Cotton is a leading plant fiber crop worldwide, grown in temperate and tropical regions of 50 countries. Cotton seed is valuable foodstuff for cattle. The present study provides a detailed summary of the nutritional content of seeds of Ajeet – III BG – II and Tulasi – 144 BG- II to give clear standards for identification of the drug. These samples were air dried for a week, powdered and then subjected to proximate analysis. Chemical analysis revealed the amount of moisture, ash, Water soluble and insoluble ash, acid soluble and acid insoluble ash, calcium, magnesium, crude fiber, lipids, crude protein, oxalates, oil, defatted seeds, carbohydrates and the presence of flavonoids, alkaloids, tannin, phenolic compounds, steroids, sterols, saponin, glycosides, amino acid and proteins by phytochemical analysis and the CHNS elemental analysis revealed the amount of carbon, nitrogen, hydrogen, sulphur. This study shows that these seeds find use in the production of therapeutic agents and domestic and industrial oil.

1. ISSN 2229-760X (Print)
ISSN 2319-7625 (Online)
Journal of Chemistry and Chemical Sciences, Vol.7(6), 425-434, June 2017
(An International Research Journal), www.chemistry-journal.org
425
Comparative Study of Seeds of Ajeet – III BG – II
and Tulasi – 144 BG- II of Cotton (Gossypium)
for the Chemical and Biochemical Contents,
Minerals and Trace Elements
Nikita K. Chamria, Arvind Singh Heer, Sajid M. Mansoori and Satish R. Ingale
Department of Chemistry,
Mithibai College, Ville Parle (West), Mumbai-400056, Maharashtra, INDIA.
email: nikskhushi1@gmail.com.
(Received on: June 10, Accepted: June 17 , 2017)
ABSTRACT
Cotton is a leading plant fiber crop worldwide, grown in temperate and
tropical regions of 50 countries. Cotton seed is valuable foodstuff for cattle. The
present study provides a detailed summary of the nutritional content of seeds of Ajeet
– III BG – II and Tulasi – 144 BG- II to give clear standards for identification of the
drug. These samples were air dried for a week, powdered and then subjected to
proximate analysis. Chemical analysis revealed the amount of moisture, ash, Water
soluble and insoluble ash, acid soluble and acid insoluble ash, calcium, magnesium,
crude fiber, lipids, crude protein, oxalates, oil, defatted seeds, carbohydrates and the
presence of flavonoids, alkaloids, tannin, phenolic compounds, steroids, sterols,
saponin, glycosides, amino acid and proteins by phytochemical analysis and the
CHNS elemental analysis revealed the amount of carbon, nitrogen, hydrogen,
sulphur. This study shows that these seeds find use in the production of therapeutic
agents and domestic and industrial oil.
Keywords: Ajeet – III BG – II and Tulasi – 144 BG- II of cottonseed, cottonseed oil,
proximate analysis, phytochemical analysis and elemental analysis.
1. INTRODUCTION
Cotton is a natural vegetable fiber obtained from the cotton plant of the genus
gossypium and belongs to Malvacae family, a shrub which grows to about 40 cm high in
tropical and subtropical areas with its flowers either red or yellow. Gossypium (cotton) is
economically the most important plant comprising of around 50 species1
. Cotton was first

