Plant Foods for Human Nutrition 56: 8395, 2001.

2001 Kluwer Academic Publishers. Printed in the Netherlands.

Bioavailability trials of -carotene from fresh and

dehydrated drumstick leaves (Moringa oleifera) in a rat
model
VANISHA S. NAMBIAR and SUBADRA SESHADRI
Department of Foods and Nutrition, Faculty of Home Science, MS University of Baroda,
Vadodara 390002, Gujarat, India
Received 20 July 1999; accepted in revised form 8 May 2000

Abstract. Male albino rats (Charles Foster, n = 40) were fed a synthetic diet deficient in
vitamin A for 4 weeks. Six rats died during the depletion period. Of the 34 surviving, 5 rats
were continued on the vitamin A deficient diet for 4 more weeks and 24 were repleted with
vitamin A (4000 IU/kg diet) in the form of vitamin A acetate (group A, n = 8), fresh drumstick
leaves (group B, n = 8) or dehydrated drumstick leaves (group C, n = 8) for 4 weeks. The
remaining 10 rats were continued on the vitamin A adequate diet for 4 (n = 5) and 8 weeks,
respectively (n = 5). A marked reduction in food intake, body weight, accompanied by clinical
signs of vitamin A deficiency and a decline in serum vitamin A (29.2 to 19.1 g/dL) and liver
vitamin A (3.7 to 2.0 g/dL) were seen at the end of 4 weeks of feeding a vitamin A deficient
diet. On repletion significant improvements in clinical signs, food intake and body weights
were noted in the three groups compared to the baseline (n = 5) and at the end of 4 weeks
of depletion. The gain in body weight was highest for the group repleted with dehydrated
drumstick leaves. Among the repleted groups, the serum vitamin A was highest for group A
(34.7 g/dL) given synthetic vitamin A, compared to group B (25.8 g/dL) and group C (28.2
g/dL) given drumstick leaves. All these were significantly higher than the serum vitamin A
values seen at the end of 4 weeks of depletion (19.1 g/dL). A significant improvement was
also observed in the liver retinol levels on repletion for 4 weeks in the three groups, compared
to the vitamin A depleted rats. These results imply that -carotene from drumstick leaves
was effective in overcoming vitamin A deficiency although serum vitamin A levels remained
somewhat lower compared to the group repleted with vitamin A acetate. In terms of growth
parameters, the fresh and dehydrated drumstick leaves were better than the synthetic vitamin
A. It is therefore concluded that in the developing countries like India, sources of vitamin A
such as drumstick leaves are valuable in overcoming the problem of vitamin A deficiency.
Key words: Bioavailability, -Carotene, Dehydrated drumstick leaves, Rat model, Serum and
liver vitamin A
Abbreviations: FER = Food Efficiency Ratio; DW = dry weight; FW = fresh weight; SVA =
synthetic vitamin A; FDL = Iresh drumstick leaves; DDL = dehydrated drumstick leaves

GSB/Prepr: QUAL 3496/TYPESET Pipsnr. 271605; Ordernr.: 7060105-JM.WEB2C (qualkap:bio1fam) v.1.2

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Table 1. Composition of drumstick leaves (Moringa
oleifera
Chemical constituents

Values dry wt basis (mg/g)

Moisture
Total carotene
-carotene
Ascorbic acid
Total iron
Calcium
Phosphorous
Oxalic acid

79.2%
1.93
0.93
6.6
0.26
22.4
6.3
11.2

Source: Nambiar & Seshadri [4].

Introduction
Food-based approaches using dietary sources of vitamin A, when adequately
implemented, can be effective in the control of vitamin A deficiency, and
contribute to alleviating the other usual accompanying nutritional deficits [1
3]. Drumstick leaves (Moringa oleifera) are one such very promising species
due to their high nutritional value [4] (Table 1), easy cultivation, fast growth
and adaptation. These leaves have been found to be the richest source of
-carotene (19210 g/100 gm fresh weight, as assessed by HPLC) among
the sixteen green leafy vegetables analyzed for -carotene in this laboratory
[5]. These leaves can retain 50% of their -carotene with shade dehydration; the dehydrated leaves can easily be rehydrated and incorporated into
traditional Western Indian recipes without altering their acceptability characteristics [6]. Several other researchers have also reported the high -carotene
content of fresh, as well as dehydrated, drumstick leaves. The younger leaflets and tender petiole stems are used in stews, soups, dals, stir-fries and a
variety of dishes alone, or in combination with other leafy vegetables [710].
However, the bioavailabilities of the -carotene in drumstick leaves in fresh
form or in the dehydrated state have not been studied.
The bioavailability of pure -carotene is not representative of the -carotene in foods as several factors such as the species of carotenoids, matrix in

