EXTRACTION OF INSULIN LIKE

COMPOUND FROM BITTER MELON

GROUP MEMBERS
NURUL HIDAYAH BT. HAMZAH D20091035090
NOR HIDAYAH BT. SAAD D20091035108
MASZAWANI BT.ABD.WAHAB D20091035110
Bittermelon

The scientific name is
Momordica charantia,

widely grown in Asia Africa
and the Caribbean

edible fruit which is among
the most bitter of all fruits

Bitter melon is worldwide
known for its effectiveness in
treating diabetes
Why does bittermelon was choosen to to
extract insulin?

Bitter melon chemically contains
a compound that is very much similar
to insulin and sometimes also referred
as p-insulin.

Researches have shown that when it
is taken continuously for some period
has the ability to substitute the insulin
in the body
What will happen when after you eat bittermelon?
Hyperglycaemic effect
is the medical term for a state produced
by a lower than normal level of blood
glucose

also refer as "under-sweet blood"

Due to the presence of charantin which
helps to lowering blood glucose level
Charantin
Composed of a mixture of -
sitosteryl glucoside and 5,25-
stigmasteryl glucoside

(Stimulates the release of insulin and blocks
the formation of glucose in the bloodstream,
which may be helpful in the treatment of
diabetes

HPLC SYSTEM
SAMPLE
PREPARATION
Washed the sample with distilled water


Cut the samples into small pieces


Dried the sample in oven at 50 degree celcius (1 day)
Pulverized the sample into fine powder in a mortar with liquid nitrogen
(400 micrometer) that will be measured by particle sizer
Store the samples at 4 degree celcius until be used


The sub-sample was weighted, dried out at 50 degree celcius


Re-weighted the subsample to obtain dry weight


Pressured Liquid
Extraction
A new technique for Charantin extraction

Factors that contribute to the efficiency of extraction

type of solvent ( acetone,dichloromethane,ethanol,water)
solvent composition (0-100% ethanol in water)
temperature (50-150 degree celcius)
solvent flow rate (2-6ml/min)

The most highly influence on extraction

-temperature: 80.34 degree celcius
-composition of solvent (50%)
Pump was used to deliver solvent to system at a specific flow rate

The solvent was preheated to the required temperature in a
preheating coil installed in the oven before entering the extraction
vessels that was preloaded with 1.0g sample

The function of back pressure regulator is to maintain the system
pressure at about 10MPa to ensure that solvent was in liquid state at all
temperature tested

The extracts was cooled in a coil immersed in water bath to prevent
possible product degradation & was collected in samples vials at
10min intervals

The sample in each vial was then evaporated under vacuum to
remove all solvent


The dried extract was then purified prior to the analysis with HPLC

Generally the colour of initial extraction was dark
green and becomes paler towards the end of extraction

The dark green colour was due to the chlorophyll
non-selectively extracted with other compound including charantin

This is a picture of the device needed for the Pressurized
Liquid Extraction
SAMPLE
PURIFICATION
Purpose : to purify crude extract
:to remove interfering compound such as chlorophyll and sugar
that may interfere with charantin peak

5ml of 50:50 (v/v) methanol-water was added to crude extract

Mixture was then sonicated for 15 minutes

Centrifuged at 3500 micrometer for 15 minutes to separate
supernatant from the precipitate



Precipitate was then add with 5ml of 70:30 (v/v) methanol-water
and the mixture was again sonicated and centrifuged

Precipitate obtain was added with 3ml of hexane & the step was
repeated

Precipitate obtain was re-dissolved in 200 L of 1:1 (v/v)
chloroform-methanol mixture

The mixture was filtered through nylon membrane filter before
being analyzed by HPLC

WHAT IS HPLC SYSTEM?

HPLC is a separation technique that involve:

The injection of a small volume of liquid sample into a tube packed
with tiny particles (3 to 5 micron (m) in diameter that called as the
stationary phase).




The individual components of the sample (charantin) are moved
down the packed tube(column) with a liquid (mobile phase) forced
through the column by high pressure delivered by a pump.

The charantin components are separated from one another by the
column packing that involves various chemical and/or physical
interactions between their molecules and the packing particles.





These separated components are detected at the exit of this tube
(column) by a flow-through device (detector) that measures their
amount. An output from this detector is called a liquid
chromatogram.
Variable Condition
Column Condition C-18 Inertsil ODS-3 column
(5 m particle, 4.6mm250mm ID).
Stationary phase Silica gel with attaching long hydrocarbon C-
18
Mobile Phase 100:2 (v/v) methanolwater
Flow rate

1 ml/min.
UV detection 204 nm
Sample injection volume 200 l.
Condition in HPLC system
HPLC condition system

Before analyze by HPLC, the purified solution was filtered through
0.45 m nylon membrane filter.

1. Column condition
Column are tubular, column dimension usually use is (4.6mm x
25m). Shorter column are generate less back pressure and form low
pressure. If a column runs at low pressure it allows the user more
flexibility to adjust the flow rate.


For small molecule like charantin, C8 and C 18 column are
usually use to capture and interact with analyte. The suitable column
will give the greatest effect on analyte during method development.
C18 column are the most widely used and tend to be most retentive
for non-polar analyte and give high retention of non-polar analyte.


Stationary Phase.