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426
cultivated in 5th millennium BC by Indus valley civilization. The capsules (seeds) are formed
as soon as the petals fall off and burst open into four parts upon maturity thereby revealing the
cottonseeds after which they are harvested mechanically. The short cellulose fibers are used
for the making of coarse yarns and many cellulose products. The outer coverings of seed are
used as roughage in ruminant animal feed and oil extracted from seed kernel is used for
cooking, salad dressing due to its flavour stability. The remaining cake is added as a
supplement in cattle and swine feed2
. Defatted cottonseed flour is used generally for animal
nutrition and only one fourth it potentially available could satisfy the present worldwide
shortage of protein3
.
Cottonseed oil is mainly used for edible purposes, a large portion of which is used in
the manufacture of margarine and a small amount is consumed as salad oil in Turkey4
. Crude
cottonseed oil from the mill requires further processing before it is used in food. The first step
in this process is refining to produce refined grades, the treatment being similar to that used
for soybean. It involves alkali refining to remove impurities, bleaching with activated clays
and finally, steaming under vacuum conditions to remove traces of odour. The refining process
also removes darker colouring materials present, leaving clear yellow oil5
. In literature review
concerning cotton seed oil, the % oil content of cotton seed is 31.33 and in the characterization
of the oil, % of unsaturated fatty acid is more of oleic and linoleic acid (74%) and the results
of physicochemical properties obtained, showed that the oil has high industrial value6
.
Cotton seeds have been used as antidote for Somal (Arsenic Trioxide) and metal
poison7
. Seeds are sweet, nerving tonic, emollient, and anodyne, anti-dysenteric, galactagogue,
expectorant, aphrodisiac, diuretic, laxative, antipyretic and tonic. They are used internally in
epilepsy, amentia, psychosomatic disorders, meningitis, tinnitus, earache, dyspepsia,
dysentery, orchitis, piles, cough, bronchitis, galactic, strangury, general debility, hyperpyrexia
with chills. Seeds are externally applied in inflammation, burns, scalds, ulcers, joint pains8
.
The seeds are also found to show pharmacological activities like antioxidant, antibacterial,
anti-fertility, anti-spermatogenic, antitumor, contraceptive, anti-diabetics, and antiviral9
.
Cotton seeds are also used in Ayurveda to cure many diseases like Kasa, Balgraha, hatura
visha, Somal Visha, Pakshaghat and for Nasya, Abhyanga10
.
Cottonseed oil finds application in the production of biscuits, crackers, doughnuts and
potato chips and the preparation of ice cream substitutes (mellorines), where the oil replaces
butter fat. Industrial uses of cottonseed oil include alkyl resins for interior paints, special
lubricants and soft soaps11
.
Hence, the seeds were both qualitatively and quantitatively analysed to determine the
nutritional content and various other parameters, to know about its properties and to validate
the compounds present.
2. EXPERIMENTAL
Sample Collection
The cottonseeds under investigation were procured from Jalgaon district (Maharashtra,
India).

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427
These seeds were authentic, healthy and matured.
2.1 Chemical analysis
Moisture Content
To determine the moisture content of the seeds, 2 g of seed powder were oven dried
for 2 hours at 1100
C and the loss in weight was recorded12
.
Crude Fibre
About 3g of finely powdered seeds were accurately weighted and transferred to an
extraction apparatus and extracted with petroleum ether (40-60)0
C for 18-20 hours, air dried
defatted powder was transferred to dry 100 ml conical flask, 200 ml of 0.25 N sulphuric acid
were added and contents were brought to the boiling point. Boiling was continued for exactly
30 minutes, maintaining a constant volume and rotating the flask after every few minutes in
order to remove the particles from the sides. The contents were filtered through Buchner’s
funnel under suction using circular filter paper (Whatman No. 41). The insoluble matter was
washed with boiling water until the washings were free from the acids. The residue was
washed back into the original flask along with 200 ml of 0.313N Sodium hydroxide. The
contents were brought to the boiling point and boiling was continued for exactly 30 minutes.
The whole insoluble matter was transferred to the filter paper by means of boiling water. It
was then washed with one percent hydrochloric acid and again with boiling water until free
from acid, and then it was washed twice with alcohol and thrice with ether. Finally, it was
transferred to a dried, previously weighed ash less filter paper and dried at 100ºC to the
constant weight. The increase in the weight of the filter paper was noted. The filter paper and
its contents were incinerated and ignited to ash in a silica crucible at dull red heat, cooled and
weighted. The weight of the ash subtracted from the increase of the weight on the paper due
to insoluble material, the difference was reported as crude fibre13
.
Lipids
Total lipids were extracted from the whole powder in the Soxhlet apparatus for 20
hours, using petroleum ether (40-60)0
C fraction as a solvent and estimated gravimetrically
after evaporating the solvent at 600
C14
.
Calcium
Prepare 50ml of acidic solution of extracts from powder of seeds with 1:1 HCl and
dilute upto 250ml in standard measuring flask with distilled water.
Pipette out 25ml of above solution in conical flask, add 2-3 drops of Patton-Reader
indicator and 8M KOH till the colour of Patton-Reader indicator appeared. Then add one and
a half test tube of 10% NH2OH.HCl solution till clear red solution is obtained and titrate
against 0.05M EDTA solution till the colour of solution changes to blue.

