140
Study on the Vitamin A Activity of Carotene in Green Fodder
EXPERIMENTAL
(1) Relation between the chemically determined carotene and its biological potency as assayed again
standard carotene.
The experimental part consisted mainly of (a) determination of the carotene content of some
green fodders by the Seshan-Sen technique, and (b) the bio-assay of the same materials by using
Coward's curative methods [1938] with albino rats. The basal diet for the rats consisted of vitamin
A-free casein 18, dried brewer's yeast 10, dextrinized starch 63, purified coconut oil 5 and MaCollum,
Simmonds and Pitz salt mixture 4 parts respectively. Ten units of calciferel in coconut oil solution
were given per os to each animal once a week. Five to ten animals were used in each group (negative
control, positive control, and test group). The standard of reference was a sample of B.D.H. carotene,
the purity of which was determined spectrographically [Morton, 1942] and a solution containing
10 mg. of true carotene in 50 c.c. of coconut or olive oil was prepared using 5 mg. of hydroquinone
as antioxidant, and this was kept in the dark at a low temperature. Carotene was stable in both
these oils for a long period and this served as stock solution from which weekly dilutions were
made for administering carotene to the positive control animals daily. The methods of dosing the
test material was first to dry quickly the green fodders in a current of air at a temperature not exceeding
62°C. It was found that the carotene content was not affected if the whole plant or leaves were dried
in this way, but there was some loss if the plants were chopped up so as to disintegrate some of the
material and expose the contents to atmospheric oxidation. The dry material was then ground to
a fine powder, analysed for its carotene content, and then mixed with a small amount of vitamin
A-free casein and fed to individual rats quantitatively before the basal ration was given to them.
This ensured a quantitative intake of the fodder carotene. In the case of barley extract, the carotene
was extracted by petroleum ether after the usual alkali alcohol digestion, evaporated to dryness,
dissolved in a small amount of oil and then fed to experimental animals. The growth experiments
were conducted for 4 weeks after the test animals reached a constant weight on the vitamin A-free
basal ration. The dosing of the positive control and the test group of animals was so arranged by
preliminary experiments that almost equal growth was obtained during the assay. In Table I
TABLE I
Vitamin A potency of some Green Fodders
Name of fodder
Barley (Hordeum vulgare) . .
Barley extract .
.
.
.
Elephant grass(Pennisetum pureum)
Oats (Avena sativa) . . .
Kollukattai grass (Pennisetum
cenchroides)
Berseem (Trifolium alexandrium) .
Lucerne (Medicago sativa) . .
Dub grass (Cynodon dactylon) .
Jowar (Andropogon Sorghum) .
Carotene
content
determin-
ed by
the
chemical
method
µg. dry
matter
mg.
414.9
1 × 10 6
203.8
400.0
148.8
286.1
327.3
245.8
214.1
Daily dose
Test
material
on dry
basis
mg.
2.41
..
4.91
5.00
13.44
6.99
6.11
8.14
9.34
Amount
of
carotene
µg.
1.0
1.0
1.0
2.0
2.0
2.0
2.0
2.0
2.0
Dose of
standard
carotene
Positive
control
µg.
1.0
1.0
1.0
2.0
2.0
2.0
2.0
2.0
2.0
Average growth rate per
week (g)
Test
material
A
6.65
6.43
4.75
9.70
8.68
8.95
8.90
8.10
8.50
Standard
Carotene
B
6.60
6.52
4.92
9.00
9.00
8.34
8.34
8.53
8.40
Average .
A/B
1.008
0.986
0.966
1.078
0.964
1.073
1.067
0.950
1.012
1.012
Vitamin A
equivalen-
ce of a µg.
of fodder
carotene
0.5040
0.4930
0.4830
0.5390
0.4820
0.5365
0.5335
0.4750
0.5060
0.5060