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Abstract [Objectives]The chlorogenic acid in Sambucus chinensis Lindl is a kind of biologically active substance with multiple pharmacological effects， but the content is relatively small. It is of great significance to find the best extraction process. Therefore， we designed single factor experiments and orthogonal experiments to screen the best conditions for extracting chlorogenic acid from S. chinensis. [Methods]With ethanol concentration， ultrasonic power， and extraction time as three single factors， single factor experiments were performed on S. chinensis， and its chlorogenic acid content was calculated from a standard curve. The contents of chlorogenic acid obtained under various conditions were compared， and then， orthogonal experiments were designed according to the orthogonal table to analyze the optimal extraction conditions of chlorogenic acid. [Results]The optimal extraction conditions of chlorogenic acid for S. chinensis were： the ethanol concentration 60%， the ultrasonic power 36 W， and the extraction time 40 min.[Conclusions]The factor that had the greatest effect on the extraction rate of chlorogenic acid in S. chinensis was ethanol concentration， followed by extraction time， and the least influencing factor was ultrasonic power. With 60% ethanol solution serving as the extractant， the ultrasonic power of 36 W， and the extraction time of 40 min， a better extraction rate was obtained. These conditions can be used as the best extraction conditions for chlorogenic acid.
Key words Chlorogenic acid; Ultrasonic extraction; Single factor; Orthogonal experiment; Ultraviolet spectrophotometry
Sambucus chinensis Lindl， also known as Jiegucao， Balengma， Zoumafeng， is a plant of Sambucus in the Caprifoliaceae family. Its stem and leaves contain chlorogenic acid[1]. S. chinensis is widely distributed in Hubei and all over China， with abundant resources. It is sweet and bitter in taste， and neutral in nature， and serves as a common herbal medicine in China， which has the effects of dispelling wind and dampness， promoting blood circulation and removing blood stasis， reducing inflammation and pain， resisting spasm and subsiding swelling， and is mainly used for icterohepatitis， bruises， rheumatism， beriberi swelling， etc.[2]. Chlorogenic acid is a kind of polyhydroxyphenolic acid compound produced by the plant body through the shikimic acid metabolism pathway during aerobic respiration. It has antibacterial and anti-inflammatory， hepatoprotective， anti-oxidant， blood pressure lowering， heart and cerebral vessel protecting， and immunity improving effects[3]， and is an important biologically active substance. It is soluble in water， easily soluble in hot water， ethanol and acetone[4]. Because the content of chlorogenic acid in plants is small and it has special pharmacological effects， it is particularly important to optimize its extraction process. In this study， single factor experiments and orthogonal optimization were used to screen the best extraction conditions of chlorogenic acid. The experimental method was ultrasonic extraction. In recent years， ultrasonic technology has been widely used in extraction of biologically active substances in plants， because of its characteristics of low energy consumption， high efficiency， and no damage to active ingredients. When ultrasonic waves propagate in a liquid medium， they can produce special cavitation between the sample and the solvent. The extreme pressure generated during ultrasonic cavitation can increase the permeability of the cell wall， and release the components in cells， which then directly contact solvents and dissolve in the solvents， which thereby increases the mass transfer rate of the components from the solid phase to the liquid phase[5]. Materials and Methods
Materials
Raw materials and reagents
S. chinensis; chlorogenic acid reference substance （Chengdu Mansite Pharmaceutical Co.， Ltd.， Chengdu Institute of Biology， Chinese Academy of Sciences， batch number： MUST-17030620）; absolute ethanol （Chengdu Jinshan Chemical Reagent Co.， Ltd.， batch number： 20160912）; distilled water.
Instruments and Equipment
Ultrasonic cleaner （Autoscience， model： AS3120A）; UV spectrophotometer （Shanghai Yuanxi Instrument Co.， Ltd.， model： UV 5800）; pocket oil-free vacuum pump （model： AP-9908S）; counter balance; volumetric flask; 5 ml pipette; dropper; analytical balance; beaker; glass rod; measuring cylinder; medicine spoon.
Methods
Extraction of chlorogenic acid from S. chinensis
A certain amount of crushed S. chinensis sample （4 g） was weighed and added into test tubes， which were then added with different volume fractions of ethanol， followed by soaking for 24 h. The sample was ultrasonically extracted for 4 times， 30 min each time， and centrifuged at 4 000 r/min for 10 min. The supernatants were diluted to constant volume， and measured with a UV spectrophotometer at 330 nm for the absorbance， which was used to calculate the extraction rate of chlorogenic acid.
