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Abstract Ultrasonic extraction conditions of polyphenols from the needles of black pine （Pinus thunbergii） were optimized by single factors analysis and BoxBehnken experiment design. The results showed that both of ethanol concentration and ultrasonic time had a significant effect on polyphenol extraction， and there was significant interaction between any two variables in the three parameters of ethanol concentration， ultrasonic temperature and ultrasonic time. The optimal parameters for polyphenol extraction were the ethanol concentration of 52.67%， ultrasonic temperature at 49.21and ultrasonic time of 30.76 min. Under these conditions， the actual content of polyphenols was close to the predicted value. Therefore， the process can be used to extract polyphenols from black pine needles.
Key words Pinus thunbergii; Needles; Polyphenols; Response surface methodology
Black pine （Pinus thunbergii） is native to Japan and the coastal areas of southern Korea. In the 1950s， black pine was planted in the Shandong Peninsula and the Liaodong Peninsula in China in large scale. It has become the main afforestation species in the coastal forest belt of the Shandong Peninsula[1]. Black pine is also a common landscape plant， which is often used in urban greening and shelterbelt construction and has a good landscape effect.
Chinas black pine needles are rich in resources and have broad prospects for development. Chinese medicine believes that pine needles are bitter， nontoxic， warm and have the effects of dispelling the wind， invigorating blood circulation， detoxifying， and relieving itching and fatigue. In recent years， many studies have found that Pinaceae plants contain a large number of polyphenolic compounds， which have a series of unique chemical and biological activity and are collectively referred to as pine polyphenols[2-3]. Pine polyphenols are a class of natural polyphenols， which are the most common secondary metabolites of plants and play an important role in the normal growth of plants against infection and injury[4-5]. Plant polyphenols have good antioxidant capacity， can protect human cells and balance free radicals， and have the effects of diminishing inflammation， alleviating pain， resisting bacteria， reducing blood sugar， resisting radiation， and preventing cardiovascular and cerebrovascular diseases[6]. There are many kinds of Pinaceae plants， but there are few reports on polyphenols in black pine needles. In this study， the ultrasonic extraction process of black pine needle polyphenols was optimized using response surface methodology on the basis of single factor experiment， with an attempt to better provide a theoretical basis for the rational development and utilization of black pine needle resources. Materials and Methods
Experimental materials
The black pine needles were collected from the Gaojia Weir black pine planting base in Matou Town， Huaiyin District， Huaian City. After the pine needles were naturally airdried， they were dried in an oven at 80， pulverized with a pulverizer and sieved with a 100 mesh sieve for later use.
Experimental methods
The extraction and determination of polyphenols were carried out according to the method of Wang[7]. A certain amount of pine needle powder （1 g） was accurately weighed， and added into a conical flask with a stopper， into which a certain concentration of ethanol solution was then added. The needle power was then ultrasonically extracted according to corresponding conditions， and filtered under reduced pressure， obtaining a filtrate， which was diluted to constant volume. The obtained solution was determined at 750 nm for absorbance， and the extraction yield of polyphenols could be calculated according to the standard curve.
Single factor experiment
Such three factors as ethanol concentration， ultrasonic temperature and ultrasonic time were selected， and the extracted polyphenol content was taken as an index to conduct a single factor experiment.
Response surface experiment design
According to the results of the previous single factor experiment， a threefactor threelevel optimization experiment was carried out by BoxBehnken design. The levels of various factors are shown in Table 1. With extracted polyphenol content as the response value， the data were processed using design expert 7.0.0 software.
Model optimization and verification
The quadratic polynomial model fitted by the response surface experiment was used to predict the optimal extraction conditions of polyphenols， and a verification experiment was carried out. The reliability of the model could be evaluated by comparing the difference between the measured value and the predicted value.
