**4.1.3 Supercritical fluid extraction**

Supercritical fluid extraction (SFE) is a rapidly developed new technology in recent years. Supercritical fluids most commonly used are carbon dioxide, ammonia, ethylene, propylene and water etc. Carbon dioxide is most frequently used due to its following properties: critical temperature and pressure easy to get, stable chemical properties, non-toxic, odorless, non-corrosiveness and reusable. Supercritical carbon dioxide extraction (SCE) is a method with bright prospect as it can obtain high purity products with no solvent residues and maintain the nutritional and functional properties of the products and need simple process, single equipment and low cost (Guan et al., 2005).

Tekerikler et al. (2001)obtained phospholipids containing 91% PC after SCE of deoiled phospholipids with 10% ethanol as entrainer at 17.2MPa and 60 degrees Celsius. Increasing the pressure to 20.7MPa increased the extraction yield and PC content (95%). Increasing the

in exchange of primary or secondary hydroxyl of some molecules with ethanolamine or choline groups of phospholipids and formation of new phospholipids. This character is called phospholipids' transfer characteristic or base exchange reaction of phospholipase D

Fractions in soybean phospholipids are isolated due to their solubilities' differences in organic solvents. PC exhibits large solubility in lower alcohol (C1-C4) whereas PE and PI have small solubilities. PC- and PI-enriched products can be obtained by their solubilities' difference. When treated with lower alcohol, PC in deoiled phospholipids is soluble in alcohol leaving insoluble matter consisting mainly of PE and PI. The ratio of PC to PE increases from 1:1 (w/w) in raw material to 3:1 (w/w), and even to 12:1 (61% PC, 5% PE). Better isolation effect on PC can be obtained by isopropanol. Mixtures of high-purity phospholipids and isopropanol with the ratios of 8:157-16:157 (12:157 is optimal) is added into the agitated- and refluxed-closed container. The extraction is conducted in thermostatic water bath or cryohydrate bath with isopropanol volume fraction of 95%-100% (100% is optimal) at -5-15 degrees Celsius (-5 degrees Celsius is optimal) for 5min-11min (5min is optimal). After the extraction, the mixture is filtered, evaporated to remove isopropanol and dried to obtain product with mass fraction of PC increased from 25.6% in raw material to 66.8%. Isopropanol extraction is the most commonly employed step to obtain high PC-

It's also an effective method to fractionate phospholipids with the property that phospholipids can react with some salts or acids and precipitate. This method is more promising than organic solvent extraction as metal ions or acids can 'recognize'

100g of phospholipids containing 45% PC is dissolved in 1L ethanol of 95% before addition of 4.5g of zinc chloride. The light yellow phospholipid-zinc chloride compound precipitate formed is centrifuged, decomposed with 250ml freezed acetone under nitrogen to obtain

Supercritical fluid extraction (SFE) is a rapidly developed new technology in recent years. Supercritical fluids most commonly used are carbon dioxide, ammonia, ethylene, propylene and water etc. Carbon dioxide is most frequently used due to its following properties: critical temperature and pressure easy to get, stable chemical properties, non-toxic, odorless, non-corrosiveness and reusable. Supercritical carbon dioxide extraction (SCE) is a method with bright prospect as it can obtain high purity products with no solvent residues and maintain the nutritional and functional properties of the products and need simple process,

Tekerikler et al. (2001)obtained phospholipids containing 91% PC after SCE of deoiled phospholipids with 10% ethanol as entrainer at 17.2MPa and 60 degrees Celsius. Increasing the pressure to 20.7MPa increased the extraction yield and PC content (95%). Increasing the

**4. Extraction and isolation of soybean phospholipids** 

containing phospholipids from deoiled phospholipids (An et al, 2001).

**4.1.2 Lower alcohol with salt or acid extraction** 

phospholipids molecules more effectively than solvents.

36.7g of phospholipids containing 99.6% PC (Ni, 1995).

single equipment and low cost (Guan et al., 2005).

**4.1.3 Supercritical fluid extraction** 

**4.1 Soybean phosphatidylcholine (SPC)** 

**4.1.1 Organic solvent extraction** 

(Song et al., 2007).

temperature to 80 degrees Celsius decreased the extraction yield and PC content which was attributed to decrease of solubility and selectivity of solvents to PC as the solvents density decrease at high temperatures.

#### **4.2 Soybean phosphatidylinositol (SPI)**

PI causes concern as it is involved in the transmission of messages in the cell. PI plays and important role in maintaining normal physiological functions of central nervous system, especially in regulating calcium homeostasis. PI on cell membrane can be hydrolyzed by phospholipase C into 1,4,5-triphosphate inositol that goes into intracellular aqueous phase as the second messenger and 1,2-diacylglycerol that stays in the cell wall. These two substances synergetically induce cell reactions such as contraction, secretion, metabolism and proliferation etc.

Soybean phospholipids are rich in PI. PI is a white amorphous solid with its sodium salt a crystal and is wet-sensitive. PI is soluble in water, chloroform and benzene, slightly soluble in methanol, diethyl ether and petroleum ether, insoluble in acetone, ethanol and water. It can be easily oxidized upon exposure to the air (Deng et al., 2003).

#### **4.2.1 Solvent method**

The solvent method is conducted to isolate and purify phospholipids and increase the content of a certain constituent with single or mixture of such solvents as methanol, ethanol, isopropyl ketone, acetone, n-hexane and chloroform etc.

