**4.3 Soybean phosphatidylethanolamine (SPE)**

Solid-liquid extraction is performed using powdery soybean phospholipids and ethanol. The parameters are as follows: ratio of phospholipids to ethanol 30g/L, ethanol concentration 95%-100% (100% is optimal), extracting temperatures -15-50 degrees Celsius (- 15 degrees Celsius is optimal) and extracting time 5min-11min (8min is optimal). PE content increases from 19.8% in raw material to 62.8% (An et al., 2006).

Phospholipids are dissolved in isopropanol below 65 degrees Celsius to reach a final concentration in the range of 1%-4% (w/v). The mixture is cooled to 26 degrees Celsius, allowed to rest and the precipitate is filtered and dried to obtain PE of 74.7%-79.9% pure (Ni, 1995).

Zhensheng Zhong et al. (2008) removed oil and fatty acids in powdery phospholipids with acetone first, and removed PC with repeated ethanol extraction due to PC has larger solubility in aliphatic alcohol than PE and PI, and finally enriched PE with petroleum ether extraction due to PE is soluble in ether while PI not.

The powdery soybean phospholipids are extracted repeatedly with acetone to remove oil and refined phospholipids containing 35% PE are obtained. PC is removed by repeated absolute ethanol extraction with heating and stirring and alcohol insolubles are obtained. The alcohol insolubles are extracted 3 times with petroleum ether in the 1:3-1:6 (1:4 is optimal, v/v) ratios at 30-60 degrees Celsius (30 degrees Celsius is optimal). PE obtained this way is 93.5% pure and has a yield of 91.9% (Zhong & Wei, 2008).

#### **4.4 Soybean phosphatidylserine (SPS)**

Pure PS is a white waxy solid. It's soluble in most of the nonpolar solvents containing little water, insoluble in anhydrous acetone and can be extracted from histiocyte with chloroform

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

Column chromatography can obtain high purity PI but the long time needed and the use of

Phospholipids can be hydrolyzed by such phospholipase as phospholipase A1, A2, C and D. When treating the ethanol-treated phospholipids (containing minor PC), phospholipase selectively catalyze hydrolization of PE and PC but not PI. More special, alkaline or acid phospholipase catalyzes hydrolization of PA and some salts produced by PE hydrolization but doesn't act on PI, PC or PE. PI products used in various fields can be obtained by this method and the purity can reach up to 99%. Lypase can be used to purify PI as well, and the

Enzymatic method has wide application prospect as it is simpler, more convenient and environmental prospective compared with solvent method and column chromatography.

Ion exchange resin may be applied to isolate PI from phospholipids mixture. The resin adsorbing PI include diethylaminoethylcellulose, diethylaminoethylagarse and

Solid-liquid extraction is performed using powdery soybean phospholipids and ethanol. The parameters are as follows: ratio of phospholipids to ethanol 30g/L, ethanol concentration 95%-100% (100% is optimal), extracting temperatures -15-50 degrees Celsius (- 15 degrees Celsius is optimal) and extracting time 5min-11min (8min is optimal). PE content

Phospholipids are dissolved in isopropanol below 65 degrees Celsius to reach a final concentration in the range of 1%-4% (w/v). The mixture is cooled to 26 degrees Celsius, allowed to rest and the precipitate is filtered and dried to obtain PE of 74.7%-79.9% pure (Ni,

Zhensheng Zhong et al. (2008) removed oil and fatty acids in powdery phospholipids with acetone first, and removed PC with repeated ethanol extraction due to PC has larger solubility in aliphatic alcohol than PE and PI, and finally enriched PE with petroleum ether

The powdery soybean phospholipids are extracted repeatedly with acetone to remove oil and refined phospholipids containing 35% PE are obtained. PC is removed by repeated absolute ethanol extraction with heating and stirring and alcohol insolubles are obtained. The alcohol insolubles are extracted 3 times with petroleum ether in the 1:3-1:6 (1:4 is optimal, v/v) ratios at 30-60 degrees Celsius (30 degrees Celsius is optimal). PE obtained

Pure PS is a white waxy solid. It's soluble in most of the nonpolar solvents containing little water, insoluble in anhydrous acetone and can be extracted from histiocyte with chloroform

dried to obtain PI of no less than 98%-99% pure (Deng et al., 2003).

quaternaryammoniumethylsephadex etc. (Deng et al., 2003).

increases from 19.8% in raw material to 62.8% (An et al., 2006).

this way is 93.5% pure and has a yield of 91.9% (Zhong & Wei, 2008).

