Assessment of Egg Yolk Oil Extraction Methods of for ShiZhenKang Oil by Pharmacodynamic Index Evaluation

To assess the extraction methods of egg yolk oil in ShiZhenKang (SZK) oil, which is used to treat eczema, a mice model of eczema was established by using 2,4-dinitrochlorobenzene (DNCB). The therapeutic effects of egg yolk oil extracted by different methods from SZK oil on the model of acute eczema in mice were evaluated. The oil yield rate of ethanol extraction is 42.06%. Its egg yolk oil is orange and has a rich, sweet, egg smell. Moreover, the SZK oil prepared from it has a very good therapeutic effect on the model of acute eczema in mice. The alcohol extraction method is the preferable method according to a comprehensive evaluation of each index of seven kinds of methods to extract the egg yolk oil.

expensive equipment [15]. The phospholipid content of egg yolk oil extracted by the sub-critical propane extraction method is low, and needs further extraction by a mixed solution of methanol and chloroform, thus it is also used rarely [16][17][18]. Besides, the explosivity of propane is also a potential risk. As for the enzymatic method, the product contains fatty acids [19]. Currently, the most commonly used industrial methods are the dry distillation method and solvent extraction method. However, there is a general lack of research on both methods [20,21]. In this study, the dry distillation method, baking method, reduced pressure distillation, and solvent extraction method were used to obtain egg yolk oil which was then used to prepare SZK oil. The effects of the different methods on egg yolk oil extraction were evaluated and the character and oil yield rate were also used to evaluate the optimal extraction method. Moreover, mice models of acute eczema were established to study the treatment efficiency using SZK oils prepared by the different methods.

Color, Smell and Oil Yield Rate of Egg Yolk Oil Extracted by Different Methods
The egg yolk oils extracted by dry distillation, baking, and reduced pressure distillation were brown and disgusting (Figure 1a-c). The oils extracted by the ether, ethanol, petroleum ether, and chloroform extraction methods were orange and egg fragrant (Figure 1d-g). The oil extracted by the ethanol extraction method was very fragrant. The oil yield with the chloroform extraction method was the highest, followed by the petroleum ether method, drying distillation method and ethanol extraction method (Table 1).
Molecules 2016, 21, 106 2 of 10 sub-critical propane extraction method is low, and needs further extraction by a mixed solution of methanol and chloroform, thus it is also used rarely [16][17][18]. Besides, the explosivity of propane is also a potential risk. As for the enzymatic method, the product contains fatty acids [19]. Currently, the most commonly used industrial methods are the dry distillation method and solvent extraction method. However, there is a general lack of research on both methods [20,21]. In this study, the dry distillation method, baking method, reduced pressure distillation, and solvent extraction method were used to obtain egg yolk oil which was then used to prepare SZK oil. The effects of the different methods on egg yolk oil extraction were evaluated and the character and oil yield rate were also used to evaluate the optimal extraction method. Moreover, mice models of acute eczema were established to study the treatment efficiency using SZK oils prepared by the different methods.

Color, Smell and Oil Yield Rate of Egg Yolk Oil Extracted by Different Methods
The egg yolk oils extracted by dry distillation, baking, and reduced pressure distillation were brown and disgusting (Figure 1a-c). The oils extracted by the ether, ethanol, petroleum ether, and chloroform extraction methods were orange and egg fragrant (Figure 1d-g). The oil extracted by the ethanol extraction method was very fragrant. The oil yield with the chloroform extraction method was the highest, followed by the petroleum ether method, drying distillation method and ethanol extraction method (Table 1).

