Investigation of Insulin Like Protein From Spirulina
41
3. Material and Methods
3.1 Collection of Microorganism
23 different Spirulina strains were procured from National Centre for Culture Collection
and Utilization of Blue Green Algae, IARI, New Delhi; Department of Botany, Universi
ty of Madras, Tamilnadu; Allahabad University, Allahabad National Facility for Marine
Cyanobacteria, Bharathidasan University, Triuchirapalli and Central Food
Technological Research Institute, Mysore.
3.2 Growth medium
Zarrouk´s medium was used for all fresh water strains (Zarrouk 1966) and ASN-III
medium was used for marine strain (Rippka 1988). Culture medium was autoclaved
(Yorco, India) maintaining 15lb/in2 or Kg/cm2 pressure for 15 minutes. Their
compositions are as follows__
A. Zarrouk´s medium
S.No. Chemicals g/liter
1. Sodium bi- carbonate 16.8
2. Di potassium hydrogen phosphate 0.5
3. Ferrous sulphate 0.01
4. Sodium nitrate 1.5
5. Potassium sulphate 1.0
6. Sodium chloride 1.0
7. Magnesium sulphate 1.2
8. Calcium chloride 0.04
Investigation of Insulin Like Protein From Spirulina
42
B. ASN-III medium
S.No. Chemicals g/liter
1. Sodium nitrate 0.75
2. Di potassium hydrogen phosphate 0.75
3. Magnesium sulphate 3.5
4. Calcium chloride 0.5
5. Citric acid 0.003
6. Ferric ammonium citrate 0.003
7. EDTA 0.005
8. Sodium carbonate 0.02
9. Sodium chloride 25.0
10. Trace metal mix 1ml
Trace Metal Mix
S.No Chemicals g/litre
1. Boric acid 2.86
2. Manganese chloride 1.810
3. Zinc sulphate 0.222
4 Sodium molybdate 0.0390
5. Copper sulphate 0.0790
6. Cobaltous nitrate 0.0494
3.3 Sterilization
All the glasswares were thoroughly washed with HCl, liquid detergent (Teepol) and tap
water. Final rinsing was done with double distilled water. Dried conical flasks were
Investigation of Insulin Like Protein From Spirulina
43
plugged with cotton and wrapped with aluminium foil and then steam sterilized in an
autoclave at 121˚C under 15Ib/cm2 pressure for 20 minutes.
3.4 Inoculation
Initial concentration of the cells was adjusted at optical density OD 560 = 0.1. For initial
inoculation 100 ml sterile medium was taken in 250 ml Erlenmeyer flasks and were
aseptically inoculated with 25 ml inoculums. For their bulk production inoculation was
done in 1L and 2L flasks.
3.5 Culture condition
The Spirulina strains were raised in BOD shaking incubator providing a light intensity
of 50 µ mol photons/m2/s light intensity 12:12 light/dark regime, 30±1ºC temperature.
Cultures were manually stirred twice daily. Spirulina biomass were harvested in their
late log phases of growth by filtration through nylon cloth. Harvested biomass were
thoroughly washed with distilled water to remove adhering salts and transferred into
sterilized Falcon tubes. These were properly corked and placed in refrigerator (- 20oC)
for one day. Stored harvested biomass was Lyophilised and then transferred in sterilized
Falcon tubes. Properly parafilm wrapped tubes were stored in refrigerator at - 20oC for
insulin like protein extraction.
3.6 Chemicals procurement
A highly purified antibody (cat no.GGHL-60P) against human insulin raised in guinea
pigs and a peroxidase conjugated guinea pig anti-IgG antibody (GTX82959) raised in
goats were purchased from GenX Bio, India, streptozotocin (STZ) from HIMEDIA
Investigation of Insulin Like Protein From Spirulina
44
(RM1758), India. Rest of the reagents (Analytical grade) used were purchased from
Merck, India & Himedia, India.
3.7 Instruments used
Autoclave (Yorco, India); UV-VIS spectrophotometer – Specord 200 (Analytikjena);
Incubator (Narang Scientific, India); Incubator shaker (JSR, Korea); Labconco
Lyophiliser (made in England); pH meter (Scientific Systems Control dynamics, India);
Balance (Sartorius Analytic, Germany); Homogenizer (Pro 200, Pro Scientific Inc.,
USA); Ice machine (Ice Flaker, Angelantoni, Italy); Centrifuge (Sigma Centrifuge 1-
15k, Germany); HPLC system (Shimadzu class –VP V 6.14 SP2); Syringe filter
(Millex®, Millipore corporation); Electrophoresis unit (BIO-RAD Laboratories, USA);
ELISA Reader (BIO-RAD Laboratories, USA); Semidry Western Blotting Device (BIO-
RAD Laboratories, USA); NMR (Bruker, Germany); Milli Q Water (SG, Ultra Clear
TWF UV plus Water system, Germany); Magnetic stirrer (Yorco, India); Gel shaker
(Rocker 100, Bangalore Genei); Molecular Imager® (BIO-RAD Laboratories, USA);
Spectropolarimeter (Jasco J-715).