Results and Analysis
Single factor analysis
The effect of single factor on the extraction of polyphenols from black pine needles was analyzed by adjusting the different levels of the single factor， with other two variables fixed. It could be seen from Fig. 1 that ethanol concentration， ultrasonic temperature and ultrasonic time all had a significant effect on the extraction of pine needle polyphenols. With the increase of ethanol concentration and ultrasonic temperature， the polyphenol yield showed a trend of increasing first and then decreasing; and ultrasonic time also had a significant effect on polyphenol yield. With the prolongation of time， the polyphenol yield gradually increased， reaching a stable level after 30 min. It could be seen that the levels and ranges of the BoxBehnken design were reasonable based on the premise of the ethanol concentration of 60%， ultrasonic temperature at 50 and ultrasonic time of 30 min. Analysis of BoxBehnken design
Establishment and analysis of response models
According to the single factor results， the threefactor threelevel BoxBehnken design was carried out for ethanol concentration A， ultrasonic temperature B and ultrasonic time C. The extraction conditions were optimized with polyphenol yield as the response value， and the design scheme and corresponding results are shown in Table 3.
The data in Table 2 were subjected to quadratic polynomial regression fitting， obtaining the regression equation of the predicted value of polyphenol yield （Y） and the three factors （A， B， C）： Y= -46.006 +0.020 A +2.343 B +0.541 C+0.003 AB +0.004 AC-0.006 BC -0.002 A2 -0.023 B2 -0.008 C2.
The analysis of variance （Table 3） showed that the regression model was highly significant （P<0.000 1）， and the P value of the test for lack of fit was 0.584 5 （>0.05）， which was not significant. The coefficient of determination （R2） of the regression model was 0.983 9， indicating that the model had a good degree of fitting with the real value and can explain 98.39% of the change. The models signaltonoise ratio （Adeq Precision） was 18.430， which was much larger than 4. These data all indicated that the model had a strong enough signal strength and the model was valid.
It could be seen from Table 3 that in the one degree terms， the effects of various factors on the polyphenol yield ranked as ethanol concentration （A） > ultrasonic time （C）>ultrasonic temperature （B）， and among them， ethanol concentration （A） and ultrasound time （C） had a very significant effect. In the interaction terms， the ethanol concentration and ultrasonic temperature （AB） and the ultrasonic temperature and ultrasonic time （BC） both had a significant effect on the polyphenol yield， and the ethanol concentration and ultrasonic time （AC） also had a significant effect. In addition， all quadratic terms （A2， B2， C2） had an extremely significant effect on the polyphenol yield. The mean square value of the models error term was small， which was 0.156， which indicated that the model was effective. Therefore， the application of the response surface methodology to optimize the extraction process was feasible.
Interaction analysis
In the set parameter ranges， the interaction between any two variables was analyzed， obtaining the response surfaces and contour maps between every two variables （Fig. 2）. A round contour indicated that the interaction between the two factors is not significant， and an elliptical contour indicated that the interaction between the two factors is significant[8]. It could be seen from Fig. 2 that the extraction yield of polyphenols had obvious response to ultrasonic temperature and ethanol concentration. With the increase of ethanol concentration and ultrasonic temperature， the yield showed a trend of increasing first and then decreasing， and the effect of ethanol concentration on polyphenol yield was greater than that of ultrasonic temperature; and the polyphenol yield significantly changed with the ethanol concentration and ultrasound temperature changing. With the increase of ethanol concentration， the extraction yield increased slowly and then gradually decreased， and with the ultrasonic temperature increasing， the extraction yield had an obvious trend of increasing first and then decreasing. Ultrasonic temperature and ultrasonic time also had a significant effect on polyphenol extraction， and the polyphenol yield showed an obvious change of increasing first and then decreasing with the increase of the two factors. The contour lines in Fig. 2 were all elliptical， indicating that the interaction between any two of the three variables （ethanol concentration， ultrasonic temperature and ultrasound time） reached a significant or extremely significant level. Determination of optimal conditions and verification of regression model
According to the principle of maximizing the polyphenol yield， the optimal conditions for extracting polyphenols from black pine needles were solved as the ethanol concentration of 52.67%， ultrasonic temperature at 49.21 and ultrasonic time of 30.76 min， under which the predicted polyphenol content was 20.49 mg/g. For convenience of the actual operation， the process conditions were corrected to be the ethanol concentration of 53%， ultrasonic temperature at 50 and ultrasonic time of 31 min. The verification experiment showed that the polyphenol content was 20.21 mg/g， which was not much different from the theoretical prediction. It showed that the model had a good fitting degree. It is reliable to analyze and predict the extraction process of polyphenols from black pine needles by regression equation， and the process conditions can be used to extract polyphenols from black pine.