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428
1000 ml of 1M EDTA = 40.08g of Ca
Magnesium
Prepare 50ml of acidic solution of extracts from powder of seeds with 4NH2SO4 and
dilute upto 250ml in standard measuring flask with distilled water.
Pipette out 25ml of above solution in conical flask, add 10ml of buffer solution and
add 2-3 drops of Eriochrome Black T indicator and titrate against 0.01M EDTA solution till
the colour of solution changes from wine red to blue.
1000 ml of 1M EDTA = 24.32g of Mg
Ash and its analysis
About 5 g of the seed powder were ignited to the ash into a previously ignited and
weighted silica crucible. It was cooled in vacuum desiccators over concentrated sulphuric acid,
weighted and the increase over the first weight of crucible was taken as the ash content.
For the determination of water-soluble ash, whole ash was boiled with 25 ml distilled
water. The suspension so obtained was filtered through an ash-less filter paper (Whatman No.
41) and the residue were thoroughly washed with hot distilled water. The filter paper
containing the residue was ignited in the original crucible, cooled and the water insoluble ash
was weighted. From these data “water soluble” ash was calculated as follows:
Water soluble ash = Total ash – Water insoluble ash
(Percent) (Percent) (Percent)
The alkalinity of water-soluble ash was determined in the filtrate so obtained after
cooling and titrating against N/10 sulphuric acid, using methyl orange as indicator. The
alkalinity was expressed in terms of sodium carbonate miliequivalents.
1 ml N/10 H2SO4 = 0.1 miliequivalents Na2CO3
The acid insoluble ash content was determined by boiling the whole ash, equivalents
to 5 g seed powder in 25 ml dilute hydrochloric acid (10 percent, v/v) for five minutes and
filtering the suspension through an ash-less filter paper (Whatman No. 41). After washing
thoroughly with hot water, the paper containing the residue was again ashed in a pre-weighed
silica crucible. It was cooled and weighed again. The difference of the two weights gave the
“acid insoluble” ash.15
Oxalates
5g of seed powder was taken with 400ml distilled water in a 600ml Pyrex beaker and
it was kept
On sand bath, while covering the top of beaker with suitable round bottom flask containing
cold water to act as condenser. After boiling for half an hour 10ml of 20% Sodium Carbonate
solution was added and contents were stirred and cooked for another half an hour.
After cooking was done, the content was filtered hot by Whatman no.41. The filtrate
was allowed to settle down and enough HCl(1:1) was added drop by drop with constant stirring

5. Nikita K. Chamria, et al., J. Chem. & Cheml. Sci. Vol.7(6), 425-434 (2017)
429
until final acid concentration became 1%. Then the precipitate was allowed to settle and
supernatant liquid was filtered off through the filter paper. Then add Ammoniacal solution and
re-acidify with glacial acetic acid. Allow precipitate to settle overnight. Remove the clear
supernatant liquid and dissolve the precipitate in Sulphuric acid and titrate against 0.05N KMnO4.
1ml of 0.05N KMno4 = 0.00225g of anhydrous oxalic acid.
Carbohydrates
The total carbohydrate was estimated by Anthrone method. 1mg of Gossypium seed
powder was hydrolysed to simple sugars by keeping it in a boiling water bath for three hours
with 5ml of 2.5N HCl and cool to room temperature. After neutralizing, the contents were
centrifuged and 0.1ml of supernatant was used for the analysis. To the sample add 4ml of
anthrone reagent and the contents were heated in a boiling water bath for 8 minutes. The tubes
were cooled and read at 630nm using spectrophotometer Schimadzu-Model-UV 1800. The
standards were developed with glucose. Standard graph plotted was used to find out the
concentration of glucose present in the hydrolyzed extract.16
2.2 Phytochemical analysis17
Test for alkaloids
Wagner’s test
To the extract add few drops of iodine solution in potassium iodide. Reddish brown precipitate
shows positive result.
Hager’s test
To the extract add few drops of saturated solution of picric acid. Yellow colour precipitation
signifies positive result.
Test for steroids and sterols
Libermann-Burchard test
To the extract add 2ml chloroform, 10 drops of acetic anhydride, and 2 drops of concentrated
sulphuric acid. Bluish red to cherry red colour in chloroform layer shows positive result.
Salwoski test
To the extract add few drops of chloroform, concentrated sulphuric acid. Bluishred to cherry
red colour.
Test for Glycosides
Legal test
To the extract added pyridine, sodium nitroprusside. Positive result shows pink red colour.
Baljet test
To the extract add picric acid. Appearance of orange colour signifies positive result.
Test for saponins
Foaming test