3.8 Extraction of insulin-like protein (Khanna et al. 1976)
Five grams of fresh Spirulina biomass was used for extraction of insulin like protein
with a solution consisting of 2 ml distilled water, 10 ml 95 % ethanol and 0.72 %
concentrated sulphuric acid. After shaking for 20 min, 13 ml water and 40 ml 95 %
ethanol were added and the pH was adjusted to 1.7 with sulphuric acid. The suspension
was then filtered and the filtrate adjusted at pH 3.0 with ammonium hydroxide. To this
suspension 150 ml of 95 % ethanol and 200 ml diethyl ether were added. After standing
Investigation of Insulin Like Protein From Spirulina
45
for 12 hours at 4oC the suspension was centrifuged at 3000 g for 10 min and the
sediment was washed with acetone and diethyl ether before dissolving in 25 % ethanol,
and pH was adjusted to 8.5. To this solution 100 µl of 1 M zinc chloride was added and
the preparation left to stand for 18 hours at 25oC to precipitate insulin like proteins. The
precipitate was collected by centrifugation after 18 hours.
3.9 Screening by ELISA (Gebara et al. 1995)
The presence of insulin like protein in Spirulina extracts were determined by qualitative
assay known as ELISA. The positive control insulin (bovine insulin, 1µg/100µl),
negative control (buffer without insulin) and the above extracts, all prepared in 0.05 M
carbonate/bicarbonate buffer, pH 9.6, were pipetted into the 96 wells of the plate (100 µl
of each solution) and left to stand for 16 hr at 4 ºC. The solution was then discarded and
the wells were washed for 1hour (changing the solution every 10 min) with Tween 20
dissolve in PBS (buffer A). After washing, 300 µl of a blocking buffer (1% BSA
dissolve in PBS) was added to the wells for 1hour. The plate was again washed for 1
hour as described above. 50 µl of the anti-human insulin antibody solution (1:5000
dilution) prepared with blocking buffer was added to the wells and left to stand at room
temperature for 45 min. The plate was then washed with buffer A for 30 min, with every
5 min changing. Then 50 µl of peroxidase-conjugated anti-IgG (1:2000) in blocking
buffer was added to the wells and incubated at room temperature for 45 min. The plate
was then washed four times with buffer A for 5 min each time. The substrate of
peroxidase was then added and the plates were left to stand for 10 min in the dark at
room temperature. The reaction was stopped by the addition of 50 µl of H2SO4 and the
absorbance was read with ELISA reader (Biorad) at 490 nm.
Investigation of Insulin Like Protein From Spirulina
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Reagents for ELISA
Carbonate buffer pH 9.6 100 ml
Sodium carbonate 159 mg
Sodium bicarbonate 293 mg
Distilled water 100 ml
Phosphate buffer saline (PBS), pH 7.4 1000 ml
Sodium chloride 8 g
Potassium dihydrogen phosphate 0.2 g
Disodium hydrogen orthophosphate 1.15 g
Potassium chloride 0.2 g
Distilled water 1000 ml
Blocking solution 100 ml
BSA 1 g
PBS 100 ml
Washing buffer 1000 ml
Tween 20 0.5 ml
Phosphate buffer saline (PBS) 1000 ml
Substrate buffer 10 ml
Citric phosphate buffer 5.5 pH (100 mM) 10 ml
H2O2 10 μl
O-Phenylene Diamine (OPD) 10 mg
Investigation of Insulin Like Protein From Spirulina
47
3.10 Characterization of insulin-like protein
3.10.1 SDS-PAGE (Laemmli. 1970)
SDS–PAGE was carried out on using a 15% (w/v) polyacrylamide gel and with 1.5 mm
thick spacer. Samples were extracted (1:10) with a solution of 4 % SDS, 12 % glycine,
50 mM Tris-HCl, 35 mM EDTA, pH 6.8, and 0.01 % bromophenol blue and manually
shaken at regular intervals (10 min) for 2 hours at 4ºC. The prepared protein extracts
(50µl) and commercial bovine insulin (10 µg, mixed in sample buffer in a ratio of 1:4)
was loaded in the wells of polyacrylamide gel. Electrophoresis was run for 1 h at 30 mA.
