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Department of Biochemistry,
*
Department of Physiology,
Laboratory Animal Service Centre, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, The People’s Republic of China
2To whom correspondence should be addressed. E-mail: zhenyuchen{at}cuhk.edu.hk.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-hydroxylase (CH) but it suppressed the activity of intestinal acyl CoA:cholesterol acyltransferase (ACAT, P < 0.05). The results suggest that the mechanism by which hawthorn fruit decreases serum cholesterol involves, at least in part, the inhibition of cholesterol absorption mediated by down-regulation of intestinal ACAT activity.
KEY WORDS: • cholesterol • Crataegus pinnatifida • hawthorn fruit • rabbits • triglycerides
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Hawthorn extract has been used to treat the early stages of congestive heart failure (2
,3
) and angina pectoris (4
). It was also clinically effective in reducing blood pressure (5
). In addition, hawthorn fruit significantly inhibited thromboxane A2 biosynthesis and platelet adhesion, thus reducing the formation of atheroma and thromobosis (6
).
Hawthorn fruit is also an excellent source of antioxidants. We previously studied the antioxidant activity of hawthorn fruit and identified seven antioxidants, namely, hyperoside, isoquercitrin, epicatechin, chlorogenic acid, quercetin, rutin and protocatechuic acid (7
). All of these compounds protected human LDL from Cu+2-mediated oxidation. They also prevented the peroxy free radical–induced oxidation of -tocopherol in human LDL. In an in vivo study, supplementation of 2% hawthorn fruit powder significantly elevated serum -tocopherol by 18–20% in rats fed a 30% polyunsaturated canola oil diet compared with the control (7
).
Hawthorn fruit is beneficial to the cardiovascular system, partially due to its effect on serum cholesterol. Previous reports showed that hawthorn decreased serum total cholesterol (TC),3
LDL cholesterol (LDL-C) and triglyceride (TG) in hyperlipidemic humans (5
,8
). The decrease in LDL-C has been attributed to an increase in LDL-receptor activity of hepatic membrane (9
,10
). The present study was carried out to examine further various possible mechanisms by which hawthorn fruit decreases serum cholesterol using New Zealand white rabbits as an animal model. First, the effect of hawthorn fruit supplementation on aortic accumulation of cholesterol was quantified. Second, we sought to determine whether supplementation of hawthorn fruit would lead to any changes in 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMG-CoA-R), cholesterol 7-hydroxylase (CH) and intestinal acyl CoA:cholesterol acyltransferase (ACAT). HMG-CoA-R is a key enzyme in cholesterol biosynthesis, whereas CH mainly regulates the conversion of cholesterol to bile acids. ACAT is believed to play an important role in intestinal cholesteryl esterification before cholesterol is absorbed and assembled in the chylomicrons. The effect of hawthorn fruit supplementation on fecal excretion of acidic and neutral sterols was also examined.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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New Zealand white male rabbits (Oryctolagus cuniculus, n = 24, Laboratory Animal Service Center, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, China) were divided into three groups and housed (one per cage) in an animal room at 25°C with a 12-h light:dark cycle. The first group (2 males and 2 females) was fed a reference diet that contained no added cholesterol (NC). The NC diet was specially prepared by Glen Forrest Stockfeeds (Western Australia, Australia) by mixing the following ingredients (g/100 g): wheat, 10.4; oats, 12, lupin, 12.5; lucerne, 20; millmix, 40; meat meal, 3.8; calcium carbonate, 1.0; vitamin and mineral mix, 0.2; and sodium chloride, 0.14. The NC diet per 100 g contained 63.1 g carbohydrate, 17.0 g protein, 4.0 g fat, 6.9 g ash and 9.0 g moisture. The second group (5 males and 5 females) was fed a high cholesterol diet (HC) that was prepared by adding 1.0 g cholesterol/100 g NC diet. The third group (5 males and 5 females) was fed the HC diet supplemented with 2.0 g/100 g hawthorn fruit powder (HC-H). Dry hawthorn fruit (Crataegus pinnatifida) was purchased from a local market. After removal of the seeds, the dry fruit flesh was ground into powder in a coffee grinder. The rabbits consumed food and tap water ad libitum. Food intake was measured and body weight was recorded weekly. The total fecal output of each rabbit was combined during all of wk 8 and 12.
