodilation was completely abolished (p < 0.001, Fig. 4). Indeed, cumulative doses of aqueous hop extract induced contraction of arterial segments from MALE group. So, maximal relaxation value significantly decreased (6.46 [+ or -] 2.95%) compared to that measured in absence of thapsigargin (88.67 [+ or -] 3.01%, p < 0.001, Table 2A).

Discussion

The main result of the present study indicates that aqueous Humulus Lupulus L. extract has a relaxant effect on endothelium intact thoracic arterial rings from Sprague-Dawley male and sham-operated female rats, whereas it has only a weak dilative effect on rings from ovariectomized female rats.

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

Ovariectomy in rats is accompanied by a typical endothelial dysfunction resembling that observed in postmenopausal women. Ovariectomized rats present a marked impairment in the L-arginine/nitric oxide (NO) pathway, characterized by a blunting production of endothelial NO (Squadrito et al., 2000). NO is the most important vasodilator released from the endothelium and in some vessels, such as the rat aorta (Vera et al., 2005). The blunted endothelial NO release might be the consequence of a decreased activity of the enzyme devoted to its regulation, the constitutive NO synthase (NOS) (Squadrito et al., 2000). The lack of aqueous hop extract effect on thoracic arterial rings from ovariectomized rats led to raise the hypothesis that, hop extract-induced relaxation is partly NO-dependent. Our results confirm this hypothesis since vasodilation produced by hop extract was antagonized by the NOS inhibitor L-NAME. L-NAME inhibits both basal NO release and ACh-stimulated NO release (Vallance et al., 1989). Here, the relaxation induced by hop extract was significantly decreased in male rats and in sham-operated female rats after L-NAME incubation. Inhibition of basal NO could antagonize the actions of aqueous hop extract if NO potentiated the effects of aqueous hop extract or vice versa. Whether the role of NO in estrogens and plant derived estrogen-induced vasodilation is well-documented (Reis et al., 1994; Gerhard et al., 1998; Mishra et al., 2000; Bahr et al., 2005), we demonstrated for the first time that the relaxation of thoracic arterial rings by aqueous hop extract is partially NO-dependent.

Although the action of aqueous hop extract seems to be partly mediated via direct activation of NO pathway, and as a consequence may depend on the presence of a functional endothelium, other mechanisms might be responsible for aqueous hop extract-induced relaxation. From a mechanistic point of view, it can be proposed that cyclooxygenase products play a key role. In our study, in presence of indomethacin, a prostacyclin inhibitor, aqueous hop extract-induced relaxation was attenuated in vessels of male and intact female rats. Prostacyclin is a prostaglandin produced from free arachidonic acid through the catalytic activity of two different cyclooxygenases (COX), termed COX-1 and COX-2. Studies performed in cultured human umbilical vein endothelial cells (HUVEC) demonstrated that prostacyclin production is stimulated after exposure to serum from postmenopausal women treated with a mixture of phytoestrogens (Garcia-Martinez et al., 2003). More recently, Hermenegildo et al. (2005) reported that genistein and daidzein, two of the most abundant substances in phytoestrogenic preparations (Ososki and Kennelly, 2003), stimulate prostacyclin production by HUVEC through estrogen receptor-mediated mechanisms, mainly involving increased protein content and COX-2 activity. We might also argue that aqueous hop extract affects the synthesis of endothelial factors such as prostacyclins which contribute to vasodilation.

Our results concerning implication of NO and cyclooxygenase products in aqueous hop extract-induced vasodilation in male and intact female rats, differ from those obtained by Gerhauser et al. in 2002. Indeed, the authors reported that xanthohumol is able to inhibit NO production and COX-1 and COX-2 activity. Thus, these findings lead us to think that aqueous hop extract used in our study probably contained very little xanthohumol because of the rapid conversion to isoxanthohumol at elevated temperatures.

