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PHYTOCHEMICAL ANALYSIS OF
MORINGA OLEIFERA SEED POWDER
214 Chapter VII
7.1 Introduction
500 mg of the dried seed sample of PKM-1, PKM-1 (A4+V+N) and wild
varieties of M. oleifera were extracted with 10ml each of phosphate buffer (39ml
of 0.1M monobasic sodium phosphate and 61 ml of o.1M dibasic sodium
phosphate).The extracts were centrifuged and the supernatants were used for
protein estimation.
Phytochemical Analysis of Moringa oleifera Seed Powder 215
iii) Alkaline solution: It was freshly prepared each time just before use by
mixing 50 ml of reagent 1 and 1ml of reagent 2.
Pipetted out 0.2, 0.4, 0.6, 0.8 and 1ml of the working standard solution of
BSA into a series of test tubes and 0.1ml of the sample extract in two other test
tubes. Made up the volume to 1ml in all test tubes. A test tube with 1ml of distilled
water served as blank. 5 ml of alkaline copper solution was added to each test tube
including the blank. Mixed well and allowed to stand for 10 minutes. Then 0.5 ml
of Folin-Ciocalteau reagent was added to each tube, mixed well and incubated at
room temperature in dark for 30 minutes. Optical density was taken at 660 nm
with spectrophotometer. A standard graph was drawn using the OD value of BSA
dilutions and the amount of protein in the sample was calculated by referring the
standard curve of BSA. Results were expressed in mg protein / gm dry weight.
The seed materials of PKM-1, PKM-1 (A4+V+N) and wild Moringa were
ground into fine powder and defatted using soxhlet apparatus with solvent hexane
for 24 hours. 100 mg each of the defatted seed powder was weighed and extracted
in 10 ml each of solvents, such as distilled water, 1% NaCl, 0.1 N NaOH and 70%
ethanol according to the procedure outlined by Harborne (1973). The extracts were
centrifuged at 5000 rpm for 15 minutes. 10 ml of ice cold TCA was added to each
of the supernatant and left for 30 minutes at 400C and centrifuged at 10,000 rpm
216 Chapter VII
for 10 minutes. Each of the sediments was dissolved in 4 ml of 0.2N NaOH. 0.1 ml
of each solution was pipetted out in different test tubes and the protein estimation
was done according to the procedure described above for total protein estimation
by Lowry’s method. 7.2.3 Assay of partially purified Moringa oleifera seed
protein on turbidity removal from synthetic turbid water
turbidity of the supernatants was measured including the control after 60 and 120
minutes of sedimentation and the NTU values were tabulated.
36.34 g Tris HCl was dissolved in distilled water; 8 ml of 10% SDS was
added to this and made up the mixture to 200 ml with distilled water.
12.1 g Tris HCl was dissolved in distilled water; 8ml of 10% SDS was
added to this and made up the mixture to 200 ml with distilled water.
It was prepared freshly at the time of use by dissolving 50mg of APS in 500
µl of distilled water.
3 g Tris HCl, 14.4 g glycine and 1g of SDS were dissolved in distilled water
and made up to 1000 ml with distilled water.
Acrylamide : 3.3 ml
Lower Tris (pH 8.8) : 2.5 ml
Distilled water : 4.1 ml
APS : 50 µL
TEMED : 7 µL
Mixed the components of separating gel just before loading into the
sandwich template. Pipetted out required quantity of this solution into sandwich
template carefully by keeping the gel margin uniform at the top. Added 1 cm of
water on the top of the separating gel solution and allowed the gel to polymerize
for 60 minutes.
Acrylamide : 1 ml
Lower Tris (pH 6.8) : 1.5 ml
Distilled water : 2.4 ml
APS : 45 µL
TEMED : 7 µL
Equal quantities of the protein sample and sample buffer were mixed in an
eppendorf tube and heated it at 1000C for 3 minutes. The protein markers were
loaded in the first well and the fractionated seed protein samples of PKM-1, PKM-
1(A4+V+N) and wild Moringa into the other wells carefully without air bubbles.
After loading the samples electrophoresis buffer was filled in the top and bottom
reservoirs.
the first well and the protein samples into the other wells carefully without air
bubbles. After loading the samples electrophoresis buffer was filled in the top and
bottom reservoirs.
7.2.3.3.4. 1 Requirements
i) Staining solution
7.2.3.3.4.2. Procedure
The gel was kept in the staining solution in a flat container with lid and
agitated for 5 hours on a slow rotary shaker. Covered the container during staining
to avoid evaporation of the staining solution. After staining the staining solution
was poured out and the gel was rinsed with distilled water. Then the destaining
solution was added into the container and kept the gel in the solution keeping the
container closed for 2 hours for destaining. Then it was rinsed with distilled water,
visualized through a luminescent box and the photograph was taken.
