Hypocholesterolemic Activity

Hawthorn fruit has hypolipidemic activity. Chen et al. (10) demonstrated that serum total cholesterol, triglyceride, and apo-B decreased by 15%, 10%, and 8%, respectively, with HDL cholesterol being unchanged, in 30 hyperlipi-demic humans who consumed hawthorn fruit drinks. In a recent unpublished study, we have also evaluated the clinical efficacy of hawthorn in lowering blood cholesterol using a randomized, double-blinded, placebo-controlled, crossover design. Seventy-three mildly hypercholesterolemic patients were asked to take a 250-mL hawthorn or placebo drink three times a day for 4 weeks. At the end of this period, a washout of 4 weeks was implemented before the crossover. Blood samples were taken at baseline and week 4, 9, and 12 for total cholesterol, LDL cholesterol, HDL cholesterol, and triglyceride for analysis. Toxicity was monitored by blood chemistry. The results of this study are shown in Table 1 and Table 2. The hawthorn group had a 7.8% reduction in total blood cholesterol and a 12.4% reduction in LDL cholesterol versus a 0.8% and 4.8% reduction, respectively, in the placebo group in the first phase of the trial. After the crossover, the hawthorn group still had a significant reduction in both total cholesterol (6.7% vs. 3.4%) and LDL cholesterol (13.8% vs. 5.0%) compared with the placebo group. Neither blood triglyceride nor HDL-cholesterol was significantly changed after the intake of hawthorn juice. Analysis of the blood chemistry results indicated

TABLE 1 Serum Total Cholesterol Level (mg/dL) After Intake of Hawthorn Juice or Placebo for 4 Weeks

Group

Baseline

Week 4

Difference

Significance

Group A:

hawthorn

268 + 57

247 + 55

-21 + 33

p < 0.05

Group C:

placebo

254 + 40

252 + 47

-2 + 35

ns

Week 9

Week 12

Difference

Significance

Group A:

placebo

235 + 39

227 + 41

-8 + 29

ns

Group C:

hawthorn

239 + 38

223 + 33

-16 + 29

p < 0.05

Subjects were given 250 mL hawthorn or placebo drink three times per day for 4 weeks. Starting at the fifth week treatment was stopped until week 9. Then the two groups of subjects were crossed over for treatment for an additional period of 4 weeks. Blood samples were analyzed at baseline, week 4, week 9, and week 12. The hawthorn drink given contained 8% water-soluble material from the fruit. The placebo drink contained artificial coloring and flavor and had the same caloric content as the hawthorn drink.

Subjects were given 250 mL hawthorn or placebo drink three times per day for 4 weeks. Starting at the fifth week treatment was stopped until week 9. Then the two groups of subjects were crossed over for treatment for an additional period of 4 weeks. Blood samples were analyzed at baseline, week 4, week 9, and week 12. The hawthorn drink given contained 8% water-soluble material from the fruit. The placebo drink contained artificial coloring and flavor and had the same caloric content as the hawthorn drink.

TABLE 2 Serum LDL Cholesterol (mg/dL) After Intake of Hawthorn Juice or Placebo for 4 Weeks

Group

Baseline

Week 4

Difference

Significance

Group A:

hawthorn

186 + 59

163 + 54

-23 + 32

p < 0.05

Group C:

placebo

168 + 38

160 + 38

-8 + 34

ns

Week 9

Week 12

Difference

Significance

Group A:

placebo

161 + 39

153 + 42

-8 + 24

ns

Group C:

hawthorn

159 + 31

137 + 24

-22 + 26

p < 0.05

See footnote to Table 1 for experimental details.

See footnote to Table 1 for experimental details.

that no significant changes in blood cell counts, liver and kidney function as well as other indexes were observed after consumption of hawthorn. Out of the 73 subjects studied, only 7 patients dropped out, 3 of them attributed to intolerance to the acidity of the juice (pH 3.5) while the remaining had problems unrelated to the trial.

In rats, the hypocholesterolemic potency of hawthorn fruit drink was even more pronounced. One of our previous studies (11) examined the hypolipidemic activity of hawthorn fruit in three groups of New Zealand white rabbits fed with one of three diets, a control diet without addition of cholesterol (NC), a 1.0% high-cholesterol diet (HC), and a HC diet supplemented with 2.0% hawthorn fruit powder (HC-H). The results showed that inclusion of 2% dry hawthorn fruit powder led to 23% lower serum total cholesterol and 22% lower serum triglyceride in rabbits (Table 3). In addition, hawthorn fruit supplementation led to 51% less cholesterol accumulation in the aorta of rabbits (Table 3). In hamsters, significant reduction in the serum total cholesterol by 10% and triglyceride by 13% was also observed after they were fed a diet supplemented with 0.5% hawthorn fruit ethanolic extract (Table 4) (12). However, supplementation of hawthorn fruit ethanolic extract had no effect on the serum HDL-cholesterol level (Table 4). All these observations confirm that hawthorn fruit modulates blood lipids favorably.