Protein bands were visualized by Coomasie Brilliant Blue R-250 (HI-MEDIA, India)
staining and followed by overnight de-staining solution. The gel was scanned by
Molecular Imager®.
Reagents for PolyAcrylamide Gel Electrophoresis
Separating gel buffer (1.5 M Tris- Cl pH 8.8)
Dissolved 181.8 g of Tris base in distilled water. Adjusted the pH to 8.8 with
concentrated HCl / 1N NaOH and made the volume to 1000 ml.
Stacking gel buffer (1 M Tris-Cl pH 6.8)
Dissolved 121.14 g of Tris base in distilled water. Adjusted the pH to 6.8 using HCl / 1N
NaOH and made the volume to 1000 ml.
Investigation of Insulin Like Protein From Spirulina
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30 % Acrlyamide solution
Dissolved 29 g acrlyamide and 1 g Bis-acrlyamide in distilled water and volume was
made up to 100 ml. Filtered the solution through activated charcoal and degassed. Stored
at 4o C.
10 % Ammonium per Sulphate (APS)
Dissolved 0.1 g ammonium per sulphate in 1 ml distilled water just before use.
Stacking gel solution (5%) 5 ml
Water
30 % Acrylamide
1.0 M Tris buffer ( pH 6.8)
10 % Sodium dodecyle sulphate (SDS)
10 % Ammonium per sulphate (APS)
TEMED
3.6 ml
0.625 ml
0.625 ml
100 µl
50 µl
5 µl
Resolving gel solution (15%) 10 ml
Water
30 % Acrylamide
1.5 M Tris buffer ( pH 8.8)
10 % Sodium dodecyle sulphate (SDS)
10 % Ammonium per sulphate (APS)
TEMED
3.45 ml
3.75 ml
2.5 ml
200 µl
100 µl
10 µl
Investigation of Insulin Like Protein From Spirulina
49
Running Buffer 1x 1000 ml
Tris buffer
Glycine
SDS
H2O
3.0 g
14.4 g
1.0 g
To 1000 final volume
After dissolution of all components volume is adjusted to 1000 ml with deionized water.
Staining solution 100 ml
Double distilled water
Methanol
Glacial Acetic acid
Coomassie brilliant blue
43 ml
50 ml
7.0 ml
0.25 g
Destaining solution 100 ml
Double distilled water
Methanol
Glacial Acetic acid
43 ml
50 ml
7.0 ml
3.10.2 Western Blotting (Towbin et al. 1979)
For immunological characterization of insulin like antigen of Spirulina sp.
Immunoprecipitation Assay (Western Blotting) was done. For Western blotting after
SDS-PAGE, the gel was washed with distilled water to remove excess SDS. The PVDF
(polyvinylidene fluoride) membrane cut to the size of gel was activated by soaking in
absolute methanol for two minute (Wear gloves at all times when handling the PVDF).
Investigation of Insulin Like Protein From Spirulina
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The other blotting pads on the membrane were arranged in order of (-) blotting pad /
SDS-PAGE gel / PVDF membrane / blotting pad (+). This was soaked in transfer buffer
before transfer. Proteins on the gel were electrophoretically transferred to the membrane
at constant current 5.5 mA/cm2 for 30 minutes. The membrane was removed from the
western blotting device and was blocked with the blocking solution (1% BSA) for 2
hours at room temperature to block unoccupied sites with gentle rotating on a horizontal
rotator. The membrane was washed with washing buffer 3 times for 5 minutes each. The
membrane was immersed in 50 ml of primary antibody solution containing 1:5000
dilution of anti-human insulin antibody diluted with PBST and 0.1% BSA. This was
incubated at room temperature under gentle shaking for 90 minutes. The primary
antibody solution was discarded and the membrane was washed 3 times with PBST, 5
minutes each. The membrane was immersed in 20 ml of secondary antibody solution
containing 1:2000 dilution of Anti- Guinea pig IgG HRP (horseradish peroxidise)
conjugate diluted with PBST and incubated at room temperature under shaking
condition for 90 minutes. The membrane was washed with PBST for 5 times, 5 minutes
each and then 5 times with PBS. The reaction was developed by placing membrane in 20
ml of PBS containing 10 mg di-amino benzidine (DAB) and 60 µl of 30 % hydrogen
peroxide solution for few minutes under shaking until the bands were clearly visible.
The reaction was stopped by discarding the developing solution and washing the
membrane in water 2 times, 5 minutes each.