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which it is incorporated and absorption modifiers may influence availability
greatly [1115].
Thus, the question of whether the -carotene from the drumstick leaves
would be equivalent to the biologically available provitamin A needs to be
answered. It is also important to determine the extent to which -carotene
can replace synthetic vitamin A through physiological investigations. Current
methods available for the assessment of vitamin A status include estimation
at the preclinical level, the overt ocular level, the circulating blood level or
the liver storage level. Rats are a standard model employed for assessing
the bioavailability of carotene/vitamin A [16, 17]. The present study was,
therefore, conducted in a rat model with the major objective of studying the
bioavailability of -carotene from fresh and dehydrated drumstick leaves,
using vitamin A depleted rats.
Materials and methods
Preliminary trials. Preliminary trials were carried out to establish the length
of time needed to induce vitamin A deficiency in the rats (as assessed by
food intake, body weight, clinical signs/symptoms and depletion of serum
and liver vitamin A levels), as well as to test the acceptability of fresh and
dehydrated drumstick leaves by the rats. A group of male weanling (50
60 g body weight) Charles Foster albino rats (n = 15) were used for the
preliminary trials. The rats were fed the basal diet for a week for acclimatization. Thereafter, 5 rats were continued on the basal diet (control) and 10
rats were shifted to the vitamin A deficient diet. All were provided food and
deionized water ad libitum. They were maintained in individual cages, on a
12-h dark/light cycle. Body weights and food intakes were recorded daily and
the animals were observed for clinical signs of vitamin A deficiency. By the
end of 4 weeks of the deficient diet, mean food intake and body weight in the
deficient animals declined to 1/3 of the control. Two rats died during the 4
week period. The 8 surviving animals manifested clinical signs of vitamin A
deficiency. At this point, the deficient rats were divided into three groups of
2, 3 and 3 and the control rats into two groups of 3 and 2. Two deficient rats
and the three control rats were sacrificed to obtain serum and liver vitamin
A values. The other group of 4 deficient rats was repleted with either fresh
(n = 2) or dehydrated drumstick leaves (n = 2) (carotene equivalent to 4000
IU vitamin A/kg diet) and observed for four weeks. The control rats (n =
2) were continued on the vitamin A adequate diet tor 4 weeks. Food intakes
and weight gains of the repleted animals were satisfactory. Significant rise
in serum and liver vitamin A values were observed on repletion compared to
the deficient rats, although they were still lower than the controls. Thus, the

