Nutraceutical Beverages - American Chemical Society

Chapter 6

Cardiovascular Protective Effects of Hawthorn 1

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Walter Κ. K . Ho , Ζ. Y. Chen , Y. Huang , Z. S. Zhang , Q. Chang , Moses Chow , Y . Liu , and Brian Tomlinson 3

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Downloaded by CORNELL UNIV on July 26, 2012 | http://pubs.acs.org Publication Date: December 1, 2003 | doi: 10.1021/bk-2004-0871.ch006

Departments of Biochemistry and Physiology, School of Pharmacy, and Department of Medicine and Therapeutics, Chinese University of Hong Kong, Shatin, N. T., Hong Kong Department of Nutrition and Food Hygiene, Beijing University Medical School, Beijing 100083, People's Republic of China 5

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Hawthorn has been used as an herbal medicine for treatment of cardiovascular disease in both the east and the west. The hawthorn fruit contains a variety of flavonoids which have been characterized; some of which possess strong antioxidant activity which may account for some of their cardiovascular protective effect. In addition to flavonoids, there are other uncharacterized compounds from the hawthorn fruit which have blood-vessel relaxant and cholesterol lowering activities. The effectiveness of hawthorn in lowering blood cholesterol has been demonstrated in both animal and human studies. The underlying mechanism on the hypocholesterolemic effect of hawthorn appears to be related to LDL receptor regulation as well as modulation of enzyme activities involved in the turnover and transport of cholesterol.

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© 2004 American Chemical Society In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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Introduction Hawthorn (Crataegus) is widely distributed throughout the northern temperate regions of the world with approximately 280 species primarily in East Asia, Europe and North America. The two major species of hawthorn in China are Crataegus pinnatifida and Crataegus cuneata. They have been used as herbal medicine in China to combat ailments such as scurvy, constipation, blood stasis and indigestion for centuries. In recent years, hawthorn has been used in China as well as Europe for treatment of cardiovascular disease (1,2,3). The beneficial effects of hawthorn appear to be associated with three major biological activities, viz., antioxidant, lipid/cholesterol lowering and blood vessel relaxation. In order to establish the medicinal effect of hawthorn, our research group has carried out extensive studies on the different mechanisms of action of the fruit. In the following sections, we shall present some of our current findings on the different modes of action of hawthorn.

Antioxidant Activity of Hawthorn Hawthorn fruits are a rich source of flavonoids (4). Flavonoid consumption has been documented to be negatively associated with coronary heart disease mortality (5). In recent years, it has been generally accepted that oxidation of serum LDL may lead to an increase risk in the development of atherosclerosis (6,7). Hence, one of the beneficial effects of hawthorn may be contributed by its high level of flavonoids as these compounds are powerful antioxidants. In this study, we characterized the major flavonoids present in the hawthorn fruit using organic solvent fractionation and HPLC techniques. The chemical structures and their contents in the fruit are described in Figure 1 and Table I. Epicatechin and procyanidin B are the most abundant, followed by chlorogenic acid, hyperoside, isoquercitrin, protocatechuic acid, rutin and quercetin. These 8 compounds demonstrated varying degrees of antioxidant activity (Table I). Procyanidin B and hyperoside were most protective to human LDL, followed by isoquercetin and quercitrin. Under the same experimental conditions, the antioxidant activity of epicatechin, rutin, cholorogenic acid and protocatechuic acid was similar but much weaker than that of procyanidin B and hyperoside. Eucomic acid, eucomic acid 4-methyl ester and urosolic acid are all found to be weak antioxidants. Although a large number of studies have been carried out on the health benefits of hawthorn, no direct study on the absorption and bioavailability of its 2

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In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.


Figure 1. Structures of 11 compounds isolatedfromhawthornfruitusing ethyl acetate extraction and HPLC techniques. The pair ofhydroxyl groups circled indicates potential site of anti-oxidant activity.


