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Abstract Ampelopsis grossedentata （HandMazz） W. T. wang is a typical edible plant with important physiological health functions. Dihydromyricetin is the main active ingredient of A. grossedentata， which has good pharmacological effects such as antibacterial， antiinflammatory， antioxidation， antitumor and liver protecting effects. This paper summarized the research progress on the extraction materials， extraction methods and separation and purification processes of dihydromyricetin in A. grossedentata to understand the best experimental method of dihydromyricetin， aiming to provide an important theoretical basis for the comprehensive development and utilization of dihydromyricetin.
Key words Ampelopsis grossedentata; dihydromyricetin; extraction method; isolation and purification; progress
Vine tea [Ampelopsis grossedentata （HandMazz） W. T. wang]， is a wild vine of Vitis in Vitaceae. Vine tea is mainly grown in Guangxi， Hunan， Yunnan， Guizhou and other places. Because of its different producing areas， it has many popular names， such as Maoyanmei， Meicha， Changshouteng， Lingzhicao， etc.[1]. It is a kind of folk medicinal plant， which has been recorded in ancient books such as "Classified Materia Medica"[2]. This tea is slightly bitter and sweet in taste and cool in nature. It has the effects of clearing heat and toxic material， resisting bacteria and inflammation， dispelling wind and eliminating dampness， strengthening bones and muscles， lowering blood pressure and protecting the liver， thus the name "the king of tea".
The most important flavonoid in vine tea with the highest content is dihydromyricetin （DMY）， which has a mass fraction as high as 30% or more in dry stems and leaves of vine tea[3]. It belongs to polyhydroxyflavonols， and has a variety of physiologically active functions， such as antibacterial[4]， antiinflammatory[5]， antioxidant[6]， antiatherosclerosis[7]， antitumor[8]， and liver protecting effects[9].
This paper summarized the research progress on extraction materials， extraction methods and separation and purification processes of dihydromyricetin in vine tea to find out the best experimental method of dihydromyricetin. This study will provide theoretical reference for comprehensive utilization and comprehensive development of dihydromyricetin.
Selection of Extracted Medicinal Materials
The same traditional Chinese medicinal material， due to different producing areas of raw material， varieties， picking positions， collection time and processing methods， the content of the same ingredient varies widely， which will affect the experimental results. Picking position
Chen[10]measured the DMY and total flavonoid contents in different parts of A. grossedentata. The experimental results showed that the part with the highest total flavonoid and DMY contents was young leaves， whose contents were 15.01% and 7.79% respectively. The part with the highest flavonoid ratio was tender stems， with a ratio of 64.31%. He et al.[11]determined that the content of DMY in vine tea leaves was 3 to 4 times higher than that in stems by HPLC， and the content was highest in May， ranging from 27.8% to 31.2%.
Collection time
Lei et al.[12]analyzed the total flavonoid and DMY contents in A. grossedentata in different seasons when using different drying methods. From April to December， the total flavonoid content was highest in April， and the highest DMY content appeared in May. And the total flavonoid and DMY contents in the vine tea ovendried after collection were highest. Yu et al.[13]found that the total flavonoid and DMY contents reached the highest in April， 8.74%and 4.69%， respectively.
Processing conditions
Wang et al.[14]found when examining the effects of different drying temperatures on the quality components of vine tea that compared with samples dried in the shade， ovendried samples showed DMY and total flavonoid contents greatly increased， and the contents of the components reached their highest values when the samples were dried at 100 ℃. Feng et al.[15]obtained a significantly higher DMY content with the ovendrying method than with natural drying， and the vine tea collected in May was best amongthe different collection periods. Yi[16]investigated the effects of drying in the shade， sun drying， and oven drying on total flavonoids and DMY in vine tea， and concluded that the best vine tea drying method was drying at 70 ℃. The research results of Yang et al.[17]showed that the DMY contents of different parts of vine tea were significantly different. The leaves （3.93 mg/g） had a DMY content significantly higher than that of roots （0.34 mg/g）， and the value got higher after being stored for two years.
Therefore， in order to ensure the highest DMY content in vine tea and facilitate experimental research， in the selection of experimental medicinal material， the best vine tea part can be selected as the leaves which are collected in May， and can be processed by oven drying， and it is better to store the material for two year before processing. Common Extraction Methods
At present， the DMY extraction methods in vine tea mainly include solvent extraction method， microwaveassisted extraction method， ultrasonic extraction method， countercurrent extraction method， supercritical fluid extraction method and some improved technologies.
Solvent extraction method
Dihydromyricetin is a polyhydroxyflavonol compound， which is easily soluble in solvents such as hot water and ethanol， and slightly soluble in ethyl acetate. Among them， water and ethanol are the most widely used to extract dihydromyricetin[18]. Guo[19]used distilled water， 30% ethanol （volume fraction）， 60% ethanol， 90% ethanol and methanol as extraction agents for DMY， and the extraction rates were 15.30%， 11.31%， 13.15%， 16.68%， 16.68% and 13.12%， respectively. From the experimental data， it could be seen that the extraction rate of 90% ethanol was the highest. However， considering that the water extraction method is simple， convenient， highly economic and has low cost and high extraction rate， distilled water can be used as the extraction solvent for DMY in the laboratory or industrial production. In the following， hot water extraction of DMY was taken as an example to summarize the best extraction process of water extraction in recent years. The details are given in Table 2 for details.
It can be known from the above table that the optimal extraction conditions for extracting DMY with hot water are as follows： the extraction temperature selected from 78 to 100 ℃， the extraction time from 60 to 120 min， the material ratio from 1∶ 15 to 1∶ 30， the pH value of about 8， and the raw material particle size of 80-100 mesh， and the secondary extraction method is more effective. The effects of the extraction factors on DMY extraction rank as extraction temperature>extraction time>materialtoliquid ratio[24-25]. A small experiment should be done first to find out the best extraction conditions in experiments according to the actual situation.