The mechanism by which dietary hawthorn fruit decreases serum cholesterol may involve multifaceted interactions of cholesterol metabolism. The decrease in cholesterol biosynthesis would lead directly to a lower blood cholesterol level. Rajendran et al. (13) followed cholesterol synthesis by measuring the incorporation of [14C]-acetate into the liver cholesterol in rats fed a diet supplemented with hawthorn ethanolic extract. It was found that supplementation of hawthorn ethanolic extract led to 33% lower cholesterol biosynthesis in rats. However, we found that inclusion of hawthorn fruit in the diet had no effect on the 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-

TABLE 3 Serum and Aortic Lipids, and Fecal Neutral and Acidic Sterols of New Zealand White Rabbits Fed a Reference Diet (NC), a High-Cholesterol Diet (HC), or a High-Cholesterol Diet Supplemented with 2.0% Dry Hawthorn Fruit Powder (HC-H) for 4 Weeks

HC-H

Serum

TABLE 3 Serum and Aortic Lipids, and Fecal Neutral and Acidic Sterols of New Zealand White Rabbits Fed a Reference Diet (NC), a High-Cholesterol Diet (HC), or a High-Cholesterol Diet Supplemented with 2.0% Dry Hawthorn Fruit Powder (HC-H) for 4 Weeks

Serum

Total cholesterol (mmol/L)

0.5

F

0.2c

24.7

F

2.8a

18.9

F

4.7b

HDL cholesterol (mmol/L)

0.3

F

0.1a

0.2

F

0.1b

0.3

F

0.1a

Triglycerides (mmol/L)

0.6

F

0.1c

2.2

F

0.5a

1.7

F

0.3b

Aorta

Total cholesterol (nmol/g)

1.5

F

0.7c

28.3

F

14.3a

13.9

F

8.1b

Triglycerides (nmol/g)

31.8

F

2.8b

52.0

F

24.2a

49.6

F

24.8a

Liver total cholesterol (nmol/g)

3.0

F

0.4c

95.0

F

24.2a

58.0

F

14.4b

Heart total cholesterol (nmol/g)

2.8

F

0.3c

7.4

F

2.4a

5.5

F

0.9b

Kidney total cholesterol (nmol/g)

6.9

F

0.6c

17.7

F

1.8a

14.2

F

2.6b

Fecal total neutral sterols (mg/g)

51.1

F

10.1c

134.1

F

19.6b

264.4

F

36.1a

Fecal total acidic sterols (mg/g)

13.2

F

2.1c

18.1

F

2.4b

35.5

F

4.8a

Means at a row with different letters differ significantly, p < 0.05.

Means at a row with different letters differ significantly, p < 0.05.

TABLE 4 Serum Lipids, Fecal Neutral and Acidic Sterols, Liver 3-Hydroxy-3-Methyl Glutaryl Coenzyme A (HMG-CoA) Reductase, Liver Cholesterol-7a-Hydroxylase (CH), and Intestinal Acyl CoA:Cholesterol Acyltransferase (ACAT), in Hamsters Fed the Control High-Cholesterol Diet or the Same High-Cholesterol Diet Supplemented with 0.5% Hawthorn Fruit Ethanolic Extract

TABLE 4 Serum Lipids, Fecal Neutral and Acidic Sterols, Liver 3-Hydroxy-3-Methyl Glutaryl Coenzyme A (HMG-CoA) Reductase, Liver Cholesterol-7a-Hydroxylase (CH), and Intestinal Acyl CoA:Cholesterol Acyltransferase (ACAT), in Hamsters Fed the Control High-Cholesterol Diet or the Same High-Cholesterol Diet Supplemented with 0.5% Hawthorn Fruit Ethanolic Extract

Control

Hawthorn

Serum total cholesterol (mmol/L)

4.6 F 0.5

4.1 F 0.5a

Serum HDL cholesterol (mmol/L)

2.3 F 0.3

2.4 F 0.3

Serum triglycerides (mmol/L)

3.3 F 0.7

2.9 F 0.4a

Fecal total neutral sterols (mg/g)

8.6 F 1.4

11.8 F 2.0a

Fecal total acidic sterols (mg/g)

3.3 F 0.9

4.8 F 1.0a

HMG-CoA reductase (pm/min/mg protein)

6.6 F 2.50

6.4 F 2.5

CH (pm/min/mg protein)

53.0 F 29.2

148.9 F 57.2a

ACAT (nm/min/mg protein)

1.0 F 0.3

0.8 F 0.2a

a Means at a row differ significantly, p < 0.05.

a Means at a row differ significantly, p < 0.05.