In analyse the charantin, the separation mode that are used to
separate most compound is reversed phase chromatography.

In this application due to silica gels polarity, the silica is
modified to make it non-polar by attaching long hydrocarbon chain to
its surface with 18 carbon atom in them.

From reversed phase, the polar will be eluted first then followed
by non-polar. Its is because there will be strong attraction between
polar solvent (MeOH/H2O) and polar compound. The reversed
phase can increase the retention time.

Re
Reversed Phase Chromatography
Condition for particle size of stationary
phase.

In order to analyse the charantin, the particle size of stationary
phase(silica) that are use is 5 micrometer.

Smaller particle will generate high pressure. It is cause from high
back pressure that are produce from small particle where the pump
need to push hard the mobile phase to move through the column and
this resistance causes a high pressure.

The smaller particles are generally give higher separation efficiency
which it gives a much greater surface area for interactions between
stationary phase and the molecules flowing past it.
Condition For Solvent Use (Mobile Phase)
In most case the HPLC that was intended to use must be able to pump and mix with
two solvent
1.Not every solvent is appropriate for HPLC. Mixing of solvents that are not
compatible with each other during a separation program is strongly forbidden
because the pump, the column and the detector cell can be damage.

Example:
Pentane is too volatile and bubble will be released from it during high pressure
change whereas ethanol are very viscous and cause high pressure in the column
when they are used at high percentage. In analyse the charantin the methanol-water
mixture was used in ratio of 100:2 (v/v). Methanol can form a strong associate with
water.
Condition for UV detector

When a substance has passed through the column, it
was detected by using several ways.

A common method is by using ultra-violet absorption.
Different compound absorbs strongly in different parts of
the UV spectrum. Methanol absorb at wavelength below
205 nm and water below 190 nm.

For methanol-water mixture that are use as solvent the
wavelength that are commonly use is 204 nm to avoid
false readings from solvent.


Condition for Flow Rate ( 1 ml/min)

Slower flow rate will allow solvent residence time was
longer and make the change for solvent-solute to be in closer contact.


Temperature.
The most common running temperature is 40C-50C
Higher temperature will lead to a shorter column lifetime. High
temperature typically results in increasing of peak efficiency and reduce
the retention time.




Sample injection volume was 200 l.
Too much sample can overload the liner and will
result some escaping through the vent giving the error
result.
M
t
1 R
t
2 R
t
Second Peak
1
11.515
R
t
=
8.924
M
t
=
1
0.215
b
w
=
2
11.515
16
0.246
(
=
(

35057.23 =
How to find the theoritical plate?
2
1
16
R
b
N
t
w
(
=
(
(

2.591 =
1' 1 R R M
t t t
=
11.515 8.924 =
2
2.591
16
0.246
(
=
(

2
1'
1
16
R
eff
b
t
N
W
(
=
(
(

1774.94 =
1) How many plates are used in chromatography
column?

2
0.29
35057.23
0.29 1
(
=
(
+

2.591
8.924
=
0.29 =
2
1
eff
K
N
K
N
(
=
(
+

1
1
R M
M
t t
k
t

=
1774.0 =
Or we can use this formula by finding the retention factor, k:
Find retention factor, k
eff N
Thus, given
by
2
1
eff
K
N
K
N
(
=
(
+

eff
N
2
20.028
R
t
=
2
0.420
B
w
=
8.924
M
t
=
2
' 11.10
R
t
=
Theoritical plate was
given by:

2
20.028
16
0.420
N
| |
=
|
\ .
36382.84 =
Third peak:
2
11.10
16
0.42
(
=
(

2
2'
2
16
R
eff
b
t
N
W
(
=
(
(

11175.5 =
2
' 11.10
R
t
=
How to find
eff
N
?
2
1
eff
K
N
K
N
(
=
(
+

2
2
'
R
M
t
k
t
=
2
1
eff
K
N
K
N
(
=
(
+

1.24
36382.84
1.24 1
=
+
11170 =
Or by using this fomula:
1)Find retention
factor,k
2)Substitute into the
above formula
11.10
8.924
=
1.24 =
How to find resolution?


2
1
k
k
o =
1.24
0.29
o =
2
1 1
4 1
s
ave
k
R N
k
o
o
| |

| |
=
|
|
+
\ .
\ .
1.24 0.29
2
ave
k
+
=
1)Separation factor
4.27 =
2)Find k, average
0.765 =
3)Substitute the value into the formula
1 4.27 1 1.24
35057.23
4 4.27 0.765 1
S R

| || |
=
| |
+
\ .\ .
25.34 =
( )
2 1
1 2
/ 2
R R
s
b b
t t
R
W W

=
+
20.028 11.515
(0.420 0.246)1/ 2

=
+
Or just simply use this formula:

25.53 =
References.

1. http://www.mendeley.com/research/new-approach-extraction-charantin-momordica-
charantia-pressurized-liquid-extraction/#page-1
2. http://www.separationprocesses.com/Adsorption/AD_Chp05a.htm
3. http://www.chemistry.adelaide.edu.au/external/soc-rel/content/expts/hplcexpt.htm









5. http://www.ionsource.com/tutorial/chromatography/rphplc.htm
4. http://www.chemguide.co.uk/analysis/chromatography/hplc.html