The extraction rate of chlorogenic acid was calculated according to the following formula：
Extraction rate of chlorogenic acid =c×V×nm×100%
Wherein c is the concentration of chlorogenic acid， μg/ml; V is the volume of the sample solution， ml; n is the dilution factor of the extract; and m is the mass of the sample， g.
Measurement and drawing of chlorogenic acid standard curve[5]
An analytical balance was used to accurately weigh 5 mg of the chlorogenic acid reference substance， which was added in a beaker. Then， 24 ml of anhydrous ethanol was prepared into a 40 ml ethanol solution with a concentration of 60%. An appropriate amount of the above diluted ethanol solution was poured into the beaker， followed by shaking to fully dissolve the chlorogenic acid reference substance. The solution was then transferred to a 25 ml volumetric flask， diluted to constant volume， and prepared into a 0.2 mg/ml reference substance solution. A 5 ml pipette was used to measure different volumes of the reference substance solution （1， 2， 3， 4， 5， 6 ml）， which were numbered 1， 2， 3， 4， 5， 6， respectively， and diluted with 60% ethanol solution to constant volume （25 ml）. The obtained solutions were measured for the absorbance at the maximum absorption wavelength （330 nm）[7]， with 60% ethanol solution as a blank control. With the concentration as the abscissa and the absorbance as the ordinate， a standard curve was drawn （Fig. 1）， obtaining a standard curve equation y=0.044 6x+0.296 1 （R2=0.992 4）. Single factor experiment
With 4.0 g of S. chinensis as the experimental material， according to the test method of "Extraction of chlorogenic acid from S. chinensis"， ethanol concentration， ultrasonic power and extraction time were used as single factors and set with different levels to extract chlorogenic acid， and the absorbance was measured to calculate the content of chlorogenic acid according to the standard curve.
Effect of ethanol concentration on the extraction rate of chlorogenic acid from S. chinensis
A 100 ml graduated cylinder was used to measure three parts of anhydrous ethanol solution， which were prepared into ethanol solutions （40 ml each） with concentrations of 50%， 60%， and 70% respectively. The prepared solutions were added into 50 ml beakers， which were added with 4 g of fresh aboveground part of S. chinensis， respectively. The obtained mixtures were extracted for 30 min using an ultrasonic extractor with an inherent frequency of 40 khz at the power of 78 W. After the extraction was completed， a vacuum filter was used to remove the filter residue， and the filtrates were diluted with the 50%， 60% and 70% ethanol solutions to 50 ml， respectively. An appropriate amount of each extract was measured at 330 nm for its absorbance， with 60% ethanol solution as a blank control. The absorbance was used to calculate the chlorogenic acid content in the sample.
Effect of extraction time on the extraction rate of chlorogenic acid from S. chinensis
Three spare parts of S. chinensis's fresh aboveground product （4 g each） were added into 50 ml beakers and added with 40 ml of 60% ethanol solution， respectively. The ultrasonic power was set to 78 W， and the extraction was performed for 20， 30 and 40 min， respectively. After the extraction was completed， a vacuum filter was used to remove the filter residue， and the filtrates were diluted with 60% ethanol solution to 50 ml， respectively. An appropriate amount of each extract was measured at 330 nm for its absorbance， with 60% ethanol solution as a blank control. The absorbance was used to calculate the chlorogenic acid content in the sample.
Effect of ultrasonic power on the extraction rate of chlorogenic acid from S. chinensis
Three spare parts of S. chinensis's fresh aboveground product （4 g each） were added into 50 ml beakers and added with 40 ml of 60% ethanol solution， respectively. The ultrasonic power was set to 36， 78 and 120 W， respectively， and the material was extracted for 40 min. After the extraction was completed， a vacuum filter was used to remove the filter residue， and the filtrates were diluted with 60% ethanol solution to 50 ml， respectively. An appropriate amount of each extract was measured at 330 nm for its absorbance， with the 60% ethanol solution as a blank control. The absorbance was used to calculate the chlorogenic acid content in the sample. Orthogonal experiment
Based on the single factor experiment， three factors of ethanol concentration， ultrasonic power， and extraction time were selected and the extraction rate of chlorogenic acid was used as an indicator to make an experimental design according to the L9 （33） orthogonal test table. The factors and levels are shown in Table 1.
