- Email: [email protected]
Hypolipidaemic activity of whole paddy in rats fed a high-fat-high-cholesterol diet
Atherosclerosis Elsevier Publishing
Company,
Amsterdam
HYPOLIPIDAEMIC ACTIVITY OF WHOLE HIGH-FAT - HIGH-CHOLESTEROL DIET
PADDY
ISOLATION
HUSK
OF AN ACTIVE
P. VIJAYAGOPALAN
AND
Department of Biochemistry, (Revised,
received August
P.
215
- Printed in The Netherlands
FRACTION
FROM
THE
IN
AND
RATS
FED
A
BRAN*
A. KURUP
University of Kerala, Trivandrum-I
(India)
16th, 197 1)
SUMMARS
Whole ground paddy has been previously found to lower cholesterol and phospholipid in serum, liver and aorta of rats fed a high fat-cholesterol diet. A polysaccharide fraction has now been isolated from the defatted bran and husk of paddy; it has a remarkable lipid-lowering effect. The yield of the material is 0.33 g from 100 g of bran and hull. It contains 2.535 ‘!,J,N and 225 mg of uranic acid per 100 g. This substance keeps the total cholesterol and phospholipid of the serum, liver and aorta in rats fed the high fat-cholesterol diet at a similar low level to that in animals fed glucose. Rats receiving this substance had the lowest level of hepatic NAD-NADP, the highest level of myocardial lipoprotein lipase and a normal blood glucose level after an oral glucose load. Rats fed boiled rice showed lower lipid levels than rats fed unboiled rice.
Key words: Atherosclerosis Myocardial
- Blood glucose - Cholesterol - Dietary
lipoprotein lipase - Polysaccharide
carbohydrate
-
fraction from rice bran and
husk
INTRODUCTION
The nature of the carbohydrate in the diet has been reported to influence the level of serum cholesterol in both man and experimental animalsi-5. In a previous communication from this laboratory 6, the effect of long-term feeding of purified rice * Patent application has been filed for patent rights for the isolation of these fractions. Reprint requests should be sent to Dr. P. A. Kurup. Atherosclerosis,
1972, 15: 215-222
216
P. vIJAYAGOPALAS,P. A. KURUP
starch and tapioca starch on the lipid level in high fat-cholesterol fed rats, as compared with the effect of glucose and sucrose was reported. It was also reported that whole ground paddy produced lipid levels significantly lower than purified rice starch. The nature of the hypolipidaemic principle or principles present in the bran and husk of whole paddy has been investigated and the results are reported. MATERIALSANDMETHODS The bran and husk from whole raw paddy was defatted with light petroleum (40-60°C). The light petroleum extract on removal of the solvent furnished the total lipids (A). Preparation
of water extract
The defatted bran and husk (B) was extracted with hot water and the extract was concentrated and evaporated to dryness (C). Preparation
oj polysaccharide
fractiola
The defatted material was subjected to papain digestion at 65-70°C in 0.1 M phosphate buffer, pH 6.5 containing 0.005 M EDTA and 0.005 M cysteine hydrochloride according to the procedure of LAURENT7. Digestion was carried out for 48 h; fresh papain was added at the end of every 16 h. NaOH solution was then added to a final concentration of 0.5 N and kept at room temperature for 18 h to complete solubilization of the polysaccharides. It was then neutralised with HCI; trichloroacetic acid was added to the neutralised solution, to a final concentration of 5%. The precipitated protein was centrifuged off and the supematant was dialysed against running tap water, until the solution was nearly neutral. It was then dialysed against distilled water and the solution concentrated. A portion was evaporated to dryness in vacua to give the total polysaccharide fraction (D). The remaining solution was treated with 3-4 volumes of absolute ethanol and the precipitate was centrifuged off and dried in vawo to furnish the polysaccharide fraction precipitated by ethanol (E). The alcohol solution was concentrated and dried, yielding the polysaccharide fraction not precipitated by alcohol (F). Purified rice starch was prepared as described befores. Boiled rice was obtained by milling boiled paddy, while unboiled rice was obtained from raw paddy. Young male albino rats (average weight 130 g) were divided into 11 groups of 10 rats each. The basal diet consisted of casein 16%, carbohydrate 56 %, hydrogenated ground nut oil 15%, cholesterol 2%, vitamin mixture O.l%, salt mixture (Hubbel, Mendel and Wakeman) 4%, cellulose 7%. The vitamin mixture contained vitamin B1,O. 125 mg; Bz, 0.25 mg; Be, 0.1 mg; niacin, 5 mg; calcium pantothenate, 1 mg; and choline chloride, 100 mg/lOOg of diet. Vitamin A and D were administered to all the rats in the form of shark liver oil (0.5 g/ 100 g of diet). Atherosclerosis,
1972, 15: 215-222
HYPOLIPIDAEMIC
ACTIVITY
217
OF WHOLE PADDY IN RATS
The rats were grouped as follows depending on the carbohydrate portion of the respective diet and the various fractions incorporated: (1) glucose; (2) sucrose; (3) purified rice starch; (4) whole ground paddy; (5) unboiled rice; (6) boiled rice; (7) same as 3 + A (0.28%); (8) same as 3 but with 40% rice starch + 16% defatted husk and bran (B); (9) same as 3 but with 54.7% rice starch + 1.23% (C); (10) same as 3 + 0.28% (D); (11) same as 3 + 5.6 mg E/l00 g; (12) same as 3 + 0.373% F. The proportion of the different fractions in the diet was based on the relative amounts present in the whole paddy. The animals were maintained on this diet for 4 months. The average daily food consumption was observed by noting the amount of food left unconsumed. The change in the weight of the animals during the period was also noted. At the end of 4 months feeding, the rats were fasted for 24 h and killed by a blow on the head. Serum, liver, aorta and heart were used for various estimations. The aorta was stripped of its adventitial adipose tissue. The liver and aorta were extracted first with ethanol:ether (3: 1, v/v) followed by chloroform:methanol (1: 1, v/v)e. The extract in each case was made up to a known volume and the solvent was evaporated from an aliquot for lipid estimation. Total cholesterol was estimated by the method of Carr-Drekters. Free cholesterol was estimated by the Schoenheimer-Sperry methodg. Phospholipid was estimated by the method given in GRADWOHL’S text
booklo.
serum
Lipoprotein
as substratell.
beforec.