Blood was collected at 0, 4, 8 and 12 wk after overnight food deprivation via the lateral ear vein. After clotting, the blood was centrifuged at 1500 x g for 10 min and the serum was then collected. At the end of wk 12, all of the rabbits were killed after overnight food deprivation under carbon dioxide anesthesia. The organs, including liver, heart, brain, kidney and adrenal, 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. The protocol was reviewed and approved by the Committee of Animal Ethics, The Chinese University of Hong Kong.
Determination of blood cholesterol and TG.
Several enzymatic kits were purchased from Sigma (St. Louis, MO) to measure serum TG (#336–20), TC (#352–20) and HDL cholesterol (HDL-C; #352–4).
Measurement of cholesterol, cholesteryl esters (CE), phospholipids (PL) and triacylglycerols (TG).
In each case, the fresh aorta was cleaned of adventitial tissue and washed in saline solution until clean. Total lipids were extracted from the aorta or other tissues with addition of stigmastanol (Sigma) as an internal standard using chloroform/methanol (2:1, v/v). The lipid extracts were then saponified with 6 mL of 1 mol/L NaOH in 90% ethanol at 90°C for 1 h, and the nonsaponified substances including cholesterol were converted to their trimethylsilyl (TMS)-ether derivatives by a commercial TMS reagent (Sigma). Analysis of the cholesterol TMS-ether derivatives was performed in a fused silica capillary column (SAC-5, 30 m x 0.25 mm, i.d.; Supelco, Bellefonte, PA) using a Shimadzu GC-14 B gas-liquid chromatograph (GLC) equipped with a flame ionization detector (Kyoto, Japan) as previously described (11
).
Total CE, TG, PL and free fatty acids (FFA) were measured as previously described (12
). Lipid classes in an aliquot of aortic extract were separated by neutral lipid TLC (20 x 20 cm plate precoated with 250 µm silica gel 60A, Macherey-Nagel, Durenm, Germany) using a developing solvent system of hexane/diethyl ether/acetic acid (80:20:1, v/v/v). CE and free cholesterol bands were recovered from the TLC plate, saponified, converted to TMS-derivative and analyzed as described above. PL, TG and FFA bands were also recovered and converted to fatty acid methyl esters, which were then analyzed in a Hewlett-Packard 5890 Series II gas chromatograph equipped with a SP-2560 flexible fused silica capillary column (100 x 0.25 mm, i.d., 20-µm film thickness; Supelco, Bellefonte, PA). The aortic TG and FFA were quantified according to the amounts of triheptadecanoin and heptadecanoic acid (internal standards) added during the extraction.
Determination of fecal neutral and acidic sterols.
Individual fecal neutral and acidic sterols were quantified as previously described (11
). In brief, stigmasterol (0.3 mg) as an internal standard for neutral sterols was added to a fecal sample (300 mg). The sample was then saponified using 9 mL of 1 mol/L NaOH in 90% ethanol containing 0.3 mg hyodeoxycholic acid as an internal standard for acidic sterols (Sigma). The total neutral sterols were extracted using 8 mL of cyclohexane and were then converted to their corresponding TMS-ether derivatives for GLC analysis.
After the cyclohexane extraction, 1 mL of 10 mol/L NaOH was added to the remaining aqueous layer and heated at 120°C for 3 h. After a cooling down period, 1 mL of distilled water and 3 mL of 3 mol/L HCl were added followed by extraction with 7 mL of diethyl ether twice. The diethyl ether layers were then pooled, followed by the addition of 2 mL methanol, 2 mL dimethoxypropane and 40 µL concentrated HCl (12 mol/L). After standing overnight at room temperature, the solvents were dried down and the acidic sterols were similarly converted to their TMS-ether derivatives at 60°C for GLC analysis.
Assays of hepatic HMG-CoA-R, CH and intestinal ACAT.
The liver microsomes were isolated according to Erickson et al. (13
). The activity of HMG-CoA-R (EC 1.1.1.34) was measured as previously described by Edwards et al. (14
). The activity of hepatic CH (EC 1.14.13.17) was measured according to Souidi et al. (15
). The activity of intestinal ACAT (EC 2.3.1.26) was measured according to the method described by Stange et al. (16
) and modified by Chautan et al. (17
).