Verfication experiment
A counter balance was used to accurately weigh three parts of fresh aboveground S. chinensis product with a weight of 4 g. The weighed three parts of materials were added into 50 ml beakers numbered 1， 2， and 3， respectively， and 40 ml of 60% ethanol solution was added into each part and mixed with the sample. The mixtures were put into an ultrasonic cleaner， the ultrasonic power of which was set to 78 W， and the extraction was performed for 40 min. After the extraction was completed， the extraction systems were vacuum filtered， separately， and each filtrate was diluted to 50 ml with 60% ethanol solution. With 60% ethanol solution as a blank control， the absorbance of each extract was determined for three times in parallel， and the average value was taken to calculate the chlorogenic acid content.
Results and Analysis
Single factor experiments
Effect of ethanol concentration on the extraction rate of chlorogenic acid from S. chinensis
It can be seen from Table 2 that the extraction rate of chlorogenic acid from S. chinensis was the highest when the ethanol concentration was 60%. Therefore， the following experiment was carried out with the ethanol concentration fixed at 60%， the ultrasonic power fixed at 78 W， and the ultrasonic extraction time as the variable.
Effect of extraction time on the extraction rate of chlorogenic acid from S. chinensis
It can be seen from Table 3 that the extraction rate of chlorogenic acid from S. chinensis was the highest under the condition of ultrasonic action time of 40 min， and the extraction rate was relatively low when the action time was less than 40 min. Therefore， the following experiment was carried out with the ethanol concentration fixed at 60%， the ultrasonic extraction time fixed at 40 min， and the ultrasonic power as the variable.
Effect of ultrasonic power on the extraction rate of chlorogenic acid from S. chinensis
It can be seen from Table 4 that the extraction rate of chlorogenic acid from S. chinensis was the highest under the condition of the ultrasonic power of 78 W， and excessive power reduced the extraction rate. Orthogonal experiments
It can be seen from Table 5 that the R value that the effects on the experimental results were in order of factor A>factor B>factor C， that is， ethanol concentration>extraction time>ultrasonic power. From the results of orthogonal experiments， it could be concluded that the extraction conditions for the highest extraction rate of chlorogenic acid in S. chinensis were A2B3C2， that is， with the ethanol concentration of 60%， the extraction time of 40 min， and the extraction power of 78 W， the extraction rate of chlorogenic acid from S. chinensis was the highest.
Verification experiments
It can be seen from Table 6 that the results of the three experiments were not much different， indicating that the previous experimental results were reliable and the experimental feasibility was high. The best chlorogenic acid extraction process in this experiment was feasible.
Discussion and Conclusions
Chlorogenic acid in S. chinensis has a variety of biological activities， but because the content of chlorogenic acid is less， it is of great significance to obtain the optimal extraction conditions. In this study， through single-factor experiments and orthogonal experiments， the highest extraction rate of chlorogenic acid was obtained under the conditions of ethanol concentration 60%， ultrasonic power 78 W and extraction time 40 min. Ethanol concentration was the most influential factor. When the ethanol concentration was within the range of 50%-60%， the extraction rate gradually increased， but the differences were not large. When it exceeded 60%， the extraction rate dropped instead， which might be because the concentration of the extractant was too high and caused S. chinensis's other chemical components to be extracted， which interfered with the extraction of chlorogenic acid， resulting in a decrease in the extraction rate. The extraction time had little effect. From the perspective of the three extraction experiments with the same ethanol concentration， the longer the extraction time， the higher the extraction rate. Because this experiment only tested 20， 30， and 40 min， it is impossible to know whether the extraction rate of chlorogenic acid will continue to increase if the extraction time exceeds 40 min. However， as far as laboratory extraction is concerned， comprehensive consideration is given to saving time and effort， and the extraction rate of 40 min was already high enough， so 40 min could be selected as the best extraction time. Ultrasonic power had the least effect， but from the experimental results， the extraction rate was the highest when the ultrasonic power was 78 W， so the best ultrasonic power could be set to 78 W. During the experiment， there might be other interfering factors. For example， in the process of shredding S. chinensis， since it was fresh， juice would flow out during the cutting process. Although not much， because the content of chlorogenic acid was not much， the cutting process might have a certain impact on the experimental results. Secondly， during the ultrasonic extraction process， the temperature gradually increased， and temperature might also be one of the factors affecting the extraction rate of chlorogenic acid. However， this study did not list temperature as an influencing factor， so it might also affect the experimental results. However， after consulting literatures， it can be seen that the three factors involved in this study are the factors that have the greatest impact on the experimental results. Therefore， in summary， the conclusion of this experiment is： the best conditions for extraction of chlorogenic acid from S. chinensis were ethanol concentration 60%， ultrasonic power 78 W， and extraction time 40 min. References