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430
Foams produce when the extract is shake with water.
Test for flavonoids
Shinoda test
To the extract added magnesium turnings, 1-2 drops of concentrated hydrochloric acid.
Appearance of red colour indicates positive result.
Zinc hydrochloride test
To the extract added zinc dust, 1-2 drops of concentrated hydrochloric acid. Appearance of
red colour indicates positive result.
Test for tannin and phenolic compounds
Ferric chloride test
To the extract add ferric chloride. Formation of greenish black colour shows positive result.
Potassium dichromate test
To the extract add potassium dichromate solution. Positive result is confirmed by a formation
of brown precipitate.
Test for protein and amino acids
Biuret test
To the extract added 4% sodium hydroxide, few drops of 15% copper sulphate gives purple
colour.
Ninhydrin test
Bluish violet colour forms when a solution of ninhydrin and extract mixture was heated.
2.3 Elemental analysis
By the use of Elemental analyser amount of Carbon, Hydrogen, Nitrogen and Sulphur
was determined and by the help of amount of nitrogen, amount of crude protein in the plant
leaves was calculated, by the formula; Wp= (WN * 12.5)/1000.
3. RESULT AND DISCUSSION
The results of physical parameters of selected varieties (Ajeet – III BG – II and Tulasi
– 144 BG- II) of cottonseeds are summarized in table 1, phytochemical screening of the same
are summarized in table 2, elemental analysis summarized in table 3.
Chemical analysis revealed the amount of moisture, ash, crude fibre, proteins,
carbohydrates, oil, defatted seed, oxalates, calcium and magnesium. The two varieties Ajeet –
III BG – II and Tulasi – 144 BG- II are considered on various accounts. It was found that Ajeet
– III BG – II (4.35%) has less moisture as compared to Tulasi – 144 BG- II (6.20%). Lower
value of moisture content in Ajeet – III BG – II indicates high calorific value and high energy
and can therefore be used as a good biofuel. Ash usually represents the inorganic part of the
cottonseed. Ash value is useful in determining authenticity and purity of sample and also these
values are important qualitative standards. In the study, there was a marginal difference in the

