EXTRACTION OF INSULIN LIKE COMPOUND FROM BITTER MELON

GROUP MEMBERSNURUL HIDAYAH BT. HAMZAH D20091035090NOR HIDAYAH BT. SAAD D20091035108MASZAWANI 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 effectis 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 β-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 (µm) 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’’.

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 gel’s polarity, the silica is modified to make it non-polar by attaching long hydrocarbon chain to it’s surface with 18 carbon atom in them.

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

ReRe

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 40°C-50°C

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 µl.

Too much sample can overload the liner and will result some escaping through the vent giving the error result.

Mt1Rt

2Rt

Second Peak

111.515

Rt

8.924Mt

10.215

bw

211.515

160.246

35057.23

How to find the theoritical plate?

2

1

16 R

b

N tw

2.591

1' 1R R Mt t t

11.515 8.924 2

2.59116

0.246

2

1'

1

16 Reff

b

tN W

1774.94

1)How many plates are used in chromatography

column?

20.29

35057.230.29 1

2.591

8.924

0.29

2

1eff

KNKN

11

R M

M

t tk t

1774.0

Or we can use this formula by finding the retention factor, k:

Find retention factor, keffNThus, given

by2

1eff

KNKN

effN

220.028

Rt

20.420

Bw

8.924Mt

2' 11.10

Rt

Theoritical plate was given by:

220.028

160.420

N

36382.84

Third peak:

211.10

160.42

2

2'

2

16 Reff

b

tN W

11175.5

2' 11.10

Rt

How to find effN ?

2

1eff

KNKN

22

'R

M

tk t 2

1eff

KNKN

1.2436382.84

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

1.24

0.29

21 1

4 1save

kR N

k

1.24 0.29

2avek

1)Separation factor

4.27

2)Find k, average

0.765

3)Substitute the value into the formula

1 4.27 1 1.2435057.23

4 4.27 0.765 1SR

25.34

2 1

1 2 / 2R R

sb b

t tR

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-12. http://www.separationprocesses.com/Adsorption/AD_Chp05a.htm3. 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