Investigation of Insulin Like Protein From Spirulina
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Reagents for western blotting
Western Blot transfer buffer 1000 ml
Tris base (25mM) 3 g
Glycine (192 mM) 14 g
Absolute methanol 200 ml
SDS 1 g
Distilled water (make upto 1000 ml) 1000 ml
Developing reagent 20 ml
Di-amino Benzidine (DAB) 10 mg
Hydrogen peroxide (30% solution) 60 µl
PBS 20 ml
Western blotting is the electrophoretic transfer of separated proteins in a gel to the
surface of a thin support membrane matrix, where they are bound and immobilized with
specific antibodies. Semi-dry blotting is performed with plate electrodes in a horizontal
configuration, sandwiching a gel and membrane between sheets of buffer-soaked filter
paper that function as the ion reservoir. During electrophoretic transfer, negatively
charged molecules migrate out of the gel and move towards the positive electrode,
where they are deposited on the membrane. The plate electrodes, separated only by the
gel and filter paper stack, provide high field strength (V/cm) across the gel, allowing for
very efficient, rapid transfers.
3.10.3 RP-HPLC (Venancio et al. 2003)
Insulin-like protein were separated and purified by RP-HPLC (Shimadzu class –VP V
6.14 SP2 chromatograph with UV-VIS detector) using a C-18 Phenomenex column
Investigation of Insulin Like Protein From Spirulina
52
(250×4.6 mm, 5 μm) with 30–80% acetonitrile (A) in a 0.1% trifluoroacetic acid (TFA)
gradient (B). The fraction obtained by zinc chloride precipitation was dissolved in 500
μL of 0.1% TFA containing 0.1 M EDTA and applied to the column. The column was
operated at 1 mL min−1 and the proteins detected by absorbance at 216 nm. Bovine
insulin (50 μg of protein in 500 μL) was used as standard to compare retention times.
HPLC Reagents
Solvent (A) : Distilled water + TFA 0.1%
Solvent (B) : Acetonitrile
High Performance Liquid Chromatography (HPLC) is one mode of chromatography,
one of the most used analytical techniques. Chromatographic process can be defined as
separation technique involving mass-transfer between stationary and mobile phase.
HPLC utilises a liquid mobile phase to separate the components of a mixture. The
stationary phase can be a liquid or a solid phase. These components are first dissolved in
a solvent, and then forced to flow through a chromatographic column under a high
pressure. In the column, the mixture separates into its components. The amount of
resolution is important, and is dependent upon the extent of interaction between the
solute components and the stationary phase. The stationary phase is defined as the
immobile packing material in the column. The interaction of the solute with mobile and
stationary phases can be manipulated through different choices of both solvents and
stationary phases. As a result, HPLC acquires a high degree of versatility not found in
other chromatographic systems and it has the ability to easily separate a wide variety of
chemical mixtures.
Investigation of Insulin Like Protein From Spirulina
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3.10.4 Circular Dichroism (Johnson. 1988)
CD spectroscopy is an adequate, convenient and well established physical technique
used for studying biomolecular conformation in solution under a wide range of
conditions. CD spectroscopy directly measures the differential absorption coefficients
between left and right handed circularly polarized light. The far UV CD bands of protein
(200–250 nm) derive primarily from the amide chromophore and they reflect the
secondary structure of protein (α-helix, β-sheets, β-turns).
Far-UV CD was measured in Jasco Spectropolarimeter (model J-715) equipped with
peltier-type temperature controller (PTC-348) and interfaced with personal computer.
Protein concentration used was in the range of 0.2-0.3mg/ml and 0.1 cm path length.
Baseline correction was carried out with the phosphate buffer in which protein was
dissolved. Measurements were taken at 25oC. N2 was continuously flushed through the
machine at the rate of 2-2.5 lit/min and higher below 200 nm to minimize the noise
level. CD data was reduced to concentration independent parameter [q] (deg cm2 dmol-
1), the mean residue ellipticity using the relation:
[q]l =M0 ql/10lc
where ql is the observed ellipticity in millidegrees at wavelength, l, M0 is the mean
residue weight of the protein, c is the protein concentration in gm/cm3, and l is the path
length of the cell in centimeters.
Since most of the proteins contain β- sheet, the observed residue ellipticity ([θ]λp) of the
protein is written as the linear combination of all structures, i.e.
