CHAPTER - VII

PHYTOCHEMICAL ANALYSIS OF
MORINGA OLEIFERA SEED POWDER
214 Chapter VII

7.1 Introduction

The M. oleifera seeds found to contain a coagulant protein which affected

the purification of drinking water (Ndabigengesere et al. 1995) and hence useful
for waste water treatment (Ndabigengesere and Narasiah,1998). The capacity to
coagulate the pollutants has made the Moringa seed powder of great interest in the
scientific world.

In the present investigation, the defatted seed powder of PKM-1, PKM-1

(A4+V+N) and wild variety of M. oleifera were extracted in four different solvents,
such as distilled water, 1% NaCl, 0.1N NaOH and 70% ethanol according to the
method suggested by Osborne (1924), based on the solubility properties of plant
proteins. The different levels of water purification showed by the different extracts
may be due to the variations in the quantity and quality of proteins present in these
extracts. Hence, the quantity of proteins in each extract and total proteins in the
seed powder were analysed and the most effective extract was partially purified by
ammonium sulphate precipitation and fractionation of the extract. The fraction
showing maximum clarification of synthetic turbid water was separated and its
molecular weight was assessed by SDS-PAGE. Along with this, some of the most
important phytochemical components in the PKM-1, PKM-1 (A4+V+N) and wild
Moringa were estimated by using non-defatted seed powder these varieties of
Moringa.

7.2 Materials and methods

7.2.1 Estimation of total protein by Lowry’s method (Lowry et al. 1951)

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

7.2.1.1 Reagents used

i) Alkaline sodium carbonate (Reagent-1): 2 g of Na2CO3 was dissolved in

0.1N NaOH and made up to 100ml using the same solution.

ii) CuSO4-Na-K-tartrate reagent (Reagent-2): 500 mg of CuSO4 was

dissolved in 100 ml of 1% Na-K- tartrate solution.

iii) Alkaline solution: It was freshly prepared each time just before use by
mixing 50 ml of reagent 1 and 1ml of reagent 2.

iv) Folin-Ciocalteau reagent : The commercial reagent was diluted with

equal volume of distilled water just before use.

7.2.1.2 Estimation of total protein

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.

7.2.2 Estimation of protein in the four extracts of PKM-1, PKM-1 (A4+V+N)

and wild Moringa seed powder

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

7.2.3 Assay of partially purified M. oleifera seed powder extracts on

turbidity removal from synthetic turbid water

7.2.3.1 Partial purification of Moringa seed extracts by ammonium sulfate

fractionation

2 gm each of defatted seed powder of PKM-1, PKM-1 (A4+V+N) and wild

Moringa were extracted in 100 ml each of distilled water and 1% NaCl for 5
minutes and incubated for 24 hours in a shaker at 100 rpm at room temperature.
The extracts were centrifuged at 10,000 rpm for 5 minutes and the supernatants
were collected.

Ammonium sulphate fractionation was done in the supernatants by

precipitating the proteins using 40, 60 and 80% solutions of ammonium sulphate.
The precipitates were collected and dissolved in 10 ml of phosphate buffer saline
(PBS). These solutions were stored in refrigerator and used for testing their ability
to coagulate the contents of synthetic turbid water.

7.2.3.2 Coagulation activity assay in synthetic turbid water

The coagulation activity assay was based on the method described by

Ghebremichael et al. (2005). 10g of bentonite clay was mixed with one litre of tap
water and stirred with a magnetic stirrer for 30 minutes and allowed to settle down
for 24 hours to achieve complete hydration. Turbid water samples of high turbidity
with 500 NTU and low turbidity with 50 NTU were prepared from this stock
solution by dilution with tap water. 100 ml each of the two types of the turbid
solutions were transferred to 250 ml beakers to which 200 µL of partially purified
protein solutions in PBS were added and stirred for 5 minutes and allowed to settle
down. A control also was kept, to which no protein solution was added. Then the
Phytochemical Analysis of Moringa oleifera Seed Powder 217

turbidity of the supernatants was measured including the control after 60 and 120
minutes of sedimentation and the NTU values were tabulated.

