- -I Food Science Mimeo Report FS 68-1
Lake Alfred, Florida 475-10/12/67 RCR
EFFECT OF FLAVONOIDS ON EXPERIMENTALLY INDUCED ATHEROSCLEROSIS AND THROMBOSIS1
R. C. Robbins
Food Science Department
University of Florida,
A number of studies have been reported which demonstrate some protective
effect of flavonoids against atherosclerosis and thrombosis. Naegeli and Matis
(7) stated that clinical evidence warranted use of flavonoids in treatment of
thromboembolism. Griffith et al. (6) reported that in a hypertensive population,
rutin therapy reduced by 65% the expected death rate due to coronary occlusion.
Smolenskii et al. (10) found that flavonoids along with vitamin C reduced athe-
roma formation in rabbits.
In view of the above evidence, a series of experiments have been conducted
to determine the effect of various flavonoids on serum cholesterol levels, sur-
vival time of animals fed atherogenic or thrombogenic regimens, and on a number
of blood parameters which are believed to be involved in either or both athero-
sclerosis and thrombosis.
Experimental Procedure and Results
To determine the effect of flavonoids on serum cholesterol, male rats were
fed the atherogenic or thrombogenic regimens described by Gresham and Howard (5).
These are semi-synthetic diets containing 40% butter or peanut oil with 5%
cholesterol, 2% cholic acid and 0.2% thiouracil. The peanut oil diet induces
atherosclerosis while the butter containing diet causes thrombosis. If the
cholesterol, cholic acid and thiouracil are omitted, the diets do not induce
vascular disease. The addition of 0.2% rutin to the diets lowered serum cho-
lesterol on the butter containing diets, but had no effect on the peanut oil
diets (Table 1).
A series of experiments were conducted to determine the effect of flavonoids
on survival time of rats fed the atherogenic or thrombogenic regimens described
above. Sixteen groups were fed the thrombogenic regimens plus a flavonoid and
all 16 groups showed an increase in survival time over controls fed similar regi-
mens without flavonoids. Chi-square analysis showed a highly significant
(P< 0.001) effect due to flavonoids. Four groups of rats were fed the athero-
genic regimen plus a flavonoid, but only 2 groups showed an increased survival
time over controls. The rats fed hesperidin showed an increased survival time,
but those fed naringin and tangeretin did not survive as long as the control
rats (Table 2).
There is experimental evidence that intravascular clumping (aggregation,
agglutination or sludging) of the formed elements of blood promotes thrombosis
and is associated with a number of conditions believed to be involved in
atherogenesis (8). Also, there is evidence that an increased capillary fragility
may predispose the subject to vascular disease. To test the hypothesis that
flavonoids and vitamin C may act on both blood cell clumping and capillary fragil-
ity, rutin, hesperidin or naringin were fed at a 20 mg per day level along with
three levels of vitamin C, (0.25, 0.3 and 8.0 mg) to vitamin C deficient guinea
1 Presented at Eighteenth Annual Citrus Processors' Meeting, Citrus Experiment
Station, Lake Alfred, Florida.
Vitamin C deficiency was accompanied by both a severe blood cell aggregation
and a decreased capillary resistance. Vitamin C added to the diet in graded
amounts reduced blood cell aggregation and increased capillary resistance pro-
portional to dose. The flavonoids brought about a further reduction in blood
cell aggregation and increased capillary resistance. Blood cell aggregation and
capillary resistance showed an inverse relationship, thus implicating aggregation
in capillary fragility. The flavonoids and vitamin C showed a synergistic re-
lationship. No difference was detected in physiological effect among the flavo-
noids (hesperidin, naringin, rutin) used. Therefore, all data were combined for
statistical analysis (Table 3).