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preliminary trials established that 4 weeks were sufficient time for vitamin A
depletion to occur and that both fresh and dehydrated drumstick leaves were
well accepted by the rats.
Experimental design. Fifty-five male (Charles Foster) rats (5060 g) were
randomly divided into three groups, baseline (n = 5); control (n = 10) and
experimental (n = 40). The control rats (n = 10) were fed a vitamin A adequate
diet for four (n = 5) and eight weeks (n = 5) at the end of which they were
sacrificed to obtain data on serum and liver vitamin A levels. The five rats
in the baseline group were sacrificed at the beginning of the study to obtain
reference values for serum and liver parameters at baseline.
The experimental group of rats (n = 40) was fed a vitamin A deficient diet
for 4 weeks, during which 6 rats died. All others developed unmistakable clinical signs of vitamin A deficiency, which included hair loss, easy pluckabiliy,
discoloration in fur and corneal changes. The surviving 34 rats were divided
randomly into five groups. One group of five rats was sacrificed and autopsied
immediately to obtain liver and serum vitamin A levels. A second group of 5
rats was continued on the vitamin A deficient diet for 4 more weeks by which
time all of them died. The three other groups of 8 animals each were repleted
with a) synthetic vitamin A (Group A), b) fresh drumstick leaves (Group B) or
c) blanched and sulphited shade dehydrated drumstick leaves (Group C). Both
fresh and dehydrated drumstick leaves were analyzed for vitamin A and the
quantities added corresponded to four times the amount of synthetic vitamin
A (4000 IU/kg diet) given to animals in Group A. The repletion period was
four weeks at the end of which the animals were sacrificed and all analyses
carried out.
During the course of the 8-week study period, food intakes and body
weights were recorded every other day. The animals were also observed for
the presence of clinical signs and symptoms of vitamin A deficiency.
Diet. A synthetic diet consisting of cornstarch, casein and groundnut oil, to
which water soluble and fat soluble vitamins [18] and mineral mix [19] were
added, was used. The control group of rats received this diet complete with
vitamin A, while the experimental group of rats had no vitamin A added.
Both diets were isocaloric and isonitrogenous. During repletion, the same
diet was fed with the addition of synthetic vitamin A or fresh or dehydrated
drumstick leaves such that the amount of leaves added was equivalent to 4000
IU vitamin A acetate/kg diet, using a conversion factor of 4 from carotene
to vitamin A. All the calculations were based on the -carotene value of
drumstick leaves analyzed in this laboratory, 19000 g/100 gm fresh weight,
using the conversion 1 IU = 0.6 g -carotene. The fresh leaves were minced
finely with a pair of stainless steel scissors and mixed with the vitamin A

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deficient diet everyday. The dehydrated leaves were prepared twice during
the study by steam blanching fresh leaves for 5 min followed by sulphiting
using 3% potassium meta bisulphite solution (3 min), and shade dehydration
as described in an earlier paper, which showed a retention of 50% -carotene
from drumstick leaves after a 3 month storage [6]. The dried leaves were
powdered using a blender, sieved and stored in an airtight double lidded jar.
Diets were made in sufficient quantities to last for a week.
Feeding and care of animals. The animals were housed in individual cages.
Food and water (deionized) were given ad libitum. A 12-h dark/light cycle
was maintained. Individual body weights and food intakes were recorded
every other day during the 8-week study period.
Tissue collection. After noting the food intake and body weight, the animals
were lightly anaesthetised with chloroform, and blood was collectcd by severing the jugular vein. If inadequate blood was obtained from the jugular vein,
the heart was punctured and blood was collected. The test tube in which blood
was collected was wrapped and covered with an aluminium foil and allowed
to stand in the dark at room temperature for about an hour to permit the blood
to clot. Thereafter, it was centrifuged (Remi, India) for 10 min at 1500 rpm
for the separation of the serum. The serum was stored in a freezer until the
analysis of retinol, which was usually done within 24 hours of sacrifice. The
liver was quickly removed. The non-hepatic tissues were removed from the
liver, which was then blotted on a filter paper and weighed. Each liver was
wrapped in aluminium foil and stored in the freezer (20 C) until analysis
of retinol content which was usually done within 24 hrs of sacrifice. The
kidneys, spleen and testes were also removed, cleaned and weighed.
Parameters studied. Food intake was measured as the difference between
food given and the sum of food spilled and food leftover. The change in
weight per day was averaged over a week. The organ weights were recorded
using an electronic balance (Sartorius). Clinical signs and symptoms were
recorded using the methods detailed by Dawson [20]. Biochemical parameters included measurement of serum and liver vitamin A using the Neeld &
Pearsons method [21].
Statistical analysis. Means and standard errors were calculated for all the
parameters. Paired and independent t-tests and ANOVA were performed to
test the differences between the control and repleted groups. Correlations
were performed to find the relationship between the serum and liver retinol
levels. All differences were accepted as significant at p 60.05.

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Figure 1. Food intake of rats fed drumstick leaves.

