Extraction of Seabuckthorn Seed Oil and Analysis of Its Fatty Acid Composition

Seabuckthorn seed oil was extracted by fat extraction method. The effects of quality of seabuckthorn seed, breaking time, soaking time and extraction solvent on the extraction rate were investigated by single factor experiment and response surface methodology. The fatty acids in seabuckthorn seed oil were determined. The research results show that the most suitable extraction conditions for using a fat extraction instrument are: seabuckthorn seed mass is 1.40 g, crushing time is 11 s, soaking time is 26 min, extraction time is 4 h, temperature is 70°C, and the extraction solvent is petroleum ether. The extraction rate can reach 6.56%; the extracted seabuckthorn seed oil mainly contains three main fatty acids: linoleic acid, linolenic acid and oleic acid, these three fatty acids are also the main sn-2 fatty acids.


Efficacy of Seabuckthorn Seed Oil
Seabuckthorn seed oil is a kind of oil extracted from seabuckthorn seeds, which is rich in nutritional value, contains more amino acids and fatty acids, and has a variety of nutritional components, such as vitamin C, vitamin E, flavonoids, calcium ions and iron ions. Seabuckthorn seed oil can protect stomach and treat gastric ulcer. Seabuckthorn seed oil can prevent arteriosclerosis and reduce liver damage caused by excessive drinking and drugs. Seabuckthorn seed oil can limit the thickness of tear film, relieve the symptoms of red eyes, and has the effect of protecting eyesight. Seabuckthorn seed oil can reduce blood lipid, soften blood vessels, and whiten teeth. Seabuckthorn seed oil also has the effect of beautifying, which can help resist wrinkles and freckles and prevent the skin from being damaged by sunlight and ultraviolet rays. Eating seabuckthorn seed oil in moderation can help prevent cancer and cancer; It can accelerate the discharge of waste in the body, promote intestinal peristalsis and improve constipation; And has the function of promoting blood circulation and removing blood stasis, and can reduce the cholesterol content in the body (Hao, He, & Zhu, 2019).

Protective Effect on Cardiovascular and Cerebrovascular Diseases
In recent years, a series of studies on seabuckthorn seed oil have shown that it has significant protective effect on cardiovascular and cerebrovascular diseases. The research by Wu Ying et al. showed that seabuckthorn seed oil can significantly reduce the serum total cholesterol of high-fat rats (Wu & Wang, 2000), and can increase the serum high-density lipoprotein cholesterol content, and can also significantly improve the mouse auricle microcirculation and the rat mesenteric microcirculation. The above research shows that linolenic acid, tocopherol and phytosterol in seabuckthorn seed oil can reduce blood lipid, inhibit platelet aggregation, improve vascular microcirculation, etc., and have certain health care effects on cardiovascular and cerebrovascular diseases.

Effects on Gastrointestinal Tract
Studies have shown that seabuckthorn seed oil has obvious preventive and therapeutic effects on various experimental gastric ulcers, such as pylorus ligation, stress and reserpine, which are characterized by reduced ulcer incidence, ulcer index and gastric juice secretion. Seabuckthorn seed oil is rich in antioxidant β-sitosterol, which can protect gastrointestinal tract and relieve gastrointestinal ulcer symptoms.

Effect on Skin
Seabuckthorn seed oil is widely used in repairing various skin injuries, including eczema, burns, severe wounds which are difficult to recover, sun burns, radiotherapy and postoperative recovery after laser cosmetic surgery (ZEB, 2004). Seabuckthorn seed oil is rich in palmitoleic acid, which is a fatty acid in skin and can effectively treat local burns and scalds. Oral seabuckthorn seed oil capsule can effectively improve the average smoothness and maximum smoothness of skin and eliminate wrinkles.

Auxiliary Protection of Chemical Liver Injury
Seabuckthorn seed oil can inhibit the increase of glutamic pyruvic transaminase and glutamic oxaloacetic transaminase in liver injury induced by carbon tetrachloride, ethanol and acetaminophen, www.scholink.org/ojs/index.php/sssr Studies in Social Science Research Vol. 2, No. 2, 2021 67 Published by SCHOLINK INC. and protect liver cell membrane. Seabuckthorn oil has auxiliary protective effect on chemical liver injury. This may be because seabuckthorn oil contains high content of tocopherols, carotenoids and other substances with strong antioxidant effect, which can reduce the activity of aspartate aminotransferase and alanine aminotransferase in blood and increase the activity of GSH in liver (Zang, Deng, & Cong, et al., 2015).