Statistics.
Data are expressed as means ± SD. ANOVA with unequal numbers where applicable followed by student’s t test (two-tailed) were used for statistical evaluation of significant differences in means among the NC (n = 4), HC (n = 10) and HC-H (n = 10) groups using SigmaStat (Jandel Scientific Software, San Rafael, CA). ANOVA was also used to compare the difference in the fecal neutral and acidic sterols between wk 8 and 12. Differences were considered significant when P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The final body weight of the NC group (4.1 ± 0.2 kg) was significantly greater than that of the HC (3.6 ± 0.2) and HC-H (3.8 ± 0.3) rabbits (P < 0.05). The HC and HC-H groups did not differ in body weight. Similarly, the NC group (127 ± 3 g/d) ate significantly more than the HC (88 ± 10) and HC-H (94 ± 7) rabbits. Three rabbits in the HC group died, presumably due to atherosclerosis (autopsy was not performed), whereas the rabbits in the other two groups appeared to be healthy.
Serum lipids.
The concentrations of serum TC and TG for the NC group were unchanged during the 12-wk feeding period. Serum TC and TG levels in the HC and HC-H groups were greater than those in the NC rabbits (Fig. 1
). Serum TC level in the HC group was 27.8% higher at wk 8 and 23.4% higher at wk12 than that in the HC-H rabbits (P < 0.05). Similarly, the serum TG level in the HC group was significantly higher by 27.6% at wk 8 (P < 0.05) and 22.2% at wk 12 than that in the HC-H rabbits. In contrast, serum HDL-C level in HC rabbits was significantly lower than that in NC and HC-H groups at wk 8 and 12 (P < 0.05). Serum HDL-C level did not differ in NC and HC-H rabbits (Fig. 1)
.
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The NC rabbits accumulated only 1.5 ± 0.2 µmol cholesterol/g aortic tissue, whereas the HC group had 28.3 ± 14.5 µmol cholesterol/g aorta (Fig. 2
). When hawthorn fruit was supplemented in the diet, the accumulation of cholesterol in HC-H rabbits was reduced to 13.9 ± 8.0 µmol/g aorta (P < 0.05 vs. the HC group). Most of the cholesterol accumulated was CE (Fig. 2)
. Compared with the NC group, both HC and HC-H had a significantly greater level of CE. However, the HC-H rabbits accumulated less CE than the HC group. No significant differences in aortic TG level were observed among the three groups although the TG in the HC and HC-H groups was slightly higher than the NC group (P < 0.07, Fig. 2
). Interestingly, the HC-H rabbits had a higher level of FFA in aorta than the NC and HC groups (P < 0.05, Fig. 2
).
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The liver accumulated <3.0 ± 0.3 µmol cholesterol/g in the NC rabbits, whereas it reached 95.2 ± 28.1 µmol cholesterol/g liver in the HC group (Fig. 3
). When hawthorn fruit was supplemented in the diet, the accumulation of cholesterol in the liver was reduced to 58.0 ± 14.4 µmol/g (P < 0.05 vs. the HC group, Fig. 3
). No differences in hepatic TG, PL or FFA were observed among the three groups (Fig. 3)
.
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). No differences in brain cholesterol were detected among the three groups. Both the HC and HC-H groups had cholesterol greater than the NC rabbits in heart (P < 0.05, Fig. 4
). Supplementation of hawthorn fruit lowered heart cholesterol in rabbits fed high cholesterol diets (P < 0.05). Similar observations were made in the renal cholesterol level among the three groups (P < 0.05, Fig. 4
); hawthorn-supplemented rabbits had renal cholesterol 21% lower than the HC group (P < 0.05). HC and HC-H rabbits did not differ but had greater cholesterol levels in muscle than the NC rabbits (P < 0.05; Fig. 4
).