Results and discussion

Food intake. The actual weekly food intake is shown in Figure 1. No significant change between the control (vitamin A sufficient diet) and experimental
group (vitamin A deficient diet) rats was seen until the 3rd week of the study
period. Thereafter, a significant decrease in the food intake was seen in the
experimental rats. Murthy et al. [22] have reported that dietary vitamin A
deprivation resulted in a gradual atrophy of the circumvillate papilla resulting
in a loss of taste. Similar observations were made by Zile et al. [23] who reported that, in animals, vitamin A deficiency manifested first by retardation in
growth and loss of appetite followed by decrease in storage depots of vitamin
A. As mentioned in the methods section, 3 groups of 8 vitamin A deficient rats
each were repleted with either synthetic vitamin A or -carotene following
the 4 week depletion period.
ln the control group of rats, the average food intake stabilized at 27 g/day
from the 5th week onwards. In the group of experimental rats continued on
the vitamin A deficient diet for 8 weeks, food intake declined from 8.4 g/day
in the 5th week to an average of 3.4 g/day in the 7th week of the study, after
which all the rats died. Therefore, for the 8-week depletion period, serum and
liver vitamin A could not be obtained.
The food intake increased significantly on repletion with various sources
of vitamin A. The highest change in food intake was recorded for Group C, in
which the rats had an average daily intake of 16.5 g in the 5th week and 36.0

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Figure 2. Body weights of rats fed drumstick leaves.

g by the end of 4 weeks of the repletion period. In comparison, the animals

in Group A increased their food intake from 16.6 g in the 5th week to 24.9 g
in the 8th week and Group B from 16.5 g to 21.8 g.
Thus, it can be interpreted that, -carotene from both fresh and dehydrated
drumstick leaves was highly accepted by the vitamin A deficient animals;
their appetite was returned to normal with no difference in the food intakes
seen between the synthetic vitamin A group (Group A) and those fed the
drumstick leaves (Groups B and C).
Body weight. As seen trom Figure 2, the control group of rats had an increase in weight from the initial mean of 58.3 g to the final of 146 g at the
end of 8 weeks of study. The experimental group of rats depleted of vitamin
A, lost weight significantly beyond the 3rd week of the study. Thereafter, the
animals fed vitamin A deficient diets continued to lose weight and none in
the group continued on the vitamin A deficient diet for 8 weeks survived.
The three groups of rats which were repleted after 4 weeks of depletion
started gaining weight by the end of the 1st week of repletion, attained body
weights similar to the control group by the end of the 3rd week of repletion
and surpassed the control group by 4 weeks of repletion. The present study
thus corroborated the results of Anzano et al. [24], who have shown a similar
pattern of body weight gain upon supplementation. It is clearly evident that
drumstick leaves in fresh as well as dehydrated forms were equally effective
in supporting growth and restoring it to normal in vitamin A deficient animals.

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Figure 3. Organ weights of rats fed drumstick leaves.

Effciency of food utilization. Efficiency of food utilization was calculated

by noting the changes in the body weights of the rats in relation to the food
intake. A marked increase was seen in the efficiency of food utilization values
in the repletion phase in which the weight gain was better than in the control
groups throughout the last 4 weeks of the study. Thus, the food efficiency
ratios suggested that the -carotene availabilities of both fresh (FER, 0.9)
and dehydrated (FER, 0.8) drumstick leaves were very good and were similar
to those of synthetic vitamin A (FER, 0.8) by the 8th week.
Organ weights. The liver weight of the control group was 2.42 g at 4 weeks
and 9.54 g at 8 weeks compared to 1.0 g at baseline. The liver weight of the
vitamin A depleted rats at 4 weeks was only 1.9 g. On repletion, a significant
increase in the liver weights occurred, restoring them to the same size as that
seen in control rats at 8 weeks (Figure 3). The weights of kidneys, spleen and
testes followed a similar pattern. Organ weights expressed as percent body
weights followed a similar trend on depletion and repletion.
Clinical signs and symptoms. Marked clinical signs and symptoms of vitamin A deficiency were observed by the end of the third week in the experimental groups, fed the vitamin A deficient diet. Apparent hair loss characterized by easy pluckability from the coat, discoloration in the fur and
corneal changes, loss of luster and small amounts of porphyrin were evident
around the eyes, which progressed as the deficiency continued. A swelling
of the eyelids was observed and the anterior segment of the eye had a char-

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Figure 4. Serum retinol levels of rats fed drumstick leaves.