The disaggregating effect of flavonoids on clumped blood cells was further
investigated using the erythrocyte sedimentation rate as a criterion. Blood was
centrifuged and the cells separated. Flavonoids were then added to saturate the
plasma. The plasma was then centrifuged to remove excess flavonoid, and the
hematocrit adjusted to 0.36 + 1; the cells were then resuspended and the settling
rate determined. The sedimentation rate of blood cells in drawn blood is due to
rouleaux formation and clumping of the formed elements. Horse blood was used in
these trials since under normal conditions it has the highest known sedimentation
rate. The flavonoids hesperidin, quercetin, rutin and tangeretin were tested
The flavonoids decreased the settling tendency of the cells of horse blood.
The effect of the flavonoids was not immediate, but was time dependent. The
flavonoids tested showed considerable differences in their ability to alter the
sedimentation rate of blood cells. Tangeretin was most effective followed by
rutin, quercetin, hesperidin chalcone and crude hesperidin in order of decreasing
Flavonoids have effect on several blood parameters that are believe to be
involved in the pathogenesis of vascular disease. Under certain conditions they
reduced serum cholesterol levels. The mechanism here is not clear. According
to Di Maggio (2) flavonoids affect the amount of cholesterol that can be main-
tained in the plasma. However, metabolic processes appear to be involved as
flavonoids lowered serum cholesterol with a saturated fat (butter), but had no
effect with an unsaturated fat (peanut oil).
Flavonoids appear to have an anti-thrombogenic effect since rats fed the
thrombogenic regimens usually die of myocardial infarction and show extensive
thrombosis (4). Flavonoids increased the survival time of rats fed the throm-
bogenic regimen (16 out of 16 trials) and every flavonoid tested had a positive
effect. These results are consistent with the observations of Naegeli and Matis
that clinical evidence warrants use of flavonoids in treatment of thrombo-
embolism (7). The effect of flavonoids on survival time of rats fed the athero-
genic regimen was inconsistent. Hesperidin showed a positive effect while
naringin and tangeretin showed a negative effect. Survival time does not appear
to be a valid criterion for test of an anti-atherogenic effect since rats fed
the atherogenic regimen do not die from atherosclerosis. Effect on plaque form-
ation would be a much more satisfactory criterion.
The blood cell disaggregating effect of flavonoids in vivo when adminis-
tered along with vitamin C to vitamin C deficient guinea pigs and in vitro when
added to drawn horse blood may provide clues as to mechanism of action. Blood
cell aggregation has been found to accompany conditions which induce athero-
sclerosis and thrombosis (8). There are several reports that blood cell aggre-
gation promotes thrombosis (4) and severe aggregation which impedes blood flow
may be involved in atherogenesis (9). Thus, a reduction of blood cell aggre-
gation might provide a protective effect (in different ways) against both
thrombosis and atherosclerosis.
Throughout the history of investigations on the flavonoids, inconsistencies
in action have been reported. These inconsistencies appear to be in evidence in
the present experiments. However, if one of the mechanisms of action (flavonoids
may have more than one physiological effect) is on blood cell aggregation some of
the inconsistencies may be explained. There are many different forces involved
in blood cell aggregation (3) (1) and there are specificities between the agents
that cause aggregation and-the specific agents that will reverse the aggregation.
It appears from present evidence that flavonoids warrant further consider-
ations as substances that could prove of value as anti-thrombogenic agents or
substances that may provide some protection against atherogenesis. Research to
define the specificities involved, a search for more active forms, or modifi-
cations or molecular structure might provide a series of substances of thera-
peutic value over a broad range of physiological conditions.
1. Bellis, C. J. and H. L. Snow. Sludged Blood: Electrokinetic Factors
Involved. Ann. West. Med. and Surg. 4: 223, 1950.
2. Di Maggio, G. Influence of the P Group on Cholesterol Metabolism.
Congr. Intern. Biochem. Resumes Communs. 3rd Congr. Brussels, 1955.
3. Easty, G. C. and E. H. Mercer. An Election Microscope Study of Model
Tissues Formed by the Agglutination of Erythrocytes. Exptl. Cell Res. 28:
4. Gelin, L. E. and B. Zederfeldt. Experimental Evidence of the Signifi-
cance of Disturbances in the Flow Properties 6f Blood. Acta Chir. Scand. 122:
5. Gresham, G. A. and A. N. Howard. The Independent Production of
Atherosclerosis and Thrombosis in the Rat. Brit. J. Exptl. Pathol. 41: 395, 1960.