The deoiled soybean phospholipids are extracted with appropriate amount of ethanol. The induced ethanol-insoluble phase is vacuum dried to obtain a mixed phospholipids with more PI and less PC which are dissolved in n-hexane before adding 55% ethanol with sodium acetate. The mixture is put into the separating funnel, shaked, allowed to rest and layered. The same procedure is carried out again except that the 55% ethanol is sodium acetate free. The PI obtained is 40%-50% pure. If sodium acetate is replaced by aqueous ammonia with a 8.0 pH and the ethanol concentration increased to 90%, PI of 85% pure can be obtained with the same method.

Purer PI can be obtained by some chemical reaction methods. Soybean phospholipids containing 40% mixed phospholipids are dissolved in such organic solvents as anhydrous pyridine, acetonitrile, dimethyl formamide (DMF) and dimethylsulphoxide (DMSO) etc. Chloride dimethyl tertiary butyl silicon, chloride trimethyl silicon or allyl bromide are added into the mixture to protect hydroxyls of PI by reacting with them. Then PI is isolated from the mixture with solvents such as acetone or ethanol-acetone and hydrolyzed by alkali or acid at room temperature to remove the blocking groups and recover the hydroxyls of PI. PI obtained this way is 98% pure and applied in treating of central nervous system disorder (Deng et al., 2003).

#### **4.2.2 Column chromatography**

The phospholipids mixture is dissolved in the mixture of chloroform and methanol in the 1:1 (v/v) ratio before adding aluminium oxide. The eluate contains PC, lysophospholipids, neutral lipids and glycolipids etc. Residues are washed and extracted with the mixture of chloroform, methanol and 1% hydroxyl ammonium acetate in the 1:1:0.3 (v/v/v) ratio and the eluate is loaded on silica column of which the dimension is 30cm. Neutral lipids are eluted with chloroform; glycolipids and PE are sequentially eluted with chloroform and

Soybean Phospholipids 497

and methanol. When PS is dissolved in water, most of the insoluble lipids form micell while very few form true solution. PS has one positive and two negative charges, resulting in a net negative charge. PS can be hydrolyzed by weak base into metal salts of fatty acids and a remained portion, and by strong alkali into fatty acids, serine and glycerol phosphate. PS is ready to oxidize upon exposure to the air, and the color gets darker from white to yellow and finally black. Natural PS practically isn't affected by alcohol while saturated PS can form interwoven catenulated gel with alcohol and dipalmiloyl-phosphatidylserine can interact

PC is dissolved in organic phase while the enzyme and serine are dissolved in aqueous phase. After preheating for a while, the two phases are combined, and reaction occurs at the interface under certain conditions. PS is obtained by isolating and extracting of the organic solvent phase and quantified by thin layer chromatography (TLC). The parameters are as follows: ratio of organic phase to aqueous phase 4:4 (v/v), PC concentration 75mg/ml, reacting temperature 40 degrees Celsius, pH of aqueous phase 4.0 and reacting time 12h. PS

Blokland et al. (1999) compared the effects of bovine cortex phosphatidylserine (BCPS), SPS and egg phosphatidylserine (EPS) on cognitive competence of middle aged rats. The dosage given to lab mice was 15mg/kg.d. Changes of emotional behavior and cognitive competence in open field experiment, Morris water maze and two-dimension active avoidance experiment were observed. Arjan Blokland discovered that SPS and BCPS exhibit similar cognitive competence-improving effects which were higher than that of EPS. SPS might be a

1 portion of powdery soybean phospholipids is mixed with 12 portions of distilled water and stirred to form colloidal dispersion liquid in boiling water bath before 1.8 times the weight of raw material of anhydrous sodium sulfate is added. The saturated sodium sulfate solution is discarded after blocky phospholipids are precipitated. Then 5 portions of distilled water and 0.8 portions of anhydrous sodium sulfate are used to repeat the salting out procedure. The salting-out soybean phospholipids are dried at reduced pressure and 70 degrees Celsius in water bath in vacuum drier, transferred into three-mouth bottle followed by addition of 8.7 times the weight of raw material of 95% ethanol and reflux extraction at 80 degrees Celsius in water bath with stirring for 1h. After cooling, the ethanol solution containing phospholipids is poured out and stored in refrigerator overnight to precipitate PC. The ethanol solution containing soybean phospholipids is poured out, heated to about 35 degrees Celsius in water bath before addition of activated aluminum oxide of 0.5 times the weight of raw material, stirred for 1h and filtered. The ratio of powdery phospholipids,

The above ethanol solution is poured out followed by addition of activated carbon of 0.22 times the weight of raw material, stirring for 1h and filtration with sintered funnel. The filtrate is transferred into the distillation flask and subjected to reduced pressure distillation at 70 degrees Celsius in water bath under nitrogen to remove ethanol. Diethyl ether of 0.75 times the weight of raw material is added into the distillation flask to dissolve the dried soybean phospholipids. The diethyl ether solution is bottled and the bottle is airtight after filling in nitrogen and stored in refrigerator overnight before ultrafiltration. The diethyl ether is removed by reduced pressure evaporation at 40 degrees Celsius in water bath under nitrogen in evaporator. Anhydrous acetone is added into the glutinous soybean

with 5% alcohol at room temperature to form regular gel.

**4.5 Preparation of phospholipids for injection** 

ethanol and aluminum oxide is 1:8:0.5 (w/w/w).

yield is 68.9% (Yang, 2010).

substitute for BCPS.

methanol in the 80:20 (v/v) ratio; PE is further removed with chloroform and methanol in the 20:80 (v/v) ratio; PI is finally eluted with chloroform, methanol and 25% ammonia in the 80:20:5 (v/v/v) and 65:25:5 (v/v/v) ratios. The PI-containing fraction is evaporated and dried to obtain PI of no less than 98%-99% pure (Deng et al., 2003).

Column chromatography can obtain high purity PI but the long time needed and the use of complex solvent mixture reduce its feasibility in the commercial world.