Alleviate Dry Eye
Although seabuckthorn seed oil can not directly affect the fatty acid composition of tear film lipid layer, the tocopherols, carotenoids and arachidonic acid derived from fatty acids in seabuckthorn seed oil may have certain anti-inflammatory and anti-cell differentiation effects on meibomian gland cells, thus having a certain effect on relieving dry eye symptoms.

Present Situation of Seabuckthorn Seed Oil
Seabuckthorn can be used as raw material in food processing to make various beverages, foods and wines, such as juice drinks, fruit wine, fruit vinegar, jam, various cakes and dairy products, etc. In medicine, medicine and health care, it has certain curative effects on cardiovascular and cerebrovascular diseases, lung, stomach, spleen, blood circulation, blood stasis, cervical erosion, burns and frostbite, etc. Seabuckthorn also has its unique value in light industry. Developed skin care and washing products that nourish skin, promote cell metabolism, promote epithelial tissue regeneration, resist allergy, resist bacteria, have strong permeability and protect natural skin color; Seabuckthorn branch is hard and can be used as raw material of plywood and other building materials. Seabuckthorn seed oil and fruit oil, as intermediates and raw materials of medicine, cosmetics and functional foods, have broad application fields and huge market potential (Zhang & Chen, 2010).
Since 1990s, the number of seabuckthorn seed oil processing enterprises in China has increased year by year. The main dosage form of seabuckthorn seed oil is oral liquid, which has the disadvantages of short shelf life, easy oxidation and rancidity, poor taste and low absorption and utilization rate. At present, the best method is to prepare powder oil by microencapsulation technology, which can overcome these defects. Although seabuckthorn oil powder oil technology has been successfully developed, it has not been transformed into productivity. Therefore, we should adopt a variety of effective cooperation methods to vigorously promote this technology.
In recent years, the main seabuckthorn extracts at home and abroad are seabuckthorn seed oil, seabuckthorn fruit powder, seabuckthorn fruit oil, seabuckthorn flavone, seabuckthorn dietary fiber and so on. If some flavonoids are dissolved into seabuckthorn seed oil by adopting appropriate technology, the efficacy of the oil can be improved. Flavonoids, as a recognized natural antioxidant, can replace synthetic antioxidants, which will be beneficial to the preservation of seabuckthorn seed oil, prevent oleic acid spoilage and peroxidation, prolong the shelf life of seabuckthorn seed oil and ensure the curative effect. Or adding a proper amount of Chinese herbal medicine extract containing natural antioxidants or peel residue extract containing flavonoids into seabuckthorn seed oil can also play the above role, and can save the tedious process of improving oil extraction, at the same time, it also strengthens the efficacy of seabuckthorn seed oil (Zhang & Ni, 2001).
Seabuckthorn resources are abundant in China. At present, China is known as "Seabuckthorn Kingdom", with seabuckthorn forest area reaching 1.4 million hectares, accounting for more than 95% of the total seabuckthorn area in the world. Drawing lessons from the experience of comprehensive utilization of seabuckthorn at home and abroad, as long as we invest time and energy and organize a strong scientific research team to comprehensively develop and utilize seabuckthorn plant resources in China, it is believed that the new generation of seabuckthorn products will bring huge economic and social benefits.

Soxhlet Extractor Method
Soxhlet extraction, also known as continuous extraction and Soxhlet extraction, is a method for extracting compounds from solid substances. Fat is widely found in the seeds and fruits of many plants.
The determination of fat content can be used as an index to identify its quality. Soxhlet extraction method is usually used to determine the crude fat content in substances. Extraction method is widely used at home and abroad, among which Soxhlet extraction method is recognized as a classic method and the preferred standard method for grain and oil analysis in China (Dubey, Ramana, & Mishra, 2018). This method takes a long time, which is usually extracted by fat analyzer in laboratory.
That is to say, low boiling point organic solvent (ether or petroleum ether) is used for reflux extraction to remove the crude fat in the sample, and the crude fat content is calculated based on the weight difference between the sample and the residue.
Therefore, at present, it is mainly aimed at the optimization of Soxhlet extraction method, so as to get as much seabuckthorn seed oil as possible under the most suitable conditions.
The matters needing attention in Soxhlet extraction method are that the determination of sample, extractant and extraction organic solvent need dehydration. Sample size should be appropriate. We must attach great importance to the safe use of extraction solvents.