Conclusions and Discussion
The ultrasonic extraction conditions of polyphenols in black pine needles were optimized by response surface methodology. The results showed that the effects of various factors on the extraction of polyphenols ranked as ethanol concentration>ultrasonic time>ultrasonic temperature; and there was significant or extremely significant interaction between any two variables in the three variables， which significantly affected the extraction of polyphenols. The optimal extraction conditions of polyphenols in black pine needles were the ethanol concentration of 52.67%， ultrasonic temperature at 49.21 and ultrasonic time of 30.76 min， under which the measured polyphenol content was 20.21 mg/g， which was close to the predicted value of 20.49 mg/g. The process can be used to extract polyphenols from black pine needles.
References
[1]XU JW， LI CR， WANG WD， et al. Biomass and productivity of Pinus thumbergii protective forests in sandy coastal area[J]. Journal of Northeast Forestry University， 2005， 33（6）： 29-32. （in Chinese）
[2]FAN ZL， CHEN KL， GAO X， et al. Study on extraction optimization and antioxidant activity of five kinds of polyphenols from pine needles[J]. Modern Food Science & Technology， 2017， 33（8）：211-220. （in Chinese）
[3]MEI J， CHEN P， PENG P. PCDD/Fs accumulation in pine needles： Variation with species and pine needle age[J]. Environmental Science & Pollution Research， 2016， 23（1）： 563-570.
[4]CIESLIK E， GREDA A， ADAMUS W. Contents of polyphenols in fruit and vegetables[J]. Food Chemistry， 2006， 94（1）： 135-142.
[5]JEREZ M， SELGA A， SINEIRO JA， et al. A comparison between bark extracts from Pinus pinaster and Pinus radiata： Antioxidant activity and procyanidin composition[J]. Food Chemistry， 2007， 99（2）： 439-444.
[6]GORLACH S， FICHNA J， LEWANDOWSKA U. Polyphenols as mitochondriatargeted anticancer drugs[J]. Cancer Letters， 2015， 366（2）：141.
[7]WANG J. Orthogonal test optimizing ultrasonic extraction of phenolic compound from Chinese pine pollen[J]. Western Journal of Traditional Chinese Medicine， 2012， 8（25）： 36-37. （in Chinese）
[8]WANG JJ， ZHANG HY， MA GY， et al. Optimization of brewing process of Zaosu pear fruit wine and analysis of its aroma components[J]. Food and Fermentation Industries， 2012， 38（3）： 123-127. （in Chinese）
Key words Pinus thunbergii; Needles; Polyphenols; Response surface methodology
Black pine （Pinus thunbergii） is native to Japan and the coastal areas of southern Korea. In the 1950s， black pine was planted in the Shandong Peninsula and the Liaodong Peninsula in China in large scale. It has become the main afforestation species in the coastal forest belt of the Shandong Peninsula[1]. Black pine is also a common landscape plant， which is often used in urban greening and shelterbelt construction and has a good landscape effect.
Chinas black pine needles are rich in resources and have broad prospects for development. Chinese medicine believes that pine needles are bitter， nontoxic， warm and have the effects of dispelling the wind， invigorating blood circulation， detoxifying， and relieving itching and fatigue. In recent years， many studies have found that Pinaceae plants contain a large number of polyphenolic compounds， which have a series of unique chemical and biological activity and are collectively referred to as pine polyphenols[2-3]. Pine polyphenols are a class of natural polyphenols， which are the most common secondary metabolites of plants and play an important role in the normal growth of plants against infection and injury[4-5]. Plant polyphenols have good antioxidant capacity， can protect human cells and balance free radicals， and have the effects of diminishing inflammation， alleviating pain， resisting bacteria， reducing blood sugar， resisting radiation， and preventing cardiovascular and cerebrovascular diseases[6]. There are many kinds of Pinaceae plants， but there are few reports on polyphenols in black pine needles. In this study， the ultrasonic extraction process of black pine needle polyphenols was optimized using response surface methodology on the basis of single factor experiment， with an attempt to better provide a theoretical basis for the rational development and utilization of black pine needle resources. Materials and Methods
Experimental materials
The black pine needles were collected from the Gaojia Weir black pine planting base in Matou Town， Huaiyin District， Huaian City. After the pine needles were naturally airdried， they were dried in an oven at 80， pulverized with a pulverizer and sieved with a 100 mesh sieve for later use.