Blood
the modification
glucose that
lipase Total
activity
NAD-NADP
was estimated a weakly
of the
heart
in the
by the method
alkaline
copper
was
liver
reagent
was
estimated
with
estimated
of ASATOOR AND
lipaemic
as described KINGI~,
with
was usedIs.
RESULTS
1. The weight of different fractions follows: Fraction
obtained from the bran and husk is as
Weight of bran and husk (g/100 g)
1. 2. 3. 4. 5.
Water extract (C) Total lipids (A) Fraction D Fraction E Fraction F
7.3 1.7 1.66 0.33 1.30
2. The e$ect of various fractions on serum, aortic and hepatic lipid levels are given in Table 1. The effect of boiled rice considerably differs from that of unboiled rice, the latter producing much higher levels of cholesterol and phospholipid in the serum, liver and aorta than even the sucrose-fed animals. Compared with purified rice starch, whole boiled rice produced lower lipid levels. The total lipid fraction from the bran and husk did not have any appreciable effect. The defatted husk and bran produced lower Atherosclerosis,
1972, 15: 215-222
E
r”
f!
G
Y. ”
2
2
x
3
E
b
OF
1
VARIOUS
FRACTIONS
ON
SERUM,
AORTIC
AND
HEPATIC
LIPIDS
(whole ground +6.5& 3.4 paddy) (unboiled $32 & 7.1 rice) (boiled rice) -1-28& 6
IV
X
IX
VIII
VII
VI
1’
(rice starch)
111
(fraction A lipid from bran and +30 5 5.5 husk) (fraction B defatted husk + 10 i 4.8 and bran) (fraction C water $35 It 7.8 extract) (fraction U total polysaccharide) j-40 & 8
+25 & 6.4
$32 * 5.1
(sucrose)
7 & 3.5
II
+
(glucose)
change in weight (g -+ SE)
Average
I
Group
5
i
i
3.8
95.06
+
171.5 rfi 5.2
201.6 4.8
306.3 7.1
4.4 378.60 & 7.1 353.42 * 6.8 168.40 _i 8.7 431.1 + 10.5 291.70 & 5.7
93.8
+:
_L
y
*
&
5
5
144.0 & 6.1
184.0 I’ 4.6
210.0 5.1
288.0 & 6.2
104.0 4.1 363.0 8.1 302.30 7.5 176.2 4.6 427.5 12.7 272.0 6.8
total phospholipid cholesterol (mg/lOO ml i SE)
Serum
16.35 & 4.1
22.36 & 3.1
23.75 & 2.9
37.17 + 4.8
29.06 i_ 6.9
58.84 -+ 7.5
4.6
Il.65 3: 4.2
15.50 IL 4.8
16.70 I
24.93 2 4.9
19.93 _i 6.8
42.53 & 8.1
15.10 + 3.7
21.61 + 2.8
4.9
27.23 f
33.10 & 3.8
6.70 & 4.0
38.54 & 6.1
51.10 & 4.2
9.28 & 3.8 3.8
28.65 & 3.7
32.07 & 3.7
28.99 & 4.1
41.54 & 6.8
36.89 & 7.1
70.86 & 5.6
27.90 & 3.2
46.39 5 5.1
62.56 f
25.15 & 2.8
3.70 & 2.0 16.34 * 3.9
6.10 + 1.4 23.11 & 4.1
5.88 & 3.9
7.72 & 4.1
7.25 & 3.6
12.75 i_ 4.1
4.41 jI 4.1
5.78 k 3.2
4.15 = 3.1
9.2 + 2.2
7.88 -‘- 1.9
9.83 & 2.1
14.37 & 4.8
11.37 1_ 3.8
13.07 & 1.6
1.59 & 1.4
18.12 5 2.1
2.50 + 1.2
& 3.6
& 4.2
12.60 -’ 4.1
15.32 :k 3.7
16.5 & 4.2
23.9
21.9
28.12 & 6.2
14.01 * 3.1
25.83 & 1.3
28.24 & 1.8
8.74 k 1.7
total free phospholipid cholesterol cholesterol (mg/g of met tissue & SE)
Aorta
10 g per animal except in the last group.