In addition to hot water extraction， DMY can be extracted with other solvents. For example， Xie et al.[27]obtained the best extraction process conditions by the response surface analysis as follows： an ethanol concentration of 81%， a liquidtomaterial ratio at 17 ml/g and reflux extraction for 95 min， under which the DMY extraction rate could reach 9.58%， which is only 0.14% different from the theoretical value of 9.72%. Yan et al.[28]confirmed that ethyl acetate was the best extraction solvent， and the optimal conditions were as follows： an extraction temperature at 50 ℃， extraction time of 50 min and a materialtoliquid ratio at 1∶ 20， with which the extraction rate of DMY was about 14.28%. Solvent extraction has the advantages of simple operation， low cost and no pollution， and is more suitable for largescale industrial production. However， it consumes more time and solvents， and has poor reproducibility， and the product contains more impurities. And the solvents are generally harm to environment and human body[24]. Microwaveassisted extraction （MAE）
The microwaveassisted extraction refers to the rapid extraction of target substances from samples using electromagnetic radiation of microwave[29]， which is a new efficient extraction technology. Microwave extraction has the characteristics of instant realization of high temperature， short extraction time， high extraction efficiency， good reproducibility， and low pollution. Zheng et al.[30]achieved the DMY extraction rate of 25.11% with following conditions： microwave irradiation for 20 min， heating for 50 min and a materialtoliquid ratio of 1∶ 10. In the smallscale extraction[31]， samples were treated with microwave at 750 W for 4 min， and extracted at a materialliquid ratio of 1∶ 20 in a water bath at 95 ℃ for 30 min， and the DMY extraction rate reached 27.53%. However， the samples free of microwave pretreatment were extracted under the same condition， showing the DMY extraction rate of 13.73%. In comparison， the microwave pretreatment improved the extraction rate by 13.80%， indicating that the effective components were dissolved more easily with the assistance of microwave. Yang[32]determined the optimum process parameters through orthogonal experiment： an extraction temperature of 95 ℃， microwave heating time of 15 min， and a materialliquid ratio at 1∶ 20， under which the DMY extraction rate reached 27.33%. After pretreatment with microwave of 300 W for 2 min， the yield of DMY reached 28.48%， which was improved by 20% compared with the traditional water extraction method. It can be seen that the extraction rate of microwave extraction is higher than that of water extraction， but it is not convenient to control parameters such as temperature and power， which may affect the extraction rate of effective ingredients.
Ultrasoundassisted extraction （UAE）
The ultrasonicassisted extraction method is an extraction method that uses ultrasonic waves to increase the speed and frequency of movement of substance molecules， thereby increasing the solvent penetrating power and increasing the dissolution rate of the extracted components[33]. The ultrasoundassisted extraction has the characteristics of simple operation， high speed， less time， low cost and high extraction rate[34]， and has become a common processing method for providing samples in the analysis of many traditional Chinese medicines. Liang et al.[35]extracted DMY with the assistance of ultrasonic waves， and the optimum process parameters were an ethanol volume fraction of 51.00 %， a materialtoliquid ratio at 1∶ 26 （g∶ ml）， and ultrasonic treatment time of 40.00 min， under which the DMY extraction rate was 28.41%， which was close to the extraction rate predicted by the regression model （28.61%）. Yao et al.[36]proposed that the optimum process for extracting DMY from A. grossedentata was to extract at 40 ℃， a liquidtomaterial ratio of 1∶ 18 and an ethanol volume fraction of 65% with the assistance of ultrasonic waves for 40 min， giving the extraction rate of the target DMY reaching 93.1%， which was better than that under other conditions， among which the extraction method of extracting at 75 ℃ for 2 h has an extraction rate of 91.9%. Zhen KE et al. Study on Extraction and Separation of Dihydromyricetin from Vine Tea [Ampelopsis grossedentata （HandMazz） W. T. wang]
At present， there are also new improved technologies. For example， Wang[37]developed a method for extracting polysaccharides and DMY from vine tea under low temperature and ultrasound， and the DMY extraction rate can reach 24.37%. This method can reduce the times of posttreatment and is beneficial to maintaining the structure and activity of dihydromyricetin. Wang et al.[38]used the response surface method to optimize the Tween80 synergistic ultrasonic extraction of dihydromyricetin in vine tea. The DMY yield was 355.8 mg/g， the theoretical value was 353.7 mg/g， and the RSD was 0.59%. These research results fully illustrate the reliability of the extraction process. This method is green and environmentally friendly， and has great practical value for the further development of dihydromyricetin.
Countercurrent extraction method
The principle of the countercurrent extraction method is that during the extraction process， materials and solvents are input from opposite directions continuously and quantitatively through mechanical transmission， and the solvent is continuously updated， leading to a large concentration gradient always existing between the active ingredients of the two， which improves the extraction efficiency. Its essence is a solidliquid mass transfer process of the solid phase raw material to a lowconcentration liquid phase[39]. Li[40]found that in countercurrent extraction， the DMY extraction rate was higher under the conditions of an extraction temperature of 100 ℃， a materialtoliquid ratio of 1∶ 10， extraction time of 60 minand an extractant being slightlyalkaline water. The countercurrent extraction method is a green， energysaving and environmentfriendly process that can save costs， reduce energy consumption， and improve safety.
