International Journal of Pharmacognosy and Phytochemistry, ISSN: 2051-7858, Vol.28, Issue.1
1140
Evaluation of Free Amino Acid and Total Protein
Content in Pollen of Some Species of Genus
Hibiscus of Family Malvaceae
Kalkar, S.A
Bhagat, I. P.
Department of Botany,
Institute of Science Nagpur – 440001
(M.S.), India
Email:surekhakalkar@gmail.com
Department of Botany,
Institute of Science Nagpur – 440001
(M.S.), India
Email:indralatapb@gmail.com
ABSTRACT
Present paper deals with analysis of free amino acids and
total protein content in pollen of some species of genus
Hibiscus of family Malvaceae. The plants under
investigation were Hibiscus rosa-sinensis L., Hibiscus
sabdariffa L.,Hibiscus schizopetalous Hook.f. & Hibiscus
panduraeformis Burms.f.. The biochemical evaluation of
these metabolites was performed by standard methods.
Amino acids were separated and identified by thin layer
chromatographic method and estimation of protein was
done by Lowry’s method. Among all the species the
highest number of amino acids were found in H.
sabdariffa(17) followed by H. rosa-sinensis(16), H.
panduraeformis(16) and H. schizopetalous(15) while the
highest protein content was found in H. sabdariffa(9.77%),
followed
by
H.
panduraeformis(5.33%),
H.
schizopetalous(4.70%) and H. rosa-sinensis(4.43%).
Keywords- Biochemical evaluation, amino acids, protein,
2. MATERIAL & METHODS
The extraction as well as qualitative analysis of the free
amino acids and estimation of protein was performed by
standard methods [2], [3]. 100mg of pollen powder sample
was homogenized with 10ml of 70% alcohol for half an
hour. Alcoholic extract was separated by centrifugation at
6000-7000 rpm and the residue was treated with a fresh
portion of the same solvent & separated. The same
procedure was repeated thrice, the pooled supernatant
were collected & the volume reduced by evaporation. This
extract was used to perform various qualitative test and
aliquot portion of the same were used for TLC study for
the analysis of free amino acid
2.1 Test for free amino acid
1.
Ninhydrin Test: Small quantity of test solution (1ml)
were kept in test tube and to it 1ml of Ninhydrin
reagent was added. If purple or yellow colour
develops, indicated the presence of amino acids.
2.
Xanthoproteic reaction: 1 ml of conc. nitric acid was
added to the test solution. Yellow colour developed, if
amino acids are present which turns bright orange on
addition of 40% NaOH.
3.
Millon’s reaction: Few drops of Millon’s reagent was
added to 1ml of test solution and heated for 10 min.
After cooling 5 drops of 1% sodium nitrite solution
was added. Amino acid particularly tyrosine was
present if brick red colour was developed.
4.
Glyoxylic reaction: In 2 ml of test solution, 2ml of
glyoxylic acid was added. After mixing well 2ml of
conc. H2SO4 was added down the sides of tube. A
violet ring appeared at the junction of two layers if
tryptophan is present.
5.
Pauly’s test: 1ml of sulphanilic acid (1% in 1% HCl)
was mixed with 2ml of test solution in the cold
condition. Then 1ml NaNO2 solution was added and
again kept in cold condition for few min.
Development of red colour indicates the presence of
amino acids.
6.
Sakaguchi test : 40% NaOH solution and test
solution were mixed (1:3) in test tube and 2 drops of
Hibiscus, Malvaceae
1. INTRODUCTION
Being the carrier of male genetic material, pollen is also
essential for the life cycle of the other living organism e.g.
forming the principle source of normal non –liquid food
for bees. Apart from this, pollen is known to have rich
source of biochemicals. Protein and amino acids are one
of the important constituents of pollen grains.