220 Chapter VII
The seeds of PKM-1 and wild Moringa were dried at 55oC in an oven and
ground into fine powder and sieved. To 400 mg of the seed powder in a conical
flask, a mixture of 40 ml of diethyl ether and 1% acetic acid (v/v) was added and
mixed thoroughly to remove the pigmented material if any present. Discarded the
supernatant after 5 minutes and 20 ml of 70% aqueous acetone was added. Sealed
the flask with cotton plug and covered it with aluminum foil and kept in a shaker
for 2 hrs for extraction. The extracts were then filtered through Whatman No.1
filter paper and the filtrates were kept in refrigerator at 40C until analysis.
50 µL of each tannin extract was taken in a test tube and made up to 1ml
with distilled water. 0.5ml of Folin-Ciocalteau reagent was added and mixed well,
and kept at room temperature for 40 minutes. OD was taken at 725 nm using
spectrophotometer. Prepared a standard graph using standard solution of tannin
and calculated the amount of tannin in the test sample with the help of standard
graph.
sodium carbonate until effervescence ceased. Made up the volume to 100 ml and
then centrifuged.
Pipetted out 0.2, 0.4, 0.6, 0.8 and 1ml of the working standard carbohydrate
solution into a series of test tubes along with 0.1 ml of each sample extract in two
other test tubes. Made up the volume to 1ml in all test tubes and added 1ml of
phenol solution to each test tube including one test tube with distilled water
serving as the blank. Added 5 ml of 96% sulphuric acid to each test tube including
the blank and mixed well. After 10 minutes shaken the contents in the test tubes
and placed in a water bath at 25-300C for 20 minutes. OD was taken at 490 nm.
Prepared a standard graph and calculated the amount of total carbohydrate present
in the test samples using standard graph.
100g of the seed powder was extracted with petroleum ether for 6h in a
soxhlet apparatus. The extract was kept on a water bath to evaporate the petroleum
ether until no odour of ether remains. Cool at room temperature. Weighed the oil
content in the flask. The percentage of lipid in the seed was calculated by the
formula:
Weight of lipid
% of lipid in the seed sample = × 100
Weight of sample
1 gm each of the two varieties of Moringa seed sample was taken and
extracted using phosphate buffer saline (PBS) solution. Centrifuged and the
supernatants were collected for phenol estimation.
1 g each of two seed samples were extracted using 4% TCA and the volume
is made up to 10 ml with the same. Centrifuged the samples at 2000 rpm for 10
minutes. A pinch of charcoal was added with the supernatant and stirred
vigorously using a cyclomixer and kept it for 5 minutes. Again centrifuged the
mixture to remove the charcoal particles and the supernatant was collected and
used for estimation. Standard ascorbate ranging between 0.2-1.0 ml was taken in a
series of test tubes along with 1 ml each of supernatants of the samples in
duplicates were taken in test tubes. The volume was made up to 2 ml with 4 %
TCA in all the samples. 0.5 ml of dinitrophenyl hydrazine (DNPH) reagent was
added in all test tubes followed by 2 drops of 10% thiourea solution. The contents
were mixed and incubated at 37°C for 3 hrs resulting in the formation of osazone
crystals. The crystals were dissolved in 2.5 ml of 85% sulphuric acid in cold.
DNPH reagent and thiourea were added to the blank alone, after the addition of
sulphuric acid Tubes were cooled in ice and the measured absorbance at 540 nm
with spectrophotometer. Prepared a standard graph and the ascorbate content in the
samples were calculated by comparing with the graph readings and the value was
converted into percentage.
7.3 Results
7.3.1 Estimation total protein and the various proteins in different samples
The total proteins and the proteins in four different solvent extracts
(distilled water, 1% NaCl, 0.1N NaOH and 70% ethanol) of the defatted seed
powder of the PKM-1 and wild varieties of M. oleifera were estimated. When the
quantity of proteins in each sample of PKM-1 seed powder was analysed and
compared with the seed powder of wild Moringa, it was found that, the PKM-1
variety of seed extracts have higher amount of protein in all the samples than the
wild variety. The total protein in the PKM-1 and PKM-1 (A4+V+N) seed powder
were 61.4±0.3 % and 61.2±0.2 % respectively, while that of wild Moringa was
56.3±0.1 % (Table-7.1). The amount of protein in mg/100mg (%) of seed powder
in different solvent extracts of PKM-1 were observed as 58.2±0.4, 52.4±0.1,
Phytochemical Analysis of Moringa oleifera Seed Powder 223
32.6±0.3 and 28.3±0.1 for distilled water, 1% NaCl, 0.1N NaOH and 70% ethanol
respectively and the same in PKM-1 (A4+V+N) were 58.1±0.2, 52.3±0.1, 32.4±0.2
and 28.1±0.3. Whereas the protein content in the defatted seed extracts of wild
Moringa was 52.5±0.5, 41.4±0.2, 24.6±0.2 and 22.4±0.3.