[1]LI HS， ZHOU ZH， CHEN Y. Study on separation of chlorogenic acid and total flavonoids from honeysuckle with macroporous resin[J]. Chemical World， 2020（11）： 760-766. （in Chinese）
[2]WANG X， LI N， QI JH， et al. Study on the extraction process of chlorogenic acid in honeysuckle[J]. Journal of Heilongjiang Hydraulic Engineering College， 2020， 35（3）： 36-39. （in Chinese）
[3]ZOU SQ， CHEN W. Research advances in Sumbucus Chinensis Lindl [J]. Journal of Anhui Agricultural Sciences， 2006， 78（13）： 3092-3094. （in Chinese）
[4]TAO JY， FANG WS. Chemical constituents from Sambucus chinensis[J]. China Journal of Chinese Materia Medica， 2015， 37（10）： 1399-1401. （in Chinese）
[5]MIAO Q， YE MG， LIU SJ， et al. Determination of contents of chlorogenic acid in Helianthus tuberosus L. leaves and Helianthus annuus L. leaves by HPLC[J]. Chemistry & Bioengineering， 2017（2）： 63-67. （in Chinese）
[6]LIU XQ， LIU BL， ZENG XY， et al. Research progress in extraction of chlorogenic acid from herba Artemisiae Scopariae[J]. Guangdong Chemical Industry， 2013， 40（21）： 118，126. （in Chinese）
[7]LI AM， YAO YZ， GUO Y， et al. Optimization of ultrasonic-assisted extraction process of chlorogenic acid from Sambucus chinensis Lindl. leaves[J]. Science and Technology of Food Industry， 2013， 030（17）： 232-235， 240. （in Chinese）
[8]YANG HB， SHI L， XIAO P， et al. Studies on the quality of Sambucus chinensis Lindl. II[J]. Journal of Hubei College of Traditional Chinese Medicine， 2010（1）： 26-27. （in Chinese）
[9]YI CH， HE JH. Determination of chlorogenic acid in Chinese herbal medicine extracts by ultraviolet spectrophotometry[J]. Veterinary Pharmaceuticals & Feed Additives， 2004， 9（1）： 24-25. （in Chinese）
[10]LIAO QF， XIE SP， CHEN XH， et al. Determination of chlorogenic acid from Sambucus chinese in various collecting periods[J]. Traditional Chinese Drug Research and Clinical Pharmacology， 2006（3）： 198-200. （in Chinese）
Key words Chlorogenic acid; Ultrasonic extraction; Single factor; Orthogonal experiment; Ultraviolet spectrophotometry
Sambucus chinensis Lindl， also known as Jiegucao， Balengma， Zoumafeng， is a plant of Sambucus in the Caprifoliaceae family. Its stem and leaves contain chlorogenic acid[1]. S. chinensis is widely distributed in Hubei and all over China， with abundant resources. It is sweet and bitter in taste， and neutral in nature， and serves as a common herbal medicine in China， which has the effects of dispelling wind and dampness， promoting blood circulation and removing blood stasis， reducing inflammation and pain， resisting spasm and subsiding swelling， and is mainly used for icterohepatitis， bruises， rheumatism， beriberi swelling， etc.[2]. Chlorogenic acid is a kind of polyhydroxyphenolic acid compound produced by the plant body through the shikimic acid metabolism pathway during aerobic respiration. It has antibacterial and anti-inflammatory， hepatoprotective， anti-oxidant， blood pressure lowering， heart and cerebral vessel protecting， and immunity improving effects[3]， and is an important biologically active substance. It is soluble in water， easily soluble in hot water， ethanol and acetone[4]. Because the content of chlorogenic acid in plants is small and it has special pharmacological effects， it is particularly important to optimize its extraction process. In this study， single factor experiments and orthogonal optimization were used to screen the best extraction conditions of chlorogenic acid. The experimental method was ultrasonic extraction. In recent years， ultrasonic technology has been widely used in extraction of biologically active substances in plants， because of its characteristics of low energy consumption， high efficiency， and no damage to active ingredients. When ultrasonic waves propagate in a liquid medium， they can produce special cavitation between the sample and the solvent. The extreme pressure generated during ultrasonic cavitation can increase the permeability of the cell wall， and release the components in cells， which then directly contact solvents and dissolve in the solvents， which thereby increases the mass transfer rate of the components from the solid phase to the liquid phase[5]. Materials and Methods
Materials
Raw materials and reagents
S. chinensis; chlorogenic acid reference substance （Chengdu Mansite Pharmaceutical Co.， Ltd.， Chengdu Institute of Biology， Chinese Academy of Sciences， batch number： MUST-17030620）; absolute ethanol （Chengdu Jinshan Chemical Reagent Co.， Ltd.， batch number： 20160912）; distilled water.