Regarding the free amino acid composition of different
pollen, all the essential amino acids have been reported to
be present in pollen and total level of free amino acids are
usually higher in pollen than in leaves and other tissues. It
is further reported that the amino acid content can vary
with climatic and nutritional condition of the plants on
which the pollen matures, as well as with storage &
handing methods. At the same time, the concentration of
all amino acids in pollen is considerably higher in bound
form than in free fraction [1].
The present study report the free amino acid & total
protein content of the pollen of four species of genus
Hibiscus of family Malvaceae viz. Hibiscus rosa-sinensis
L., Hibiscus sabdariffa L., Hibiscus schizopetalous Hook.f.
& Hibiscus panduraeformis Burms.f.
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1% alpha naphthol in alcohol and a few drops of
bromine water were added to the mixture.
Development of red colour confirms the presence of
arginine in the test solution.
4.
Qualitative analysis of the free amino acids by thin layer
chromatography (TLC) was carried out on square silica
gel coated glass plate of 20cm+20cm. using n-butanol,
acetic acid & water in ratio of 4:1:1 (v/v) respectively as
eluant. Then, 0.3% ninhydrin in butanol containing 3ml of
acetic acid solution was used for the detection of amino
acid by heating the plates at 1100c for 5 to 10 minutes &
the Rf values were calculated.
The free amino acid of the pollen of the four investigated
taxa is presented in Table 1 & 2. The qualitative test like
Ninhydrin
test,
Million’s
reaction,
Glyoxylic,
xanthoproteic test, Sakaguachi test have shown the
positive results in all investigated taxa.Positive results
confirmed the presence of amino acids like tyrosine,
tryptophan & arginene.
The TLC of pollen extract of all investigated plants along
with the samples of authentic amino acids indicated the
presence of various amino acids in different pollen grains
of studied plants. The common free amino acids like
cystein, valine, leucine, methionine, aspartic acid,
asparagines, lysine, proline, tyrosine, tryptophan &
histidine were found in all the studied taxa.
2.2 Tests for Proteins
Biurette test: Equal amount of test solution and 10%
sodium hydroxide was mixed thoroughly followed by
1% copper sulphate solution was added drop by drop.
A violet or pink colour is formed if proteins are
present.
2.
Xanthoproteic reaction Same as in amino acids.
3.
Glyoxylic test: Same as in amino acids.
Formaldehyde test: Equal volumes of test solution
and dilute formaldehyde solution were mixed in a test
tube and a few drops of conc. H2SO4 were added
carefully to the mixture. A violet ring is developed, if
proteins containing tryptophan is present.
3. RESULTS
Extraction of protein from pollen was carried out with
trichloroacetic acid, then with this extract various
qualitative test and quantitative estimation of protein were
performed by standard methods[3], [4].
1.
1141
The amount of protein present in the pollen of four
investigated taxa is presented in Table 3. The highest
amount of protein was fount in H. sabdariffa (9.77%)
followed by H. panduraeformis (5.33%), H.
schizopetalous (4.70%) and H. rosa-sinensis (4.43%)
Table.1.Qualitative analysis of free amino acids & protein by preliminary biochemical screening of pollen grains of
studied plants
Test
H. rosa- sinensis
H. sabdariffa
H. schizopetalous
H. panduraeformis
Test for Amino acids
1) Ninhydrin test
+
+
+
+
2) Xanthoproteic test
+
+
+
+
3)Millon's reaction
+
+
+
+
4) Glyoxylic test
+
+
+
+
5) Pauly test
+
+
+
-
6) Sakaguchi test
+
+
+
+
1) Biurette test
+
+
+
-
2) Xanthoproteic test
+
+
-
-
3) Glyoxylic test
+
+
+
+
4) Formaldehyde test
+
+
+
+
Test for Proteins
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International Journal of Pharmacognosy and Phytochemistry, ISSN: 2051-7858, Vol.28, Issue.1
1142
Table.2. Analysis of free amino acids by thin layer chromatography of studied plants.