Experimental methods
The extraction and determination of polyphenols were carried out according to the method of Wang[7]. A certain amount of pine needle powder （1 g） was accurately weighed， and added into a conical flask with a stopper， into which a certain concentration of ethanol solution was then added. The needle power was then ultrasonically extracted according to corresponding conditions， and filtered under reduced pressure， obtaining a filtrate， which was diluted to constant volume. The obtained solution was determined at 750 nm for absorbance， and the extraction yield of polyphenols could be calculated according to the standard curve.
Single factor experiment
Such three factors as ethanol concentration， ultrasonic temperature and ultrasonic time were selected， and the extracted polyphenol content was taken as an index to conduct a single factor experiment.
Response surface experiment design
According to the results of the previous single factor experiment， a threefactor threelevel optimization experiment was carried out by BoxBehnken design. The levels of various factors are shown in Table 1. With extracted polyphenol content as the response value， the data were processed using design expert 7.0.0 software.
Model optimization and verification
The quadratic polynomial model fitted by the response surface experiment was used to predict the optimal extraction conditions of polyphenols， and a verification experiment was carried out. The reliability of the model could be evaluated by comparing the difference between the measured value and the predicted value.
Results and Analysis
Single factor analysis
The effect of single factor on the extraction of polyphenols from black pine needles was analyzed by adjusting the different levels of the single factor， with other two variables fixed. It could be seen from Fig. 1 that ethanol concentration， ultrasonic temperature and ultrasonic time all had a significant effect on the extraction of pine needle polyphenols. With the increase of ethanol concentration and ultrasonic temperature， the polyphenol yield showed a trend of increasing first and then decreasing; and ultrasonic time also had a significant effect on polyphenol yield. With the prolongation of time， the polyphenol yield gradually increased， reaching a stable level after 30 min. It could be seen that the levels and ranges of the BoxBehnken design were reasonable based on the premise of the ethanol concentration of 60%， ultrasonic temperature at 50 and ultrasonic time of 30 min. Analysis of BoxBehnken design
Establishment and analysis of response models
According to the single factor results， the threefactor threelevel BoxBehnken design was carried out for ethanol concentration A， ultrasonic temperature B and ultrasonic time C. The extraction conditions were optimized with polyphenol yield as the response value， and the design scheme and corresponding results are shown in Table 3.
The data in Table 2 were subjected to quadratic polynomial regression fitting， obtaining the regression equation of the predicted value of polyphenol yield （Y） and the three factors （A， B， C）： Y= -46.006 +0.020 A +2.343 B +0.541 C+0.003 AB +0.004 AC-0.006 BC -0.002 A2 -0.023 B2 -0.008 C2.
The analysis of variance （Table 3） showed that the regression model was highly significant （P<0.000 1）， and the P value of the test for lack of fit was 0.584 5 （>0.05）， which was not significant. The coefficient of determination （R2） of the regression model was 0.983 9， indicating that the model had a good degree of fitting with the real value and can explain 98.39% of the change. The models signaltonoise ratio （Adeq Precision） was 18.430， which was much larger than 4. These data all indicated that the model had a strong enough signal strength and the model was valid.
It could be seen from Table 3 that in the one degree terms， the effects of various factors on the polyphenol yield ranked as ethanol concentration （A） > ultrasonic time （C）>ultrasonic temperature （B）， and among them， ethanol concentration （A） and ultrasound time （C） had a very significant effect. In the interaction terms， the ethanol concentration and ultrasonic temperature （AB） and the ultrasonic temperature and ultrasonic time （BC） both had a significant effect on the polyphenol yield， and the ethanol concentration and ultrasonic time （AC） also had a significant effect. In addition， all quadratic terms （A2， B2， C2） had an extremely significant effect on the polyphenol yield. The mean square value of the models error term was small， which was 0.156， which indicated that the model was effective. Therefore， the application of the response surface methodology to optimize the extraction process was feasible.