7. Nikita K. Chamria, et al., J. Chem. & Cheml. Sci. Vol.7(6), 425-434 (2017)
431
ash content of Ajeet – III BG – II (4.947%) and Tulasi – 144 BG- II (5.105%) which was
subjected to further treatment to determine the amount of water soluble and insoluble ash, acid
soluble and insoluble ash. The seeds of Ajeet – III BG – II were found to contain higher water
soluble ash and so the acid soluble ash values also showed a hike. Water soluble ash in Ajeet
– III BG – II (46.81%) was much more than found in Tulasi – 144 BG- II (27.50%). Water
insoluble ash in Ajeet – III BG – II was 53.19% and in Tulasi – 144 G- II was 72.50%. Both
the varieties were found to be highly soluble in acid and the variation in acid soluble ash
content was negligible; the amount being 97.99% in Ajeet – III BG – II and 97.92% in Tulasi
– 144 G- II. The oil content in Ajeet – III BG – II (28.10%) was higher than that of Tulasi –
144 BG- II (20.10%).Cottonseed oil is controlled by multiple genes and also influenced by
environmental factors. The percentage of crude fibre and proteins in both varieties has minimal
differences. Amount of Crude fibre in Ajeet – III BG – II and Tulasi – 144 BG- II is 0.100%
and 0.175% respectively. Similarly, protein content in Ajeet – III BG – II and Tulasi – 144
BG- II is 35.56% and 36.34% respectively, the percentage of defatted seed in the former
(70.35%) is greater than the latter (67.41%). The amount of calcium in Ajeet – III BG – II is
0.802% and in Tulasi – 144 BG- II is 1.06% and the amount of magnesium in Ajeet – III BG
– II is 0.81% and in Tulasi – 144 BG- II is 0.97%.
The result of elemental analysis showed that both Ajeet – III BG – II and Tulasi – 144
BG- II had marginal differences in their carbon (52.78%, 51.953%), nitrogen (5.689%,
5.815%) and hydrogen (7.588%, 7.653%) contents respectively. Also, the amount of sulphur
in both the varieties was found to be zero.
Phytochemical analysis indicated the presence of alkaloids, flavonoids, tannins, amino
acids, glycosides, saponins, phenolic compounds and absence of steroids and sterols.
The result of proximate analysis and the elemental analysis of cottonseeds are
represented graphically in Fig No.1 and Fig No.2.
Table No. 1 Chemical analysis of cottonseeds
Constituent (%) Ajeet – III BG – II Tulasi – 144 BG- II
Moisture 4.35±0.0009 6.20±0.005
Ash content 4.947±0.002 5.105±0.018
Water soluble ash 46.81±0.003 27.50±0.004
Water insoluble ash 53.19±0.003 72.50±0.004
Acid soluble ash 97.99±0.004 97.92±0.0008
Acid insoluble ash 2.01±0.004 2.08±0.0008
Crude fibre 0.1 0.175±0.004
Crude protein 35.56 36.34
Defatted seed 70.35±0.049 67.41±0.304
Oil 28.10±0.001 20.10±0.001
Lipids 5.21±0.014 5.1±0.008
Carbohydrates 27.04 32.24
Oxalates 2.742±0.002 2.511±0.009
Calcium 0.802 1.06
Magnesium 0.81 0.97
*Values are means± SD (n=3), calculated as percentage on dry weight basis.

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432
Table 2: Phytochemical analysis of cottonseeds
Name of the test Ajeet – III BG – II Tulasi – 144 BG- II
Test for alkaloids:
a) Wagner test
b) Hagers test
+
+
+
+
Test for steroids and sterols:
a) Libermann – Burchard test
b) Salwoski test
-
-
-
-
Test for Glycosides:
a) Legal test
b) Baljet test
++
+
+
+
Test for saponins:
a) Saponin test + +
Test for flavonoids:
a) Shinoda test
b) Zinc hydrochloride test
+
+
+
+
Test for tannin and phenolic compounds:
a) Ferric chloride test
b) Potassium dichromate test
+
+
+
+
Test for amino acid and protein:
a) Biuret test
b) Ninhydrin test
+
+
+
+
+ Present, - Absent
Table 3: Elemental analysis of cottonseeds
Element (%) Ajeet – III BG – II Tulasi – 144 BG- II
Carbon 52.78 51.953
Nitrogen 5.689 5.815
Hydrogen 7.588 7.653
Sulphur 0.0 0.0
Figure 1: Graphical Representation of Chemical Analysis of Cottonseeds

9. Nikita K. Chamria, et al., J. Chem. & Cheml. Sci. Vol.7(6), 425-434 (2017)
433
Figure 2: Graphical Representation of Elemental Analysis of Cottonseeds
CONCLUSION
From the results of the analysis carried out, the seed and oil extracts of gossypium
exhibited good physiochemical properties and could be useful for industrial application. Low
moisture content is advantageous in terms of storage stability since the lower the moisture
content, the better the storability and suitability to be preserved for a longer period. It can be
concluded that Gossypium seeds can be used as a natural remedy for many ailments. The
different physicochemical parameters of the seeds were observed. The preliminary
phytochemical test of the crude extracts indicated the presence of different phytochemical
constituents and these can act as diagnostic tool for identification and authentication of raw
drug samples and play an important role in quality control and detection of adulteration.
ACKNOWLEDGEMENT
We wish to express our sincere gratitude to Mithibai College and Chemistry
Department, to give us chance to do research work, Head of the Chemistry Department, Mrs.
Rajeshwari Mirji, for providing all facilities to work in Laboratory, SAIF Department, IIT
Bombay; for analysing CHNS Estimation.
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