7.2.3.3 Separation of proteins and determination of molecular weight of

partially purified protein by SDS-PAGE

7.2.3.3.1 Development of SDS-PAGE system

7.2.3.3.1.1 Reagents Required

i) Acrylamide stock ( 30%)

29.2 g Acrylamide and 0.8 g bis acrylamide were dissolved in distilled

water and made up the volume to 100 ml with distilled water. The solution was
filtered through Whatman No.1 Filter paper.

ii) Lower Tris (pH 8.8)

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.

iii) Upper Tris (pH 6.8)

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.

iv) Ammonium persulphate (APS)

It was prepared freshly at the time of use by dissolving 50mg of APS in 500
µl of distilled water.

v) Electrophoresis Buffer (pH 8.3)

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.

vi) Sample buffer

It was prepared by dissolving 150 mg of SDS and 10 mg Bromothymol blue

in a mixture of 0.5 ml β-mercaptoethanol, 1 ml of Glycerol, 1.25 ml of Upper Tris
(pH 6.8) and 7.25 ml of distilled water.
218 Chapter VII

7.2.3.3.1.2 Composition of separating gel

(Final percentage of Acrylamide (10%)

Acrylamide : 3.3 ml
Lower Tris (pH 8.8) : 2.5 ml
Distilled water : 4.1 ml
APS : 50 µL
TEMED : 7 µL

7.2.3.3.1.3 Preparation and loading of separating gel

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.

7.2.3.3.1.4 Composition of Stacking Gel (Final percentage of Acrylamide (10%)

Acrylamide : 1 ml
Lower Tris (pH 6.8) : 1.5 ml
Distilled water : 2.4 ml
APS : 45 µL
TEMED : 7 µL

7.2.3.3.1.5 Preparation and loading of stacking gel

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.

7.2.3.3.2 Preparation and loading of samples

Mixed equal quantities of the protein sample and sample buffer in an

eppendorf tube. Heated it at 1000C for 3 minutes. Loaded the protein markers in
Phytochemical Analysis of Moringa oleifera Seed Powder 219

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.

7.2.3.3.3 Running of the system

Attached the electrode plugs to appropriate electrodes and the power

supply was turned on to 60 V until the sample entered into the separating gel and
continued at 100V till the end of separating gel. The migration of the samples
continued for about 2 hours. Then the power supply was turned off and carefully
removed spacers from the gel plate and stained the gel with coomassie brilliant
blue (R-250).

7.2.3.3.4 Staining of proteins in Gels

7.2.3.3.4. 1 Requirements

i) Staining solution

Dissolved 1g of coomassie brillient blue (R-250) in a mixture of 250 ml

methanol, 35 ml glacial acetic acid and 215 ml distilled water.

ii) Gel destainer

Prepared a mixture of 300 ml methanol,70 ml glacial acetic acid and 630 ml

distilled water.

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

7.2.4 Estimation of different phytochemical compounds in Moringa seed

7.2.4.1 Estimation of tannins

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.

7.2.4.2 Estimation of free amino acids

500 mg of seed samples of PKM-1 and wild Moringa were extracted in 10

ml of 80% ethanol, centrifuged the contents and the supernatants were collected
and concentrated by evaporation. To 0.1ml of extract 1ml of ninhydrin solution
was added and made up the volume to 2ml with distilled water. Boiled the mixture
in a boiling water bath for 20 minutes. After cooling for 15 minutes in room
temperature the OD was measured at 570 nm. By using standard solution of
leucine a standard graph was prepared and the free amino acids content in the
samples were calculated using the standard graph.

7.2.4.3 Estimation of total carbohydrate by phenol sulphuric acid method

Extracted 100 mg samples of PKM-1 and wild Moringa in 5ml of 2.5 N

HCl and kept in a water bath for hydrolysis and neutralized the samples with solid
Phytochemical Analysis of Moringa oleifera Seed Powder 221

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.

7.2.4.4 Estimation of lipids

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

7.2.4.5 Estimation of phenol by O-Phenanthroline method

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.