Table 7.1 Quantity of total protein and proteins in the four extracts of the of
defatted seed powder of M. oleifera varieties
PKM-1 PKM-1(A4+V+N) WM
% % %
7.3.2 Partial purification of water and NaCl extracted protein and the assay
of their turbidity removal efficiency
As the water and NaCl extracts were the effective ones in all varieties of
Moringa seeds, an attempt was also made for the partial purification of seed
protein of these extracts by ammonium sulphate precipitation and fractionation,
using 40, 60 and 80% ammonium sulphate solutions. The different fractions were
tested for their purification efficiency using synthetic turbid waters made with
bentonite clay. The high and low turbidity solutions of bentonite powder were used
for the analysis and the turbidity of non-treated (control) and treated samples were
measured. It was found that the highest turbidity removal occurred in samples
treated with 60% fractions of water and NaCl extract of PKM-1 as well as wild
Moringa seeds (Tables-7.2&3). Among the water and NaCl extract fractions, NaCl
fractions were found to be more effective than water fractions. Among the
Moringa varieties, the NaCl fraction of PKM-1 types were observed to be better
than wild Moringa seed powder fractions (Tables-7.2&3).
224 Chapter VII
Fig. 7.1 SDS-PAGE showing separation of 13.4 kda protein from NaCl extracts of
wild, PKM-1 and PKM-1(A4+V+N) varieties of M. oleifera Lam.
226 Chapter VII
PKM-1 and wild Moringa seeds were analysed for other components apart
from proteins (Table-7.4). Carbohydrate content was found to be higher in wild
Moringa (7.68±1.2%) than PKM-1 and PKM-1 (A4+V+N) (6.05±1.5%&6.02±2.2).
Similarly, free amino acids also were higher in wild variety (4.31±0.01%) than
PKM-1 and PKM-1 (A4+V+N) (3.29±0.01&3.31±0.02%). On the contrary, the
quantity of lipids was found to be higher in PKM-1 and PKM-1 (A4+V+N)
(43.2±0.2 & 42.8 ±0.3%) than wild Moringa (38.7±0.2%). Tannins also were higher
in amount in the PKM-1 and PKM-1 (A4+V+N) (0.69±0.02&0.69±0.03 %) than
wild Moringa (0.55±0.02%). Phenol content was found to be more in PKM-1 and
PKM-1 (A4+V+N) (0.325±0.02&0.316±0.03%), while in wild variety it was less
(0.184±0.02%). The amount of ascorbic acid was not having much difference
(0.93±0.02, 0.90±0.03 and 0.87±0.03% in PKM-1, PKM-1 (A4+V+N) and wild
Moringa respectively) (Table-7.4). β carotene content was analysed, but it was
found to be insignificant in both the varieties and the result is not included here.
Carbo Free
Lipids Tannins Phenol Ascorbic
hydrates amino acids
% % % acid %
% %
6.05 43.2 3.29 0.69 0.325 0.93
PKM-1
±1.5 ±0.2 ±0.01 ±0.02 ±0.02 ±0.02
PKM-1 6.02 42.8 3.31 0.69 0.316 0.90
(A4+V+N) ±2.2 ±0.3 ±0.02 ±0.03 ±0.03 ±0.03
7.68 38.7 4.31 0.55 0.184 0.87
WM
±1.2 ±0.2 ±0.01 ±0.02 ±0.02 ±0.03
7.4 Discussion
Comparative study of the seed protein content of the defatted seed powder
of PKM-1 types and wild Moringa extracts, in the four solvents studied here were
not reported anywhere in literature. But there are studies regarding the content of
protein in the crude extract and water extract. The water soluble kernel crude
Phytochemical Analysis of Moringa oleifera Seed Powder 227
protein of wild Moringa was reported as 36.8% by Foidl et al. (2001). They also
found that the loss of dry matter from kernel and meal (fat free kernel) after water
extraction was 20.5 and 41.8% respectively. But they added that the residues left
after water extraction of kernels or meal had crude protein contents of 35.3 and
70.3 % respectively. This is an evidence for the presence of other proteins in the
kernel and meal (defatted seed powder), which are not water soluble. Compaore et
al. (2011) observed that he seeds of M. oleifera are rich in proteins having
35.37±0.07 g/100 g in the crude kernel. The seed material in the present study was
the seed meal after oil removal, in which the amount in the water extract of wild
Moringa was 52.5%. The protein content in four different extracts on comparison
found that, among the studied varieties water extracts contain the highest quantity
of protein, which is followed by NaCl, NaOH and ethanol extracts in a descending
order. The least content of protein was found to be present in the ethanol extract
(Table-7.1).
that the major active components responsible for the water purification and
antimicrobial ability of M. oleifera seed extract are proteins.