Patent Application
20080009449
The effectiveness of lignan complex from flax for the prevention of atherosclerosis in subjects that show no symptoms of the disease has previously been demonstrated by the inventor of the present invention in U.S. Pat. No. 6,673,773, the disclosure of which is incorporated herein by reference.
The inventor has now established that lignan complex is not only effective in preventing the development of hypercholesterolemic atheroschlerosis in healthy mammals, but can actually slow the progression of the disease once it has developed in a subject
Lignan complex from flax, and a method for its extraction, is described in U.S. Pat. No. 6,264,853, issued Jul. 24, 2001, the disclosure of which is incorporated herein by reference. The process may comprise obtaining an aqueous aliphatic alcohol extract of commercial flax, e.g. flaxseed or flax meal. The extract is then subjected to ultrafiltration, whereby low molecular weight species remain with the filtrate and higher molecular weight species are retained. By proper selection of the filtration medium, it is possible to retain a lignan complex in substantially pure form comprising secoisolariciresinol diglucoside (SDG), cinnamic acid glucosides (CAG), and hydroxymethylglutaric acid (HMGA). The ultrafiltration is preferably carried out for a size exclusion of 30,000 Daltons or greater, generally in the range of 30,000 to 100,000 Daltons, and more preferably 30,000 to 50,000 Daltons.
The complex used according to the present invention typically contains 34 to 38% by weight of secoisolariciresinol diglucoside (SDG), and optionally 15 to 21% by weight cinnamic acid glucosides (CAG), and 9.6 to 11% by weight hydroxymethylglutaric acid (HMGA). The CAG component typically includes coumaric acid glucoside and/or ferulic acid glucoside, normally present in the complex in amounts of about 9.5 to 16.0% by weight coumaric acid glucoside and 4.5 to 5.0% by weight ferulic acid glucoside.
The complex typically contains about 59 to 70% by weight of the three stated ingredients, the balance comprising protein, ash and water of crystallization.
In the present invention, the complex is preferably employed at a purity of 95% by weight or more.
As noted above, the lignan complex has been found effective for slowing the progression of atherosclerosis and preventing the progression of atherosclerosis that occurs after removal of high cholesterol diet (normalizing the serum cholesterol), and hence may be useful for the treatment of related diseases, such as coronary artery disease, stroke, intermittent claudication, restenosis following coronary artery percutaneous intervention (PCI), coronary bypass grafts closure, and other peripheral vascular disease, as well as reducing complications associated with such diseases.
The lignan complex may be administered orally in any suitable form, e.g. as a medicament for suitable for treating atherosclerosis in a mammal, preferably mixed with a pharmaceutically-acceptable diluent or carrier, optionally prepared, for example, as a tablet, capsule or food. Suitable dosages may be determined by trail and experimentation, but dosages of 20-60 mg per kg body weight of the mammal are normally most effective, particularly when the mammal is a rabbit.
The invention is further illustrated by the following non-limiting Examples.
The studies below were conducted in New Zealand white female rabbits weighing between 1.2 and 1.5 g. (6 to 8 wks old) after a one-week period of acclimatization. The rabbits were housed individually under constant climate conditions (temperature 20 to 22° C., relative air humidity 50±5%). The rabbits were housed under a 12-hour light and 12-hour dark cycle. The studies were approved by the Ethics Committee of the University of Saskatchewan, Saskatchewan, Canada, and the animal care was according to approved standard for Laboratory Animal Care. Food and water were provided ad libitum.
(i) For the study of the slowing of progression of atherosclerosis:
(ii) For the study of the regression of atherosclerosis:
At the end of the protocol, rabbits were anesthetized with pentobarbital sodium (40 mg/kg, intravenously) and the aortas were removed for the assessment of atherosclerotic changes. The assessment of atherosclerotic changes in the aorta was made by using Herxheimer's solution containing Sudan® IV for lipid staining (1). Photographs of the stained intimal surface of the aorta were taken and color slides were prepared. The slides were projected on a Caramate® Projector screen with a grid in mm2. The extent of atherosclerosis was expressed as a percentage of total intimal surface area.
Results are expressed as mean±SEM. The Kruskal-Wallis test was used to determine the differences in the atherosclerotic changes in the four groups. The Mann-Whitney U test was used to determine the significance of differences between any two groups. Type-1 error for multiple comparison was controlled by the Bonferroni correction.
This study was conducted to see if lignan complex would slow the progression of atherosclerosis. Representative photographs of the atherosclerotic changes in the intimal surface of the aortas of the four groups are shown in
The original photographs for
The numerical values in brackes above the bars in
* p<0.05, 0.25% cholesterol (2 months) vs. 0.25% cholesterol (4 months), or 0.25% cholesterol (2 months)+0.25% cholesterol and lignan complex (2 months).
† p<0.25, 0.25% cholesterol (4 months) vs. 0.25% cholesterol (2 months)+0.25% cholesterol and lignan complex (2 months).
The intimal surface of the aorta of Groups II, III and IV were covered with atherosclerotic plaques to the extent of 37.76±7.96% and 76.6±9.04% and 52.95±10.29%, respectively. As expected 0.25% cholesterol diet for four months produced a 103% greater extent of atherosclerosis than a similar diet for two months. The atherosclerotic process progressed with hypercholesterolemic diet. The rabbits in group IV, which received 0.25% cholesterol and lignan complex for two months following a 0.25% cholesterol diet for two months, had a lesser extent of atherosclerotic plaques compared to Group III that received 0.25% cholesterol for 4 months (52.95±10.3% vs. 76.59±9.04%). Lignan complex group (Group IV) had 40% greater atherosclerosis as compared to Group II, but 31% less than that of Group III. Lignan slowed the progression of atherosclerosis by 31%. These results suggest that lignan complex slows the progression of atherosclerosis.
This study was conducted to determine if lignan complex can produce regression of already-developed atherosclerosis. The representative photographs of the atherosclerotic changes in the intimal surface of the aortas from the four groups are shown in
The original photographs for
The results shown in
The numerical values in brackets above the bars in
In
* p<0.05, 0.25% cholesterol (2 months) vs. 0.25% cholesterol (2 months)+regular diet (4 months), or 0.25% cholesterol (2 months)+regular diet and Lignan complex (4 months)
† p<0.05, 0.25% cholesterol (2 months)+regular diet (4 months) vs. 0.25% cholesterol (2 months)+regular diet and lignan complex (4 months).
The atherosclerotic plaques were absent in the group of rabbits (Group I) on a regular diet. It was found that 37.75±7.96% of the intimal surface of the aortas of rabbits fed on 0.25% cholesterol for two months was covered with atherosclerotic plaques. The extent of atherosclerosis in group of rabbits on a regular diet for 4 months following 0.25% cholesterol diet was 57.0% greater than those on 0.25% cholesterol diet for 2 months (atherosclerosis 59.29±6.71 vs. 37.76±7.96%). This shows that the atherosclerotic process continued even on regular diet for 4 months following a 0.25% cholesterol diet. The extent of atherosclerosis in the group of rabbits on lignan complex and a regular diet (Group IV) was not significantly different from those of group II (40.26±4.42 vs. 37.76±7.96), but was 32.1% lower compared to Group III. These results suggest that the lignan complex did not produce regression of already present atherosclerotic lesions, however it completely prevented the progression of atherosclerosis that developed after removal of high cholesterol diet (normalization of serum lipids).
The results suggest that lignan complex is very effective in slowing the progression (28-31%) of atherosclerosis. It did not produce regression of atherosclerosis. However, it completely prevented the further development of atherosclerosis that occurs after removal of a high cholesterol diet.