total free phosfihocholesterol cholesterol lipid (mglg of wet tissue f SE)
Liver
The average daily food consumption in all the groups was approximately
EFFECT
TABLE
5 S
b 5 3 P 1 2 E _G’
(rractlon L polysaccharide, ppted. by ethanol) (fraction F not precipitated by ethanol)
+35
I II IV V VI VII VIII IX X XI XII
f
-
*
32.054 2.5618 16.756 6.210 6.956 4.793 18.237 21.251 33.167 32.918 22.475
23.200 5.499 14.333 8.488 2.993 1.470” 10.177 13.446 16.375 19.218 12.389
4.071 1.696b 2.522~ 2.1OOd 1.029c 0.176~ 2.190& 2.365& 3.019 3.295 -
total cholesterol
total
10.98 & 3.4
4.2
5.48 * 2.8
3.63 5 2.6
10.03 + 2
1
7.986 1.085~ 3.516 1.971“ 0.894C 0.504c 2.122& 2.68Oa 3.076 6.397 -
3.265 1.2270 2.1148 1.470” 0.689= 0.208C 1.517” 1.058~ 1.177c 1.855d -
2.3998 0.716~ 1.654b 1.385” 0.49oc 0.257~ 1.187~ 1.053c 1.373” 1.613b -
3.584 0.96Oc 1.9764 1.606b 0.871C 0.332~ 1.567” 1.710b 2.4148 2.7258 -
3.651 2.5428 3.071 3.303 1.087~ 0.571c 2.6598 2.2738 2.498& 2.668& -
free phosphocholesterol lipids Cm& + SE)
total cholesterol
A orta
All the animals in this group died after the 2nd month
27.76 f
free phosphocholesterol lipids Cm& i SE)
(at the end of 2 months) -
15.84 &- 3.2
phosphocholesterol lipids (mg/lOO ml f SE)
173.50 -& 7.2
128.0 5.1
Liver
166.36 * 4.8
83.17 f 4.6
Serum
6.6
Where values are not marked with 8, b, c or d, P < 0.01. a P 0.01 < 0.05. b P 0.1 < 0.2. c P > 0.2. d P 0.05 < 0.1.
III III III III III III III III III III III
Values for Students’ t-tests between groups
XII
Al
220 TABLE EFFECT
P. VIJAYAGOPALAN, 2 OF DIFFERENT
LIPOPROTEIN
LIPASE
FRACTIONS AND
BLOOD
Oral dose of 4 g of glucose/kg
Group
ON TOTAL GLUCOSE,
FREE
NAD AND
HEPATIC
FASTING
AND
1 H AFTER
Fraction
Hepatic NAD-NADP
ofwet
III SE)
Myocardial lipoprotein lipase (pm01 of glycerol liberated/g of wet tissue zt SE)’
____ II III IV V VI VII VIII IX X XI
XII
(glucose) (sucrose) (purified starch) (whole ground paddy) (unboiled rice) (boiled rice) (total lipids) (defatted bran and husk) (water extract) (total polysaccharide fraction D) (polysaccharide fraction E ppted. by alcohol) (soluble fraction (F))
Values for Students’ t-tests between groups
III III III III III III III III III III
1 II IV V VI VII VIII IX X XI
LEVELS,
GLUCOSE
MYOCARDIAL LOAD
Blood glucose 1 1~after glucose load (mg/lOO ml k SE)
fasting
~_~
228 i 416 f 375 f
6.4 7.6 4.8
0.98 5 0.15 0.51 f 0.18 0.70 f 0.17
75.6 84.5 87.6
+ 3.1 & 3.6 f 4.1
94.2 132.8 141.2
i_ 4.1 + 6.5 $: 4.8
315 483 378 390
6.9 6.1 5.8 7.1
0.99 0.57 0.74 0.73
0.19 0.20 0.12 0.17
84.0 92.6 91.4 89.1
:k & & &
103.2 165.5 130.2 135.4
2 & i: &
301 & 5.8 333 f 6.5
0.75 & 1.16 0.76 f 0.12
72.5 71.8
_t 3.8 i 4.4
250 i
7.2
0.98 & 0.19
81.50 f
201 f
6.2
1.28 3 0.13 -
72.5
Hepatic NAD-NADP
& & k &
& & f &
4.1 4.6 3.8 4.6
3.1
106.42 + 4.2
& 4.2
98.35 1 4.1
(pglg of wet tissue + SE)
18.375 4.561 7.138 13.914 0.398c 1.750d 9.829 5.198 14.445 22.191
1.235~ 0.767~ 1.137c 0.495c 0.192c 0.125~ 0.214~ 0.288c 1.098c 2.710a
1972, 15: 215-222
2.335a 0.568c 0.621~ 0.811~ 0.68oc 0.243~ 2.7018 2.627s 1.187C 2.573a
3.7 4.1 3.8 3.6
121.5 & 3.4 118.38 & 3.1
Myocardial Blood glucose --______ lipoprotein 1 h after .fasting lipase (,umol of glucose load glycerol (mg/lOO ml f SE) liberated/g of wet tissue & SE)
Where values are not marked with a, b, c or d, P < 0.01. & P 0.01 < 0.05. b P 0.1 < 0.2. c P > 0.2. d P 0.05 < 0.1.
Atherosclerosis,
NADP ORAL
body weight.