65 Table I. Flavonoid Content of Hawthorn Fruit and Their Protective Activities on LDL Oxidation IC50 Value on LDL Content in Fruit (mg/100g, n=3) Oxidation (μΜ) Procyanidin B 0.75 + 0.03 194.02 + 4.28 Hyperoside 0.95 + 0.02 24.56 + 1.06 Isoquercitrin 1.14 + 0.01 16.44 + 0.51 Quercetin 1.17 + 0.02 0.88 + 0.05 Epicatechin 1.38 + 0.01 178.27 + 6.54 Rutin 1.40 + 0.02 2.57 + 0.06 Chlorogenic acid 1.50 + 0.01 64.86 + 2.02 Protocatechuic acid 2.63 + 0.02 3.21 + 0.06 Eucomic acid >100 nd Eucomic acid 4-methyl ester >100 nd Ursolic acid >100 nd Ascorbic acid (positive control) 37.6 + 0.28 Note: The LDL oxidation protection assay was performed by the TBARS method as described in reference 21. nd, not determined. Compound


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active components has been investigated. To correlate the pharmacological action of the hawthorn flavonoids with their potential health benefit, we studied the absorption kinetics and excretion of epicatechin, chlorogenic acid, hyperside and isoquercitrin after oral administration in rats. For comparison, the individual pure compounds as well as a hawthorn phenolics extract (ΗΡΕ) were studied. Rats were randomly divided into 6 groups. In Groups 1 and 2, the rats received ΗΡΕ at a single oral dose of 220 mg/kg for determination of epicatechin (Group 1) and chlorogenic acid, hyperoside and isoquercitrin (Group 2) simultaneously. The separate determination of epicatechin from others was needed due to the highly different pharmacokinetics between epicatechin and the three other flavonoids. For rats in Groups 3, 4, 5 and 6, the pure compounds were administered individually at a single oral dose of 34.8 (epicatechin), 4.5 (isoquercitrin), 7.5 (chlorogenic acid) and 6.0 (hyperoside) mg/kg, respectively. These amounts were equivalent to the individualflavonoidcontent administered as ΗΡΕ in Groups 1 and 2. As chlorogenic acid and hyperoside could not be detected in the plasma, urine or feces after oral administration, their pharmacokinetics parameters could not be assessed. The pharmacokinetic parameters of epicatechin and isoquercitrin, after oral administration in an extract mixture or pure form are shown in Table II. No significant difference in kinetic parameters were noticed when epicatechin and isoquercitrin were administered in either a mixture



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Table II. Pharmacokinetic Parameters of Isoquercitrin and Epicatechin in Rats Following Oral Administration Compound Administered Isoquercitrin (n = 9) Pure form (4.5 mg/kg) Mixture Epicatechin (n = 7) Pure form (34.8 mg/kg) Mixture

Tmax

Cmax

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fag/ml)

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9.4 ±1.7 10.6 ±1.7

1.31 ±0.45 0.92 + 0.34

10.0 ±2.0 12.3 ±2.7

64± 11 68+14

1.60 ±0.53 1.26 + 0.28

71 ± 1 3 78 ± 1 4

Note: The number of animals used in the experiments is indicated by the value n. Value presented are mean ± SD.



67 formulation or a pure form. Isoquercitrin was absorbed and entered the systemic circulation very rapidly with a T , ^ of approximately 10 min. In contrast, epicatechin was absorbed much more slowly reaching a Tmax at 66 min. The absolute bioavailability of epicatechin and isoquercitrin were 34 and 61%, respectively, as estimated from our data. Based on the above limited study, it is apparent that different flavonoids from hawthorn may have very different oral absorption and clearance characteristics when administered to either animals or human. More detailed investigations are needed to delineate the pharmacological benefits of these flavonoids as some of them might have limited bioavailability. Structurally, isoquercitrin and hyperoside are very similar except the former one is a glucoside and the other is a galactoside (Figure 1). Yet, isoquercitrin is absorbed into the bloodstream quickly while hyperoside is not. The reason for this observation is not clear at the moment. Out of the four compounds studied, isoquercitrin was observed to be least stable when incubated in the presence of small intestinal and colon contents. In contrast, all compounds were stable in the presence of stomach contents. Thus, the difference in absorption between hyperoside and isoquercitrin is likely due to rapid uptake of the latter in the stomach and unlikely due to degradation of the compounds in the GI tract. Moreover, we have also evaluated whether hyperoside was absorbed in a modified chemical form by measuring the concentration of quercetin in the plasma after enzymic hydrolysis. The result showed that no quercetin was detected. Hence, it is likely that certainflavonoidsmay be preferentially uptake or degraded in the GI tract while others may not.