Supercritical fluid extraction （SFE）
The supercritical fluid extraction method uses a supercritical fluid as an extractant to separate components to be separated from a solid or liquid material at a critical point state. This method has strong extraction ability， fast extraction speed， and high purity of the obtained extract. It is an efficient rapid component analysis method. However， there are few reports at home and abroad on the extraction of dihydromyricetin from vine tea by the supercritical fluid method. Ying[41]extracted bioactive substances flavonoids and total phenol from vine tea with supercritical CO2 and obtained following optimum process parameters： 95% ethanol solution as entrainer， an extraction pressure of 200 bar， a temperature at 50 ℃， extraction time of 80 min. Shen[42]used the CO2 supercritical fluid extraction technology to extract DMY， and the best extraction conditions obtained through orthogonal experiments included an extraction pressure of 25 MPa， a temperature at 50 ℃，extraction time of 1.5 h and a CO2 flow rate at 20 L/h， under which the extraction yield of DMY was 84.3%. Therefore， researchers can conduct indepth research from this aspect， but this method also faces many application difficulties， such as high equipment costs， poor recovery rates， and difficult largescale production. Therefore， it is currently used in laboratory research and industrial production less. Other extraction methods
In addition to some of the abovementioned extraction methods of dihydromyricetin， a few scholars have adopted more advanced techniques to optimize the extraction and purification of dihydromyricetin.
Aqueous twophase extraction （ATPE）
The principle of the aqueous twophase extraction is that after the target substance enters the aqueous twophase system， due to the molecular force of the separated substance， such as the influences of hydrogen bonds， charges and ionic bonds， it is selectively distributed between the upper and lower phases to achieve separation[43]. This method has the advantage of simple operation， direct connection to subsequent purification processes， no need for special treatment and mild separation environment and can maintain the original activity of organisms. It can be used for largescale industrial production， and its various parameters can be changed accordingly without changing the product yield， which is unmatched by other technologies[44]. Li[45]established an ammonium sulfateethanol twoaqueous phase ultrasonicassisted extraction system for dihydromyricetin in vine tea， and the optimal extraction process parameters for DMY in vine tea were as follows： an ammonium sulfate mass fraction of 26.96%， an ethanol mass fraction of 21.84% and an extraction temperature at 51 ℃， under which the yield was （360.4±5.8） mg/g. Moreover， in order to investigate the advantages and disadvantages of the ultrasonicassisted aqueous twophase extraction process， the author compared the effects of ultrasonicassisted aqueous twophase extraction， ultrasonic extraction and reflux extraction with heating on the yield of DMY in vine tea. The yields followed an order of ultrasoundassisted aqueous twophase extraction （360.4 mg/g）>ultrasonic extraction （313.6 mg/g）>reflux extraction with heating （303.4 mg/g）. Compared with other two methods， the ultrasoundassisted aqueous twophase extraction is greatly improved， showing better extraction efficiency.
Microwave dynamic multistage countercurrent extraction
During the process of microwave dynamic multistage countercurrent extraction， the material and solvent flow continuously in opposite directions to continuously update the solidliquid twophase interface， such that the materials are in a higher concentration gradient difference， and through the strengthening effect of microwave， the dissolution of the effective ingredients in the material into the solvent is accelerated， thereby achieving the purpose of efficiently extracting effective ingredients in the medicinal material[46]. This method can overcome the shortcoming of small difference in the concentration of the effective ingredients between the material and the solvent when they are close to equilibrium during microwave extraction， thereby effectively shortening the extraction time of the effective ingredients and improving the extraction efficiency. Li et al.[47]used this technology to extract DMY under following optimum conditions： extraction time of 10 min， an extraction temperature at 110 ℃， a materialtoliquid ratio at 1∶ 30 and a microwave power at 600 W， with which the extraction rate of DMY was 92.2%， which was significantly higher than the extraction rate of microwave static intermittent extraction （67.4%）， indicating that the technology has good extraction efficiency. Li et al.[48]obtained significant extraction efficiency by the microwaveassisted countercurrent extraction technology. Due to high extraction efficiency and small consumption of extraction solvents， the method might be proven to be a promising extraction technology that can be used for extracting other biologically active substances from natural substances. Enzyme extraction
The enzyme extraction method is a new extraction technology that uses highefficiency specific enzymes， such as cellulase that hydrolyzes cellulose， pectinase that hydrolyzes pectin， to destroy the cell wall of the medicinal material and quickly dissolve its active ingredients[49]. Chen et al.[50]used the enzyme method to extract dihydromyricetin from waste vine tea stems， and obtained following optimum extraction process conditions： a compound enzyme addition amount of 2.0%， an enzymolysis temperature at 45 ℃， a solution pH of 4.46 and a materialtoliquid ratio at 1∶ 20 （g/ml）， under which the DMY extraction rate was 30.65%. Compared with the conventional hot extraction method， the extraction rate and purity are improved to a certain extent. Enzyme extraction has the advantages of high extraction rate， good selectivity， short extraction time， no residual organic solvents， and no pollution. Meanwhile， complex enzymes are cheap and easy to obtain. Therefore， this method is a simple， costeffective method to improve the quality of DMY， which worth promoting. Whether some key enzymatic reactions occur in the enzyme extraction method is worth further exploration.