**4.3 Soybean phosphatidylethanolamine (SPE)** 

extraction due to PE is soluble in ether while PI not.

**4.4 Soybean phosphatidylserine (SPS)** 

**4.2.3 Enzymatic method** 

**4.2.4 Other methods** 

1995).

purifies are 60%-70% (Deng et al., 2003).

complex solvent mixture reduce its feasibility in the commercial world.

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

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 yield is 68.9% (Yang, 2010).

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 substitute for BCPS.

#### **4.5 Preparation of phospholipids for injection**

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

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

Soybean Phospholipids 499

**Food industry**: The amount of phospholipids used in food is usually 0.1%-2% of the fat in food. Phospholipids are used in margarines, shortenings, candies, soup bases, pot foods, instant foods (e.g. milk powder), bakery products (e.g. bread, cookie, dessert, biscuit and cracknel) and processed foods of meat and seafood etc. They are also used as coatings of

**Feed industry**: Phospholipids are applied in animal feed such as milk replacer for calf and feed of cattle, pig, poultry, hairy animals, pets and aquatic animal (e.g. fish and crustaceans) etc. Phospholipids are the essential additives of the eel feed as they decrease the diseases of

**Agriculture**: Phospholipids can inhibit the growth of powdery mildew on cucumber, eggplant, green pepper and strawberry. The solution of 0.1% phospholipids-sodium carbonate can effectively inhibit orange green mold, cucumber powdery mildew and rice blights. Phospholipids are used as the coating components after harvesting of fruits and vegetables to improve the storage effect. Phospholipids are additives of pesticide formulae which can improve the adhesivity and permeability of pesticide and reduce their toxicity to

**Daily chemical industries**: Phospholipids are applied in such cosmetics as moisturizer, facial cleanser, sunblocking cream, soap, bath oil, shampoo, hair care agent, shaving cream, shave clean agent, nail polish, makeup powder, blush, rouge, eye shadow, lipstick and hairspray etc. Applying of phospholipids in detergent can improve the dirt-removing power

**Other chemical industries**: Phospholipids are widely used in various paints, wax, shoes polish, wood preservatives, mold spray, tape coating, printing ink, ink, toner, additives of photographic materials and polyamide coating etc. as well as in papermaking and printing. They are also widely used in cement, pitch, tar shingle, surface sealant of linoleum and

An, H.; Wang, M.L. & Cheng, L.B. (2001). Fractionation of Soya Lecithin With High

An, H.; Zhang, L. & He, X.F. (2006). The Fractionation of Phosphatidylethanolamine. *Food* 

Blokland, A.; Honig, W.; Browns, F. & Jolles, J. (1999). Cognition-Enhancing Properties of

Deng, Q.G.; Qi, L. & An, H. (2003). Study On Separating Technique of Soybean Lipositol.

Gu, L.W.; Gao, X.R. & Zhao, J.L. (1999). Recent Advances in Enzymatic Modification of Phospholipids. *China Oils and Fats*, Vol.24, No.6, pp. 60-62, ISSN 1003-7969 Guan, R.L.; Zou, J. & Zhu, H. (2005). Recent Advances in Isolation and Purification of Soybean Lecithin. *China Food Additives*, No.5, pp. 44-48, ISSN 1006-2513 Huang, G.W.; Zhao, J.T. & Gu, K.R. (2003). Hydrogenation of Soybean Powder Lecithin.

*Science*, Vol.27, No.12, pp. 343-346, ISSN 1002-6630

*Chemical Engineer*, No.6, pp. 14-16, ISSN 1002-1124

*China Oils and Fats*, Vol.28, No.2, pp. 51-54, ISSN 1003-7969

Phosphatidylcholine Content Using Isopropanol. *Fine Chemicals*, Vol.18, No.7, pp.

Subchronic Phosphatidylserine (PS) Treatment in Middle-Aged Rats: Comparison of Bovine Cortex PS with Egg PS and Soybean PS. *Nutrition*, Vol.15, No.10, pp. 778-

can, soup packaging and casing of meat such as sausages etc.

the eel and improve their growth.

plants.

of anionic detergents.

putty gum etc.

**7. References** 

385-387, ISSN 1003-5214

783, ISSN 0899-9007

phospholipids in the evaporator. The mixture is pestled and embathed for several times to remove the residual oil and moisture. Then a powdery parenteral soybean phospholipids product is produced. It exhibits the following characteristics: AV 9.9, IV 91.29, nitrogen content 1.9%, phosphorus content 4.08% and AI 99.3% (PC content is 96.7%) (Shao et al., 2000).