$T$
I
P. A. KURUP
7.445 1.040r 6.270 3.849 1.797” 0.967c 3.349 3.994 5.453 6.78%
_~~_
HYPOLIPIDAEMIC
ACTIVITY
OF WHOLE PADDY
221
IN RATS
levels of cholesterol and phospholipid in the serum, liver and aorta when compared with the rice starch group, which shows that the hypolipidaemic effect of whole ground paddy is contained in the defatted bran and husk. The water extract of the bran and husk also produced lower lipid levels. The soluble complex polysaccharide obtained after papain digestion lowered lipid levels still further and this was due to the fraction precipitated from it by ethanol. Cholesterol and phospholipid in serum, liver and aorta of the animals receiving this fraction compared quite favourably with those in the animals fed glucose. Thus, the hypolipidaemic activity of whole ground paddy appears to be due to this polysaccharide fraction. The soluble polysaccharide fraction (that is not precipitated by alcohol) was found to be toxic. It showed some cholesterol- and phospholipid-lowering effect at the end of 2 months, but all the animals in this group died soon afterwards. Debility and weight loss, reduced food intake, oedema, dermatitis of the nose, paws and tail, alopecia and anaemia were some of the signs observed. The intestinal wall at necropsy appeared to be atrophied. Thus, polysaccharide (E) obtained by alcohol precipitation appears to be the active hypolipidaemic
factor in the whole bran and husk of paddy. It is soluble in
water, but more soluble in dilute alkali. It contains 2.535% N and 225 mg of uranic acid per 100 g of material. The animals receiving glucose, whole ground paddy and defatted husk showed minimum gain in weight and in these cases, the increase in the lipids of the serum, liver and aorta was also lower. But in the case of the active polysaccharide fraction, even though there was considerable decrease in the lipids, the gain in weight was appreciable. levels, myocardial 3. E$ect of di$erent fractions on the free hepatic NAD-NADP lipoprotein lipase and blood glucose levels fast&g and 1 h after an oral glucose load. The results are given in Table 2. The results agree with those reported previously, i.e. total free NAD-NADP increased, myocardial lipoprotein lipase decreased and blood glucose increased 1 h after the oral glucose load, depending on the extent of the hyperlipaemia. The rats receiving unboiled rice showed the highest level of hepatic
NAD-
NADP, the lowest level of myocardial lipoprotein lipase and,the highest level of blood glucose 1 h after the oral glucose load. The animals receiving the active polysaccharide fraction had the lowest level of NAD-NADP, the highest level of myocardial lipoprotein lipase and a normal blood glucose level after the glucose load. DISCUSSION
The hypolipidaemic effect of whole paddy appears to be due to a polysaccharide fraction which has now been isolated from the bran and husk. The substance contains nitrogen and uranic acid and detailed investigation on its chemical nature is in progress. It has a pronounced effect in lowering cholesterol and phospholipid in serum, liver and aorta, even at the low dose given (5.6 mg/lOOg of diet). At this level the dose per rat per day is only 0.56 mg. Animals receiving this material daily for a period of Atherosclerosis, 1972, 15: 215-222
P. VIJAYAGOPALAN,
222 4 months
did not show any untoward
P. A. KUKUP
signs and their weight gain was quite
satis-
factory. FISHER AND GRIMINGER~~
reported
that oat hull was more effective
in lowering
lipid levels in chicks fed a hypercholesterolaemic diet than oat starch and they suggested that this effect was due to some complex carbohydrate, even though they did not isolate any such material.
A similar hypocholesterolaemic
effect has been clemonstrat-
ed for complex carbohydrates including pectin, various gums and scleroglucansl5. The material that has now been isolated from the bran and hull of paddy has been obtained pronounced
in a fairly
good yield (0.33 g/100 g of bran
hypolipidaemic
effect in very low concentration
and hull). In view of its and its apparently
non-
toxic nature, this substance seems to be promising for preventing atherosclerosis. Detailed investigations on the chemistry and pharmacological action of this substance are in progress.
REFERENCES 1 STAUB, R. W. AND R. THIESSEN, JR., Dietary carbohydrate and serum cholesterol in rats, Fed. Proc., 1967, 26: 490. 2 PORTMAN, 0. W., E. Y. LAWRY AND D. BRUNO, Effect of dietary carbohydrate on experimentally induced hypercholesteremia and hyperbetalipoproteinemia in rats, Proc. Sm. exp. Biol. Med., 1956, 91: 321. 3 KRITCHEVSKY, D., P. SALLATA AND S. A. TEPPER, Experimental atherosclerosis in rabbits fed cholesterol free diets, J. Atheroscler. Res., 1968, 8: 697. 4 BAILEY, IX., C. B. TAYLOR AND W. BARTLEY, Effect of dietary carbohydrate on hepatic lipogenesis in the rat, Nature (Land.), 1968, 217: 471. 5 ANDERSON, T. A., Effect of carbohydrate source on serum and hcpatic cholesterol levels in the cholesterol fed rats, PYOC.Sot. up. Biol. Med., 1969, 130: 884. 6 VIJAYAGOPALAN, P. AND P. A. KURUP, Effect of dietary starches on the serum, aorta and hepatic lipid levels in cholesterol-fed rats, Atherosclerosis, 1970, 11: 257. 7 LAURENT, T., quoted in SCOTT, J. E., Aliphatic ammonium salts in the assay of acidic polysaccharides from tissues. In: D. GLICK (Ed.), Methods in Biochemical Anal.ysis, Vol. 8, Interscience, London, 1960, p. 154. s ACKERMANN, P. G. AND G. TORO, Blood lipids total cholesterol. In: S. FRANKEL AND S. REITMAN (Eds.), Gradwohl’s Clinical Laboratory Methods and Diagnosis, Mosby, St. Louis, MO., 1963, p. 255. 9 ACKERMANN, P. G. AND G. TORO, Blood lipids, total and free cholesterol. In: S. FRANKEL z~x~) S. REITMAN (Eds.), Gradwohl’s Clinical Laboratory Methods and Diaposis, Mosby, St. Louis, MO., 1963, p. 258. 10 ACKERMANN, P. G. AND G. TORO. Blood lipids Phospholipids. In: S. FRANKEL .*NU S. REITMAN (Eds.), Gradwohl’s Clinical Laboratory Methods and Diagnosis, Mosby, St. Louis, MO., 1963, p. 258. 11 AUGUSTI, K. T. AND P. A. KURU~, Effect of tolbutamide on the lipolytic response of adipose tissue to adrenaline and ACTH, Indian J. Biochem., 1968, 5: 76. 12 ASATOOR, A. AND E. J. KING, Simplified calorimetric blood sugar method, Bioclzen&. J., 1954, 56: xliv. 13 SOMOGYI, M., Note on sugar determination, J. Biol. C/Tens.,1952, 795: 19. 14 FISHER, H. AND P. GRIMINGER, Cholesterol lowering effect of certain grains and of oat fractions in the chick, Proc. Sot. exp. Biol. Med., 1967, 126: 108. 15 FAHRENBACH, M. J., B. A. RICCARDI AND W. C. GRANT, Hypocholesterolemic activity of mucilaginous polysaccharides in white Leghorn cockerels, Proc. Sot. exp. Biol. Med., 1966, 123:
321.