Microwaveassisted semibionic extraction
The semibionic extraction method is a new type of traditional Chinese medicine extraction technology， which simulates the environment in which oraltakendrugs are transported and absorbed through the gastrointestinal tract from the perspective of biopharmaceutics and selected acidic water and alkaline water to sequentially and continuously extract active mixtures containing higher effective ingredients[33]. It can significantly improve the extraction rate of medicinal herbs， with shortened production cycle and reduced costs[51]. However， at present， the semibionic extraction method still follows the hightemperature cooking method and the extraction time is long. Therefore， microwave radiation can be used instead of the traditional cooking method. Depending on its advantages of uniform heating and shortened heating time， the two can be effectively combined to avoid the destruction of active ingredients caused by long heating time， thereby generating a new efficient and environmentallyfriendly extraction method[52]. Ni et al.[53]used this method to obtain the best extraction process of extracting three times with water at pH of 6.0， 7.5 and 9.5， respectively， accompanied by microwave treatment for 3.0， 1.5 and 1.5 min， respectively. The results showed that the microwaveassisted semibionic extraction method can effectively and rapidly extract the effective components in vine tea. In order to confirm the effect of the semibionic extraction method on the extraction of vine tea， scholars once designed the scheme in which the extraction solvent was changed to purified water without adjusting the pH for the three times of extraction under the same conditions， and it was found that the obtained extraction rate was only about 40% of the semibionic extraction method， indicating that the continuous acidbase extraction of the semibionic extraction method can effectively increasethe content of active ingredients from vine tea. In other methods， there is no emphasis on designing the effect of changing the pH value of the solvent on the extraction of DMY during multipleextractions， so researchers can conduct related experiments from this aspect to investigate whether the extraction efficiency can be improved through continuous acidbase extraction realized by changing the pH value without changing other experimental conditions. Separation and Purification Method
Recrystallization
Recrystallization is one of the common and important separation methods for the separation and purification of solid compounds. It uses the obvious difference in the solubility of a target component in a certain solvent in the solid mixture with temperature. Generally， the higher the temperature， the larger the solubility， and the lower the temperature， the smaller the solubility[18]. The target component can be recrystallized through heating， cooling and filtration， thereby achieving separation and purification. Dihydromyricetin is a polar compound， the solubility of which in hot water at 100 ℃ （1.502 g/100 g） is about 18 times that in cold water at 20 ℃ （0.084 g/100 g）[19]， so dihydromyricetin can be heated in water and recrystallized to remove impurities such as myricetin， proteins and sugars. This method has the characteristics of simple operation， low cost， and high safety. Meng[54]performed recrystallization three times， the yield of DMY was 34.59%， and the purity could reach more than 90%. Yang[55]concluded that the best purification conditions were crystallization with water three times and then crystallization with 10% ethanol protected by nitrogen. The purity of DMY increased from 63.18% to 98.06%， and the yield was 14.60%. Yuan[56]found that compared with simple water recrystallization， recrystallization with ethanolwater dual solvent can obviously reduce the times of recrystallization. A purity of more than 98% could be achieved by only 4 times of recrystallization， which greatly improved the purification efficiency and yield of DMY. The method has the advantages of simple operation， low cost and environmental protection， and is suitable for industrialized largescale production.
Macroporous resin adsorption
The macroporous resin adsorption method is a purification method which removes impurities using a macroporous resin with a porous structure that does not contain exchange groups as an adsorbent to selectively adsorb active ingredients physically through elution and recovery. The method is simple in process， high in separation efficiency， mild in operating conditions. Moreover， the resin can be recycled and reused， thereby reducing costs， and the method is thus nontoxic and pollutionfree[57]. In order to obtain the highquality quercetin raw material， Huang et al.[58]investigated the process conditions for the separation and purification of quercetin from the leaves of A. grossedentata by macroporous adsorption resin， and finally achieved a quercetin mass fraction of 95.5% with the optimum SP700 macroporous resin through dynamic elution under optimum conditions. Tan et al.[59]obtained samples with a purity more than 95% from 40.1% sample solution through NKA9 macroporous adsorption resin column. Zhang et al.[60]used D16 macroporous resin to purify DMY， and followed "heating to increase solubility， passing through the column at controlled temperature， and desorbing with warm water" in the experiment. The suitable temperature of this method is 50-70 ℃， preferably 60 ℃， and DMY with a purity of about 70% can be purified to 80%. However， this method has the disadvantage of blind resin selection in separation and application. Due to lack of effective theoretical guidance and lack of pertinence in molecular design during synthesis， it has become an obstructive factor for the research on separation and development of macroporous resins[61]. Highspeed countercurrent chromatography （HSCCC）
Highefficiency countercurrent chromatography separation technology is based on the principle of dynamic liquidliquid partition. It uses two immiscible solvent systems to achieve unidirectional hydrodynamic equilibrium in a highspeed rotating spiral tube and achieves separation of the components which have different distribution coefficients in the two phases. Since no solid support or carrier is needed， sample loss， inactivation， and denaturation caused by irreversible adsorption are avoided. The method has high separation efficiency and large separation amount， and is especially suitable for the separation of natural biologically active components[62]. It is an ideal preparation and separation means. Zhang et al.[63]applied highspeed countercurrent chromatography to purify dihydromyricetin， which well purified crude dihydromyricetin with a purity of about 80% to more than 90%; and the highspeed countercurrent chromatography combining GS10A countercurrent chromatography and AKTAprime plus chromatography system simultaneously purified DMY and myricetin in the crude extract to more than 99% within 5 h[64]， and 50 g of crude vine tea was separated continuously， exhibiting the separation yields of 85.5% and 87.6%， respectively.
Prospect
There is not only one fixed way to extract a certain natural product， and different methods have their own advantages and disadvantages. Due to the influences of uncontrollable factors such as the variety of vine tea， producing area， picking position and experimental conditions used in these methods in literatures， the extraction rates are slightly different. Among the above DMY extraction methods， microwaveassisted， ultrasonicassisted， countercurrent extraction， and supercritical fluid extraction technologies have the characteristics of shorter extraction time and higher extraction efficiency than traditional water extraction and ethanol extraction. These advanced technologies are green， environmentally friendly， economic and efficient， and is more suitable for largescale production. This paper summarized the extraction and purification methods of dihydromyricetin， in order to explore new energysaving efficient methods based on the original method.