Blood Vessel Relaxant Properties of Hawthorn Hawthorn extract is well recognized in Europe as an antihypertensive remedy, particularly useful in the treatment of mild forms of cardiac insufficiency and angina pectoris which are usually associated with decreased coronary blood flow (3). Animal studies with the isolated heart have verified that hawthorn extract increased coronary blood flow (8). The cardioprotective effect of hawthorn extract was also reported on the ischemic-reperfused rat heart (9). This protection may be partly associated with the anti-oxidative activity of hawthorn extract, which inhibits formation of free radicals and subsequent damage to the cardiac tissue (10,11). We have recently demonstrated that hawthorn extract induced concentration-dependent relaxation primarily through endothelium-dependent action in rat isolated mesenteric arteries and aortas. Our results showed that the relaxant effect of hawthorn extract in rat mesenteric arteries was concentration dependently reduced by N -nitro-L-arginine methyl ester, a competitive inhibitor G


68 of nitric oxide synthase or by methylene blue, an inhibitor of guanylate cyclase. Pretreatment of endothelium-intact artery rings with L-arginine, the nitric oxide precursor, partially reversed the effect of N -nitro-L-arginine methyl ester (12). Neither indomethacin nor glibenclamide affected the relaxant response to hawthorn extract, indicating that relaxing prostanoids or activation of ATPsensitive K channels are not involved. The endothelial nitric oxide-mediated relaxation induced by hawthorn extract was further supported by the stimulatory effect on tissue content of cyclic GMP in endothelium-intact rat aortic rings. Hawthorn extract dose-dependently increased cyclic GMP levels and this effect was completely abolished following pretreatment with N -nitro-L-arginine (another nitric oxide synthase inhibitor) or 1 H-[ 1,2,4] oxadiazolo [4,2a]quinoxalin-l-one (selective guanylate cyclase inhibitor) or in the absence of the functional endothelium (unpublished results). Pretreatment with small doses of hawthorn extract significantly enhanced the relaxant response to acetylcholine, an endothelium/nitric oxide-dependent vasodilator. However, we also found a differential role of endothelial nitric oxide in hawthorn fruit extractinduced relaxation of other three different rat arteries. In cerebral, coronary and carotid arteries, the endothelium does not seem to participate in the extractinduced relaxation since neither N -rutro-L-arginine methyl ester nor 1H[l,2,4]oxadiazolo[4,2-a]quinoxalin-l-one altered the relaxant effect of hawthorn extract. In contrast, endothelial nitric oxide mediates the extract-induced endothelium-dependent relaxation in rat aorta (Chan et al, unpublished observation). It is suggested that some active ingredients in hawthorn extract may have a direct muscle relaxant effect, e.g., possible inhibition of Ca influx in vascular smooth muscle cells (12). The reported anti-oxidant activity of hawthorn fruit (13), the relaxing effect on the carotid, cerebral and coronary arteries suggests a potential preventative effect of hawthorn fruit against cerebral or coronary circulation-related disease such as cerebral vasospasm and stroke. The endothelial nitric oxide-dependent effect indicates that hawthorn fruit extract (12) may possess a broad spectrum of beneficial effects on the cardiovascular function. G

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Hypocholesterolemic Activity of Hawthorn

Animal Studies Hawthorn fruit is beneficial to the cardiovascular system, partially due to its effect on serum cholesterol. To investigate the mechanisms by which hawthorn fruit decreases serum cholesterol, two animal models, using the rabbit and


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hamster, were used in the present study. First, the effect of hawthorn fruit supplementation on the accumulation of cholesterol in different organs were quantified. Second, we sought to determine whether supplementation of hawthorn fruit would lead to any changes in major enzyme activities, viz., liver 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) redutase, liver cholesterol-7a-hydroxylase (CH) and intestinal acyl CoArcholesterol acyltransferase (ACAT), which may affect cholesterol turnover or metabolism. The effect of hawthorn supplementation on fecal excretion of acidic and neutral sterols was also examined.

Rabbits New Zealand white rabbits (n=24, 3.8-4.4kg) were divided into three groups and housed in an animal room at 25 °C with 12:12-h light-dark cycles. The first group (n=4) was fed a reference diet (Glen Forrest Stockfeeds, Western Australia, Australia) that contained no added cholesterol (NC). The second group (n=10) was fed a high-cholesterol diet (HC) that was prepared by addingl.Og cholesterol per 100 g of the NC diet. The third group (n=10) was fed a HC diet supplemented with 2.0g/100g hawthorn fruit powder (HC-H). All the rabbits were killed after overnight food deprivation under carbon dioxide anesthesia and the blood was collected. The total fecal output of each rabbit was combined for whole week 12. The organs including liver, heart, and kidney were removed, washed with saline, and stored at - 80°C. The thoracic aorta from the aortic bulb to the branching of the celiac artery was then removed and saved for measurement of cholesterol. Serum total cholesterol (TC) and triacylglycerol (TG) levels in the HC-H was 23.4% and 22.2% lower, respectively, than those in the HC rabbits (p

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