Name of plants
S. No
Amino Acids
H. rosa- sinensis
H. sabdariffa
H. schizopetalous
H. panduraeformis
1
Glysine
+
+
-
-
2
Alanine
-
+
-
+
3
Serine
-
-
-
-
4
Crysteine
+
+
+
+
5
Threonine
+
+
+
+
6
Valine
+
+
+
+
7
Leucine
+
+
+
+
8
Isoleucine
-
-
-
-
9
Methionine
+
+
+
+
10
Asparatic acid
+
+
+
+
11
Asparagine
+
+
+
+
12
Glutamic acid
+
+
+
+
13
Glutamine
+
+
+
+
14
Arginine
+
+
+
+
15
Lysine
+
+
+
+
16
Proline
+
+
+
+
17
Phenylalanine
-
-
-
-
18
Tyrosine
+
+
+
+
19
Tryptophan
+
+
+
+
20
Histidine
+
+
+
+
16
17
15
16
Total Amino acids
- = Absent
+ = Present
Table3. Quantitative estimation of protein by Lowery Method of studied plants
S. N.
1
Name of plants
Hibiscus
sinensis
rosa-
2
Hibiscus sabdariffa
3
Hibiscus
schizopetalous
4
Hibiscus
panduraeformis
Percentage
of proteins
Average(%)
±
Standard
Deviation
4.43
0.21
9.77
0.15
4.70
0.26
5.33
0.15
4.5
4.6
4.2
9.9
9.8
9.6
4.6
4.5
5
5.2
5.5
5.3
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1143
about 50% of the species studied had 10 or more amino
acids. Results of present work showed that at least 15
amino acids were found in all taxa studied.
5.
CONCLUSION
The present findings indicate that proline and other
common essential free amino acids were present in all the
examined taxa of the family Malvaceae. This conforms
with the results of other studies on the composition of
amino acids in pollen. Among all four H. sabdariffa has
more amount of protein as well as amino acids it shows
that its ethanobotanical value.
REFERENCES
Fig.1. Estimation of Amino acids & protein in pollen of
studied plants
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4. DISCUSSION
In present investigation for analysis of amino acids,
proline and histidine was found in pollen of all
investigated taxa. Tupy in 1964 has reported that the
histidine – proline quotient in pollen represent an
important criterion of pollen fertility [5]. The potential role
of proline in pollen tube growth has also been suggested
[6].
Amino acids like arginine in certain pollen may have a
role in storage and transport [7], [8]. It is reported that the
levels of arinine, amides (aspagines & glutamine) and
proline significantly in pollen increased nitrogen in
fertilization.
It is very difficult to draw any conclusion on evolution
based upon the data on free amino acid content only, as
free amino acid composition greatly varies with climatic
and nutritional conditions as well as with storage and
handling patterns [9].
Proline in pollen is closely associated with their fertility
and is involved in pollen tube formation and in other
fundamental metabolic reactions associated with the
sexual process [1]. Proline have also reported in role of
protein synthesis [10].
Protein is one of the chief constituent of all parts of plants.
They are the chief body building materials. Besides they
also perform the function of antibody. The presence of
protein in the pollen grains has been reported by some
workers [1], [10]. The precise relationship between
various amino acids levels and the physiological status of
pollen is not very clear. Higher quantities of protein have
been found in fertile anther than in sterile.
The taxa which having more amino acid having more
amount of proteins. The protein and amino acid
complement not differ greatly in different species is also
proved [11]. In present work, pollen grains of H.
sabdariffa have also shown more amount of amino acid &
protein content as compared with other studied taxa.
Free amino acids from pollen of 107 different species have
also been studied by Barbier [12] and he was found that
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International Journal of Pharmacognosy and Phytochemistry, ISSN: 2051-7858, Vol.28, Issue.1
[13]
M. Barbier. Chemistry and Biochemistry of
pollen .Progress in Phytochemistry, Vol.2,
Reinhold & Liwschitz (Eds).Pub. Interscience,
London. 1970.
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