Miss Anchana Anjinta and Associate Professor Artiwan Shotipruk, Ph.D.

Separation ofSeparation of γγ-oryzanol from -oryzanol from by-products of rice bran oil by-products of rice bran oil

processing by semi-preparative processing by semi-preparative chromatographychromatography

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Properties of γ-oryzanol White or slightly yellow crystalline powder Molecular formula: C40H58O4

Molecular weight: 602.89

Melting point: 137.5-138.5°C

What is γ-oryzanol? γ-oryzanol consists of a mixture of ferulic acid ester of sterols and triterpene alcohols.

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(Nattawuth Kittiruangthong. Extraction and Purification of gamma-oryzanol from rice bran oil soap stock and acid oil., 2005.)

The main purpose of the neutralization step is to remove free fatty acids and

other impurities.

A large amount of γ-oryzanol is lost fromthe oil into the soapstock about 83-95% .Soapstock is reacted with sulfuric acid.

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Semi-preparative column chromatography

Liquid-liquid extraction

Remove glycerides

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Comparison of soapstock (non-saponified and saponified) and acid oil (non-hydrolyzed and hydrolized) in order to find the suitable starting material for purifing γ-oryzanol in semi-preparative chromatography.

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Soapstock100 grams +

NaOH 2.4 grams

Stirred at 100 ˚C for 2 hours

Original dried soapstock or dried saponified soapstock

15 grams +

Ethyl acetate 200 mlfor 4 hours

Measure the pH

Moisture content

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Measure the pH

Acid oil 1 ml +

2 N NaOH 5 ml

Vortex 1 minute

Heat at 80 ˚C for 10 minutes

Add HCl

Original acid oil or hydrolyzed acid oil 5 ml

+ Ethyl acetate 5 ml

Vortex 1 minute

Centrifuge 4000 rpm for 5 minutes

Collect ethyl acetate phase

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Semi-preparative column chromatography

Stationary phase: Silica gel(25-40 μm) 10 grams

Sample injection: 1ml

Mobile phase: A mixture of hexane and ethyl acetate(70:30 v/v)

Each fraction was collected every 5 minutes.

High Performance LiquidChromatography(HPLC)

C18 column reverse phase HPLC

ELSD detector

Methanol: isopropanol (70:30 v/v) as a mobile phase

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Soapstock and acid oil sample properties

Sample pH Moisture content

Soapstock 10.5 31%

Acid oil 5.0 Max 2.0%

12RP-HPLC chromatograms of a) non-saponified soaptock extracted sample, b) saponified soapstock extracted sample c) non-hydrolyzed acid oil extracted sample and d) hydrolyzed acid oil extracted sample

a)

RP-HPLC Analysis of sample extractsb)

d)

vv vc)

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Purification of γ-oryzanol

Sample Non-saponified soapstock

Saponified soapstock

Non-hydrolyzed

acid oil

Hydrolyzed acid oil

% Recovery 23% 44% 39% 74%

% Purity 69% 79% 17% 77%

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Saponification

Extraction (Leaching)

Semi-preparative column

chromatography

DehydrationLiquid-liquid

extraction

Semi-preparative column

chromatography

%Recovery: 44%

%Purity: 79%

%Recovery: 23%

%Purity: 69%

%Recovery: 39%

%Purity: 17%

%Recovery: 74%

%Purity: 77%

Soapstock