CoA) reductase activity in hamsters and rabbits (11,12), suggesting that the cholesterol-lowering effect of hawthorn fruit is not mediated by a down-regulation of HMG-CoA reductase.

The inhibition of cholesterol absorption in the intestine could also be responsible for the hypocholesterolemic activity of hawthorn fruits. As shown in Tables 3 and 4, supplementation of hawthorn fruit in the form of either crude water-soluble extract powder or ethanolic extract significantly increased cholesterol excretion in the rabbit and hamster. The effect of hawthorn fruit supplementation on intestinal acyl CoA:cholesterol acyltrans-ferase (ACAT) activity was studied because intestinal ACAT may play a key role in the absorption of cholesterol by esterification of cholesterol prior to absorption (14). The results in hamsters demonstrated that supplementation of hawthorn fruit ethanolic extract was associated with a lower intestinal ACAT activity (12), suggesting that inhibition of cholesterol absorption of dietary cholesterol is at least partly mediated by downregulation of intestinal ACAT activity.

Bile acids are the major metabolites of cholesterol. Greater excretion of bile acids could also lead to a lower level of serum cholesterol. We found that the fecal excretion of both primary (cholic and chenodeoxycholic) and secondary (lithocholic and deoxycholic) bile acids was greater in hamsters and rabbits (11,12) fed diets supplemented with hawthorn fruit (Tables 3 and 4). The liver cholesterol 7a-hydroxylase (CH) is a regulatory enzyme in the metabolic pathway from cholesterol to bile acids. Hawthorn fruit supplementation in the diet significantly increased the liver CH activity compared with the control group (Table 4), suggesting that the increased excretion of bile acids is partly mediated by upregulation of this enzyme.

Blood total and LDL-cholesterol level is maintained in a steady balance in which the rate of entry of cholesterol into the blood is equal to the removal of cholesterol from the blood. A reduced serum cholesterol level indicates a shift in this steady state, resulting from either a decrease in the rate of entry or an increase in the rate of removal by peripheral tissues. The rate by which LDL cholesterol is taken up by peripheral tissues is mediated by LDL receptors. Upregulation of LDL receptors is probably an alternative mechanism responsible for the hypocholesterolemic activity of hawthorn fruits. We have investigated the effect of hawthorn extract on LDL receptor level in HepG2 cells and found that hawthorn fruit extract could prevent the down-regulation of LDL receptors by LDL in a dose-dependent manner (Fig. 1) (15). A similar effect was observed in a study by Rajendran et al. (13), who showed that supplementation of 0.5 mL ethanolic extract per 100 g body weight per day for 6 weeks was associated with a 25% increase in hepatic LDL-receptor activity, resulting in greater influx of plasma cholesterol into the liver. It is concluded that hawthorn fruit lowers serum cholesterol by a

Cone, of hawthorn extract (mg/mL)

Figure 1 Inhibition of LDL-receptor downregulation by hawthorn water-soluble extract. HepG2 cells were incubated in the presence and absence of hawthorn with and without 500 Ag LDL/mL. In the absence of hawthorn, LDL receptor was downregulated by LDL to maximum level. The presence of 0.5 and 1.0 mg/mL of hawthorn extract prevented this downregulation in a proportional manner.

Cone, of hawthorn extract (mg/mL)

Figure 1 Inhibition of LDL-receptor downregulation by hawthorn water-soluble extract. HepG2 cells were incubated in the presence and absence of hawthorn with and without 500 Ag LDL/mL. In the absence of hawthorn, LDL receptor was downregulated by LDL to maximum level. The presence of 0.5 and 1.0 mg/mL of hawthorn extract prevented this downregulation in a proportional manner.

combination of mechanisms involving increasing LDL receptor activity and reducing cholesterol absorption and bile acid reabsorption.