In order to well elucidate the mechanisms involved in vasorelaxant activity of aqueous hop extract, we measured the effects of [K.sub.Ca] channels. Our findings demonstrated that enhanced relaxation with hop extract was significantly decreased in presence of TEA (a nonselective inhibitor of [K.sup.+] channels) in male and sham-operated female rats. On the other hand, iberiotoxin and apamin, had no effect. Thus, in our study, EDHF mainly via activation of [K.sub.Ca] channels did not play a major role in aqueous hop extract-induced vasodilation of thoracic arterial rings from male and intact female rats. Our data do not agree with those previously reported by Nevala et al. (2001). The authors investigated whether [K.sup.+] channels participate in the genistein-and daidzein-induced arterial relaxation, like they do in the case of 17[beta]-estradiol. They found that, in rat mesenteric arteries, the relaxation caused by 17[beta]-estradiol, genistein and daidzein were antagonized by large (iberiotoxin and charybdotoxin) and small conductance [K.sub.Ca] channel inhibitors (apamin). Thus, we may argue that, the main phytoestrogens present in our experimental aqueous hop extract (isoxanthohumol for example) do not act similarly on thoracic arteries than genistein and daidzein on mesenteric arteries.

Finally, we investigated the involvement of SERCAs in aqueous hop extract-induced vasodilation. In presence of thapsigargin, relaxation induced by hop extract was significantly decreased in intact female rats and completely abolished in male rats. Thapsigargin inhibits the activity of ATP-dependent [Ca.sup.2+] pumps of the sarcoplasmic reticulum (SR) or endoplasmic reticulum (ER), termed SERCAs. SERCAs transport [Ca.sup.2+] ions accross a lipid membrane from the cell cytosol into distinct regions of the sarcoplasmic/endoplasmic reticulum. Because aqueous hop extract-induced vasodilation was attenuated after thapsigargin incubation, we may suggest that SERCAs contributed to aqueous hop extract-induced beneficial effects on arterial rings from male and intact female rats.

In the light of our results, we could suggest that the effect of aqueous hop extract in enhancing vascular reactivity was independent of gender. Indeed, vasorelaxant effect of aqueous hop extract appeared in both male and intact female rats. Similar gender-independent relaxation induced by plant derived estrogens has also been reported in rat mesenteric arteries (Nevala et al., 1998), in rabbit (Figtree et al., 2000) or in porcine coronary arteries (Lee and Man, 2003). On the other hand, in ovariectomized female rats the relaxation response to aqueous hop extract was very weak. Contrary to the literature about phytoestrogens supplementation (Squadrito et al., 2000; Altavilla et al., 2001; Catania et al., 2002), our experimental hop extract did not improve endothelial dysfunction induced by ovariectomy in rats. This suggests that in vitro effect of aqueous hop extract on vasodilation may depend on the presence of some circulating estrogens.

In conclusion, this study indicates for the first time, that aqueous hop extract is a potent vasodilator of endothelium-intact thoracic arterial rings from male and sham-operated female rats and that this vasodilation seems to depend on hormonal status. Moreover, aqueous hop extract induced-vasorelaxation was strongly inhibited by NOS, prostacyclin and SERCAs inhibitors, suggesting the implication of NOS activation, cyclooxygenase products and [Ca.sup.2+] pathways in hopinduced vasodilation.

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H. Figard (a,b,*), C. Girard (c), F. Mougin (a,b), C. Demougeot (b), A. Berthelot (b)

(a) UFR STAPS Besancon, 31 chemin de l'Epitaphe, Universite de Franche--Comte, 25000 Besancon, France

(b) Laboratoire de Physiologie Pharmacologie, Nutrition Preventive Experimentale, EA 3921, Faculte de Medecine et de Pharmacie, Place Saint Jacques, Universite de Franche-Comte, 25030 Besancon Cedex, France

(c) Laboratoire de Pharmacognosie, EA 3921, Faculte de Medecine Pharmacie, Place Saint Jacques, Universite de Franche-Comte, 25030 Besancon Cedex, France

*Corresponding author. Laboratoire de Physiologie et de Pharmacologie, Nutrition Preventive Experimentale, Faculte de Medecine et de Pharmacie, Place Saint Jacques, 25030 Besancon Cedex. France. Tel.: [phone]; fax: [phone].

E-mail address: [e-mail] (H. Figard).