To 100 µl of each sample solution 300 µl of 0.2 M sodium acetate

(CH3COONa) was added to keep the pH of the solution in between 3 and 4. Then
200 µl of 0.1M FeCl3 and 200 µl of 0.5% O-Phenanthroline solutions were added
and incubated for 24 hrs in the dark. OD was measured at 500 nm. A standard
calibration graph was prepared by using standard phenol solution and the phenol
contents in the samples were estimated with the help of standard graph.
222 Chapter VII

7.2.4.6 Estimation of Ascorbic acid (Vitamin C)

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
% % %

Total proteins 61.4±0.3 61.2±0.2 56.3±0.1

Water extract 58.2±0.4 58.1±0.2 52.5±0.5

1% NaCl extract 52.4±0.1 52.3±0.1 41.4±0.2

0.1 N NaOH extract 32.6±0.3 32.4±0.2 24.6±0.2

Ethanol extract 28.3±0.1 28.1±0.3 22.4±0.3

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

Table 7.2 Effect of partially purified NaCl extracts of M. oleifera in turbidity

removal from highly turbid synthetic turbid water (500 NTU)

Turbidity after Turbidity after Turbidity after

PKM-1 protein PKM-1 (A4+V+N) W.M. protein
treatment protein treatment treatment
Protein fractions After After After After After After
60 min. 120 min. 60 min. 120 min. 60 min. 120 min.
Control 498.3 480.5 498.3 480.5 498.3 480.5
±2.3 ±4.5 ±2.1 ±4.5 ±2.1 ± 4.5
40% fraction of 361.4 306.2 361.6 306.5 469.1 437.1
water extract ±2.1 ±2.2 ±2.3 ±2.5 ±3.1 ±2.3
60% fraction of 307.2 295.3 307.5 295.4 385.3 344.2
water extract ±1.2 ±1.5 ±1.5 ±1.2 ±2.2 ±2.4
80% fraction of 333.1 325.2 333.4 325.5 457.2 393.1
water extract ±2.3 ±3.4 ±2.2 ±3.5 ±1.2 ±3.3
40% fraction of 346.2 308.1 345.8 307.8 374.2 329.1
1% NaCl extract ±2.4 ±2.1 ±2.4 ±2.2 ±2.2 ±1.1
60% fraction of 278.2 246.2 278.4 246.4 299.1 276.2
1% NaCl extract ±3.1 ±4.1 ±2.2 ±2.2 ±1.3 ±2.3
80% fraction of 328.4 312.3 328.6 312.2 352.4 321.3
1% NaCl extract ±2.2 ±2.1 ±2.2 ±2.2 ±2.2 ±2.3

Table 7.3 Effect of partially purified NaCl extracts of M. oleifera in turbidity

removal from highly turbid synthetic turbid water (50 NTU)

Turbidity after Turbidity after Turbidity after

PKM-1 protein PKM-1 (A4+V+N) W.M. protein
treatment protein treatment treatment
Protein fractions After After After After After After
60 min. 120 min. 60 min. 120 min. 60 min. 120 min.
Control 49.2 47.3 49.2 47.3 49.2 47.3
±1.2 ± 1.3 ±1.2 ± 1.3 ±1.2 ± 2.3
40% fraction of 32.3 27.5 32.5 27.2 48.2 43.8
water extract ±2.1 ±1.5 ±2.5 ±1.2 ±2.4 ±1.2
60% fraction of 24.6 18.2 24.4 18.1 32.4 29.3
water extract ±2.3 ±2.1 ±2.2 ±1.1 ±1.2 ±1.1
80% fraction of 30.6 32.5 30.8 32.6 45.6 37.8
water extract ±2.2 ±2.5 ±2.2 ±2.3 ±1.3 ±2.2
40% fraction of 34.6 26.2 34.6 26.5 37.4 32.9
1%NaCl extract ±1.3 ±2.1 ±1.3 ±2.5 ±1.2 ±2.3
60% fraction of 20.4 15.3 20.6 15.5 25.8 22.3
1%NaCl extract ±2.2 ±1.3 ±1.3 ±0.5 ±2.2 ±2.1
80% fraction of 30.2 25.4 30.5 25.2 32.5 29.1
1%NaCl extract ±2.2 ±1.2 ±2.5 ±1.2 ±2.1 ±1.1
Phytochemical Analysis of Moringa oleifera Seed Powder 225

7.3.3 Isolation and determination of molecular weight of 60% NaCl fraction

of protein by SDS-PAGE

As the 60% ammonium sulphate fraction of NaCl extract was found

efficient among different fractions in all varieties of M. oleifera, the 60%
ammonium sulphate precipitated fraction of NaCl alone was selected for further
analysis using SDS-PAGE. The figure 7.1 shows that the seed fractions of all types
of Moringa seeds contain the same type of proteins with a molecular weight
around 13.4 kda. A broad prominent band of protein found to be isolated in the gel
at this region. No other bands were isolated in the gel from this fraction. Hence a
single protein or a group of proteins around the molecular weight of 13.4 kda may
be present in this fraction and active in the purification.

