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.

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: 602.89 Melting point: 137.5-138.5°C

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.)

Chromatography

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

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.

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

Liquid- liquid extraction Experimental Extraction of γ-oryzanol from acid oil Acid oil 1 ml + 2 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

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.

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

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

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

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%

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%

Thank you for your attention

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

Introduction

Introduction γ-oryzanol

Introduction

Introduction Saponification Hydrolysis

Introduction Saponification

Introduction Hydrolysis

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

Results and discussion Time (min) concentration of γ-oryzanol (mg/ml) γ-oryzanol (mg) - 5.62575672 95 0.002583544 0.005167087 100 0.058425354 0.116850708 105 0.249944616 0.499889232 110 0.358563812 0.717127623 115 0.257763195 0.51552639 120 0.128247741 0.256495482 125 0.072573858 0.145147716 130 0.048115701 0.096231402 135 0.031469526 0.062939052 140 0.014099864 0.028199727 145 0.003700224 0.007400448 sum 2.450974867 % Recovery 43.56702554 Time (min) concentration of γ-oryzanol (mg/ml) γ-oryzanol (mg) - 3.04103331 95 100 0.0011745 0.002349 105 0.089040114 0.178080228 110 0.185984109 0.371968218 115 0.22831736 0.456634719 120 0.199659002 0.399318003 125 0.121280589 0.242561178 130 0.116752424 0.233504847 135 0.07577082 0.15154164 140 0.059254245 0.11850849 145 0.039249945 0.07849989 150 0.00866538 0.01733076 200 0.001315881 0.002631762 250 0.002675111 0.005350221 300 0.0014697 0.0029394 sum 2.261218356 % Recovery 74.35690851 The extract of saponified soapstock The extracted of hydrolyzed acid oil

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

Evaporative Light Scattering Detector (ELSD)

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.