acteristic dry appearance. A small white spot was observed in two of the
experimental group rats. Dawson et al. [20] reported that all mammalian species depleted of vitamin A show rod dysfunction, conjunctival and corneal
xerosis. These changes occur only after liver and serum retinol levels are
substantially depleted.
On repletion with either vitamin A or drumstick leaves, the ocular signs
disappeared by 3 weeks of repletion indicating that the -carotene from the
fresh and dehydrated drumstick leaves was efficiently absorbed and was able
to reverse the signs of xerophthalmia. Mariath et al. [25] have also shown a
reversal of clinical xeropthalmia after feeding fruits of palm tree (Mauritia
vinifera) providing 134 g of retinol equivalents for 20 days to rats.
Serum vitamin A. Serum levels are altered markedly in extreme conditions
of hypo- and hypervitaminosis A [13]. The serum retinol levels measured at
the 4th week revealed that a rise from the baseline value of 29.2 g/dL to 34.8
g/dL was evident in the control group of rats, whereas a significant decline
to 19.1 g/dL was seen in the rats depleted of vitamin A (Figure 4). The
rats continued on the control diet for another 4-week period showed a small
increase in serum retinol level. On repletion, the maximum rise was seen
in Group A (given vitamin A acetate) (34.7 g/dL), followed by Group C
(given dehydrated drumstick leaves) (28.2 g/dL) and group B (fresh drumstick leaves) (25.8 g/dL). Though the serum retinol levels of the groups

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Figure 5. Liver retinol levels of rats fed drumstick leaves.

supplemented with -carotene in the form of drumstick leaves were lower

than the synthetic vitamin A group; they were signifcantly higher than the
serum retinol levels seen after 4 weeks of depletion. The serum retinol levels
by 4 weeks of repletion in Groups B and C approached the value seen in the
animals at baseline (29.2 g/dL) to support the argument that foods containing provitamin carotene, especially leafy vegetables are valuable as a source
of vitamin A.
Liver vitamin A. Since 90% of the bodys reserves of the vitamin A are
stored in the liver and these are readily depleted during vitamin A deficiency,
measurement of hepatic vitamin A content yields the most accurate information about the vitamin A status [17]. Figure 5 data reveals that the deposition
of vitamin A in the control group of rats at 4 weeks (7.1 g/g liver) and
8 weeks (6.2 g/g liver), significantly exceeded that at baseline (3.7 g/g
liver). In the experimental group at 4 weeks, the liver retinol was 2.0 g/g
liver, less than 1/3 of the control rats. A decline in the hepatic retinol levels
in vitamin A deficiency have been reported by Mejia et al. [27] and Chauhan
& Kansal [28]. Significant increases in the liver retinol levels were observed
in all the repleted groups, wherein group C had the maximal value of 6.0
g/g liver, approaching the value seen in the control rats at 8 weeks (6.2 g/g

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liver). The coefficient correlation between the serum vitamin A and the liver
vitamin A of the repleted animals was 0.16 which was not significant at 5%
level. These results show that the serum vitamin A values did not increase in
tandem with liver vitamin A upon repletion [29].
From these results, it is apparent that the drumstick leaves (both fresh and
dehydrated) produced an increase in food intake and weight gain comparable
or superior to that in animals fed synthetic vitamin A in vitamin A depleted
rats. The serum and liver levels also rose significantly upon repletion with dehydrated and fresh drumstick leaves, although the magnitude of rise in serum
vitamin A levels was somewhat lower compared to those noted in synthetic
vitamin A fed rats. When the fresh and dehydrated drumstick leaf fed groups
were compared, the latter was found to be better as a source of vitamin A.
The higher bioavailability of -carotene in dehydrated drumstick leaves seen
in the present study could have been due to:
1. The partial cooking of dehydrated leaves during blanching and sulphiting.
2. Reduction in the matrix effect as a function of the dehydrated leaves
being finely powdered.

Conclusions
These results highlight the importance of plant sources of vitamin A in diets and underscore their equivalence to synthetic vitamin A. Since the food
supplements of -carotene had a therapeutically similar effect to vitamin A
acetate in the vitamin A deficient rats, the study has important public health
implications for the control of vitamin A deficiency. Fresh or dehydrated
drumstick leaves can be good sources of vitamin A in supplementary feeding
program and need to be promoted.
Acknowledgments
The authors are grateful to the University Grants Commission, New Delhi,
India, for awarding the junior and senior research fellowship to Dr Vanisha
Nambiar.
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