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The total fecal samples were collected only during wk 8 and 12 from the three groups. Some bacteria in the colon can degrade and convert cholesterol into other sterols and metabolites (18
,19
). We did not quantify the amount of cholesterol degraded or converted during the passage through the large intestine but quantified total fecal sterols present in the fecal samples. The excretions of fecal sterols were compared among the three groups at a given week. As shown in Table 1
, total fecal neutral and acidic sterols in the NC rabbits were significantly lower than the HC and HC-H rabbits in both wk 8 and 12 (P < 0.01). The HC-H group had higher fecal excretions of neutral sterols including cholesterol, coprostanol, coprostanone and dihydrocholesterol per se during wk 8 and 12 compared with the HC group (P < 0.05, Table 1
). The output of total fecal bile acids in the HC-H group was greater than in the HC rabbits at both wk 8 and 12 (Table 1)
. Lithocholic and deoxycholic acids were significantly higher in the feces of HC-H rabbits than in those of the HC group. Excretions of fecal sterols also were evaluated over time for a given group. At wk 12, both the HC and HC-H groups had greater excretions of total neutral and acidic sterols than at wk 8 (Table 1)
.
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There were no significant differences in the activity of liver HMG-CoA-R among the NC [58.1 ± 18.5 nmol/(g protein · min)], the HC (39.1 ± 15.6) and HC-H (42.9 ± 15.6) rabbits. Similarly, no significant differences in the activity of liver CH were observed among the NC [25.8 ± 4.1 nmol/(g protein · min)], the HC (18.6 ± 4.1) and HC-H (21.9 ± 4.4) rabbits. However, the activity of intestinal ACAT in the HC group [3.4 ± 0.8 µmol/(g protein · min)] was greater than that of the NC group [1.5 ± 1.4 µmol/(g protein · min), P < 0.05]. Supplementation of hawthorn fruit powder significantly suppressed the activity of intestinal ACAT [1.5 ± 1.7 µmol/(g protein · min)], which was significantly lower than that of the HC group and not different from the NC group.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The present study demonstrated clearly that hawthorn fruit possessed hypolipidemic activity. Supplementation of hawthorn fruit lowered not only serum TC but also serum TG in New Zealand white rabbits fed a diet containing 1 g/100 g cholesterol (Fig. 1)
. However, hawthorn supplementation did not affect serum HDL-C, consistent with several previously published reports. Chen et al. (8
) demonstrated that TC and TG levels were decreased by 15 and 10%, respectively, but HDL-C was unchanged in 30 hyperlipidemic subjects who consumed hawthorn drinks for 1 mo. In the same report, replacing tap water with hawthorn drinks reduced both serum TC and TG in rats. However, the HDL-C level was greater in the hawthorn group than in those drinking tap water. Ho et al. (10
) showed that oral administration of hawthorn extract in experimental rats significantly suppressed the increase in the level of serum cholesterol that occurred in control rats fed the same high cholesterol diet. Together with these previous reports in humans and rats, this study supports the claim that hawthorn fruit may serve as a health supplement to treat hypolipidemia, especially for those patients whose cholesterol is marginally high and does not warrant the prescription of cholesterol-lowering drugs.
The underlying mechanisms for hypocholesterolemic activity of hawthorn fruit are poorly understood. One of these may be related to its "up-regulation" effect of LDL receptors on cell surfaces (9
,10
). The serum 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 lowered serum cholesterol 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. Ho et al. (10
) investigated the effect of hawthorn extract on LDL receptor level in HepG2 cells and found that hawthorn fruit extract up-regulated LDL receptors. A similar effect was found in a study by Rajendran et al. (9
), who demonstrated that there was a significant increase in the binding of LDL to liver plasma membranes in vitro when hawthorn ethanol extract was administered to rats fed an atherogenic diet. A decrease in cholesterol biosynthesis would also lead to a lower blood cholesterol level. However, there is no study that has investigated cholesterol biosynthesis in relation to the consumption of hawthorn fruit. The present study is the first examination of the effect of consumption on the HMG-CoA-R activity; consumption of hawthorn fruit had no action on this enzyme, at least in rabbits, suggesting that inhibition of cholesterol synthesis is unlikely to be a part of the hypocholesterolemic mechanism of hawthorn fruit.
