| Pinaceae is one of the largest species in world which is widely distributed and having rich resource. There are 10 genera including 230 different kinds. China has very rich resource and variety in pine tree, which is widely distributed and relatively concentrated. Pine needle is a straight needle-shaped leaves of pinaceae plant. Through modern test technology found that pine needle extract is composed by carbohydrates, crude protein, crude fat, trace mineral elements, vitamins, biological flavonoids, chlorophyll and other substances. But currently, researching and processing on pine needle are limited to simple and rough producing, such as feed, and so on. It can not reflect the great value in medicine and food, even can not obtain better economic interest. Therefore, on the basis of protection of forest resources, we need to adopt an advanced technology to extract pine needle.This paper studies dry pine needles shedding in autumn by HPEF technology for the effective extraction and a preliminary separation of flavonoid substances. Besides, it will also compare HPEF and other conventional extraction methods, which reflects the obvious advantages of PEF in the field of extraction broad and application prospects. And the main conclusions are as follows:1.Through the single factor experiment, the paper separately studied the effect of electric field intensity, pulse number, solid/liquid ratio on the extraction yield of flavonoids.⑴When the solid/liquid ratio was 1:30, the ethanol concentration was 60% and the pulsed number was 6, with increase of the electric field intensity, the extraction yield of flavonoids was increasing until the electric field intensity was 20kV/cm, then the extraction yield of flavonoids was decreasing. So the better electric field intensity is 20kV/cm for extraction of flavonoids from pine needle by PEF.⑵When the solid/liquid ratio was 1:30, the ethanol concentration was 60% and the electric field intensity was 20kV/cm, with increase of the pulsed number, the extraction yield of flavonoids was increasing until pulsed number was 8, then the extraction yield of flavonoids was decreasing. So the better pulsed number is 8 for extraction of flavonoids from pine needle by PEF.⑶When the electric field intensity was 20kV/cm, the pulsed number was 8, the ethanol concentration was 60% with increase of the solid/liquid ratio, the extraction yield of flavonoids was increasing. It has a obvious effect on the extraction yield of flavonoids. But it will take a lot of trouble for the next separate process, and increase the cost. So generally considered, solid/liquid ratio of 1:40 is better for extraction of flavonoids from pine needle by PEF.2.Through orthogonal test based on single factor experiment, a maximum of the extraction yield was achieved when electric field intensity was 20kV/cm, pulsed number was 8 and solid/liquid ratio was 1:40. The electric field intensity and solid/liquid ratio were notable factors. Solid/liquid ratio was the most important factor, with significant level of 0.01.Then the significant level of electric field intensity was 0.05.The conclusion by orthogonal test is that the optimal level was electric field intensity of 20kV/cm, pulsed number of 8 and solid/liquid ratio of 1:40.Trough the experimental verification, the extraction rate of pine needle was 9.491%.3.In order to achieve the purpose of precise optimization, a quadratic general rotary unitized design was arranged near the best parameter which was fixed by orthogonal test, and the Mathematics model was established. The regression equation was:So, the regression equation and regression coefficient were notable and level of significance was 0.01. The best combination parameters were fixed exactly: pulse number of 8, electric field intensity of 20kV/cm, solid/liquid rate 1:43.4. Trough the combination analysis of single factor analysis and orthogonal test by the means of return by ethanol extraction, it turned out that under the conditions of the ethanol concentration of 60%, solid/liquid ratio of 1:50 and time of 130 min, the flavonoid substances had maximum extraction rate of 6.219%. The ethanol concentration and solid/liquid ratio were notable factors. Both of them were with significant level of 0.05.The effect of time of extraction was not obvious. 5. Trough the combination analysis of single factor analysis and orthogonal test by the means of microwave-assisted extraction, it turned out that under the conditions of the ethanol concentration of 60%, solid/liquid ratio of 1:40, microwave power of 500W and microwave time of 45s, the flavonoid substances had maximum extraction rate of 8.417%. The solid/liquid ratio, microwave power and time were notable factors. The solid/liquid ratio was the most important factor with significant level of 0.05.The second important factor was microwave power with significant level of 0.05.The microwave time also had effect to extraction rate of flavonoid with significant level of 0.10.6. Trough the combination analysis of single factor analysis and orthogonal test by the means of return by ultrasonic-assisted extraction, it turned out that under the conditions of the ethanol concentration of 60%, solid/liquid ratio of 1:40, Ultrasonic power of 90W and time of 40 min, the flavonoid substances had maximum extraction rate of 7.801%. The ethanol concentration and solid/liquid ratio were notable factors. Both of them were with significant level of 0.05.The effect of time of extraction was not obvious.7. Compared the extraction rates of flavonoid from pine needle which were extracted by the four methods of PEF, return by ethanol extraction, ultrasonic-assisted extraction and microwave-assisted extraction, on each best condition, the comparison of the extraction yields of flavonoid were as follows: ultrasonic> PEF> microwave> return by ethanol.⑴The extraction rate of PEF was the highest one which was 9.627%.It was 1.548 times by the extraction rate of return by ethanol which was 6.219%; was 1.234 times by ultrasonic-assisted which was 7.801%; was 1.144 times by the extraction rate of microwave-assisted which was 8.417%.⑵The extraction time of PEF was less than 1s; the extraction time of return by ethanol was 130 min; the extraction time of ultrasonic-assisted was 40min; the extraction time of microwave-assisted was 15min. Generally speaking, PEF saved a lot of time, reduced the cost of experiment, besides that it was safe and had a good extraction yield.⑶PEF is a low-temperature extraction technology, which works at low temperature and with short time. That gives the maximize protection of the flavonoid substances of dry pine needles in physical and chemical properties.8. With time increasing of storage, the extraction rate of flavonoid showed a downward trend. Similarly, with the extract went to red-brown color to deepen direction, the longer time, the deeper color. It was supposed that the flavonoid which was extracted from pine needle contained tannins which caused the color browning.9. Using different solvents with different nature to extract flavonoids substances from pine needles, it was found that ethanol of 60% with a certain polarity had the largest extraction rate 7.519%, while the extraction rate of 0.592% with petroleum ether as solvent; the extraction rate of 0.803% with water as solvent.10. After using different treatment methods to concentrated extract, dried and milled the extract separately. Tested each content of flavonoid on the base of rutin. The powder which was static two days and dried from the upper after centrifugation had a higher content of flavonoid, at 63.02%.In conclusion, PEF was the better method for extracting flavonoid from pine needle. It also, reflects the obvious advantages in the field of extraction broad and application prospects compared with other conventional extraction methods, such as safe, convenient quick and low-cost especially it had a good extraction yield and an application prospects.
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