Atherosclerosis,
1972, 15: 215-222
Company,
Amsterdam
HYPOLIPIDAEMIC ACTIVITY OF WHOLE HIGH-FAT - HIGH-CHOLESTEROL DIET
PADDY
ISOLATION
HUSK
OF AN ACTIVE
P. VIJAYAGOPALAN
AND
Department of Biochemistry, (Revised,
received August
P.
215
- Printed in The Netherlands
FRACTION
FROM
THE
IN
AND
RATS
FED
A
BRAN*
A. KURUP
University of Kerala, Trivandrum-I
(India)
16th, 197 1)
SUMMARS
Whole ground paddy has been previously found to lower cholesterol and phospholipid in serum, liver and aorta of rats fed a high fat-cholesterol diet. A polysaccharide fraction has now been isolated from the defatted bran and husk of paddy; it has a remarkable lipid-lowering effect. The yield of the material is 0.33 g from 100 g of bran and hull. It contains 2.535 ‘!,J,N and 225 mg of uranic acid per 100 g. This substance keeps the total cholesterol and phospholipid of the serum, liver and aorta in rats fed the high fat-cholesterol diet at a similar low level to that in animals fed glucose. Rats receiving this substance had the lowest level of hepatic NAD-NADP, the highest level of myocardial lipoprotein lipase and a normal blood glucose level after an oral glucose load. Rats fed boiled rice showed lower lipid levels than rats fed unboiled rice.
Key words: Atherosclerosis Myocardial
- Blood glucose - Cholesterol - Dietary
lipoprotein lipase - Polysaccharide
carbohydrate
-
fraction from rice bran and
husk
INTRODUCTION
The nature of the carbohydrate in the diet has been reported to influence the level of serum cholesterol in both man and experimental animalsi-5. In a previous communication from this laboratory 6, the effect of long-term feeding of purified rice * Patent application has been filed for patent rights for the isolation of these fractions. Reprint requests should be sent to Dr. P. A. Kurup. Atherosclerosis,
1972, 15: 215-222
216
P. vIJAYAGOPALAS,P. A. KURUP
starch and tapioca starch on the lipid level in high fat-cholesterol fed rats, as compared with the effect of glucose and sucrose was reported. It was also reported that whole ground paddy produced lipid levels significantly lower than purified rice starch. The nature of the hypolipidaemic principle or principles present in the bran and husk of whole paddy has been investigated and the results are reported. MATERIALSANDMETHODS The bran and husk from whole raw paddy was defatted with light petroleum (40-60°C). The light petroleum extract on removal of the solvent furnished the total lipids (A). Preparation
of water extract
The defatted bran and husk (B) was extracted with hot water and the extract was concentrated and evaporated to dryness (C). Preparation
oj polysaccharide
fractiola
The defatted material was subjected to papain digestion at 65-70°C in 0.1 M phosphate buffer, pH 6.5 containing 0.005 M EDTA and 0.005 M cysteine hydrochloride according to the procedure of LAURENT7. Digestion was carried out for 48 h; fresh papain was added at the end of every 16 h. NaOH solution was then added to a final concentration of 0.5 N and kept at room temperature for 18 h to complete solubilization of the polysaccharides. It was then neutralised with HCI; trichloroacetic acid was added to the neutralised solution, to a final concentration of 5%. The precipitated protein was centrifuged off and the supematant was dialysed against running tap water, until the solution was nearly neutral. It was then dialysed against distilled water and the solution concentrated. A portion was evaporated to dryness in vacua to give the total polysaccharide fraction (D). The remaining solution was treated with 3-4 volumes of absolute ethanol and the precipitate was centrifuged off and dried in vawo to furnish the polysaccharide fraction precipitated by ethanol (E). The alcohol solution was concentrated and dried, yielding the polysaccharide fraction not precipitated by alcohol (F). Purified rice starch was prepared as described befores. Boiled rice was obtained by milling boiled paddy, while unboiled rice was obtained from raw paddy. Young male albino rats (average weight 130 g) were divided into 11 groups of 10 rats each. The basal diet consisted of casein 16%, carbohydrate 56 %, hydrogenated ground nut oil 15%, cholesterol 2%, vitamin mixture O.l%, salt mixture (Hubbel, Mendel and Wakeman) 4%, cellulose 7%. The vitamin mixture contained vitamin B1,O. 125 mg; Bz, 0.25 mg; Be, 0.1 mg; niacin, 5 mg; calcium pantothenate, 1 mg; and choline chloride, 100 mg/lOOg of diet. Vitamin A and D were administered to all the rats in the form of shark liver oil (0.5 g/ 100 g of diet). Atherosclerosis,
1972, 15: 215-222
HYPOLIPIDAEMIC
ACTIVITY
217
OF WHOLE PADDY IN RATS
The rats were grouped as follows depending on the carbohydrate portion of the respective diet and the various fractions incorporated: (1) glucose; (2) sucrose; (3) purified rice starch; (4) whole ground paddy; (5) unboiled rice; (6) boiled rice; (7) same as 3 + A (0.28%); (8) same as 3 but with 40% rice starch + 16% defatted husk and bran (B); (9) same as 3 but with 54.7% rice starch + 1.23% (C); (10) same as 3 + 0.28% (D); (11) same as 3 + 5.6 mg E/l00 g; (12) same as 3 + 0.373% F. The proportion of the different fractions in the diet was based on the relative amounts present in the whole paddy. The animals were maintained on this diet for 4 months. The average daily food consumption was observed by noting the amount of food left unconsumed. The change in the weight of the animals during the period was also noted. At the end of 4 months feeding, the rats were fasted for 24 h and killed by a blow on the head. Serum, liver, aorta and heart were used for various estimations. The aorta was stripped of its adventitial adipose tissue. The liver and aorta were extracted first with ethanol:ether (3: 1, v/v) followed by chloroform:methanol (1: 1, v/v)e. The extract in each case was made up to a known volume and the solvent was evaporated from an aliquot for lipid estimation. Total cholesterol was estimated by the method of Carr-Drekters. Free cholesterol was estimated by the Schoenheimer-Sperry methodg. Phospholipid was estimated by the method given in GRADWOHL’S text
booklo.
serum
Lipoprotein
as substratell.
beforec.