Extraction of Seabuckthorn Seed Oil
Seabuckthorn seed oil was extracted by Soxhlet extraction method.
Weigh the extraction cup with an electronic balance and record the weight of the cup. Accurately weigh a certain amount of seabuckthorn seed powder and put it into a filter paper bag, put the wrapped sample into a filter paper frame, and fix the filter paper frame on a magnet fixture. Measure 60 mL petroleum ether with a measuring cylinder and pour it into the extraction cup. Soak the filter paper frame in the extraction cup, and lift the filter paper frame to suspend it after soaking for a certain time. Open the condensed water, and set the parameters of the fat analyzer to start heating and extracting. The parameters were set as follows: the extraction temperature was 70℃ and the extraction time was 4 hours.
After extraction, stop heating and remove the extraction cup. Recover petroleum ether. Shut down, cut off water and power. Put the removed extraction cup in a fume hood for a certain time until petroleum ether is completely volatilized. Weigh the extraction cup again to make it constant weight to obtain the total mass of the extraction cup and grease. Finally, the quality of oil was obtained and the extraction rate was obtained.

Effect of Seabuckthorn Seed Quality on Extraction Rate of Seabuckthorn Seed Oil
To explore the effect of seabuckthorn seed quality on the extraction rate of seabuckthorn seed oil. The crushing time of seabuckthorn seeds was set as 5 s, the extraction solvent was petroleum ether, the solvent was taken as 60 mL, soaking for 20 min, extracting for 4 h and the temperature was set at 70℃.
Seabuckthorn seeds with the mass of 1 g, 2 g, 3 g, 4 g and 5 g were taken for experiments. Each group did two parallel experiments.

Effect of Crushing Time on Extraction Rate of Seabuckthorn Seed Oil
To explore the effect of crushing time on the extraction rate of seabuckthorn seed oil. 2 g seabuckthorn seeds were extracted with petroleum ether and 60 mL solvent, soaked for 20 min and extracted for 4 h at 70℃. The extraction experiments of seabuckthorn seed oil were carried out with crushing time of 3 s, 5 s, 7 s, 9 s, 11 s and 13 s respectively.

Effect of Reagent Types on Extraction Rate of Seabuckthorn Seed Oil
To explore the effect of reagent types on the extraction rate of seabuckthorn seed oil. Take 2 g seabuckthorn seeds, crush for 5 s, take 60 mL solvent, soak for 20 min, extract for 4 h, and set the temperature at 70℃. Three reagents, ether, n-hexane and petroleum ether, were used to extract seabuckthorn seed oil.

Effect of Soaking Time on Extraction Rate of Seabuckthorn Seed Oil
To explore the effect of soaking time on the extraction rate of seabuckthorn seed oil. 2 g seabuckthorn seeds were crushed for 5 s, extracted with petroleum ether and 60 mL of solvent for 4 h, and the temperature was set at 70℃. The extraction experiments of seabuckthorn seed oil were carried out with soaking time of 10 min, 20 min, 30 min and 40 min respectively.

Response Surface Methodology for Extraction of Seabuckthorn Seed Oil
There are three factors and three levels in this experiment. See Table 3 for factor level and Table 4 for experimental table.

