| Pine needles are straight needle-shaped leaves of pinaceae plant. It is found,through modern examine method, that pine needle extracts contain much nutrients, such as carbohydrates, crude protein, crude fat, trace mineral elements, vitamins, as well as biological flavonoids, chlorophyll and other substances. Dry pine needle fallen in autumn still has these valuable components, most of which was deserted in forest. A kind of new plant source will be found and pine needle value will be increased through extracting and using its biological substances.Biological flavonoids of fallen pine needle was extracted by pulsed electric field (PEF)technology and enzymatic hydrolysis method respectively in this paper. Extracts were purified with macroporous resin. And its in vitro antioxidant activity was examined too.PEF extraction conditions of pine needle flavonoids were investigated. PEF technology increased the extraction ratio remarkably. Reasonable ranges of parameters were achieved through single factor experiments. Solvent concentration, electric field intensity, pulse number and liquid to solid ratio were 60%(v/v) ethanol solution , 15~25 kV/cm, 6~10 and 30:1~60:1 respectively. Optimal conditions of extraction were determined by orthogonal tests, which were as follows: electric field intensity was 20 kV/cm, pulse number was 8, liquid/solid ratio was 50:1. By prediction, true value of extraction ratio would lie between 9.335% and 10.145%,and its confidence level was 95%.Extraction conditions were further studied based on Central Composite Design principle. And mathematics model of the correlation between extraction ratio(Y) and influencing factors was establishedIn the formula, A, B and C refer to electric field intensity, pulse number and liquid to solid ratio respectively. AB, AC and BC refer to interactive effect between different two factors. A~2, B~2 and C~2 are quadric terms of former factors.As for factors in the model, A, B, C, AB, AC, A~2, B~2 and C~2 are all significant. According to the model, maximum extraction ratio (9.527%) would be gained under the combination conditions, which was as follows: electric field intensity was 20.2 kV/cm, pulse number was 8, liquid/solid ratio was 41:1Effects of different extraction methods were compared. Optimal conditions of ethanol solvent extraction (reflux extraction) were: 60%(v/v) ethanol solution, 50:1 of liquid/solid ratio, 130 min of reaction time. Optimal conditions of microwave-assisted extraction were: 60%(v/v) ethanol solution, 40:1 of liquid/solid ratio, 500w of microwave power, 45s of microwave time.And that of ultrasonic-assisted extraction were: 60%(v/v) ethanol solution, liquid/solid ratio 40:1, ultrasonic power 500w, treating time 40 min. Extraction efficiency decreased in the order: PEF method, microwave-assisted extraction, ultrasonic-assisted extraction and reflux extraction. DPPH radical and hydroxyl radical scavenging activities of pine needle extracts,treated by the former three methods, were respectively similar to each other. In addition, that of reflux method extracted were much weaker.PEF was the better method for extracting flavonoids from pine needle. In conclusion, PEF 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. It also demonstrated the obvious advantages in the field of extraction.Enzymatic extraction method was also studied. Flavonoids extraction ratio increased along with the addition of compound enzyme (cellulose to pectinase ratio was 1:3). Influencing factors were solvent, treating time, reaction temperature, enzyme addition. Mathematics model of the correlation between extraction ratio(Y) and influencing factors was established according to Central Composite Design principle. In the formula, A, B, C and D refer to enzyme addition, treating time, reaction temperature and liquid/solid ratio respectively. AB, AC, AD, BC, BD and CD refer to interactive effect between different two factors. A~2, B~2, C~2 and D~2 are quadric terms of former factors. Among these terms, A, B, C, D,AD,BC,BD ,CD,A2, C~2 and D~2 are all significant. According to the model, maximum extraction ratio (8.28%) would be obtained under the combination conditions, which was as follows: enzyme addition was 0.23%, treating time was 0.75h, reaction temperature was 32℃, liquid/solid ratio was 45:1.Condensed extracts solution was precipitated with ethanol first. Purity of dried supernatant was promoted than crude products. Four different macroporous resin (S-8, D3520, AB-8, NKA-9) were screened with regard to absorption and desorption capacity for pine needle flavonoids. And AB-8 resin was determined to be the best one to purify pine needle flavonoids,due to its relatively higher absorption and desorption ability. Sample concentration, pH value, dynamic leakage curve, eluent concentration as well as eluent quantity were determined. Reasonable absorption and eluting conditions of pine needle flavomoids were as follows: sample concentration was 1.491~3.479mg/mL,pH was 5~6,sample quantity was 1~4 times BV, eluent was 75% (v/v) ethanol solution, eluent quantity was 4 times BV.Flavonoids of pine needle had showed strong antioxidant activity. It displayed moderate DPPH radical scavenging activity, which increased with the promotion of flavonoids concentration. At the point 4.266μg/mL,DPPH radical scavenging activity of ascorbic acid is only 75% of that with pine needle extracts,but it chased up at higher concentration. Hydroxyl radical and super-oxide anion radical of pine needle flavonoids were as 4 times and 2 times as that of ascorbic acid respectively. The reducing power, EC50(defined as amount of material inμg or mg per mL reaction volume that produces 0.5absorbance unit atλ700nm ) of pine needle extracts was 17.20μg/mL,which was as 1/2.8 times as that of ascorbic acid.Water solution of pine needle flavonoids was classified into different fractions by ethyl acetate and butanol successively. The ethyl acetate phase showed strongest antioxidant activity .DPPH radical scavenging activity and reducing power of water phase were higher than that of butanol phase. On the contrary, hydroxyl radical and super-oxide radical scavenging activity of butanol phase were stronger than that of water phase.β-glucosidase is used to hydrolyze pine needle flavonoids to increase its in vitro antioxidant effect. It is found that sound ranges for reaction temperature, time, enzyme addition and extracts substance concentration were 40~50℃, 1~3h, 1/1500～1/500 and 0.3～0.9mg/mL respectively. Optimal conditions were achieved through orthogonal tests. They are as follows: reaction temperature was 40℃, enzyme addition was 1/1000, extracts concentration was 0.6 mg/mL, time was 5h. True value of the indicator, DPPH radical scavenging activity, lies between 17.79% and 20.43%(confidence level was 95%).It is demonstrated, through validation tests, that enzymatic hydrolysis of pin needle flavonoids could improve its antioxidant effect.In general, PEF technology and enzymatic extraction are both convenient and safe methods to extract pine needle flavonoids. Purification conditions of pine needle extracts by macroporous resin could be expanding to industrial field. Antioxidant effects investigated in the paper establish theory foundation for developing nature pine needle antioxidant. All the results achieved here can provide reference with academic and practical meanings for the further research of plant extracts.
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