III. ANTIOXIDANT ACTIVITY

Pharmacological studies of hawthorn fruits focus on its cardiovascular protective, hypotensive, and cholesterol-lowerig activity (1,4-7,9). However, mechanisms of these beneficial effects are still being investigated. Dietary antioxidants may reduce the initiation and propagation of free radicals in vivo, and therefore minimize the free-radical-induced damage to the heart tissue and cardiovascular vessels. In recent years, it has been generally accepted that oxidation of human LDL is one of the risk factors in the development of cardiovascular disease (16-19). In vitro and in vivo experiments support the view that hawthorn fruit has strong antioxidant activity (20-22).

Hawthorn fruit is a rich source of phenolic antioxidants (22). To quantify these phenolic antioxidants present in hawthorn fruits, a HPLC

method was developed in our laboratory. As shown in Figure 2, at least eight flavonoids were identified in hawthorn fruit. The structures of these compounds are shown in Figure 3. The HPLC analysis found that epicatechin was most abundant (1.78 g/kg dry fruit) followed by chlorogenic acid (0.65 g/kg), hyperoside (0.25 g/kg), isoquercitrin (0.13 g/kg), protocatechuic acid (0.03 g/ kg), rutin (0.03 g/kg), and quercetin (0.01 g/kg). The eight flavonoids purified from hawthorn fruit demonstrated varying antioxidant activity (Fig. 4). When incubated with LDL, ursolic acid showed no antioxidant activity while

Figure 2 High-performance liquid chromatographic profile of hawthorn fruit phenolics. See Ref. 22 for the conditions.
Figure 3 Chemical structures of chlorogenic acid, epicatechin, hyperoside, isoquercitrin, protocatechuic acid, quercetin, rutin, and usolic acid.

hyperoside was most protective to human LDL followed by quercetin and isoquercitrin (Fig. 4). Under the same experimental conditions, the antioxidant activity of epicatechin, chlorogenic acid, and rutin was similar but it was weaker than that of hyperoside, quercetin, and isoquercitrin (Fig. 4).

a-Tocopherol is the major antioxidant in human LDL. The flavonoids purified from hawthorn fruit were also effective in protecting a-tocopherol from free-radical-induced degradation in human LDL (22). Supplementation of hawthorn fruit in the diet (2%) significantly increased serum a-tocopherol in rats (Fig. 5). At the end of 3 weeks, serum a-tocopherol in the hawthorn-fruit-supplemented group was increased by 18% as compared with that of the control rats. At the end of 6 weeks, serum a-tocopherol in the hawthorn-fruit-supplemented group was increased by 20% as compared with that of the control rats (Fig. 5). Epidemiological studies showed that flavonoid consumption was negatively associated with coronary heart disease mortality (23). If the consumption of hawthorn fruit is associated with a significantly

Figure 4 Effect of hawthorn fruit phenolics on production of thiobarbituric acid-reactive substances (TBARS) in Cu2 + -mediated oxidation of human LDL.

Figure 5 Effect of hawthorn fruit powder supplementation (2%) in diet on serum a-tocopherol in rats. Means at a given time point differ significantly. < 0.05; **p < 0.01. See Ref. 22 for the experimental conditions.

*p lower risk of cardiovascular disease in humans, part of the mechanism may also involve the protective role of these antioxidants to a-tocopherol and human LDL from oxidation.

To correlate the pharmacological action of the hawthorn flavonoids with their apparent health benefits, we also studied the absorption kinetics and excretion of four major hawthorn flavonoids, viz., epicatechin, chloro-genic acid, hyperoside, and isoquercitrin, after oral administration to rats. As chlorogenic acid and hyperoside could not be detected in the plasma, urine, or feces after oral administration, their pharmacokinetics could not be assessed. For isoquercitrin, the systemic absorption rate was very rapid and maximum level was observed in the blood after 10 min. In contrast, epicatechin was absorbed much slower reaching a Tmax at 66 min. The absolute bioavailability of the two compounds was 61% and 34%, respectively. Based on this limited study, different flavonoids from hawthorn may have very different oral absorption and clearance characteristics. More detailed studies are needed to delineate the pharmacological benefits of these compounds as some of them might have limited bioavailability. Isoquercitrin and hyperoside are structurally very similar except one is a glucoside and the other is a galactoside. Yet, one of them is absorbed into the bloodstream quickly while the other is not. Hence, it is likely that some flavonoids may be preferentially uptaken in the gastrointestinal tract and this information would be essential to determine the health benefits of dietary supplements even though they may contain high amounts of flavonoids.

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Dieting Dilemma and Skinny Solutions

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