Another possible mechanism for the hypocholesterolemic activity of hawthorn fruit may be either its inhibition of cholesterol and bile acid absorption or increased excretion of these neutral and acidic sterols. In fact, the greater excretions of both fecal neutral and acidic sterols in the HC-H group compared with the HC group was consistent at both wk 8 and 12. Thus, the reduced absorption of dietary cholesterol and increased excretion of bile acids was directly associated with a lower blood cholesterol. Intestinal ACAT may play a key role in the absorption of cholesterol by esterification of cholesterol before absorption (21
). To determine whether hawthorn fruit reduced the absorption of cholesterol, ACAT activity in the intestine of HC-H rabbits was compared with that of the HC group. We found that hawthorn supplementation suppressed intestinal ACAT activity, suggesting that hawthorn fruit supplementation decreased the absorption of dietary cholesterol due mainly to its down-regulation of intestinal ACAT activity.
Hawthorn fruit is not only hypolipidemic but also an excellent source of antioxidants. The protective activity of hawthorn fruit on the cardiovascular system may also be attributed to these antioxidants because they reduce the production of free radicals, alleviate subsequent damage to the heart tissue and decrease deposition of oxidized LDL-C (7
). The oxidative modification of LDL may play a pathogenic role in the development of atherosclerosis (22
–24
). We previously identified seven antioxidants in hawthorn and found that they were very potent in protecting human LDL from oxidation (7
). The hawthorn antioxidants are mainly proanthocyanidin and flavonoids. Bahorum et al. (25
) examined the antioxidant activity of hawthorn extract and found that it could scavenge hydrogen perioxide and superoxide species. The present study did not measure total antioxidant capacity in serum because the diets contained 56 mg -tocopherol/kg diet. However, our previous study demonstrated that supplementation of 2% hawthorn fruit extract in rats could increase serum -tocopherol by 20% compared with the control rats (7
), suggesting that hawthorn fruit antioxidants had sparing effects on serum -tocophcerol. In addition, hawthorn fruit is rich in fructose. It will be interesting to study further the effect of hawthorn fruit supplementation on serum glucose.
It has been shown that consumption of flavonoid antioxidants is inversely correlated with risk of coronary heart disease (26
). It is not known whether the flavonoids are active components in reducing blood cholesterol. The supplementation with 2 g hawthorn fruit powder/100 g diet represented an amount of total flavonoids similar to that consumed by humans. Hertog et al. (26
) showed that the flavonoid intake in humans could reach 40 mg/d, i.e., 4 mg flavonoids/MJ provided that a total of 10 MJ is ingested per person (40 mg/10 MJ = 4). In the present study, the rabbits consumed 
90 g food/d, which is equivalent to 1.34 MJ/d. The total flavonoid intake would be 5.0 mg/d based on the previous study (7
) showing the total flavonoid concentration in hawthorn fruit to be 2.8 mg/g dry fruit (2% x 90 g diet x 2.8 mg/g = 5.0). Thus, 2 g/100 g hawthorn fruit supplementation would achieve a flavonoid intake of 3.7 mg/kJ (5.0 mg ÷ 1.34 MJ = 3.7), close to the level consumed by humans. We have developed a hawthorn fruit beverage and tested its potency in reducing cholesterol level in hypercholesterolemic humans. The preliminary data supported the claim that hawthorn fruit is hypolipidemic (unpublished data). We are currently studying the chemical composition of hawthorn fruit to identify the active components that are responsible for this hypocholesterolemic activity.
The present results in rabbits, although not directly applicable to humans, suggest that inclusion of hawthorn fruit in diets may be an effective way of lowering serum cholesterol level and the accumulation of cholesteryl esters in artery. The present study was the first to explore the mechanism by which hawthorn fruit alters the serum lipoprotein cholesterol profile. The results demonstrated that hawthorn fruit decreased intestinal ACAT activity and thereby increased both neutral and acidic sterol excretion.
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FOOTNOTES |
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3 Abbreviations used: ACAT, acyl CoA:cholesterol acyltransferase; CE, cholesteryl esters; CH, cholesterol 7-hydroxylase; FFA, free fatty acids; GLC, gas-liquid chromatograph; HC, high cholesterol diet; HC-H, high cholesterol diet with hawthorn fruit powder; HDL-C, HDL cholesterol; HMG-CoA-R, 3-hydroxy-3-methyl glutaryl coenzyme A reductase; LDL-C, LDL cholesterol; NC, diet with no added cholesterol; PL, phospholipids; TC, total cholesterol; TG, triacylglycerols; TMS, trimethylsilyl.
Manuscript received 25 April 2001. Initial review completed 25 June 2001. Revision accepted 21 September 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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