[θ]λp = ƒα[θ]λ
α + ƒβ[θ]λβ + ƒt [θ]λ
t + ƒr[θ]λr (1)
Investigation of Insulin Like Protein From Spirulina
54
Where ƒα, ƒβ, ƒt and ƒr are the fractions of α-helix, β-sheet, turn and random coil,
respectively, and [θ]λα
, [θ]λβ
, [θ]λt, and [θ]λ
r are the mean residues ellipticities of pure of
α-helix, β-sheet, turn and random coil at wavelength λ, respectively. Due to the
uncertainty of [θ]λt of proteins and inaccessibility of measurements beyond 200 nm for
all pH values, equation (1) was reduced to the following form,
[θ]λp = ƒα[θ]λ
α + ƒβ[θ]λβ + ƒr[θ]λ
R (2)
Where R represent everything which is not the part of α-helix and β-sheet. Using data
provided by Yang et al., (1986) for the reference spectra, we analysed each far –UV CD
curve for different pH values for the elements of secondary structures according to
equation (2).
CD spectroscopy plays an important role in the study of characterisation of secondary
structures of proteins. Circular dichroism (CD) is an instrumentation technology for
elucidating the secondary structure of the proteins and peptides. It also allows the
detection and quantitation of the chirality of molecular structures. CD is a variant of
absorption spectroscopy which measures the difference in absorption of left and right
polarized light in the ultraviolet (UV) band by a medium or sample. Although the
peptide bond is planar and hence symmetric, there is usually an asymmetric alpha-
carbon on either side; hence the peptide bond transitions interact to give a CD signal
which is very sensitive to secondary structure. In the far UV region (200- 250 nm) the
CD of a protein is primarily that of the amide chromophores along the backbone. With
the introduction of an optically active sample, a preferential absorption is seen during
one of the polarization periods and the intensity of the transmitted light now varies
during the modulation cycle. The variation is directly related to the circular dichroism
Investigation of Insulin Like Protein From Spirulina
55
of the sample at that wavelength. Successive detection is performed at various
wavelengths leads to the generation of the full CD spectrum. CD uses very little sample
(200 μl of 0.5 mg/ml solution in standard cells), it is non-destructive and relative
changes due to influence of environment on sample (pH, denaturants, temperature etc.)
can be monitored very accurately.
3.10.5 MALDI-TOF (Sequencing)
Proteins, either in solution or trapped in a band from a gel, can be analyzed by MALDI-
TOF mass spectrometry. Isolated protein was run on SDS-PAGE; for analysis protein
band was cut and submitted in commercial lab (TCGA) for amino acid sequence. In in-
solution digest the protein sample was mixed with an aqueous buffer (NH4HCO3) and
trypsin (5%) was added. In-gel digestion is a little more complicated and involves a
number of different steps; first the gel spots have to be washed. This was done using
50% acetonitril for 2 x 15 min. Then the gels were treated with 100% acetonitril (the gel
pieces will shrink!) and rehydrated in the digestion buffer (NH4HCO3). The washing
step was repeated using 50% acetonitril and then the gel pieces were dried down in the
speedway. To reduce potential disulfide bridges the proteins in the spot were treated
with 10 mM DTT (in 0.1 M digestion buffer) and incubated for 45 min. at 56 0C. To
prevent reconstruction of the bridges the free cysteins were alkylated; the gel pieces
were incubated in 55 mM iodoacetamid (in 0.1 M digestion buffer) for 30 min. in a dark
place. The washing steps were then repeated. Loading of the sample/matrix mixture onto
the MALDI-target directly from a micro-column The protein was then cut into smaller
pieces (peptides) by a protease (e.g. 12.5 ng/µl trypsin in 50 mM NH4HCO3, 5 mM
CaCl2). This was done on ice. After 45 min. the enzyme was replaced by digestion
Investigation of Insulin Like Protein From Spirulina
56
buffer and left for digestion at 37 0C overnight. If necessary, the peptides were extracted
from the gel using acetonitril and 5% formic acid. Although MALDI-TOF generally
tolerant towards contaminants, such as salts and detergents, these significantly lower the
sensibility of the spectra. When analyzing small amounts of biological analytes using
MALDI-TOF, optimizing sample preparation and removing contaminants from the
sample can improves the spectra a lot. The breakthrough of cleaning up contaminant
from samples came with the introduction of low price self-assembled nano-scale sample
preparation methods, e.g. Micro-column purification. Here, a small column of a
chromatographic material was packed inside a small GelLoader tip. By loading the
sample onto this column the peptides were bind to the chromatographic resin and the
contaminant washed away. By adding the matrix (dissolved in acetonitril) onto the
column, the peptides were elute together with the matrix. The sample/matrix mixture
was then eluted directly onto the target and is ready for analysis by MALDI-TOF.