Pharmacodynamic Index of Egg Yolk Oils Extracted by Different Methods
The mouse eczema model was established by application of 2,4-dinitrochlorobenzene (DNCB) solution to the dorsal skin of mice before administration of the different egg yolk oils. According to the visual inspection of the mice's backs, the mice had varying degrees of eczema, which indicated that the model was established successfully. The eczema symptoms of the reduced pressure distillation group worsened three days after the administration and aggravated obviously five days after the administration, while the eczema symptoms of the other groups began to improve. The curative effect was similar to that obtained with paeonol (control drug group) ( Table 2). It could been seen that obvious erythema, edema, oozing, and auricle swelling appeared on the right ear of mice after stimulating the Delayed Type Hypersensitivity (DTH) reaction. Mouse auricle swelling was obviously improved after treatment with SZK oil obtained by the dry distillation, baking, and ethanol extraction methods and in the paeonol control, but the mice degree of auricle swelling of the reduced pressure distillation method group grew worse. This confirmed that except for the reduced pressure distillation method, the SZK oils prepared from egg yolk oil extracted by different methods had a good effect on the degree of mice auricle swelling, which had significant differences compared with the model group (Table 3). The influence of eczema on the thymus index of each group (Table 4) was: reduced pressure distillation group > chloroform extraction group > ethanol extraction group > paeonol control group > ether extraction method group > baking method group. The spleen index was not significantly different among groups.

Auricle Tissue Pathological Section
The histological images of auricle tissue confirmed that the mice model of eczema was successfully established (

Auricle Tissue Pathological Section
The histological images of auricle tissue confirmed that the mice model of eczema was successfully established (Figure 2b

Discussion
The use of egg yolk oil for topical treatment (to heal fire sores) appeared in the "set proved recipe" of the Northern Zhou Dynasty for the first time, and there are documents about treating sores, fistulas, dysentery and other diseases by oral treatment with egg yolk oil. This has been documented in ancient monographs, such as "Rihuazi Bencao", "Compendium of Materia Medica", "Chinese Eaglewood" and "Bencao Pinhui Jingyao" [14,[22][23][24]. The document "Chinese herbal medicine" summarizes that egg yolk oil mainly treats burns and scalds, otitis media, chapped skin, trauma and parasite sores, and so on [25]. Modern pharmacological studies have shown that egg yolk oil possesses analgesic, wound healing promoting, anti-oxidation, anti-aging, improving memory, lowering blood pressure and other effects [26].
This study evaluated the extraction methods of egg yolk oil according to the effects and characteristics of the egg yolk oil in eczema rehabilitation prescription (Table 5). At present, most approaches aimed at extracting egg yolk oil are based on oil yield rate and chemical ingredients content. However, Chinese medicine uses medicines made of more than one ingredient, and there is no direct linear relationship between the efficacy and one of chemical ingredient or oil yield rate index. In this study, egg yolk oil obtained by different extraction methods was used to prepare SZK oil. Its treatment efficacy was evaluated pharmacodynamically by using the mice model with acute eczema, combined with oil yield rate, characteristics, color and other indicators which provided safe and effective evidences for clinical application. The oil yield rates by the chloroform extraction method, petroleum ether extraction method, baking method, and the ethanol extraction method were all above 40%. However, oil extracted by baking method is black brown and has a burnt smell, so it is not suitable for the preparation of eczema liniments. The ethanol extraction and petroleum ether extraction method product is orange in color. In particular, the ethanol extraction method resulted in a rich egg fragrance and therefore is expected to be more easily accepted by patients. Zhang [22] prepared egg yolk oil by the ethanol extraction method, and obtained a good product in terms of odor, character and pharmacological activity.
As for the pharmacodynamics, the oils extracted by the ether, ethanol and petroleum ether extraction methods showed good curative effect on eczema skin lesions in mice. The carbonization method, baking method and ethanol extraction method products showed a good effect on the auricle swelling of the mice eczema model, which was significantly different compared with the model groups. The ether, chloroform and ethanol extraction method oils can reduce the mice thymus index that is the comparable to that of the control drug paeonol. The pathological section of auricle tissue in the mice model of eczema also confirmed that curative effect and the repair effect on the epithelial defects. The pathology images of auricle tissue showed that except for the reduced pressure distillation method oil, the SZK oils prepared from egg yolk oil extracted by different methods could reduce edema and oozing, alleviate keratinization, lighten denaturalization and putrescence of epithelial keratinocytes and decrease infiltration of inflammation cells.
For economics and safety, chloroform and ethyl ether are chemically dangerous reagents, so both of them should be carefully considered if used for production, and petroleum ether is expensive, while ethanol, compared with ethyl ether, chloroform and petroleum ether, is both economic and safe. Jia [27] studied the application of ethanol in the extraction of egg yolk oil, and found that the oil yield reached 60%. The egg yolk oil was of high quality and the extraction cost was 86007 RMB/t (oil). Thus the ethanol extraction method is suitable for industrial production.
Overall, the ethanol extraction method showed high oil yield rate, orange color, rich egg fragrance and good therapeutic effect on acute eczema. The ethanol extraction method is thus the optimal egg oil extraction method to prepare SZK thanks to its relative safety and economic feasibility. In further experiments, in order to achieve a better application to clinical studies, the unique ingredients and main components in egg yolk oil extracted by the ethanol extraction method should be studied, and the number and variety of experimental animals should be increased.