Blood
the modification
glucose that
lipase Total
activity
NAD-NADP
was estimated a weakly
of the
heart
in the
by the method
alkaline
copper
was
liver
reagent
was
estimated
with
estimated
of ASATOOR AND
lipaemic
as described KINGI~,
with
was usedIs.
RESULTS
1. The weight of different fractions follows: Fraction
obtained from the bran and husk is as
Weight of bran and husk (g/100 g)
1. 2. 3. 4. 5.
Water extract (C) Total lipids (A) Fraction D Fraction E Fraction F
7.3 1.7 1.66 0.33 1.30
2. The e$ect of various fractions on serum, aortic and hepatic lipid levels are given in Table 1. The effect of boiled rice considerably differs from that of unboiled rice, the latter producing much higher levels of cholesterol and phospholipid in the serum, liver and aorta than even the sucrose-fed animals. Compared with purified rice starch, whole boiled rice produced lower lipid levels. The total lipid fraction from the bran and husk did not have any appreciable effect. The defatted husk and bran produced lower Atherosclerosis,
1972, 15: 215-222
E
r”
f!
G
Y. ”
2
2
x
3
E
b
OF
1
VARIOUS
FRACTIONS
ON
SERUM,
AORTIC
AND
HEPATIC
LIPIDS
(whole ground +6.5& 3.4 paddy) (unboiled $32 & 7.1 rice) (boiled rice) -1-28& 6
IV
X
IX
VIII
VII
VI
1’
(rice starch)
111
(fraction A lipid from bran and +30 5 5.5 husk) (fraction B defatted husk + 10 i 4.8 and bran) (fraction C water $35 It 7.8 extract) (fraction U total polysaccharide) j-40 & 8
+25 & 6.4
$32 * 5.1
(sucrose)
7 & 3.5
II
+
(glucose)
change in weight (g -+ SE)
Average
I
Group
5
i
i
3.8
95.06
+
171.5 rfi 5.2
201.6 4.8
306.3 7.1
4.4 378.60 & 7.1 353.42 * 6.8 168.40 _i 8.7 431.1 + 10.5 291.70 & 5.7
93.8
+:
_L
y
*
&
5
5
144.0 & 6.1
184.0 I’ 4.6
210.0 5.1
288.0 & 6.2
104.0 4.1 363.0 8.1 302.30 7.5 176.2 4.6 427.5 12.7 272.0 6.8
total phospholipid cholesterol (mg/lOO ml i SE)
Serum
16.35 & 4.1
22.36 & 3.1
23.75 & 2.9
37.17 + 4.8
29.06 i_ 6.9
58.84 -+ 7.5
4.6
Il.65 3: 4.2
15.50 IL 4.8
16.70 I
24.93 2 4.9
19.93 _i 6.8
42.53 & 8.1
15.10 + 3.7
21.61 + 2.8
4.9
27.23 f
33.10 & 3.8
6.70 & 4.0
38.54 & 6.1
51.10 & 4.2
9.28 & 3.8 3.8
28.65 & 3.7
32.07 & 3.7
28.99 & 4.1
41.54 & 6.8
36.89 & 7.1
70.86 & 5.6
27.90 & 3.2
46.39 5 5.1
62.56 f
25.15 & 2.8
3.70 & 2.0 16.34 * 3.9
6.10 + 1.4 23.11 & 4.1
5.88 & 3.9
7.72 & 4.1
7.25 & 3.6
12.75 i_ 4.1
4.41 jI 4.1
5.78 k 3.2
4.15 = 3.1
9.2 + 2.2
7.88 -‘- 1.9
9.83 & 2.1
14.37 & 4.8
11.37 1_ 3.8
13.07 & 1.6
1.59 & 1.4
18.12 5 2.1
2.50 + 1.2
& 3.6
& 4.2
12.60 -’ 4.1
15.32 :k 3.7
16.5 & 4.2
23.9
21.9
28.12 & 6.2
14.01 * 3.1
25.83 & 1.3
28.24 & 1.8
8.74 k 1.7
total free phospholipid cholesterol cholesterol (mg/g of met tissue & SE)
Aorta
10 g per animal except in the last group.
total free phosfihocholesterol cholesterol lipid (mglg of wet tissue f SE)
Liver
The average daily food consumption in all the groups was approximately
EFFECT
TABLE
5 S
b 5 3 P 1 2 E _G’
(rractlon L polysaccharide, ppted. by ethanol) (fraction F not precipitated by ethanol)
+35
I II IV V VI VII VIII IX X XI XII
f
-
*
32.054 2.5618 16.756 6.210 6.956 4.793 18.237 21.251 33.167 32.918 22.475
23.200 5.499 14.333 8.488 2.993 1.470” 10.177 13.446 16.375 19.218 12.389
4.071 1.696b 2.522~ 2.1OOd 1.029c 0.176~ 2.190& 2.365& 3.019 3.295 -
total cholesterol
total
10.98 & 3.4
4.2
5.48 * 2.8
3.63 5 2.6
10.03 + 2
1
7.986 1.085~ 3.516 1.971“ 0.894C 0.504c 2.122& 2.68Oa 3.076 6.397 -
3.265 1.2270 2.1148 1.470” 0.689= 0.208C 1.517” 1.058~ 1.177c 1.855d -
2.3998 0.716~ 1.654b 1.385” 0.49oc 0.257~ 1.187~ 1.053c 1.373” 1.613b -
3.584 0.96Oc 1.9764 1.606b 0.871C 0.332~ 1.567” 1.710b 2.4148 2.7258 -
3.651 2.5428 3.071 3.303 1.087~ 0.571c 2.6598 2.2738 2.498& 2.668& -
free phosphocholesterol lipids Cm& + SE)
total cholesterol
A orta
All the animals in this group died after the 2nd month
27.76 f
free phosphocholesterol lipids Cm& i SE)
(at the end of 2 months) -
15.84 &- 3.2
phosphocholesterol lipids (mg/lOO ml f SE)
173.50 -& 7.2
128.0 5.1
Liver
166.36 * 4.8
83.17 f 4.6
Serum
6.6
Where values are not marked with 8, b, c or d, P < 0.01. a P 0.01 < 0.05. b P 0.1 < 0.2. c P > 0.2. d P 0.05 < 0.1.