3.11 Hypoglycemic activity (In-vivo studies)
3.11.1 Animals and housing conditions
Albino Wistar rats (both sexes) aged seven to eight weeks (150-200g) were housed in a
well-ventilated animal unit (25±2°C, 60-70% relative humidity, 12h light/dark cycle)
provided by the Central Animal House Facility, Jamia Hamdard, New Delhi, India. The
animals were fed standard chow and filtered tap water ad libitum. All the protocols were
performed in the accordance with the Institutional Animal Ethical Committee (IAEC) as
per the directions of the Committee for the Purpose of Control and Supervision of
Experiments on Animals (CPCSEA).
Investigation of Insulin Like Protein From Spirulina
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3.11.2 Aqueous extraction
Ten grams of dried Spirulina was extracted in distilled water for 6 h at slow heat. Every
2 h it was filtered through 2 layers of muslin cloth and centrifuged at 5000 g for 15 min.
The supernatant was collected. This procedure was repeated twice and after 6 h the
supernatant was concentrated to make the final volume one-fifth of the original volume.
The extract concentration was determined by gravimetric method. The extract was then
stored at 4 °C for further experiment.
3.11.3 Ethanol extraction
Ten grams of dried Spirulina was extracted with 100 ml of 80 % ethanol kept on a rotary
shaker for 24 h. Thereafter, it was filtered and centrifuged at 5000 g for 15 min. The
supernatant was collected and the solvent was evaporated to make the final volume one-
fifth of the original volume. The dried extract was resuspended with 1 ml of phosphate
buffer and administered through orally (50 mg / kg body weight) to the experimental
rats. It was stored at 4 °C in airtight bottles for further studies.
3.11.4 Induction of diabetes mellitus
A freshly prepared solution of streptozotocin (45mg/kg) in 0.1M cold citrate buffer, pH
4.5 was injected intraperitoneally. After 72 h of STZ administration, blood was drawn
from the fasting STZ-treated animals. The rats with blood glucose above 250 mg/dL
were considered to be diabetic and used for the experiment. Control rats were given
citrate buffer (pH 4.5) only.
Investigation of Insulin Like Protein From Spirulina
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3.11.5 Experimental design
In the experiment, a total number of 36 rats (30 diabetic and 6 normal rats) were used.
The rats were divided into six groups of six rats each: group 1, normal untreated rats;
group 2, diabetic control rats; group 3, diabetic rats given crude Spirulina (50mg/kg
body weight); group 4, diabetic rats given aqueous extract of Spirulina (50mg/kg body
weight); group 5, diabetic rats given ethanolic extract of Spirulina (50mg/kg body
weight); group 6, diabetic rats given insulin-like protein from Spirulina (50 μg of
insulin-like protein dissolved in 100μl saline).
3.11.6 Biochemical analysis
At the end of 35 days, the animals were deprived of food overnight and anaesthezied and
sacrificed by cervical dislocation. Blood was collected in two different tubes, i.e. one
with anticoagulant, potassium oxalate for plasma, and another without anticoagulant for
serum separation. Serum was separated by centrifugation.
For blood glucose test, blood was collected from the tail of the rats. The blood sugar was
estimated using the ACCU-CHEK® Go, Glucometer (Roche, Germany). The glucose
estimation was made weekly throughout the study. Glycosylated hemoglobin (HbA1C)
was estimated by the method (Bannon 1982), LFT (SGOT, SGPT, ALP and Bilrubin
total) and KFT (Creatinine Serum, Uric acid Serum and Blood urea nitrogen) analysis
were estimated by using commercial kits.
Investigation of Insulin Like Protein From Spirulina
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3.11.6.1 Liver Functional Test
SGOT
SGOT (AST) catalyzes the transfer of amino group between L-Aspartate and a
Ketoglutarate to form Oxaloacetate and Glutamate. The Oxaloacetate formed reacts with
NADH in the presence of Malate Dehydrogenase to form NAD. The rate of oxidation of
NADH to NAD is measured as a decrease in absorbance which is proportional to the
SGOT activity in the sample.
SGOT L-Aspartate + a Ketoglutarate ———–––———> Oxaloacetate + L-Glutamate MDH Oxaloacetate + NADH + H+ ———–––———> Malate + NAD+
Reagent1:
L-Aspartate > 200 mmol/l
Malate Dehydrogenase > 2000 IU/L
Reagent2:
Alpha-Ketoglutarate >35 mmol/l
NADH 1.05 mmol/l
Investigation of Insulin Like Protein From Spirulina
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Procedure
Particulars Sample Volume
Reagent 1 800 µl
Reagent 2 200 µl
Mix and incubate at 37°C for 2 minutes then add Sample 100 µl
AST activity is determined by measuring the rate of oxidation of NADH at 340 nm.