There are rich vine tea resources in China and has good prospects for development， but most of the resources are mainly used for the preparation of coarsely processed tea beverage products. The deep processing and comprehensive application of vine tea are still in the early stages[65]， and its development and utilization level is not high. Now that the demand for dihydromyricetin is increasing， how to vigorously promote the cultivation of vine tea and improve the quality of vine tea is a problem we need to solve in the future. [37] WANG HB， XIONG W， HU JW， et al. Study on ultrasoundlow temperature extraction of polysaccharide and dihydromyricetin from Ampelopsis grossedentata[J]. The Food Industry， 2015， 36（8）： 81-85. （in Chinese）
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Key words Ampelopsis grossedentata; dihydromyricetin; extraction method; isolation and purification; progress
Vine tea [Ampelopsis grossedentata （HandMazz） W. T. wang]， is a wild vine of Vitis in Vitaceae. Vine tea is mainly grown in Guangxi， Hunan， Yunnan， Guizhou and other places. Because of its different producing areas， it has many popular names， such as Maoyanmei， Meicha， Changshouteng， Lingzhicao， etc.[1]. It is a kind of folk medicinal plant， which has been recorded in ancient books such as "Classified Materia Medica"[2]. This tea is slightly bitter and sweet in taste and cool in nature. It has the effects of clearing heat and toxic material， resisting bacteria and inflammation， dispelling wind and eliminating dampness， strengthening bones and muscles， lowering blood pressure and protecting the liver， thus the name "the king of tea".
The most important flavonoid in vine tea with the highest content is dihydromyricetin （DMY）， which has a mass fraction as high as 30% or more in dry stems and leaves of vine tea[3]. It belongs to polyhydroxyflavonols， and has a variety of physiologically active functions， such as antibacterial[4]， antiinflammatory[5]， antioxidant[6]， antiatherosclerosis[7]， antitumor[8]， and liver protecting effects[9].
This paper summarized the research progress on extraction materials， extraction methods and separation and purification processes of dihydromyricetin in vine tea to find out the best experimental method of dihydromyricetin. This study will provide theoretical reference for comprehensive utilization and comprehensive development of dihydromyricetin.
Selection of Extracted Medicinal Materials
The same traditional Chinese medicinal material， due to different producing areas of raw material， varieties， picking positions， collection time and processing methods， the content of the same ingredient varies widely， which will affect the experimental results. Picking position
Chen[10]measured the DMY and total flavonoid contents in different parts of A. grossedentata. The experimental results showed that the part with the highest total flavonoid and DMY contents was young leaves， whose contents were 15.01% and 7.79% respectively. The part with the highest flavonoid ratio was tender stems， with a ratio of 64.31%. He et al.[11]determined that the content of DMY in vine tea leaves was 3 to 4 times higher than that in stems by HPLC， and the content was highest in May， ranging from 27.8% to 31.2%.
Collection time
Lei et al.[12]analyzed the total flavonoid and DMY contents in A. grossedentata in different seasons when using different drying methods. From April to December， the total flavonoid content was highest in April， and the highest DMY content appeared in May. And the total flavonoid and DMY contents in the vine tea ovendried after collection were highest. Yu et al.[13]found that the total flavonoid and DMY contents reached the highest in April， 8.74%and 4.69%， respectively.
Processing conditions
Wang et al.[14]found when examining the effects of different drying temperatures on the quality components of vine tea that compared with samples dried in the shade， ovendried samples showed DMY and total flavonoid contents greatly increased， and the contents of the components reached their highest values when the samples were dried at 100 ℃. Feng et al.[15]obtained a significantly higher DMY content with the ovendrying method than with natural drying， and the vine tea collected in May was best amongthe different collection periods. Yi[16]investigated the effects of drying in the shade， sun drying， and oven drying on total flavonoids and DMY in vine tea， and concluded that the best vine tea drying method was drying at 70 ℃. The research results of Yang et al.[17]showed that the DMY contents of different parts of vine tea were significantly different. The leaves （3.93 mg/g） had a DMY content significantly higher than that of roots （0.34 mg/g）， and the value got higher after being stored for two years.
Therefore， in order to ensure the highest DMY content in vine tea and facilitate experimental research， in the selection of experimental medicinal material， the best vine tea part can be selected as the leaves which are collected in May， and can be processed by oven drying， and it is better to store the material for two year before processing. Common Extraction Methods
At present， the DMY extraction methods in vine tea mainly include solvent extraction method， microwaveassisted extraction method， ultrasonic extraction method， countercurrent extraction method， supercritical fluid extraction method and some improved technologies.
Solvent extraction method
Dihydromyricetin is a polyhydroxyflavonol compound， which is easily soluble in solvents such as hot water and ethanol， and slightly soluble in ethyl acetate. Among them， water and ethanol are the most widely used to extract dihydromyricetin[18]. Guo[19]used distilled water， 30% ethanol （volume fraction）， 60% ethanol， 90% ethanol and methanol as extraction agents for DMY， and the extraction rates were 15.30%， 11.31%， 13.15%， 16.68%， 16.68% and 13.12%， respectively. From the experimental data， it could be seen that the extraction rate of 90% ethanol was the highest. However， considering that the water extraction method is simple， convenient， highly economic and has low cost and high extraction rate， distilled water can be used as the extraction solvent for DMY in the laboratory or industrial production. In the following， hot water extraction of DMY was taken as an example to summarize the best extraction process of water extraction in recent years. The details are given in Table 2 for details.
It can be known from the above table that the optimal extraction conditions for extracting DMY with hot water are as follows： the extraction temperature selected from 78 to 100 ℃， the extraction time from 60 to 120 min， the material ratio from 1∶ 15 to 1∶ 30， the pH value of about 8， and the raw material particle size of 80-100 mesh， and the secondary extraction method is more effective. The effects of the extraction factors on DMY extraction rank as extraction temperature>extraction time>materialtoliquid ratio[24-25]. A small experiment should be done first to find out the best extraction conditions in experiments according to the actual situation.