III III III III III III III III III III III
Values for Students’ t-tests between groups
XII
Al
220 TABLE EFFECT
P. VIJAYAGOPALAN, 2 OF DIFFERENT
LIPOPROTEIN
LIPASE
FRACTIONS AND
BLOOD
Oral dose of 4 g of glucose/kg
Group
ON TOTAL GLUCOSE,
FREE
NAD AND
HEPATIC
FASTING
AND
1 H AFTER
Fraction
Hepatic NAD-NADP
ofwet
III SE)
Myocardial lipoprotein lipase (pm01 of glycerol liberated/g of wet tissue zt SE)’
____ II III IV V VI VII VIII IX X XI
XII
(glucose) (sucrose) (purified starch) (whole ground paddy) (unboiled rice) (boiled rice) (total lipids) (defatted bran and husk) (water extract) (total polysaccharide fraction D) (polysaccharide fraction E ppted. by alcohol) (soluble fraction (F))
Values for Students’ t-tests between groups
III III III III III III III III III III
1 II IV V VI VII VIII IX X XI
LEVELS,
GLUCOSE
MYOCARDIAL LOAD
Blood glucose 1 1~after glucose load (mg/lOO ml k SE)
fasting
~_~
228 i 416 f 375 f
6.4 7.6 4.8
0.98 5 0.15 0.51 f 0.18 0.70 f 0.17
75.6 84.5 87.6
+ 3.1 & 3.6 f 4.1
94.2 132.8 141.2
i_ 4.1 + 6.5 $: 4.8
315 483 378 390
6.9 6.1 5.8 7.1
0.99 0.57 0.74 0.73
0.19 0.20 0.12 0.17
84.0 92.6 91.4 89.1
:k & & &
103.2 165.5 130.2 135.4
2 & i: &
301 & 5.8 333 f 6.5
0.75 & 1.16 0.76 f 0.12
72.5 71.8
_t 3.8 i 4.4
250 i
7.2
0.98 & 0.19
81.50 f
201 f
6.2
1.28 3 0.13 -
72.5
Hepatic NAD-NADP
& & k &
& & f &
4.1 4.6 3.8 4.6
3.1
106.42 + 4.2
& 4.2
98.35 1 4.1
(pglg of wet tissue + SE)
18.375 4.561 7.138 13.914 0.398c 1.750d 9.829 5.198 14.445 22.191
1.235~ 0.767~ 1.137c 0.495c 0.192c 0.125~ 0.214~ 0.288c 1.098c 2.710a
1972, 15: 215-222
2.335a 0.568c 0.621~ 0.811~ 0.68oc 0.243~ 2.7018 2.627s 1.187C 2.573a
3.7 4.1 3.8 3.6
121.5 & 3.4 118.38 & 3.1
Myocardial Blood glucose --______ lipoprotein 1 h after .fasting lipase (,umol of glucose load glycerol (mg/lOO ml f SE) liberated/g of wet tissue & SE)
Where values are not marked with a, b, c or d, P < 0.01. & P 0.01 < 0.05. b P 0.1 < 0.2. c P > 0.2. d P 0.05 < 0.1.
Atherosclerosis,
NADP ORAL
body weight.