Table 1. Maximal relaxation and pD2 values in response to Humulus
Lupulus L. in aortic rings from 20-week-old Sprague-Dawley males,
sham-operated female and ovariectomized female rats

          Max %                   pD2

MALE      88.67 [+ or -] 3.01**   6.44 [+ or -] 0.47**
Sham OVX  99.00 [+ or -] 0.54***  8.17 [+ or -] 0.48***
OVX       51.53 [+ or -] 10.59    3.74 [+ or -] 0.79

The hop-induced maximal relaxation (Max %) on aortic rings was
determined by linear regression analysis. p[D.sub.2] value was expressed
as the negative log of the dose required to produce a half-maximal
response. Values are expressed as means [+ or -] SEM (n = 8 rats per
group).
**p < 0.01 and ***p < 0.001 vs OVX rats.

Table 2

(A) Effects of L-NAME ([10.sup.-4] M), indomethacin ([10.sup.-5] M) and
thapsigargin ([10.sup.-4] M) on maximal relaxation and pD2 values in
response to hop on aortic rings from 20-week-old Sprague-Dawley male
rats and sham-operated female rats

                           Hop
          Max %                pD2

MALE      88.67 [+ or -] 3.01  6.44 [+ or -] 0.47
Sham OVX  99.00 [+ or -] 0.54  8.17 [+ or -] 0.48

                           L-NAME
          Max %                     pD2

MALE      24.79 [+ or -] 4.67***    4.08 [+ or -] 0.28**
Sham OVX  83.27 [+ or -] 3.32 (##)  5.10 [+ or -] 0.5 (##)

                          Indomethacin
          Max %                      pD2

MALE      31.25 [+ or -] 5.63***     4.69 [+ or -] 0.46*
Sham OVX  41.46 [+ or -] 10.68 (##)  5.56 [+ or -] 0.76 (#)

                        Thapsigargin
          Max %                     pD2

MALE       6.46 [+ or -] 2.95***    Not calculated
Sham OVX  56.76 [+ or -] 10.61 (#)  5.88 [+ or -] 0.57 (#)

(B) Effects of iberiotoxin ([3.10.sup.-8] M), apamin ([3.10.sup.-8] M)
and TEA ([10.sup.-7] M) on maximal relaxation and pD2 values in response
to hop on aortic rings from 20-week-old Sprague-Dawley male rats and
sham-operated female rats

                           Hop
          Max %                pD2

MALE      88.67 [+ or -] 3.01  6.44 [+ or -] 0.47
Sham OVX  99.00 [+ or -] 0.54  8.17 [+ or -] 0.48

                     Iberiotoxin
          Max %                pD2

MALE      85.85 [+ or -] 5.72  6.66 [+ or -] 0.45
Sham OVX  97.61 [+ or -] 0.85  7.87 [+ or -] 0.48

                         Apamin
          Max %                 pD2

MALE      83.46 [+ or -] 10.67  6.24 [+ or -] 0.49
Sham OVX  98.91 [+ or -] 0.81   7.40 [+ or -] 0.37

                           TEA
          Max %                    pD2

MALE      77.61 [+ or -] 1.65*     6.40 [+ or -] 0.62
Sham OVX  89.18 [+ or -] 2.29 (#)  6.69 [+ or -] 0.43

The hop-induced maximal relaxation (Max %) of aortic rings was
determined by linear regression analysis. [pD.sub.2] value expressed
negative log of the dose required to produce a half-maximal response.
Values are expressed as means [+ or -] SEM (n = 8 rats per group).
*p < 0.05, **p < 0.01 and ***p < 0.001, significantly different from
values obtained in MALE group after cumulative doses of hop in absence
of inhibitor. (#) p < 0.05, (##) p < 0.01 and (###) p < 0.001,
significantly different from values obtained in Sham OVX group after
cumulative doses of hop in absence of inhibitor.
Because [EC.sub.50] values (concentration of aqueous hop extract that
induced 50% of maximum relaxation response to contraction elicited by
[10.sup.-7] M NE) obtained in MALE group after thapsigargin incubation
were negative, pD2 values were not calculated.