Instruments and Equipment
Ultrasonic cleaner （Autoscience， model： AS3120A）; UV spectrophotometer （Shanghai Yuanxi Instrument Co.， Ltd.， model： UV 5800）; pocket oil-free vacuum pump （model： AP-9908S）; counter balance; volumetric flask; 5 ml pipette; dropper; analytical balance; beaker; glass rod; measuring cylinder; medicine spoon.
Methods
Extraction of chlorogenic acid from S. chinensis
A certain amount of crushed S. chinensis sample （4 g） was weighed and added into test tubes， which were then added with different volume fractions of ethanol， followed by soaking for 24 h. The sample was ultrasonically extracted for 4 times， 30 min each time， and centrifuged at 4 000 r/min for 10 min. The supernatants were diluted to constant volume， and measured with a UV spectrophotometer at 330 nm for the absorbance， which was used to calculate the extraction rate of chlorogenic acid.
The extraction rate of chlorogenic acid was calculated according to the following formula：
Extraction rate of chlorogenic acid =c×V×nm×100%
Wherein c is the concentration of chlorogenic acid， μg/ml; V is the volume of the sample solution， ml; n is the dilution factor of the extract; and m is the mass of the sample， g.
Measurement and drawing of chlorogenic acid standard curve[5]
An analytical balance was used to accurately weigh 5 mg of the chlorogenic acid reference substance， which was added in a beaker. Then， 24 ml of anhydrous ethanol was prepared into a 40 ml ethanol solution with a concentration of 60%. An appropriate amount of the above diluted ethanol solution was poured into the beaker， followed by shaking to fully dissolve the chlorogenic acid reference substance. The solution was then transferred to a 25 ml volumetric flask， diluted to constant volume， and prepared into a 0.2 mg/ml reference substance solution. A 5 ml pipette was used to measure different volumes of the reference substance solution （1， 2， 3， 4， 5， 6 ml）， which were numbered 1， 2， 3， 4， 5， 6， respectively， and diluted with 60% ethanol solution to constant volume （25 ml）. The obtained solutions were measured for the absorbance at the maximum absorption wavelength （330 nm）[7]， with 60% ethanol solution as a blank control. With the concentration as the abscissa and the absorbance as the ordinate， a standard curve was drawn （Fig. 1）， obtaining a standard curve equation y=0.044 6x+0.296 1 （R2=0.992 4）. Single factor experiment
With 4.0 g of S. chinensis as the experimental material， according to the test method of "Extraction of chlorogenic acid from S. chinensis"， ethanol concentration， ultrasonic power and extraction time were used as single factors and set with different levels to extract chlorogenic acid， and the absorbance was measured to calculate the content of chlorogenic acid according to the standard curve.
Effect of ethanol concentration on the extraction rate of chlorogenic acid from S. chinensis
A 100 ml graduated cylinder was used to measure three parts of anhydrous ethanol solution， which were prepared into ethanol solutions （40 ml each） with concentrations of 50%， 60%， and 70% respectively. The prepared solutions were added into 50 ml beakers， which were added with 4 g of fresh aboveground part of S. chinensis， respectively. The obtained mixtures were extracted for 30 min using an ultrasonic extractor with an inherent frequency of 40 khz at the power of 78 W. After the extraction was completed， a vacuum filter was used to remove the filter residue， and the filtrates were diluted with the 50%， 60% and 70% ethanol solutions to 50 ml， respectively. An appropriate amount of each extract was measured at 330 nm for its absorbance， with 60% ethanol solution as a blank control. The absorbance was used to calculate the chlorogenic acid content in the sample.
