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Department of Food Science, University of Sonora, Hermosillo, Sonora State, Mexico and * Department Nutritional Sciences, University of Connecticut, Storrs, CT 06269
2To whom correspondence should be addressed. E-mail: maria-luz.fernandez{at}uconn.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-hydroxylase, the regulatory enzyme of cholesterol catabolism to bile acids was 33% higher in the PO groups (P < 0.02). Fecal bile acids were 3 times higher in the PO groups than in the control group. These results suggest that PO exerts its hypolipidemic effect by affecting bile acid absorption and altering hepatic cholesterol metabolism.
KEY WORDS: • Plantago ovata • bile acids • plasma triglycerides • plasma cholesterol • guinea pigs
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Elevated levels of plasma cholesterol and triglycerides (TG) have been implicated as a causative factor in the development of CHD (2
). Dietary interventions that lower plasma cholesterol concentrations, mainly atherogenic LDL, are highly recommended. High intake of dietary fiber has been associated with reductions in plasma LDL cholesterol (LDL-C) concentrations (3
) and a decreased risk for CHD (4
).
Reports demonstrating the beneficial effects of fiber on plasma lipids have addressed the utilization of conventional and unconventional sources of fiber. Some conventional sources are pectin (5
), seeds from Plantago ovata (psyllium) (6
–8
), ß-glucan from oat bran (9
), guar gum (10
) or rye bread (11
). Among the unconventional fiber sources, we can include pectin derived from prickly pear (12
), ß-glucan from yeast (13
) or fiber from the husks of lime-treated corn (14
).
Psyllium’s effect in lowering plasma LDL-C has been confirmed in recent studies (6
,15
, 16
), which demonstrated that psyllium was an effective adjunct therapy for hypercholesterolemic men and women by reducing LDL-C by 
7% (15
,16
). In addition, the safety and efficacy of psyllium for individuals with type 2 diabetes was also established by showing no adverse effects in the participating subjects and a 13% lowering of LDL-C (15
). Animal studies have also shown significant reductions in plasma LDL-C by psyllium intake (17
, 18
). Although a substantial number of studies on the effects of psyllium husks in reducing plasma LDL-C have been reported (6
,15
–18
), to our knowledge there are no reported studies investigating the seeds of this plant.
Northern Mexico has adequate climatologic and soil conditions for increasing the production of Plantago ovata, and the seeds from this plant have the potential to be utilized for human consumption. At present, the seeds are used mainly as feed for farm animals. The composition of the seeds is unusual because they contain high concentrations of total carbohydrates (75 g/100 g), which include a high percentage of gums (40 g/100 g), other sources of soluble fiber and sugars. The potential for increasing the production of these seeds in Northern Mexico, their unusual composition and the established hypocholestesterolemic action of the husks prompted us to evaluate their effects on plasma lipid metabolism.
The purpose of the present study was to test whether the seeds of Plantago ovata, similar to what has been observed in the husks from this plant, would lower plasma LDL-C concentrations. A second objective was to evaluate the potential mechanisms of hypocholesterolemia associated with the intake of Plantago ovata seeds. On the basis of similarities in lipoprotein cholesterol distribution (19
) and in their response to psyllium (20
) and other sources of fiber including gums (10
), guinea pigs were chosen as the experimental animal model.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Cholesterol oxidase, cholesterol esterase, peroxidase, cholesterol and TG kits were purchased from Boehringer Mannheim (Indianapolis, IN). Free cholesterol and phospholipid kits were obtained from Wako (Osaka, Japan). Glucose-6-phosphate, glucose-6-phosphate dehydrogenase, NADP, phosphatidlycholine and the bile acid kit were obtained from Sigma Chemical (St. Louis, MO). 7-Hydroxycholesterol and 7ß-hydroxycholesterol were purchased from Steraloids (Newport, RI). 14C cholesterol was obtained from Perkin Elmer (Boston, MA). [1-14C] cholesterol (1.8 GBq/mmol) and DL-3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) were from Amersham (Clearbrook, IL).). Aluminum and glass silica gel plates were purchased from EM Science (Gibbstown, NJ). Plantago ovata (PO) seeds were provided by Mr. Molina (Cd. Obregon, Mexico).
Diets.