In addition to hot water extraction， DMY can be extracted with other solvents. For example， Xie et al.[27]obtained the best extraction process conditions by the response surface analysis as follows： an ethanol concentration of 81%， a liquidtomaterial ratio at 17 ml/g and reflux extraction for 95 min， under which the DMY extraction rate could reach 9.58%， which is only 0.14% different from the theoretical value of 9.72%. Yan et al.[28]confirmed that ethyl acetate was the best extraction solvent， and the optimal conditions were as follows： an extraction temperature at 50 ℃， extraction time of 50 min and a materialtoliquid ratio at 1∶ 20， with which the extraction rate of DMY was about 14.28%. Solvent extraction has the advantages of simple operation， low cost and no pollution， and is more suitable for largescale industrial production. However， it consumes more time and solvents， and has poor reproducibility， and the product contains more impurities. And the solvents are generally harm to environment and human body[24]. Microwaveassisted extraction （MAE）
The microwaveassisted extraction refers to the rapid extraction of target substances from samples using electromagnetic radiation of microwave[29]， which is a new efficient extraction technology. Microwave extraction has the characteristics of instant realization of high temperature， short extraction time， high extraction efficiency， good reproducibility， and low pollution. Zheng et al.[30]achieved the DMY extraction rate of 25.11% with following conditions： microwave irradiation for 20 min， heating for 50 min and a materialtoliquid ratio of 1∶ 10. In the smallscale extraction[31]， samples were treated with microwave at 750 W for 4 min， and extracted at a materialliquid ratio of 1∶ 20 in a water bath at 95 ℃ for 30 min， and the DMY extraction rate reached 27.53%. However， the samples free of microwave pretreatment were extracted under the same condition， showing the DMY extraction rate of 13.73%. In comparison， the microwave pretreatment improved the extraction rate by 13.80%， indicating that the effective components were dissolved more easily with the assistance of microwave. Yang[32]determined the optimum process parameters through orthogonal experiment： an extraction temperature of 95 ℃， microwave heating time of 15 min， and a materialliquid ratio at 1∶ 20， under which the DMY extraction rate reached 27.33%. After pretreatment with microwave of 300 W for 2 min， the yield of DMY reached 28.48%， which was improved by 20% compared with the traditional water extraction method. It can be seen that the extraction rate of microwave extraction is higher than that of water extraction， but it is not convenient to control parameters such as temperature and power， which may affect the extraction rate of effective ingredients.
Ultrasoundassisted extraction （UAE）
The ultrasonicassisted extraction method is an extraction method that uses ultrasonic waves to increase the speed and frequency of movement of substance molecules， thereby increasing the solvent penetrating power and increasing the dissolution rate of the extracted components[33]. The ultrasoundassisted extraction has the characteristics of simple operation， high speed， less time， low cost and high extraction rate[34]， and has become a common processing method for providing samples in the analysis of many traditional Chinese medicines. Liang et al.[35]extracted DMY with the assistance of ultrasonic waves， and the optimum process parameters were an ethanol volume fraction of 51.00 %， a materialtoliquid ratio at 1∶ 26 （g∶ ml）， and ultrasonic treatment time of 40.00 min， under which the DMY extraction rate was 28.41%， which was close to the extraction rate predicted by the regression model （28.61%）. Yao et al.[36]proposed that the optimum process for extracting DMY from A. grossedentata was to extract at 40 ℃， a liquidtomaterial ratio of 1∶ 18 and an ethanol volume fraction of 65% with the assistance of ultrasonic waves for 40 min， giving the extraction rate of the target DMY reaching 93.1%， which was better than that under other conditions， among which the extraction method of extracting at 75 ℃ for 2 h has an extraction rate of 91.9%. Zhen KE et al. Study on Extraction and Separation of Dihydromyricetin from Vine Tea [Ampelopsis grossedentata （HandMazz） W. T. wang]
At present， there are also new improved technologies. For example， Wang[37]developed a method for extracting polysaccharides and DMY from vine tea under low temperature and ultrasound， and the DMY extraction rate can reach 24.37%. This method can reduce the times of posttreatment and is beneficial to maintaining the structure and activity of dihydromyricetin. Wang et al.[38]used the response surface method to optimize the Tween80 synergistic ultrasonic extraction of dihydromyricetin in vine tea. The DMY yield was 355.8 mg/g， the theoretical value was 353.7 mg/g， and the RSD was 0.59%. These research results fully illustrate the reliability of the extraction process. This method is green and environmentally friendly， and has great practical value for the further development of dihydromyricetin.
Countercurrent extraction method
The principle of the countercurrent extraction method is that during the extraction process， materials and solvents are input from opposite directions continuously and quantitatively through mechanical transmission， and the solvent is continuously updated， leading to a large concentration gradient always existing between the active ingredients of the two， which improves the extraction efficiency. Its essence is a solidliquid mass transfer process of the solid phase raw material to a lowconcentration liquid phase[39]. Li[40]found that in countercurrent extraction， the DMY extraction rate was higher under the conditions of an extraction temperature of 100 ℃， a materialtoliquid ratio of 1∶ 10， extraction time of 60 minand an extractant being slightlyalkaline water. The countercurrent extraction method is a green， energysaving and environmentfriendly process that can save costs， reduce energy consumption， and improve safety.