Determination of Extraction Rate of Seabuckthorn Seed Oil
Weighing of seabuckthorn seed oil: Weigh the original quality of the extraction cup and the quality of the extraction cup after constant weight, and the twice quality difference is the extraction amount of seabuckthorn seed oil (Zhao & Liu, 2019).
In the equation: m1 is the mass of seabuckthorn seed powder taken at the beginning of the experiment, g; m2 is the quality of seabuckthorn seed oil obtained by extraction, g.
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Determination of Total Fatty Acid Composition in Seabuckthorn Seed Oil
Take 5 μL seabuckthorn seed oil into a 10 mL gland centrifuge tube, add 1 mL chromatographic pure n-hexane, 200 μL potassium hydroxide methanol solution (2 mol/L) and swirl for 1 min, then add 1 g sodium bisulfate, and swirl until no sodium bisulfate is attached to the centrifuge tube wall. After vortexing, let stand, take 1 mL supernatant, transfer it to the injection brown vial, and perform GC analysis.
Parameters of GC analysis instrument are: Agilent 7820A gas chromatograph; FID detector; Chromatographic column DB-23(30m×0.250mm); Carrier gas: nitrogen. Programmed temperature rise: keep the temperature of the column box at 50℃ for 2 min, raise it to 180℃ at a rate of 10℃/min, keep it for 5 min, and then raise it to 230℃ at a rate of 5℃/min, and keep it for 5 min. The temperature of the rear inlet is 250℃; 12.97kpa pressure; The split ratio is 15: 1; The flow rate is 12ml/min; Total flow rate is 14.4ml/min; Septum sweeping 1.2; Carrier gas saving: off. The post detector temperature is 280℃. The flow rate of hydrogen is 30ml/min; The air flow rate is 300ml/min; Exhaust gas (nitrogen) 25ml/min; Sample injection needle size 10 μ l; Sample injection volume is 2 μL. Mark the sequence table, start the analysis after the instrument is stable, mark each fatty acid in the running chart, and calculate the average value and standard deviation between the two experimental groups.

Determination of sn-2 Fatty Acid Composition in Seabuckthorn Seed Oil
Add 50 μL seabuckthorn seed oil into a 10 mL centrifuge tube with stopper, add 0.5 mL analytically pure n-hexane, 2 mL Tris-HCl buffer (C=1 mol/L,PH=8), 0.5 mL sodium cholate solution (C=1 g/L), 0.2 mL calcium chloride solution (C=220 g/L), and then add 20 mg sodium cholate solution. Seal the periphery of the cover with a sealing film, put it into a 40℃ thermostatic magnetic stirring bath for heating in water bath, shake it up and down by hand for 10 min, finally add 1 mL hydrogen chloride solution (C=6 mol/L), add 0.8 mL anhydrous ether after shaking it evenly, then put it into a centrifuge for 5 min at the speed of 3500 r/min, after centrifugation, transfer in a fume hood, take out the supernatant, evaporate to 0.5 mL, and use a 10 μL pipette gun to spot samples (Bo & Qin, 2008;Zeng, Deng, & Yu, 2020).
Adding about 1 cm of chromatographic solution (n-hexane analytical grade: ether: formic acid = 130: 70: 3) into the chromatographic cylinder, and standing for 30-40 min to homogenize the chromatographic solution. After homogenization, the plated boards (triplicate boards with the size of 10 cm*10 cm) were placed in a chromatography cylinder, and the chromatography plate was taken out to dry in a fume hood when the chromatography liquid ran to 1-2 cm from the top of the chromatography plate; If the separation of triester and fatty acid was not obvious during observation, the isochromatographic solution ran to the top and waited for 3-5 min to separate it, the chromatographic plate was taken out to dry, stained with staining agent (anhydrous ethanol: 2 ′, 7 ′-dichlorofluorescein C=4 g/L), and observed by ultraviolet light after staining and drying.
The chromatographic plate was observed by ultraviolet light. The silica gel on the monoglyceride strip was scraped off and placed in a 10 mL gland-covered centrifuge tube. Then 0.7 mL chromatographic pure normal hexane and 200 μL potassium hydroxide methanol solution (2 mol/L) were successively added and vortexed for 1 min. Then 1 g sodium hydrogen sulfate was added and vortexed until no sodium hydrogen sulfate was attached to the wall of the centrifuge tube. After vortexing, allow to stand and transfer 1 mL of the supernatant to a brown vial for GC analysis.
Parameters of GC analysis instrument refer to those during determination of total fatty acids.