Diets were prepared and pelleted by Research Diets (New Brunswick, NJ). All diets were equal in composition except for the amount of PO seeds. The chemical composition of PO is as follows (g/100 g): protein, 15; fat, 5; minerals, 3; fiber, 55 (including gums and other components); and sugars, 20. Fat was evaluated for fatty acid composition by gas chromatography and the major fatty acids were as follows (g/100 g): linoleic, 39; oleic, 35; palmitic, 12; stearic, 3; linolenic, 0.8; and arachidonic, 0.5. The concentration of PO used for diet formulations was 0, 7.5 or 10 g/100 g (Table 1)
. The control diet contained 10 g/100 g cellulose and 2.5 g/100 g guar gum for a final ratio of insoluble:soluble fiber of 3:1. The amount of fiber in the PO diets was adjusted to 12.5 g/100 g with cellulose. Dietary cholesterol was maintained at 0.17 g/100 g to raise plasma cholesterol concentrations to more readily detect PO effects on plasma lipids. This amount of dietary cholesterol corresponds to an absorbed amount equal to the daily cholesterol synthesis rates (21
) in guinea pigs and is equivalent to 1200 mg/d for a human diet. The fat mix was rich in lauric and myristic acids, which cause endogenous hypercholesterolemia in guinea pigs (22
). The composition of the fat mix was as follows (g/100 g): lauric acid, 23.8; myristic, 7.8; palmitic, 9.2; stearic, 8.6; oleic, 19.9; linoleic, 26.4; and other, 4.
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Male Hartley guinea pigs weighing between 250 and 300 g were purchased from Harlan SpragueDawley (Indianapolis, IN). They were randomly allocated to one of the three treatments, 10 guinea pigs per group, for a total of 4 wk, an amount of time that results in a steady-state plasma cholesterol level. Two guinea pigs were housed per cage, in a light cycle room (light from 700 to 1900h) at 22°C. Food and water were consumed ad libitum. To measure fecal bile acids and food consumption, guinea pigs from each group were housed individually for 48 h. During this time, feces were collected and diets were weighed daily to determine the amount of food consumed. All animal experiments were conducted in accordance with U.S. Public Health Service/U.S. Department of Agriculture guidelines. Experimental protocols were approved by the University of Connecticut Institutional Animal Care and Use Committee.
Lipoprotein isolation.
Nonfood-deprived guinea pigs were anesthetized under halothane vapors and blood was obtained via heart puncture. Plasma samples were collected and preservation cocktail was added to the samples (aprotonin, 0.5 mL/100 mL, phenylmethylsulfonyl fluoride, 1 mL/L and sodium azide, 1 mL/L). Plasma from each guinea pig (1 mL) was stored at 4°C for analysis of plasma lipids; the remainder was used for lipoprotein isolation.
Lipoprotein isolation was done by sequential ultracentrifugation in an L8-M ultracentrifuge (Beckman Instruments, Fullerton, CA). VLDL were isolated in a density range of 1.006 to 1.019 kg/L at 125,000 x g at 15° for 19 h in a Ti50 rotor. LDL were isolated in a density range of 1.019 to 1.09 kg/L at 150,000 x g for 3 h using a Vti 65.5 rotor (23
). LDL samples were dialyzed in 0.09% NaCl, 0.01% EDTA, pH 7.2 for 24 h and stored at 4°C for composition analysis.
Plasma and hepatic lipids.
Plasma total cholesterol, TG and HDL-C were determined using enzymatic analysis (Boheringer Mannheim kits) (24
). Plasma TG were determined by blanking free glycerol (25
) HDL-C was analyzed after precipitation of apolipoprotein (apo) B–containing lipoproteins with dextran sulfate (26
) with a modification (23
).
Livers were excised from guinea pigs after exsanguination and were stored at -20°C for lipid analysis. Liver lipids were extracted from 1 g of liver sliced into small pieces and combined with 10 mL of chloroform/methanol (2:1) (27
) overnight. The mixture was then filtered by gravity filtration and the filtrate mixed with acidified water and separated into two phases with a separatory funnel. An aliquot of 0.2 mL, taken from the lower phase, was evaporated completely and homogenized in 0.2 mL of ethanol for enzymatic determination of total and unesterified cholesterol (24
)
Lecithin cholesterol acyltransferase (LCAT) and cholesterol ester transfer protein (CETP) determinations.