Supercritical fluid extraction （SFE）
The supercritical fluid extraction method uses a supercritical fluid as an extractant to separate components to be separated from a solid or liquid material at a critical point state. This method has strong extraction ability， fast extraction speed， and high purity of the obtained extract. It is an efficient rapid component analysis method. However， there are few reports at home and abroad on the extraction of dihydromyricetin from vine tea by the supercritical fluid method. Ying[41]extracted bioactive substances flavonoids and total phenol from vine tea with supercritical CO2 and obtained following optimum process parameters： 95% ethanol solution as entrainer， an extraction pressure of 200 bar， a temperature at 50 ℃， extraction time of 80 min. Shen[42]used the CO2 supercritical fluid extraction technology to extract DMY， and the best extraction conditions obtained through orthogonal experiments included an extraction pressure of 25 MPa， a temperature at 50 ℃，extraction time of 1.5 h and a CO2 flow rate at 20 L/h， under which the extraction yield of DMY was 84.3%. Therefore， researchers can conduct indepth research from this aspect， but this method also faces many application difficulties， such as high equipment costs， poor recovery rates， and difficult largescale production. Therefore， it is currently used in laboratory research and industrial production less. Other extraction methods
In addition to some of the abovementioned extraction methods of dihydromyricetin， a few scholars have adopted more advanced techniques to optimize the extraction and purification of dihydromyricetin.
Aqueous twophase extraction （ATPE）
The principle of the aqueous twophase extraction is that after the target substance enters the aqueous twophase system， due to the molecular force of the separated substance， such as the influences of hydrogen bonds， charges and ionic bonds， it is selectively distributed between the upper and lower phases to achieve separation[43]. This method has the advantage of simple operation， direct connection to subsequent purification processes， no need for special treatment and mild separation environment and can maintain the original activity of organisms. It can be used for largescale industrial production， and its various parameters can be changed accordingly without changing the product yield， which is unmatched by other technologies[44]. Li[45]established an ammonium sulfateethanol twoaqueous phase ultrasonicassisted extraction system for dihydromyricetin in vine tea， and the optimal extraction process parameters for DMY in vine tea were as follows： an ammonium sulfate mass fraction of 26.96%， an ethanol mass fraction of 21.84% and an extraction temperature at 51 ℃， under which the yield was （360.4±5.8） mg/g. Moreover， in order to investigate the advantages and disadvantages of the ultrasonicassisted aqueous twophase extraction process， the author compared the effects of ultrasonicassisted aqueous twophase extraction， ultrasonic extraction and reflux extraction with heating on the yield of DMY in vine tea. The yields followed an order of ultrasoundassisted aqueous twophase extraction （360.4 mg/g）>ultrasonic extraction （313.6 mg/g）>reflux extraction with heating （303.4 mg/g）. Compared with other two methods， the ultrasoundassisted aqueous twophase extraction is greatly improved， showing better extraction efficiency.
Microwave dynamic multistage countercurrent extraction
During the process of microwave dynamic multistage countercurrent extraction， the material and solvent flow continuously in opposite directions to continuously update the solidliquid twophase interface， such that the materials are in a higher concentration gradient difference， and through the strengthening effect of microwave， the dissolution of the effective ingredients in the material into the solvent is accelerated， thereby achieving the purpose of efficiently extracting effective ingredients in the medicinal material[46]. This method can overcome the shortcoming of small difference in the concentration of the effective ingredients between the material and the solvent when they are close to equilibrium during microwave extraction， thereby effectively shortening the extraction time of the effective ingredients and improving the extraction efficiency. Li et al.[47]used this technology to extract DMY under following optimum conditions： extraction time of 10 min， an extraction temperature at 110 ℃， a materialtoliquid ratio at 1∶ 30 and a microwave power at 600 W， with which the extraction rate of DMY was 92.2%， which was significantly higher than the extraction rate of microwave static intermittent extraction （67.4%）， indicating that the technology has good extraction efficiency. Li et al.[48]obtained significant extraction efficiency by the microwaveassisted countercurrent extraction technology. Due to high extraction efficiency and small consumption of extraction solvents， the method might be proven to be a promising extraction technology that can be used for extracting other biologically active substances from natural substances. Enzyme extraction
The enzyme extraction method is a new extraction technology that uses highefficiency specific enzymes， such as cellulase that hydrolyzes cellulose， pectinase that hydrolyzes pectin， to destroy the cell wall of the medicinal material and quickly dissolve its active ingredients[49]. Chen et al.[50]used the enzyme method to extract dihydromyricetin from waste vine tea stems， and obtained following optimum extraction process conditions： a compound enzyme addition amount of 2.0%， an enzymolysis temperature at 45 ℃， a solution pH of 4.46 and a materialtoliquid ratio at 1∶ 20 （g/ml）， under which the DMY extraction rate was 30.65%. Compared with the conventional hot extraction method， the extraction rate and purity are improved to a certain extent. Enzyme extraction has the advantages of high extraction rate， good selectivity， short extraction time， no residual organic solvents， and no pollution. Meanwhile， complex enzymes are cheap and easy to obtain. Therefore， this method is a simple， costeffective method to improve the quality of DMY， which worth promoting. Whether some key enzymatic reactions occur in the enzyme extraction method is worth further exploration.