Single Factor Experimental Results and Analysis of Extracting Seabuckthorn Seed Oil by Soxhlet
Extraction 3.1.1 Effect of Seabuckthorn Seed Quality on Extraction Rate of Seabuckthorn Seed Oil

Figure 1. Effect of Seabuckthorn Seed Quality on Extract Rate of Seabuckthorn Seed Oil
It can be seen from Figure 1 that with the increase of seabuckthorn seed quality, the extraction of seabuckthorn seed oil first increased and then decreased. When the mass of seabuckthorn seeds is 2 g, the extraction rate of seabuckthorn seed oil is the highest, which is 7.89%. When the mass is 3 g, the extraction rate tends to decrease obviously. The SPSS software was used to analyze its significance.
There was a significant difference between the extraction rate of seabuckthorn seed oil when it was 2 g and when the seabuckthorn seed quality was 1 g, 3 g, 4 g and 5 g (p<0.05), so the seabuckthorn seed quality was finally determined to be 2 g for experiment.

Figure 2. Effect of Broken Time on Extract Rate of Seabuckthorn Seed Oil
It can be seen from fig. 2 that the extraction rate of seabuckthorn seed oil tends to increase slowly with the increase of the breaking time of seabuckthorn seeds. When the crushing time of seabuckthorn seeds is 11 s, the extraction rate of seabuckthorn seed oil is higher. The extraction rate was 8.22% in 11 s. By SPSS software, the extraction rate of seabuckthorn seed oil was significantly different between the crushing time of 11 s and the crushing time of 3 s (p<0.05), but there was no significant difference among other crushing times. Therefore, when extracting seabuckthorn seed oil, the crushing time of seabuckthorn seeds was 11 s.

Figure 3. Effect of Reagent Types on Extract Rate of Seabuckthorn Seed Oil
As shown in Figure 3, when petroleum ether was used as the extraction solvent, the extraction rate of seabuckthorn seed oil was the highest, reaching 7.89%. Statistical analysis was performed using SPSS software for statistical significance. When petroleum ether and n-hexane were used, there was a significant difference in the extraction rates of seabuckthorn seed oil (p<0.05). Therefore, when seabuckthorn seed oil was extracted, petroleum ether was selected as the extraction solvent for the experiment.

Figure 4. Effect of Soaking Time on Extract Rate of Seabuckthorn Seed Oil
As shown in Figure 4, SPSS software was used for significance analysis. The results showed that there was a significant difference in extraction rates of seabuckthorn seed oil when the soaking time was 10 min, and when the soaking time was 20 min or 30 min (P < 0.05). There was significant difference in extraction rates of seabuckthorn seed oil when the soaking time was 40 min, and when the soaking time was 20 min or 30 min (P < 0.05). When the soaking time was 20 min and 30 min, there was no significant difference in the extraction rates of seabuckthorn seed oil. When the soaking time was 20 min, the extraction rate of seabuckthorn seed oil increased significantly, and the extraction rate was 7.79%. The change of extraction rate was gentle and almost unchanged from 20 to 30 min. The extraction rate of seabuckthorn seed oil decreased significantly when the soaking time was 40 min.
Therefore, when seabuckthorn seed oil was extracted, the soaking time of 30 min was taken for the experiment.

Determination of the Best Conditions for Extracting Seabuckthorn Seed Oil by Soxhlet Extraction
Through single factor experiment, Design-Expert 8.06 was used to design response surface methodology, and the three factors that greatly affected the extraction rate of seabuckthorn seed oil, including seed quality, crushing time and soaking time, were selected as Box-Behnken design. With the extraction rate of seabuckthorn seed oil as the response value, the design and results are shown in the following  The analysis of variance of the regression model is shown in Table 6. It can be seen from ANOVA of regression model in Table 6 that the P value of the model selected in the experiment was 0.0020, which was extremely significant (P<0.05 was reasonable in theory), indicating that the linear relationship between each factor described in the regression equation and the response value was significant, and also indicating the reliability of this test method. The missing term P=0.3828>0.05 was not significant, indicating that the equation had good fitting condition for the experiment and small experimental error. The method could be used to predict the extraction rate of seabuckthorn seed oil under different reaction conditions.
By analyzing the p-values of the primary, interactive and quadratic terms, we can find that the quadratic term, the primary term and the interactive term have the most significant effects on the equation.

Figure 5. Response Surface Diagram of Crushing Time and Seabuckthorn Seed Quality
As shown in Figure 5, the crushing time had little effect on the extraction rate of seabuckthorn seed oil, and the curve was smooth, while the quality of seabuckthorn seed was significantly affected, and the curve was steep.