LCAT and CETP activities were determined according to Ogawa and Fielding (28
). CETP activity was calculated by measuring the mass transfer of cholesterol ester between HDL and apo B–containing lipoproteins. Physiologic CETP activity was determined without inhibition of LCAT. Samples were incubated at 37°C for 6 h in a shaking water bath; total and plasma free total cholesterol and HDL-C were measured. LCAT activity was determined by mass analysis of the decrease in plasma free cholesterol between 0 and 6 h at 37°C. Assays were carried out concurrently with measurements of CETP. Both of these methods have been well standardized for guinea pig plasma (29
).
Hepatic microsome isolation.
Microsomes were isolated as previously described (23
). Briefly, livers obtained from guinea pigs fed the different diets were pressed through a tissue grinder, placed in a cold buffer (50 mmol/L KH2PO4, 0.1 mol/L sucrose, 50 mmol/L KCL, 50 mmol/L NaCl, 30 mmol/L EDTA and 2 µmol/L dithiothreitol, pH 7.2), and homogenized with a Potter-Elvehjem homogenizer. The microsomal fraction was obtained after two centrifugations at 10,000 x g for 15 min (JA-20 rotor in a J2–21 centrifuge, Beckman Instruments), and 1 h centrifugation at 100,000 x g at 4°C. Samples were further homogenized and centrifuged for one additional hour at 100,000 x g at 4°C. Microsomal pellets were resuspended in buffer, homogenized and stored at -70°C. Protein content in microsomes was measured according to Markwell (30
).
Hepatic microsomal lipids.
Free cholesterol was measured in hepatic microsomes isolated from guinea pigs fed the different diets; microsomes (100 µL) were treated with 20 volumes of chloroform/methanol (2:1) overnight. After filtration the next day, 1 mL of acidified water was added and phases were separated by a separatory funnel. The lower phase was collected and the volume was adjusted to 2 mL with chloroform/methanol (2:1). Samples were dried under nitrogen and lipids were solubilized with 1 mL of water with Triton 100X (1 g/100 g). Free cholesterol was determined by enzymatic methods.
HMG-CoA reductase assay.
The activity of HMG-CoA reductase (EC 1.1.1.34) was measured in hepatic microsomes according to Shapiro et al. (31
). Microsomes were incubated with 50 µL of a solution containing 7.5 nmol (0.33 GBq/nmol) [3-14C] HMG-CoA, 4.5 mmol glucose-6-phosphate, 3.6 mmol EDTA, 0.45 mmol NADP and 0.3 IU glucose-6-phosphate dehydrogenase. [3H]mevalonic acid (0.024 GBq) was added as a recovery standard.
The reaction was stopped after 15 min with 10 mol/L HCl (0.025 mL/tube). An excess of mevalonic acid was added and samples were incubated for another 30 min at 37°C to allow for the conversion of mevalonic acid to mevalonalactone. After incubation, microsomes were pelleted by centrifugation for 1 min at 1000 x g. An aliquot of the supernatant (0.1 mL) was applied to silica gel TLC plates and developed with acetone/benzene (1:1, v/v); the area containing the mevalonate (Rf = 0.6–0.9) was scraped and mixed with 5 mL aquasol. Radioactivity was measured using a liquid scintillation counter (Packard, Downers Grove, IL). HMG-CoA activity is expressed as pmol [14C]mevalonate produced/(min · mg microsomal protein). Recoveries of [3H]mevalonate were 60–70%.
Cholesterol 7-hydroxylase (CYP7) activity.
CYP7 (EC 1.14.13.7) activity was assayed according to Jelinek et al. (32
). [14C]cholesterol was used as a substrate and delivered as cholesterol/phosphatidylcholine liposomes (1:8 by weight). After preparation by sonification, an NADPH-regenerating system (glucose-6-phosphate dehydrogenase, NADP, and glucose-6-phosphate) was included as a source of NADPH. After addition of glucose-6-phosphate dehydrogenase (0.3 IU), samples were incubated for an additional 30 min. The reaction was stopped by the addition of 5 mL of chloroform/methanol (2:1) and 1 mL acidified water (0.05% sulfuric acid). Tubes were mixed, the top layer was discarded and samples were dried under nitrogen. Samples and 7- and 7ß-hydroxycholesterol standards were dissolved in 100 µL of chloroform, applied to silica gel TLC plates, and developed with ethyl acetate/toluene (3:2). The plate was placed in iodine vapors to mark the 7- and 7ß-hydroxycholesterol standards and placed on XAR-5 film overnight. Using the film as a guide, the locations of the [14C]7-hydroxycholesterol spots were determined, scraped from the plate and counted in a liquid scintillation counter.