SGPT
SGPT (ALT) catalyzes the transfer of amino group between L-Alanine and a
Ketoglutarate to form Pyruvate and Glutamate. The Pyruvate formed reacts with NADH
in the presence of Lactate Dehydrogenase to form NAD. The rate of oxidation of NADH
to NAD is measured as a decrease in absorbance which is proportional to the SGPT
activity in the sample.
SGPT L-Alanine + a Ketoglutarate ———–––———> Pyruvate + L-Glutamate LDH Pyruvate + NADH + H+ ———–––———> Lactate + NAD+
Reagent1:
L-Alanine > 200 mmol/l
Lactate Dehydrogenase > 2000 IU/L
Investigation of Insulin Like Protein From Spirulina
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Reagent2:
Alpha-Ketoglutarate >35 mmol/l
NADH 1.05 mmol/l
Procedure
Particulars Sample Volume
Reagent 1 800 µl
Reagent 2 200 µl
Mix and incubate at 37°C for 2 minutes then add Sample 100 µl
ALT activity is determined by measuring the rate of oxidation of NADH at 340 nm.
ALP
This method utilizes 4-nitrophenylphosphate as the substrate. Under the optimised
conditions ALP present in the sample catalyses the following transphosphorylation
reaction.
ALP 4 - NPP + H2O ———–––———> 4-NPO + Phosphate Mg / Alkaline pH
At the pH of the reaction 4-nitrophenoxide has an intense yellow colour. The reagent
also contains a metal ion buffer system to ensure that optimal concentrations of Zinc and
Magnesium are maintained. The metal ion buffer can also chelate other potentially
inhibitory ions which may be present. The reaction is monitored by measuring the rate
Investigation of Insulin Like Protein From Spirulina
62
of increase in absorbance at 405 nm which is proportional to the activity of ALP in the
serum. It is almost always measured in units/litre (U/L).
Reagent1:
p-Nitrophenylphosphate 15 mmol/L
Reagent2:
2-Amino-2-methyl-1- propanol
350 mmol/L
Procedure
Particulars Sample Volume
Reagent 1 800 µl
Reagent 2 200 µl
Mix and incubate at 37°C for 2 minutes then add Sample 100 µl
Bilrubin Total
Bilibuin is coupled with diazotized sulphanilic acid in acidic medium to form the pink
colored azobilirubin.The intensity of the color produced is directly proportional to
bilirubin concentration present in the sample. Direct bilirubin or conjugated bilirubin is
water soluble and it directly reacts in acidic medium. However, for indirect bilirubin or
unconjugated bilirubin a surfactant is used for dissolving it in water and then the
dissolved material reacts similar to direct bilirubin.
Investigation of Insulin Like Protein From Spirulina
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Reagent1:
Sulphanilic acid
4 mmol/l
Surfactant
0.6 mmol/l
Reagent2:
Sodium nitrite
144 mmol/L
Procedure
Particulars Sample Volume
Reagent 1 500 µl
Reagent 2 10 µl
Mix and incubate at 37°C for 2 minutes then add Sample 25 µl
Read the absorbance at 546/670 nm.
3.11.6.2 Kidney Functional Test
Creatinine
In alkaline solution, creatinine forms a yellow-orange complex with picrate. The color
intensity is directly proportional to the creatinine concentration and can be measured
photometrically. Assays using rate-blanking minimize interference by bilirubin. Serum
and plasma samples contain proteins which react non specifically in the Jaffe method.
Serum and plasma results must be reduced by 0.3 mg/dl (26 µmol/l) to obtain accurate
values.
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64
NaOH creatinine + picric acid ———–––———> creatinine - picric - acid - complex
Reagent1:
Sodium hydroxide
0.2 mmol/L
Reagent2:
Picric acid 25 mmol/L
Procedure
Particulars Sample Volume
Reagent 1 500 µl
Reagent 2 100 µl
Sample 200 µl
Mix and incubate at 37°C for 60 second
Read the absorbance at 492/500 nm.
Uric acid Serum
Uric acid is oxidized by phosphotungstic acid in alkaline medium containing the
tetrasodium salt of ethylenedinitrilotetraacetic acid (EDTA-Na4), sodium tungstate, and
hydrazine sulfate.