Animals and Diet
All procedures in this study were approved by the Hunan University of Chinese Medicine Animal Care and Use Committee and were in compliance with the Guide for the Care and Use of Laboratory Animals. One hundred Kuming mice (50 males and 50 females, four weeks of age, n = 10 for each group) were obtained from East innovation experimental animal science and Technology Services Department (Changsha, China). Animal qualified number was SCXK (Xiang) 2009-0013. They were allowed to acclimate for one week. All mice were maintained at 21˝C on a 12-h light/12-h dark cycle with free access to water and food.

Extraction of Egg Yolk Oil
Fresh eggs were bought from a local Chinese market, and then they were cooked by bringing tap water to a boil and then kept simmering for 20 min. After being cooled in tap water for 15 min, eggs were shelled and egg yolk and egg white were separated. Wet pellets from egg were produced, through 14 mesh sieves, and then placed into the constant temperature drying box at 37˝C for 12-20 h until the yolk granules dried. The experimental samples were kept in sealed plastic bags in refrigerator at a temperature of +4˝C until experiment. The egg yolk oil was extracted by different methods as follows: 1.
Dry distillation method: egg yolk powder (174 g) was gently heated in the crucible until the water evaporated, and then heated with high heat to 275˝C until the egg yolk oil precipitated out. The oil obtained was separated from the residue with an asepsis gauze and weighed with an electronic balance.

2.
Baking method: egg yolk powder (100 g) was put onto a Buchner funnel lined with 60 mesh barbed wire and covered with a dish. The bottom of the funnel was put into a beaker, and placed in a constant temperature drying box, which was heated slowly up to 240˝C for 30 min. The obtained egg yolk oil was collected and weighed in the beaker.

3.
Reduced pressure distillation method: egg yolk (100 g) was put into four round bottom flasks and distilled on the electric heating set, which was adjusted to 250˝C. The egg yolk was distilled under reduced pressure (0.05-0.06 MPa) for 4 h in. The oil was collected and weighed. 4.
Ether extraction method: egg yolk powder (70 g) was added to ether (560 mL) and the oil was extracted under water bath heating at room temperature for 1.5 h. The remaining egg yolk powder was extracted again by ether (420 mL) for 1 h. After the removal of ether, the oil was collected and weighed.

5.
Ethanol extraction method: egg yolk powder (100 g) was added to 95% ethanol (800 mL) and extracted under water bath heating at 85˝C for 1.5 h, followed by addition of 95% ethanol (600 mL) to the remaining powder for 1 h. After the removal of ethanol, the oil was collected and weighed. 6.
Petroleum ether extraction method: egg yolk powder (100 g) was added to petroleum ether (800 mL) and extracted under water bath heating at 65˝C for 1.5 h, and then more petroleum ether (600 mL) was added to the remaining powder for extraction for 1 h more. After removal of petroleum ether, the oil was collected and weighed. 7.
Chloroform extraction method: egg yolk powder (100 g) was placed in a Soxhlet extractor filled with chloroform (200 mL) and heated in a 65˝C water bath for 1 h. The extraction was repeated four times until the egg yolk was white. After the removal of chloroform, the oil was collected and weighed.
All these extraction experiments were repeated five times according to the above extraction methodx. Oil yield rate % = (mass of egg yolk oil/mass of egg yolk powder)ˆ100%.