Fecal bile acids.
Fecal bile acids were assayed by a colorimetric method of Mashinge et al. (33
). Feces were weighed, dried for 5 h at 37°C, pulverized and weighed again. t-Butanol/water (4 mL) (1:1, v/v) was added to 0.2 g of fecal samples and heated at 37°C for 15 min with continuous agitation. The samples were then centrifuged at 3,000 x g for 10 min (JA-20 rotor in a J2–21 centrifuge, Beckman Instruments) and the supernatant was removed and discarded. Each sample (200-µL aliquot) was analyzed in duplicate, and a blank reagent was added to a third aliquot to correct for the color provided by each sample. Samples were incubated at 37°C for 5 min. The reaction was stopped and color was read in a spectrophotometer at 530 nm. The amount of fecal bile acids was calculated as mmol/(kg · d) after subtracting the blank.
Statistical analysis.
One-way ANOVA was used to evaluate significant differences in plasma and hepatic lipids, LCAT, CETP, HMG-CoA reductase and CYP7 activities and fecal bile acids. Differences with P-values < 0.05 were considered significant. The Newman-Keuls test was used as post-hoc analysis. Data are presented as means ± SD.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Guinea pigs fed the 7.5 and the 10 g/100 g PO diets had 22% lower plasma cholesterol than those fed the control diet (Table 2)
. There was no dose effect because both the 7.5 and the 10 g/100 g diets had similar hypocholesterolemic properties. The reduction of plasma cholesterol was associated with 23% lower plasma LDL-C concentrations in the PO groups compared with the control (P < 0.05). In contrast, VLDL- and HDL-C were not modified by PO intake.
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). However, PO intake resulted in 34% lower plasma TG concentrations (P < 0.001) compared with the control group (Table 2)
.
We measured the activity of two proteins involved in the remodeling of lipoproteins, LCAT and CETP. The activity of both proteins was affected by PO treatment. Guinea pigs fed the control diet had 100 and 36% higher activities of LCAT and CETP, respectively (Table 3)
.
|
. However cholesteryl ester concentrations were 50% lower in guinea pigs fed the PO compared with controls (P < 0.01). In addition, hepatic microsomal free cholesterol was 23–40% lower in the PO groups compared to the control. To further explore the mechanisms responsible for the lower plasma cholesterol and triglyceride concentrations, some of the regulatory enzymes of cholesterol homeostasis were evaluated. Hepatic HMG-CoA reductase, the regulatory enzyme of cholesterol synthesis, was 37% higher in guinea pigs fed the PO diet (Table 5)
. Similarly, the activity of CYP7, the regulatory enzyme of cholesterol catabolism to bile acids, was up-regulated by 32% by PO intake (P < 0.05). In agreement with these results, the excretion of fecal bile acids was 3 times higher in PO-treated guinea pigs compared with controls (P < 0.02) (Table 5)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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,20
). Psyllium intake resulted in 25% lower LDL-C (with a low cholesterol diet) or 53% lower LDL-C (with a high cholesterol diet) (17
, 20
) compared with control guinea pigs. Further, the observed metabolic alterations in hepatic cholesterol and lipoprotein metabolism, as a result of PO intake, followed a pattern similar to that of psyllium or other sources of dietary soluble fiber in this animal model (17
). Plantago ovata effects on fecal bile acids and hepatic cholesterol metabolism.