Reagent1:
Sodium tungstate 10% w/v
Investigation of Insulin Like Protein From Spirulina
65
Reagent2:
Sulphuric acid 1/12 N
Reagent3:
EDTA 5% w/v
Reagent4:
Hydrazine sulfate 0.025% w/v
Reagent5:
EDTA-Tungstate 5% EDTA and 60% Sodium tungstate
Reagent6:
Phospho-Tungstate 40 g Sodium tungstate and 32 ml of 85% orthophosphoric acid
Reagent7:
Saline 0.9% NaCl
Procedure
Particulars Sample Volume
Reagent 2 1.6 ml
Serum 200 µl
Reagent 1 200 µl
Mix vigorously allow to stand for 10 min and centrifuge
Investigation of Insulin Like Protein From Spirulina
66
Supernatant 0.5 ml
Reagent 4 1 ml
Reagent5 4 ml
Reagent6 1 ml
Mix and incubate at 37°C for 30 minutes
Read the absorbance at 700 nm
Blood urea nitrogen
Urea in an acidic medium condenses with Diacetyl monoxime at 100°C to form a red
coloured complex. Intensity of the colour formed is directly proportional to the amount
of urea present in the sample.
100°C
Urea + Diacetyl monoxime ———–––———> Red Coloured Complex
Reagent1:
Urea
0.2 mmol/L
Reagent2:
Acid
0.2 mmol/L
Reagent3:
DAM
0.2 mmol/L
Investigation of Insulin Like Protein From Spirulina
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Procedure
Particulars Sample Volume
Reagent 1 1 ml
Reagent 2 1 ml
Reagent 3 1 ml
Sample 100 µl
Mix and incubate at 100°C for 10 minutes
Read the absorbance at 520 nm.
3.12 Growth measurement (as protein)
The growth of selected Spirulina strain(s) was determined as protein (Lowry et al.
1951). The algal biomass (1mg) was taken in a test tube. 1mL 1N NaOH was added to it.
The test tube was placed in a boiling water bath for 10 minutes. The blank / sample
solution were taken and added 5mL of Reagent-A (prepared by adding 1mL freshly
prepared 1% Na-K tartarate solution containing 0.5% CuSO4 into 50mL 2% Na2CO3
solution) and incubated at room temperature for 10 minutes. Then added 0.5 mL
Reagent-B (Folin reagent) and again incubated at room temperature for 15 minutes. The
absorbance of the supernatant was observed at λ650 nm. Protein content was evaluated
from the concentration of BSA solution known from standard curve.
3.13 Culture condition manipulation
S. platensis (CFTRI, Mysore) was grown in different concentrations of NaNO3, K2HPO4,
NaHCO3 and Calcium in Zarrouk’s medium for culture condition manipulation.
Investigation of Insulin Like Protein From Spirulina
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NaNO3
30 mM as in regular Zarrouk’s medium was considered as control. Five different
concentrations (15, 25, 35, 45 and 55 mM) of nitrate added to medium.
K2HPO4
3 mM as in regular Zarrouk’s medium was considered as control. Five different
concentrations (1.5, 2.5, 3.5, 4.5 & 5.5mM) of phosphate added to medium.
NaHCO3
0.2 M as in regular Zarrouk’s medium was considered as control. Five different
concentrations (0.06, 0.12, 0.18, 0.24 & 0.3M) of carbonate added to medium.
For sulphate effect
K2SO4 (6 mM), MgSO4 (0.8 mM) and FeSO4 (0.036 mM) as in regular Zarrouk’s
medium were considered as controls.
K2SO4+MgSO4+FeSO4 were added in a dose dependent manner [K2SO4 (4.5, 5.5, 6.5,
7.5 and 8.5 mM); MgSO4 (0.65, 0.75, 0.85, 0.95 and 1.05 mM); FeSO4 (0.015, 0.025,
0.035, 0.045 and 0.055 mM)].
Calcium
0.4 mM as in regular Zarrouk’s medium was considered as control. Five different
concentrations (0.2, 0.6, 0.8, 1.0, 1.2 mM) of calcium added to medium.
Investigation of Insulin Like Protein From Spirulina
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Selenium
The effect of selenium stress was examined by adding calculated amount of Sodium
selenite in the culture medium to have varying concentrations (0.15, 0.30, 0.45, 0.6, 0.75
mM) of the selenium.
Glucose
The effect of exogenous glucose stress was examined by adding calculated amount in
the culture medium to have varying concentrations (5, 7.5, 10, 12.5, 15 mM) of the
glucose.
pH stress
The effect of pH on the growth S.platensis CFTRI, Mysore was examined by growing
the test strain at different pH scale like - 8, 10 and 12. pH of the control was 9. The
different pH values were adjusted by adding N/10 HCl or N/10 NaOH into the culture
medium.
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