Effect of extraction time on the extraction rate of chlorogenic acid from S. chinensis
Three spare parts of S. chinensis's fresh aboveground product （4 g each） were added into 50 ml beakers and added with 40 ml of 60% ethanol solution， respectively. The ultrasonic power was set to 78 W， and the extraction was performed for 20， 30 and 40 min， respectively. After the extraction was completed， a vacuum filter was used to remove the filter residue， and the filtrates were diluted with 60% ethanol solution to 50 ml， respectively. An appropriate amount of each extract was measured at 330 nm for its absorbance， with 60% ethanol solution as a blank control. The absorbance was used to calculate the chlorogenic acid content in the sample.
Effect of ultrasonic power on the extraction rate of chlorogenic acid from S. chinensis
Three spare parts of S. chinensis's fresh aboveground product （4 g each） were added into 50 ml beakers and added with 40 ml of 60% ethanol solution， respectively. The ultrasonic power was set to 36， 78 and 120 W， respectively， and the material was extracted for 40 min. After the extraction was completed， a vacuum filter was used to remove the filter residue， and the filtrates were diluted with 60% ethanol solution to 50 ml， respectively. An appropriate amount of each extract was measured at 330 nm for its absorbance， with the 60% ethanol solution as a blank control. The absorbance was used to calculate the chlorogenic acid content in the sample. Orthogonal experiment
Based on the single factor experiment， three factors of ethanol concentration， ultrasonic power， and extraction time were selected and the extraction rate of chlorogenic acid was used as an indicator to make an experimental design according to the L9 （33） orthogonal test table. The factors and levels are shown in Table 1.
Verfication experiment
A counter balance was used to accurately weigh three parts of fresh aboveground S. chinensis product with a weight of 4 g. The weighed three parts of materials were added into 50 ml beakers numbered 1， 2， and 3， respectively， and 40 ml of 60% ethanol solution was added into each part and mixed with the sample. The mixtures were put into an ultrasonic cleaner， the ultrasonic power of which was set to 78 W， and the extraction was performed for 40 min. After the extraction was completed， the extraction systems were vacuum filtered， separately， and each filtrate was diluted to 50 ml with 60% ethanol solution. With 60% ethanol solution as a blank control， the absorbance of each extract was determined for three times in parallel， and the average value was taken to calculate the chlorogenic acid content.
Results and Analysis
Single factor experiments
Effect of ethanol concentration on the extraction rate of chlorogenic acid from S. chinensis
It can be seen from Table 2 that the extraction rate of chlorogenic acid from S. chinensis was the highest when the ethanol concentration was 60%. Therefore， the following experiment was carried out with the ethanol concentration fixed at 60%， the ultrasonic power fixed at 78 W， and the ultrasonic extraction time as the variable.
Effect of extraction time on the extraction rate of chlorogenic acid from S. chinensis
It can be seen from Table 3 that the extraction rate of chlorogenic acid from S. chinensis was the highest under the condition of ultrasonic action time of 40 min， and the extraction rate was relatively low when the action time was less than 40 min. Therefore， the following experiment was carried out with the ethanol concentration fixed at 60%， the ultrasonic extraction time fixed at 40 min， and the ultrasonic power as the variable.
Effect of ultrasonic power on the extraction rate of chlorogenic acid from S. chinensis
It can be seen from Table 4 that the extraction rate of chlorogenic acid from S. chinensis was the highest under the condition of the ultrasonic power of 78 W， and excessive power reduced the extraction rate. Orthogonal experiments
It can be seen from Table 5 that the R value that the effects on the experimental results were in order of factor A>factor B>factor C， that is， ethanol concentration>extraction time>ultrasonic power. From the results of orthogonal experiments， it could be concluded that the extraction conditions for the highest extraction rate of chlorogenic acid in S. chinensis were A2B3C2， that is， with the ethanol concentration of 60%， the extraction time of 40 min， and the extraction power of 78 W， the extraction rate of chlorogenic acid from S. chinensis was the highest.
Verification experiments
It can be seen from Table 6 that the results of the three experiments were not much different， indicating that the previous experimental results were reliable and the experimental feasibility was high. The best chlorogenic acid extraction process in this experiment was feasible.