One of the primary mechanisms by which soluble fiber decreases plasma LDL-C concentrations is an interruption of the enterohepatic circulation of bile acids (35
). The threefold increase in fecal bile acids observed in guinea pigs fed the PO diets suggests a major effect in disrupting bile acid metabolism. This bile acid loss will affect hepatic cholesterol metabolism in two distinctive ways: 1) by decreasing lipid absorption and 2) by enhancing the conversion of hepatic cholesterol to bile acids to maintain bile acid homeostasis. Both of these mechanisms result in the depletion of hepatic cholesterol. In guinea pigs, significant reductions of hepatic cholesterol have been observed by different sources of soluble fiber (10
,12
,20
). In the present study, we observed a decrease in hepatic cholesteryl ester concentrations in the PO groups. We also observed increased fecal bile acid excretion and higher activity of CYP7 in guinea pigs fed the PO diets, which agree with the postulated mechanisms for the action of dietary fiber.
Conversion of hepatic cholesterol to bile acids represents the major regulatory pathway by which the body eliminates excess cholesterol (36
). This step is catalyzed by cholesterol 7-hydroxylase (CYP7), the main regulatory enzyme in the classical bile acid synthesis pathway (37
). Studies in hamsters and guinea pigs (18
,38
) have demonstrated that there is an up-regulation in the activity and the mRNA abundance of CYP7 after psyllium intake, indicating a role of psyllium in increasing the catabolism of cholesterol to bile acids. Similar to these observations, we observed an up-regulation of CYP7 by PO intake, which is in agreement with the higher excretion of fecal bile acids and with the reported effects of psyllium husks on bile acid metabolism.
Another important metabolic alteration that takes place in liver after soluble fiber treatment is increased synthesis of cholesterol within the hepatocyte as demonstrated by higher activity of HMG-CoA reductase (39
) or measurement of sterol synthesis in vivo (40
). Such a compensatory increase in hepatic synthesis occurs when intestinal cholesterol absorption is impaired or when bile acid synthesis is stimulated. Our results in the present study are in agreement with this mechanism because an up-regulation of HMG-CoA reductase was observed in the PO groups.
Plantago ovata effects on plasma lipids and lipoprotein metabolism.
In addition to the expected lowering in plasma cholesterol, PO intake also resulted in significant reductions in plasma TG. This was an unexpected finding that may be related to the hypertriglyceridemia initially present in this group of guinea pigs. Similar to these observations, clinical studies conducted in our laboratory showed that men whose plasma TG levels were elevated initially experienced a reduction of this plasma lipid after intake of a psyllium supplement for 1 mo (41
).
As a result of depleted hepatic pools due to fiber intake, lipoprotein metabolism is altered. The decreases in hepatic cholesterol have been related to lower rates of hepatic apo B secretion in hamsters (42
), guinea pigs (17
) and nonhuman primates (43
) and to faster LDL turnover rates in rats (44
) and guinea pigs (17
).
In this study, we also observed decreases in LCAT and CETP activities with PO intake. These results indicate that PO may decrease the esterification of cholesteryl ester in HDL and the subsequent transfer to VLDL and LDL, which is considered to be an atherogenic process (45
). Lower CETP activity may contribute to the mechanisms of hypocholesterolemia attributed to soluble fiber (29
). Although there is controversy regarding the pro- or antiatherogenic role of CETP, human studies suggest that if CETP deficiency is associated with decreased concentrations of HDL-C, the role of CETP appears to be proatherogenic (45
). In the present study, PO lowered plasma LDL-C and CETP activity without affecting HDL-C levels; thus a beneficial effect of PO in decreasing pro-atherogenic lipoproteins can be postulated.
In conclusion, the seeds from Plantago ovata have hypocholesterolemic properties, although not to the same extent as the husks (17
,20
,29
). The major mechanisms involved in the lowering of LDL-C, including interruption of the enterohepatic circulation of bile acids and alterations in hepatic cholesterol and lipoprotein metabolism, are in agreement with those observed in guinea pigs (17
, 20
,29
) and in humans (8
) treated with psyllium. These findings suggest that the seeds of Plantago ovata could be used as hypocholesterolemic agents in humans.
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FOOTNOTES |
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3 Abbreviations used: apo, apolipoprotein; C, cholesterol; CETP, cholesteryl ester transfer protein; CHD, coronary heart disease; CYP7, cholesterol 7-hydroxylase; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; LCAT, lecithin:cholesterol acyltransferase; TG, triglycerides.
Manuscript received 11 January 2002. Initial review completed 26 January 2002. Revision accepted 13 February 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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