$T$
I
P. A. KURUP
7.445 1.040r 6.270 3.849 1.797” 0.967c 3.349 3.994 5.453 6.78%
_~~_
HYPOLIPIDAEMIC
ACTIVITY
OF WHOLE PADDY
221
IN RATS
levels of cholesterol and phospholipid in the serum, liver and aorta when compared with the rice starch group, which shows that the hypolipidaemic effect of whole ground paddy is contained in the defatted bran and husk. The water extract of the bran and husk also produced lower lipid levels. The soluble complex polysaccharide obtained after papain digestion lowered lipid levels still further and this was due to the fraction precipitated from it by ethanol. Cholesterol and phospholipid in serum, liver and aorta of the animals receiving this fraction compared quite favourably with those in the animals fed glucose. Thus, the hypolipidaemic activity of whole ground paddy appears to be due to this polysaccharide fraction. The soluble polysaccharide fraction (that is not precipitated by alcohol) was found to be toxic. It showed some cholesterol- and phospholipid-lowering effect at the end of 2 months, but all the animals in this group died soon afterwards. Debility and weight loss, reduced food intake, oedema, dermatitis of the nose, paws and tail, alopecia and anaemia were some of the signs observed. The intestinal wall at necropsy appeared to be atrophied. Thus, polysaccharide (E) obtained by alcohol precipitation appears to be the active hypolipidaemic
factor in the whole bran and husk of paddy. It is soluble in
water, but more soluble in dilute alkali. It contains 2.535% N and 225 mg of uranic acid per 100 g of material. The animals receiving glucose, whole ground paddy and defatted husk showed minimum gain in weight and in these cases, the increase in the lipids of the serum, liver and aorta was also lower. But in the case of the active polysaccharide fraction, even though there was considerable decrease in the lipids, the gain in weight was appreciable. levels, myocardial 3. E$ect of di$erent fractions on the free hepatic NAD-NADP lipoprotein lipase and blood glucose levels fast&g and 1 h after an oral glucose load. The results are given in Table 2. The results agree with those reported previously, i.e. total free NAD-NADP increased, myocardial lipoprotein lipase decreased and blood glucose increased 1 h after the oral glucose load, depending on the extent of the hyperlipaemia. The rats receiving unboiled rice showed the highest level of hepatic
NAD-
NADP, the lowest level of myocardial lipoprotein lipase and,the highest level of blood glucose 1 h after the oral glucose load. The animals receiving the active polysaccharide fraction had the lowest level of NAD-NADP, the highest level of myocardial lipoprotein lipase and a normal blood glucose level after the glucose load. DISCUSSION
The hypolipidaemic effect of whole paddy appears to be due to a polysaccharide fraction which has now been isolated from the bran and husk. The substance contains nitrogen and uranic acid and detailed investigation on its chemical nature is in progress. It has a pronounced effect in lowering cholesterol and phospholipid in serum, liver and aorta, even at the low dose given (5.6 mg/lOOg of diet). At this level the dose per rat per day is only 0.56 mg. Animals receiving this material daily for a period of Atherosclerosis, 1972, 15: 215-222
P. VIJAYAGOPALAN,
222 4 months
did not show any untoward
P. A. KUKUP
signs and their weight gain was quite
satis-
factory. FISHER AND GRIMINGER~~
reported
that oat hull was more effective
in lowering
lipid levels in chicks fed a hypercholesterolaemic diet than oat starch and they suggested that this effect was due to some complex carbohydrate, even though they did not isolate any such material.
A similar hypocholesterolaemic
effect has been clemonstrat-
ed for complex carbohydrates including pectin, various gums and scleroglucansl5. The material that has now been isolated from the bran and hull of paddy has been obtained pronounced
in a fairly
good yield (0.33 g/100 g of bran
hypolipidaemic
effect in very low concentration
and hull). In view of its and its apparently
non-
toxic nature, this substance seems to be promising for preventing atherosclerosis. Detailed investigations on the chemistry and pharmacological action of this substance are in progress.
REFERENCES 1 STAUB, R. W. AND R. THIESSEN, JR., Dietary carbohydrate and serum cholesterol in rats, Fed. Proc., 1967, 26: 490. 2 PORTMAN, 0. W., E. Y. LAWRY AND D. BRUNO, Effect of dietary carbohydrate on experimentally induced hypercholesteremia and hyperbetalipoproteinemia in rats, Proc. Sm. exp. Biol. Med., 1956, 91: 321. 3 KRITCHEVSKY, D., P. SALLATA AND S. A. TEPPER, Experimental atherosclerosis in rabbits fed cholesterol free diets, J. Atheroscler. Res., 1968, 8: 697. 4 BAILEY, IX., C. B. TAYLOR AND W. BARTLEY, Effect of dietary carbohydrate on hepatic lipogenesis in the rat, Nature (Land.), 1968, 217: 471. 5 ANDERSON, T. A., Effect of carbohydrate source on serum and hcpatic cholesterol levels in the cholesterol fed rats, PYOC.Sot. up. Biol. Med., 1969, 130: 884. 6 VIJAYAGOPALAN, P. AND P. A. KURUP, Effect of dietary starches on the serum, aorta and hepatic lipid levels in cholesterol-fed rats, Atherosclerosis, 1970, 11: 257. 7 LAURENT, T., quoted in SCOTT, J. E., Aliphatic ammonium salts in the assay of acidic polysaccharides from tissues. In: D. GLICK (Ed.), Methods in Biochemical Anal.ysis, Vol. 8, Interscience, London, 1960, p. 154. s ACKERMANN, P. G. AND G. TORO, Blood lipids total cholesterol. In: S. FRANKEL AND S. REITMAN (Eds.), Gradwohl’s Clinical Laboratory Methods and Diagnosis, Mosby, St. Louis, MO., 1963, p. 255. 9 ACKERMANN, P. G. AND G. TORO, Blood lipids, total and free cholesterol. In: S. FRANKEL z~x~) S. REITMAN (Eds.), Gradwohl’s Clinical Laboratory Methods and Diaposis, Mosby, St. Louis, MO., 1963, p. 258. 10 ACKERMANN, P. G. AND G. TORO. Blood lipids Phospholipids. In: S. FRANKEL .*NU S. REITMAN (Eds.), Gradwohl’s Clinical Laboratory Methods and Diagnosis, Mosby, St. Louis, MO., 1963, p. 258. 11 AUGUSTI, K. T. AND P. A. KURU~, Effect of tolbutamide on the lipolytic response of adipose tissue to adrenaline and ACTH, Indian J. Biochem., 1968, 5: 76. 12 ASATOOR, A. AND E. J. KING, Simplified calorimetric blood sugar method, Bioclzen&. J., 1954, 56: xliv. 13 SOMOGYI, M., Note on sugar determination, J. Biol. C/Tens.,1952, 795: 19. 14 FISHER, H. AND P. GRIMINGER, Cholesterol lowering effect of certain grains and of oat fractions in the chick, Proc. Sot. exp. Biol. Med., 1967, 126: 108. 15 FAHRENBACH, M. J., B. A. RICCARDI AND W. C. GRANT, Hypocholesterolemic activity of mucilaginous polysaccharides in white Leghorn cockerels, Proc. Sot. exp. Biol. Med., 1966, 123:
321.
Atherosclerosis,
1972, 15: 215-222