1. Separation of γ-oryzanol from by-products of rice bran oil processing by semi-preparative chromatography
Miss Anchana Anjinta and Associate Professor Artiwan Shotipruk, Ph.D.

2. γ-oryzanol and applications
What is γ-oryzanol?
γ-oryzanol consists of a mixture of ferulic acid ester of sterols and triterpene alcohols. .
Properties of γ-oryzanol
White or slightly yellow crystalline powder
Molecular formula: C40H58O4
Molecular weight:
Melting point: °C

3. Soapstock is reacted with sulfuric acid.
Rice bran oil refining
The main purpose of the neutralization step is to remove free fatty acids and other impurities.
การทำให้บริสุทธิ์ด้วยวิธีการทางเคมี
Soapstock is reacted with sulfuric acid.
A large amount of γ-oryzanol is lost from the oil into the soapstock about 83-95% .
(Nattawuth Kittiruangthong. Extraction and Purification of gamma-oryzanol from rice bran oil soap stock and acid oil., 2005.)

4. Chromatography

5. Pretreatment and Extraction
Soapstock
Acid oil
Saponification
Hydrolysis
Remove glycerides
Dehydration
Liquid-liquid extraction
Extraction (Leaching)
Semi-preparative column chromatography

6. Objectives
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.

7. Extraction (Leaching)
Experimental
Extraction of γ-oryzanol from soapstock
Soapstock
Saponification
Dehydration
Extraction (Leaching)
Measure the pH
Soapstock100 grams + NaOH 2.4 grams
Stirred at 100 ˚C for 2 hours
Moisture content
Original dried soapstock or dried saponified soapstock 15 grams + Ethyl acetate 200 ml
for 4 hours

8. Liquid- liquid extraction
Experimental
Extraction of γ-oryzanol from acid oil
Acid oil 1 ml N NaOH 5 ml Vortex 1 minute
Heat at 80 ˚C for 10 minutes
Add HCl
Acid oil
Hydrolysis
Liquid- liquid extraction
Measure the pH
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

9. Purification of γ-oryzanol
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.

10. Analysis of γ-oryzanol
High Performance Liquid
Chromatography(HPLC)
C18 column reverse phase HPLC
ELSD detector
Methanol: isopropanol (70:30 v/v) as a mobile phase

11. Results and discussion
Soapstock and acid oil sample properties
Sample pH
Moisture content
Soapstock
10.5
31%
Acid oil
5.0
Max 2.0%

12. Results and discussion
RP-HPLC Analysis of sample extracts a) b) c) d) v v v
RP-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

13. Results and discussion
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%

14. Conclusions Soapstock Acid oil Non-saponification Saponification
Non-hydrolyzed acid oil
Hydrolyzed acid oil
Dehydration
Liquid-liquid extraction
Extraction (Leaching)
Semi-preparative column chromatography
Semi-preparative column chromatography
%Recovery: 39%
%Purity: 17%
%Recovery: 74%
%Purity: 77%
%Recovery: 23%
%Purity: 69%
%Recovery: 44%
%Purity: 79%

15. Thank you for your attention

16. Introduction
What is rice bran?
(Thai Edible Oil Co., Ltd.)

17. Introduction

18. Introduction
γ-oryzanol

19. Introduction

20. Introduction
Saponification
Hydrolysis

21. Introduction
Saponification

22. Introduction
Hydrolysis

23. The extract of saponified soapstock
γ-oryzanol
Fatty acid
The extract of saponified soapstock
The extracted of hydrolyzed acid oil

24. Results and discussion
Time (min)
concentration of γ-oryzanol (mg/ml)
γ-oryzanol (mg) - 95
100
105
110
115
120
125
130
135
140
145
sum
% Recovery
Time (min)
concentration of γ-oryzanol (mg/ml)
γ-oryzanol (mg) - 95
100
105
110
115
120
125
130
135
140
145
150
200
250
300
sum
% Recovery
The extract of saponified soapstock
The extracted of hydrolyzed acid oil

25. Results and discussion
Hydrolyzed acid oil
% Recovery: 74%
%Purity: 77%
RP-HPLC chromatogram of purified fraction of semi-preparative chromatography purification of acid oil

26. Evaporative Light Scattering Detector (ELSD)

27. Results and discussion
γ-oryzanol content in rice bran oil soapstock
Absorbance of γ-oryzanol at 320 nm in extract extracted from dehydrated soapstock (3g/200 ml solvent) after soxhlet extraction at various times.