Figure 6. Contour Lines of Breaking Time and Seabuckthorn Seed Quality
As shown in Figure 6, when the crushing time was 11 s, the contour line was elliptical, indicating that the interactive effect of crushing time and seed quality on the extraction rate of seabuckthorn seed was significant.

Extraction Rate of Seabuckthorn Seed Oil
As shown in Figure 7, the image presented a saddle shape, indicating that the effects of soaking time and seed quality on the extraction rate of seabuckthorn seed oil were similar.

Figure 8. Contour Lines of Soaking Time and Seabuckthorn Seed Quality
As shown in Figure 8, compared with the effect of soaking time and the quality of seabuckthorn seed on the extraction rate of seabuckthorn seed oil, the effect of the quality of seabuckthorn seed was more significant, and the contour line was closer to the quality of seabuckthorn seed.
Figures 9 and 10 showed the interaction between soaking time and crushing time on the extraction rate of seabuckthorn seed oil when the quality of seabuckthorn seed was 2 g.

Figure 9. Response Surface Plot of Soaking Time and Crushing Time to the Extraction Rate of Seabuckthorn Seed Oil
As shown in Figure 9, soaking time had little effect on the extraction rate of seabuckthorn seed oil, manifested as a smooth curve, while crushing time had greater effect, manifested as a steep curve.

Figure 10. Contours of Soaking Time and Crushing Time
As shown in Figure 10, compared with the effect of soaking time and crushing time on the extraction rate of seabuckthorn seed oil, the effect of crushing time was more significant, and the contour line was closer to the crushing time.

Verification Test
The theoretical optimum extraction conditions and response values were obtained by optimizing the extraction conditions with the experimental assistant software Design Expert as follows: seabuckthorn seed mass is 1.37 g, crushing time is 11.27 s, soaking time is 25.62 min, and the extraction rate of seabuckthorn seed oil is 6.73%.
Verification of the model: considering and analyzing the good operability of the experiment, the experiment was carried out under the conditions of 1.40 g seabuckthorn seed mass, 11 s crushing time and 26 min soaking time. The extraction rate of seabuckthorn seed oil calculated by the extraction rate calculation method is 6.56%, and the relative error is 2.82%, less than 10%. It is proved that it is feasible to optimize the extraction rate of seabuckthorn seed oil by response surface methodology.

Fatty Acid Composition Analysis of Seabuckthorn Seed Oil
The gas chromatographic diagram of total fatty acids in seabuckthorn seed oil is shown in Figure 11.

Figure 11. Chromatogram of Fatty Acid Composition of Seabuckthorn Seed Oil
Chromatograms of sn-2 fatty acid in seabuckthorn seed oil are shown in Figure 12.

Note. ND indicates that the result is not detected
The results in Table 7 showed that the seed oil of Hippophae rhamnoides contained more linoleic acid, linolenic acid and oleic acid, 30.17%, 30.37% and 20.21% respectively, which were also the main sn-2 fatty acids, with the contents of 39.31%, 36.98% and 18.44%, respectively.
We can calculate the proportion of sn-1 or sn-3 by using the formula: (3x-y)/2=z through Table 1 and the 39.31% linoleic acid at sn-2. X: linoleic acid in total acid; Y: linoleic acid% ratio in sn-2 position; Z: linoleic acid at position sn-1 or sn-3. The calculated linoleic acid content at the sn-1 or sn-3 position was 25.6%. Therefore, we could know that the linoleic acid in seabuckthorn seed oil was mainly present at the sn-2 position, while the linoleic acid at the sn-1, sn-3 positions was less (Zhao & Liu, 2019).
From 18.44% oleic acid at the sn-2 position, we can calculate an oleic acid content of 21.10% at the sn-1 or sn-3 position. Therefore, we could know that the oleic acid in seabuckthorn seed oil was mainly present at the sn-1 and sn-3 positions, while the oleic acid at the sn-2 position was less.
We can calculate the linolenic acid content at sn-1 or sn-3 as 27.07% from 36.98% at sn-2. Therefore, we could know that the linolenic acid in seabuckthorn seed oil was mainly present at the sn-2 position, while the linolenic acid at the sn-1, sn-3 positions was less.