Discussion and Conclusions
Chlorogenic acid in S. chinensis has a variety of biological activities， but because the content of chlorogenic acid is less， it is of great significance to obtain the optimal extraction conditions. In this study， through single-factor experiments and orthogonal experiments， the highest extraction rate of chlorogenic acid was obtained under the conditions of ethanol concentration 60%， ultrasonic power 78 W and extraction time 40 min. Ethanol concentration was the most influential factor. When the ethanol concentration was within the range of 50%-60%， the extraction rate gradually increased， but the differences were not large. When it exceeded 60%， the extraction rate dropped instead， which might be because the concentration of the extractant was too high and caused S. chinensis's other chemical components to be extracted， which interfered with the extraction of chlorogenic acid， resulting in a decrease in the extraction rate. The extraction time had little effect. From the perspective of the three extraction experiments with the same ethanol concentration， the longer the extraction time， the higher the extraction rate. Because this experiment only tested 20， 30， and 40 min， it is impossible to know whether the extraction rate of chlorogenic acid will continue to increase if the extraction time exceeds 40 min. However， as far as laboratory extraction is concerned， comprehensive consideration is given to saving time and effort， and the extraction rate of 40 min was already high enough， so 40 min could be selected as the best extraction time. Ultrasonic power had the least effect， but from the experimental results， the extraction rate was the highest when the ultrasonic power was 78 W， so the best ultrasonic power could be set to 78 W. During the experiment， there might be other interfering factors. For example， in the process of shredding S. chinensis， since it was fresh， juice would flow out during the cutting process. Although not much， because the content of chlorogenic acid was not much， the cutting process might have a certain impact on the experimental results. Secondly， during the ultrasonic extraction process， the temperature gradually increased， and temperature might also be one of the factors affecting the extraction rate of chlorogenic acid. However， this study did not list temperature as an influencing factor， so it might also affect the experimental results. However， after consulting literatures， it can be seen that the three factors involved in this study are the factors that have the greatest impact on the experimental results. Therefore， in summary， the conclusion of this experiment is： the best conditions for extraction of chlorogenic acid from S. chinensis were ethanol concentration 60%， ultrasonic power 78 W， and extraction time 40 min. References
[1]LI HS， ZHOU ZH， CHEN Y. Study on separation of chlorogenic acid and total flavonoids from honeysuckle with macroporous resin[J]. Chemical World， 2020（11）： 760-766. （in Chinese）
[2]WANG X， LI N， QI JH， et al. Study on the extraction process of chlorogenic acid in honeysuckle[J]. Journal of Heilongjiang Hydraulic Engineering College， 2020， 35（3）： 36-39. （in Chinese）
[3]ZOU SQ， CHEN W. Research advances in Sumbucus Chinensis Lindl [J]. Journal of Anhui Agricultural Sciences， 2006， 78（13）： 3092-3094. （in Chinese）
[4]TAO JY， FANG WS. Chemical constituents from Sambucus chinensis[J]. China Journal of Chinese Materia Medica， 2015， 37（10）： 1399-1401. （in Chinese）
[5]MIAO Q， YE MG， LIU SJ， et al. Determination of contents of chlorogenic acid in Helianthus tuberosus L. leaves and Helianthus annuus L. leaves by HPLC[J]. Chemistry & Bioengineering， 2017（2）： 63-67. （in Chinese）
[6]LIU XQ， LIU BL， ZENG XY， et al. Research progress in extraction of chlorogenic acid from herba Artemisiae Scopariae[J]. Guangdong Chemical Industry， 2013， 40（21）： 118，126. （in Chinese）
[7]LI AM， YAO YZ， GUO Y， et al. Optimization of ultrasonic-assisted extraction process of chlorogenic acid from Sambucus chinensis Lindl. leaves[J]. Science and Technology of Food Industry， 2013， 030（17）： 232-235， 240. （in Chinese）
[8]YANG HB， SHI L， XIAO P， et al. Studies on the quality of Sambucus chinensis Lindl. II[J]. Journal of Hubei College of Traditional Chinese Medicine， 2010（1）： 26-27. （in Chinese）
[9]YI CH， HE JH. Determination of chlorogenic acid in Chinese herbal medicine extracts by ultraviolet spectrophotometry[J]. Veterinary Pharmaceuticals & Feed Additives， 2004， 9（1）： 24-25. （in Chinese）
[10]LIAO QF， XIE SP， CHEN XH， et al. Determination of chlorogenic acid from Sambucus chinese in various collecting periods[J]. Traditional Chinese Drug Research and Clinical Pharmacology， 2006（3）： 198-200. （in Chinese）