Microwaveassisted semibionic extraction
The semibionic extraction method is a new type of traditional Chinese medicine extraction technology， which simulates the environment in which oraltakendrugs are transported and absorbed through the gastrointestinal tract from the perspective of biopharmaceutics and selected acidic water and alkaline water to sequentially and continuously extract active mixtures containing higher effective ingredients[33]. It can significantly improve the extraction rate of medicinal herbs， with shortened production cycle and reduced costs[51]. However， at present， the semibionic extraction method still follows the hightemperature cooking method and the extraction time is long. Therefore， microwave radiation can be used instead of the traditional cooking method. Depending on its advantages of uniform heating and shortened heating time， the two can be effectively combined to avoid the destruction of active ingredients caused by long heating time， thereby generating a new efficient and environmentallyfriendly extraction method[52]. Ni et al.[53]used this method to obtain the best extraction process of extracting three times with water at pH of 6.0， 7.5 and 9.5， respectively， accompanied by microwave treatment for 3.0， 1.5 and 1.5 min， respectively. The results showed that the microwaveassisted semibionic extraction method can effectively and rapidly extract the effective components in vine tea. In order to confirm the effect of the semibionic extraction method on the extraction of vine tea， scholars once designed the scheme in which the extraction solvent was changed to purified water without adjusting the pH for the three times of extraction under the same conditions， and it was found that the obtained extraction rate was only about 40% of the semibionic extraction method， indicating that the continuous acidbase extraction of the semibionic extraction method can effectively increasethe content of active ingredients from vine tea. In other methods， there is no emphasis on designing the effect of changing the pH value of the solvent on the extraction of DMY during multipleextractions， so researchers can conduct related experiments from this aspect to investigate whether the extraction efficiency can be improved through continuous acidbase extraction realized by changing the pH value without changing other experimental conditions. Separation and Purification Method
Recrystallization
Recrystallization is one of the common and important separation methods for the separation and purification of solid compounds. It uses the obvious difference in the solubility of a target component in a certain solvent in the solid mixture with temperature. Generally， the higher the temperature， the larger the solubility， and the lower the temperature， the smaller the solubility[18]. The target component can be recrystallized through heating， cooling and filtration， thereby achieving separation and purification. Dihydromyricetin is a polar compound， the solubility of which in hot water at 100 ℃ （1.502 g/100 g） is about 18 times that in cold water at 20 ℃ （0.084 g/100 g）[19]， so dihydromyricetin can be heated in water and recrystallized to remove impurities such as myricetin， proteins and sugars. This method has the characteristics of simple operation， low cost， and high safety. Meng[54]performed recrystallization three times， the yield of DMY was 34.59%， and the purity could reach more than 90%. Yang[55]concluded that the best purification conditions were crystallization with water three times and then crystallization with 10% ethanol protected by nitrogen. The purity of DMY increased from 63.18% to 98.06%， and the yield was 14.60%. Yuan[56]found that compared with simple water recrystallization， recrystallization with ethanolwater dual solvent can obviously reduce the times of recrystallization. A purity of more than 98% could be achieved by only 4 times of recrystallization， which greatly improved the purification efficiency and yield of DMY. The method has the advantages of simple operation， low cost and environmental protection， and is suitable for industrialized largescale production.
Macroporous resin adsorption
The macroporous resin adsorption method is a purification method which removes impurities using a macroporous resin with a porous structure that does not contain exchange groups as an adsorbent to selectively adsorb active ingredients physically through elution and recovery. The method is simple in process， high in separation efficiency， mild in operating conditions. Moreover， the resin can be recycled and reused， thereby reducing costs， and the method is thus nontoxic and pollutionfree[57]. In order to obtain the highquality quercetin raw material， Huang et al.[58]investigated the process conditions for the separation and purification of quercetin from the leaves of A. grossedentata by macroporous adsorption resin， and finally achieved a quercetin mass fraction of 95.5% with the optimum SP700 macroporous resin through dynamic elution under optimum conditions. Tan et al.[59]obtained samples with a purity more than 95% from 40.1% sample solution through NKA9 macroporous adsorption resin column. Zhang et al.[60]used D16 macroporous resin to purify DMY， and followed "heating to increase solubility， passing through the column at controlled temperature， and desorbing with warm water" in the experiment. The suitable temperature of this method is 50-70 ℃， preferably 60 ℃， and DMY with a purity of about 70% can be purified to 80%. However， this method has the disadvantage of blind resin selection in separation and application. Due to lack of effective theoretical guidance and lack of pertinence in molecular design during synthesis， it has become an obstructive factor for the research on separation and development of macroporous resins[61]. Highspeed countercurrent chromatography （HSCCC）
Highefficiency countercurrent chromatography separation technology is based on the principle of dynamic liquidliquid partition. It uses two immiscible solvent systems to achieve unidirectional hydrodynamic equilibrium in a highspeed rotating spiral tube and achieves separation of the components which have different distribution coefficients in the two phases. Since no solid support or carrier is needed， sample loss， inactivation， and denaturation caused by irreversible adsorption are avoided. The method has high separation efficiency and large separation amount， and is especially suitable for the separation of natural biologically active components[62]. It is an ideal preparation and separation means. Zhang et al.[63]applied highspeed countercurrent chromatography to purify dihydromyricetin， which well purified crude dihydromyricetin with a purity of about 80% to more than 90%; and the highspeed countercurrent chromatography combining GS10A countercurrent chromatography and AKTAprime plus chromatography system simultaneously purified DMY and myricetin in the crude extract to more than 99% within 5 h[64]， and 50 g of crude vine tea was separated continuously， exhibiting the separation yields of 85.5% and 87.6%， respectively.
Prospect
There is not only one fixed way to extract a certain natural product， and different methods have their own advantages and disadvantages. Due to the influences of uncontrollable factors such as the variety of vine tea， producing area， picking position and experimental conditions used in these methods in literatures， the extraction rates are slightly different. Among the above DMY extraction methods， microwaveassisted， ultrasonicassisted， countercurrent extraction， and supercritical fluid extraction technologies have the characteristics of shorter extraction time and higher extraction efficiency than traditional water extraction and ethanol extraction. These advanced technologies are green， environmentally friendly， economic and efficient， and is more suitable for largescale production. This paper summarized the extraction and purification methods of dihydromyricetin， in order to explore new energysaving efficient methods based on the original method.
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