Interaction analysis
In the set parameter ranges， the interaction between any two variables was analyzed， obtaining the response surfaces and contour maps between every two variables （Fig. 2）. A round contour indicated that the interaction between the two factors is not significant， and an elliptical contour indicated that the interaction between the two factors is significant[8]. It could be seen from Fig. 2 that the extraction yield of polyphenols had obvious response to ultrasonic temperature and ethanol concentration. With the increase of ethanol concentration and ultrasonic temperature， the yield showed a trend of increasing first and then decreasing， and the effect of ethanol concentration on polyphenol yield was greater than that of ultrasonic temperature; and the polyphenol yield significantly changed with the ethanol concentration and ultrasound temperature changing. With the increase of ethanol concentration， the extraction yield increased slowly and then gradually decreased， and with the ultrasonic temperature increasing， the extraction yield had an obvious trend of increasing first and then decreasing. Ultrasonic temperature and ultrasonic time also had a significant effect on polyphenol extraction， and the polyphenol yield showed an obvious change of increasing first and then decreasing with the increase of the two factors. The contour lines in Fig. 2 were all elliptical， indicating that the interaction between any two of the three variables （ethanol concentration， ultrasonic temperature and ultrasound time） reached a significant or extremely significant level. Determination of optimal conditions and verification of regression model
According to the principle of maximizing the polyphenol yield， the optimal conditions for extracting polyphenols from black pine needles were solved as the ethanol concentration of 52.67%， ultrasonic temperature at 49.21 and ultrasonic time of 30.76 min， under which the predicted polyphenol content was 20.49 mg/g. For convenience of the actual operation， the process conditions were corrected to be the ethanol concentration of 53%， ultrasonic temperature at 50 and ultrasonic time of 31 min. The verification experiment showed that the polyphenol content was 20.21 mg/g， which was not much different from the theoretical prediction. It showed that the model had a good fitting degree. It is reliable to analyze and predict the extraction process of polyphenols from black pine needles by regression equation， and the process conditions can be used to extract polyphenols from black pine.
Conclusions and Discussion
The ultrasonic extraction conditions of polyphenols in black pine needles were optimized by response surface methodology. The results showed that the effects of various factors on the extraction of polyphenols ranked as ethanol concentration>ultrasonic time>ultrasonic temperature; and there was significant or extremely significant interaction between any two variables in the three variables， which significantly affected the extraction of polyphenols. The optimal extraction conditions of polyphenols in black pine needles were the ethanol concentration of 52.67%， ultrasonic temperature at 49.21 and ultrasonic time of 30.76 min， under which the measured polyphenol content was 20.21 mg/g， which was close to the predicted value of 20.49 mg/g. The process can be used to extract polyphenols from black pine needles.
References
[1]XU JW， LI CR， WANG WD， et al. Biomass and productivity of Pinus thumbergii protective forests in sandy coastal area[J]. Journal of Northeast Forestry University， 2005， 33（6）： 29-32. （in Chinese）
[2]FAN ZL， CHEN KL， GAO X， et al. Study on extraction optimization and antioxidant activity of five kinds of polyphenols from pine needles[J]. Modern Food Science & Technology， 2017， 33（8）：211-220. （in Chinese）
[3]MEI J， CHEN P， PENG P. PCDD/Fs accumulation in pine needles： Variation with species and pine needle age[J]. Environmental Science & Pollution Research， 2016， 23（1）： 563-570.
[4]CIESLIK E， GREDA A， ADAMUS W. Contents of polyphenols in fruit and vegetables[J]. Food Chemistry， 2006， 94（1）： 135-142.
[5]JEREZ M， SELGA A， SINEIRO JA， et al. A comparison between bark extracts from Pinus pinaster and Pinus radiata： Antioxidant activity and procyanidin composition[J]. Food Chemistry， 2007， 99（2）： 439-444.
[6]GORLACH S， FICHNA J， LEWANDOWSKA U. Polyphenols as mitochondriatargeted anticancer drugs[J]. Cancer Letters， 2015， 366（2）：141.
[7]WANG J. Orthogonal test optimizing ultrasonic extraction of phenolic compound from Chinese pine pollen[J]. Western Journal of Traditional Chinese Medicine， 2012， 8（25）： 36-37. （in Chinese）
[8]WANG JJ， ZHANG HY， MA GY， et al. Optimization of brewing process of Zaosu pear fruit wine and analysis of its aroma components[J]. Food and Fermentation Industries， 2012， 38（3）： 123-127. （in Chinese）