Preparation of SZK Oil
SZK oil was obtained by mixing kuh-seng extracts (1.5 g), and borneol (0.5 g) with egg yolk oil (20 g) extracted according to the different extraction methods.

1.
Grouping and experimental preparation: after one week of adaptive feeding, the 100 mice were randomly divided into 10 groups, including blank control group, model control group, dry distillation method group, baking method group, reduced pressure distillation method group, ether extraction group, ethanol extraction group, petroleum ether extraction group, chloroform extraction group and paeonol (as positive drug) control drug group. Each group included five male mice and five female mice. All mice were sheared with an area of 3 cmˆ3 cm on the abdomen and 1 cmˆ2 cm on the left back.

2.
Sensitization: Except for the blank control group, 50 µL 5% DNCB acetone solution was evenly smeared onto the mice abdomen sheared zone to sensitize on the 1st day, and repeated on the 2nd day. 3.
DTH reaction stimulation: 10 µL and 15 µL of 1% DNCB were evenly smeared on the right ear in mice (both sides) and left back sheared zone on the 7th day, respectively. The ear and back skin of the blank control group were smeared similarly with DNCB All mice were stimulated again with a double dose on the 9th and 10th day.

Oil Administration
After 24 h of stimulation, each group of mice was treated with the corresponding drugs on the right ear and back sheared zone. The inside of the right ear and back sheared zone were treated with 0.05 mL and 0.1 mL SZK oil, respectively, twice per day for 5 days. The mice were killed 2 h after the last treatment.

Visual Observation
The skin changes, such as erythema and edema, on the back of experimental mice were observed daily since the start of drug treatment. The mice were scored two times per week according to the severity of erythema (No erythema as 0, slight erythema, edema and oozing or keratinization as 1, obvious erythema, edema and oozing as 2, moderately severe erythema and edema, obvious oozing as 3, severe erythema and edema, serious oozing, obvious keratinization and escharosis as 4).

Auricle Swelling Degree
The auricular tissue samples were immediately removed using a 7 mm hole puncher when the mice were executed and then weighed. The weight difference between the left and right ear was defined as swelling degree, and the ratio (%) of the weight difference of the left ear and right ear was defined as swelling rate: Swelling degree " Right ear piece weight´Left ear piece weight, Swelling rate " Weight difference of left and right ear tissue{Left ear.

Thymus Index and Spleen Index
The thymus and spleen of each group of mice were weighed after the mice were executed and the thymus index and spleen index were calculated, respectively: Thymus index " Thymic weight{Mice weight, Spleen index " Spleen weight{Mice weight.

Pathological Section of Auricle Tissue
The auricle tissues from the right ear of mice were cut into slices, dyed by hematoxylin and eosin (HE) staining, and observed under microscope.

Statistical Analysis
Results are expressed as means˘standard deviation of the mean (X˘SD) for five independent experiments. A two-tailed Student's t test was used for statistical comparisons between any two specific experimental groups. Multiple group comparisons were done by ANOVA using a least significant difference post hoc analysis (SPSS 16.0 SPSS Inc., Chicago, IL, USA). p < 0.05 was considered statistically significant.

Conclusions
Using the egg yolk powder as raw material, egg yolk oil was prepared by the distillation, baking, reduced pressure distillation, ether extraction, ethanol extraction, petroleum ether extraction and chloroform extraction methods, respectively. SZK oil was then made by using the obtained egg yolk oils to evaluate the egg yolk oil extraction methods by their pharmacodynamic indexes combined with oil yield rate, characteristics, color, smell, etc. The results indicated that ethanol extraction was the optimal extraction method. This method preserves the main functions of the eczema rehabilitation prescription, is suitable for preparation production and has a good pharmacodynamic performance.