Single Factor Experimental Conclusion
Through single factor test, the experimental conditions of high extraction rate of seabuckthorn seed oil were determined as follows: seabuckthorn seed mass 2.00 g, crushing time 11 s and soaking time 30 min. Subsequently, the experimental conditions were optimized by response surface methodology and the optimal experimental conditions were obtained.

Response Surface Experimental Conclusion
The optimum extraction conditions of seabuckthorn seed oil were determined by Box-Behnken response surface methodology: the weight of seabuckthorn seed was 1.40 g, the crushing time was 11 s and the soaking time was 26 min. Under these conditions, the extraction rate of seabuckthorn seed oil reached 6.56%.

Fatty Acid Composition Analysis of Seabuckthorn Seed Oil
The seed oil of Hippophae rhamnoides contains more linoleic acid, linolenic acid and oleic acid, 30.17%, 30.37% and 20.21% respectively, which are also the main sn-2 fatty acids, with the contents of 39.31%, 36.98% and 18.44% respectively.
The results showed that linoleic acid in seabuckthorn seed oil mainly existed at sn-2 position; The oleic acid in seabuckthorn seed oil mainly existed at sn-1 and sn-3 positions. Linolenic acid in seabuckthorn seed oil mainly existed at sn-2 position.

Summary and Experience
From the middle of December, my experimental subject was determined as extraction of seabuckthorn seed oil and analysis of its fatty acid composition. Now I have successfully completed the experiment.
In these two short months, I have learned many things that I cannot learn in class or from books. I have made great progress in both theoretical knowledge and practical ability. More importantly, I have learned how to use some instruments and equipment. I have benefited a lot.
After I determine the design topic, under the guidance of teacher Cao Xi I access to a large number of papers related to the subject, translation of literature, in the process of my research content and subject related research progress also have more understanding, let me from the beginning at a loss to the back slowly in-depth understanding, ideas gradually clear.
In these two months, we have paid a lot, but we have also gained a lot. Through this graduation project, I have been further familiar with and studied the correct use of basic instruments in the laboratory and the relevant precautions, and deepened the understanding and experience of the knowledge learned in the four years of college. The process of experiment is a process of continuous exploration, finding new problems and solving problems in the exploration. In this process, I learned a lot of knowledge by going to the library to look for materials and reading books. I expanded my knowledge and, more importantly, improved my ability to think, analyze and solve problems. In the experiment, sometimes to get a set of data and a conclusion, we need to constantly repeat it. After several failures, we can get a better result. This makes me deeply realize that the experiment needs scientific analysis, rigorous method and correct operation. It also needs a persistent and persistent spirit.
Through this experiment, I have deeply realized that "learning from paper is like learning from experience, and you never know how to practice it". I thought I knew enough about the experimental operation by consulting the literature, but when I conducted the experiment, I found that these were far from enough. Only by putting the theory into practice can the theory be tested. Faced with difficulties and challenges, to actively face and solve, active learning, exercise their ability to find and solve problems. However, at the same time, "knowing it is knowing it, and not knowing it is not knowing it." When facing problems that one cannot solve, one should communicate with the teacher in time to sort out the thinking, instead of relying on imagination alone, which wastes time and is useless. This I believe that this graduation project process will benefit me for my whole life.

Acknowledgements
First of all, I would like to express my sincere thanks to my supervisor, teacher Cao Xi, for her careful guidance and the teacher elder sister Pan Yayu from the laboratory for her warm-hearted help. Thank you from the bottom of my heart! In these two months of experiments, I have encountered many difficulties and doubts. Fortunately, you are here, not only to help me, to reassure me, but also to encourage me and support me. Cao teacher meticulous style affects me, make me in the analysis of the problem, more rigorous when dealing with problems; The experimental operation is more careful. In the whole process of graduation design, each set of data is the result of my strict operation, repeated many times and careful verification. At the same time, teacher Cao's persuasion also makes my understanding of the problem more clear and profound, and strive to do extrapolate, which also let me benefit a lot. When I encounter doubts and problems in the process of experiment, there is always the teacher elder sister's enthusiastic help; When I encountered difficulties and setbacks in the process of experiment, there was always their encouragement and support. All of these are the power source that inspired me to persist in the meticulous and rigorous completion of the experiment and complete the graduation project successfully.
Finally, I would also like to thank all the teachers for their teaching and all the students for their care and help in the past four years. Thank you!