6. Griffith, Jr., J. Q., C. F. Krewson and J. Naghski. Rutin and Related
Flavonoids. Mack Pub. Co., Easton, Pa., 1955.
7. Naegeli, T. and P. Matis. Significance of Several Vitamins for the
Theory of Thromboembolism. Intern. Z. Vitaminforsch. 27: 324, 1957.
8. Robbins, R. C. Intravascular Aggregation of the Cellular Elements of
Blood in Rats Fed Thrombogenic or Atherogenic Regimens. J. Atheroscler. Res.,
6: 467, 1966.
9. Schlichter, J. G., L. N. Katz and J. Meyer. The Occurrence of
Atheromatous Lesions After Cauterization of the Aorta Followed by Cholesterol
Administration. Am. J. Med. Sci. 218: 603, 1949.
10. Smolenskii, V. S., N. N. Erofeeva, N. F. Pankratova and M. N. Zaprometov.
The Influence of Vitamin C and P Administration on The Development of Experimental
Arteriosclerosis. Vitamin resursy i I spol. Sb. Nauch. Akad Inst. Biokkim.,
4: 158, 1959.
Table 1. Effect of rutin on body weight and serum cholesterol in male
rats fed high fat diets1 with and without adjuncts2.
Average animal3 weight-gms Cholesterol
Dietary fat Start End Change Mg %
Butter 156 360 +204 142
Butter + Rutin 194 275 + 81 89
Peanut oil 195 333 +138 135
Peanut oil + Rutin 193 331 +138 132
Butter + Adjuncts 384 175 -109 1552
Butter + Adjuncts + Rutin 288 187 -101 1015
Peanut oil + Adjuncts 282 197 85 868
Peanut oil + Adjuncts + Rutin 307 168 -139 869
Diets semi-synthetic containing 40% butter or peanut oil.
2Adjuncts 5 cholesterol + 2 cholic acid + 0.2 thiouracil.
Adjuncts 5% cholesterol + 2% cholic acid + 0.2% thiouracil.
Ten animals per treatment.
Table 2. Effect of flavonoids on survival time
thrombogenic or atherogenic regimens.
of rats fed
Flavonoid Gms per kg diet Survival time
Naringin 2 +20
Tangeretin 2 +16
Hesperidin 2 +14
Hesperidin 1 +24
Hesperidin (HMCC)1 1 +24
S" 3 +48
Rutin 1 +51
Hesperidin 2 +105
Naringin 2 -45
Tangeretin 2 -42
SHesperidin methylene carboxy chalcone.
Table 3. Effect of three vitamin C levels with and without flavonoids on
blood cell aggregation and capillary resistance in vitamin C deficient guinea
Vitamin C Flavonoids Capillary resistance, Blood cell aggregation2
cm Hg1 (24 day period)
Mg per day Mg per day Average Standard deviation 0 1+ 2+ 3+ 4+
0.25 0 7.7 2.2 0 0 20 49 31
0.25 20 8.6 3.1 0 3 41 50 6**
3.0 0 9.3 5.5 42 33 16 2 7*
3.0 20 11.8 4.5 55 19 8 8 10**
8.0 0 8.2 5.6 19 17 17 27 20*
8.0 20 9.0 2.8 36 10 23 26 5**
1The higher the value the greater the capillary resistance.
2 Blood cell aggregation rated from 0 (no aggregation) to 4+ (very severe).
Statistically significant at the P<0.01 level due to vitamin C.
Statistically significant at the P<0.01 level due to flavonoids.
Table 4. Effect of flavonoids on erythrocyte sedimentation rate.
Quer. Rutin Tangeretin
Sedimentation rates ml per hr.
SHesperidin methylene carboxy chalcone.