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

7.3.4 Estimation of some components other than proteins in M. oleifera seed

powder

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.

Table 7. 4 Quantities of different phytochemical components in the seeds of M.

oleifera varieties.

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).

This difference in the protein content is found to be closely related to the

variations exhibited in the water purification and antimicrobial properties of the
extracts. The extracts having lower quantities of protein were less effective in
these processes. Ethanol extracts in higher doses were effective than NaOH
extracts. Eventhough, the protein content in the NaCl extracts is lesser than water
extract; it was observed to be more efficient than water extract. In antibacterial
studies the water and NaCl extracts only were found effective.

The ammonium sulphate precipitation, fractionation and turbidity reduction

analysis showed significant difference in the clarification of turbid water by the
different fractions. It clearly expressed the fact that there are various types of
proteins in the seed extract having different degrees of water purification
efficiency. Another observation was that when the proteins were fractionated the
degree of clarification of turbid waters was reduced than that by the direct extracts
of the seeds without fractionation. Hence it can be inferred that the cumulative
effect of all the types of proteins in the extracts will be more effective than the
fractions.
228 Chapter VII

On comparison of the concentration of proteins in water and NaCl extracts

and their water clarifying ability, no direct correlation was observed. Eventhough,
water extract contain higher protein content than NaCl extract, NaCl protein
fractions were found to be superior in the efficiency of turbidity removal. This may
be due to the type of protein extracted by the solvents, i.e. not only the quantity of
protein, but type of protein also is important in the ability of flocculating the
impurities in the polluted water or turbid water. All the fractions of Ammonium
sulphate precipitation showed some degree of clarification of the turbid water.
Hence, it can be concluded that proteins are the main components in coagulating
the pollutants. No significant change was observed in the effectiveness of PKM-1
and PKM-1(A4+V+N) seed extracts in the clarification of turbid bentonite clay
water. This proved that there is no change between the protein content of PKM-1
and PKM-1(A4+V+N) seed extracts.

There are reports regarding the isolation and purification of proteins of

different molecular weights from M. oleifera seed. Ghebremichael et al. (2005)
and Gassenschmidt et al. (1995) studied and purified a flocculating cationic
protein with a molecular weight less than 6.5 KDa. Sahni et al (2008) fractionated
five prominent cationic protein bands of different intensities in the molecular
weight range of 157-158 kDa, 114-116 kDa, 97-98 kDa, 34-40 kDa and 6-16 kDa
from M. oleifera seed. The protein fractions having molecular weight in the range
6-16 KDa, was the prominent one and they suggested that it might be playing
major role in removing arsenic from water. Ndabigengesere, et al. (1995)
described dimeric cationic proteins with 12-14 Kda molecular mass. They have the
opinion that the active protein is actually composed of two 6.5 Kda subunits
connected with an S-S bond, that is cleaved when protein extraction occurs in
reducing conditions. Kwaambwa & Maikokera (2008) also separated a protein
having a molecular weight 12-14 after ammonium sulphate precipitation and
column chromatography. The findings of Ndabigengesere, et al. (1995),
Kwaambwa & Maikokera (2008) and Sahni et al. (2008) support the present
observation that the most effective protein in the purification of polluted water is
the one with a molecular weight of around 13.4 KDa. This finding clearly proved
Phytochemical Analysis of Moringa oleifera Seed Powder 229

that the major active components responsible for the water purification and
antimicrobial ability of M. oleifera seed extract are proteins.

One fact to remember is that large number of proteins with different

molecular weight having lower and higher efficiencies is present in the Moringa
seed. The combined effect of all these proteins is responsible for the water
purification and antibacterial properties of seed extracts in different solvents. The
effectiveness of the purification was reduced when they are fractionated into
groups based on molecular weight. In a single solvent extract, proteins of different
molecular weight are present. Hence instead of fractionating the proteins, the best
practice would be to purify the protein in the extract showing higher efficiency by
removing non-protein substances which may reduce the efficiency of purification.
In the present study the 1% NaCl extracts showed better efficiency. From the
analysis it can be concluded that the proteins in this extract are responsible for its
efficiency in the purification of polluted water and antibacterial property.
230 Chapter VII