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Abstract Resveratrol is a ployphenolic compound which has a wide variety of pharmacological activity. This paper reviewed the physicochemical properties of resveratrol and its purification technology including macroporous resin method， high-speed countercurrent chromatography method， molecular imprinting method， membrane separation method， column chromatography method， two-aqueous phase extraction method and the combination of purification methods， so as to provide certain reference for further research and development.
Key words Resveratrol; Physiology activity; Purification
Received： February 25， 2020 Accepted： April 27， 2020
Supported by Hebei Provincial Phase II Modern Agricultural Industry Technology System Innovation Team Construction Project （HBCT2018120207）; Tangshan Science and Technology Planning Project （19150204E）; Hebei Province Innovation Ability Promotion Plan Project （20567673H）.
Lei WANG （1982-）， male， P. R. China， associate researcher， devoted to functional ingredients and food additives.
Di WANG （1989-）， female， P. R. China， associate researcher， devoted to functional ingredients and food additives. E-mail： [email protected].
#These authors contributed equally to this work.
Corresponding author. E-mail： [email protected]; [email protected].
Resveratrol is an antitoxin produced by plants under fungal infection， ultraviolet radiation or pathological conditions， and also acts as an antioxidant and anti-mutagenic agent[1]. Resveratrol has anti-tumor， anti-atherogenesis， anti-free radical， anti-oxidation， anti-viral， anti-inflammatory， estrogen-like， liver protection and other effects[2-4]. Resveratrol is rich in plant sources and widely exists in Polygonum cuspidatum， peanut， grape， mulberry， cassia and other plants. It has been found in at least 72 plants of 31 genera in 21 families[5-8]. The resveratrol circulating on the market is mainly derived from the extraction and purification of plants， and the highest purity of resveratrol has reached 99%. However， there are few finished products of high-purity resveratrol on the market， and the price is high. Resveratrol with a purity of 99% in China has been sold for 32 000 yuan/kg.
In recent years， the industry has made great efforts to develop resveratrol， but the current processes and methods have problems of low extraction rate and relatively high production cost. Therefore， improving the extraction efficiency of resveratrol is one of the problems to be solved in the pharmaceutical and food industries. This paper reviewed the current status of resveratrol separation and purification technology， providing a reference for its development and utilization value. Physical and Chemical Properties of Resveratrol
Resveratrol is a non-flavonoid polyphenolic compound with a molecular formula of C14H12O3 and a relative molecular weight of 228.25. It is white needle-like crystals， which is difficult to dissolve in water and soluble in organic solvents. It has a melting point of 256-257 ℃ and sublimates at 261 ℃. It can produce fluorescence when irradiated by ultraviolet light with a wavelength of 365 nm， and can react with ferric chloride-potassium ferricyanide[9]. Resveratrol exists in cis and trans forms in plants. Resveratrol forms with sugar， a glycoside， which releases resveratrol under the action of glycosidase in the intestine. Resveratrol mainly exists in trans in plants， and its physiological activity is stronger than its cis isomer[10].
Separation and Purification of Resveratrol
At present， there are many methods for the separation and purification of resveratrol， and the specific method should depend on the extraction conditions and the purpose of purification.
Column chromatography
Column chromatography is a chromatography method that uses a solid adsorbent as the stationary phase and an organic solvent or buffer solution as the mobile phase. This technique gradually separate components in a mixture depending on the differences in physical and chemical properties of substances such as the molecular shape， size， charged state， solubility， adsorption capacity， molecular polarity and affinity. According to different substance separation mechanisms， column chromatography can be divided into adsorption column chromatography， ion exchange column chromatography， gel column chromatography， distribution column chromatography， and electrofocusing column chromatography[11].
Jiang et al.[12]used silica gel column chromatography to purify resveratrol in peanut roots， with chloroform-methanol （9∶1） as the eluting solvent and a ratio of silica gel adsorbent to sample volume at 50∶1. The purity of resveratrol increased from 39% to more than 99%. Chen[13]used silica gel column chromatography to purify resveratrol in peanut roots. The thin layer chromatography and silica gel column plate and HPLC purity detection were used. When the ratio of chloroform to methanol was 9∶1， the purity of resveratrol in the eluate of silica gel column chromatography increased to more than 99%. Zhang et al.[14]investigated the separation effect of resveratrol samples on Sephadex LH-20 gel chromatography column under different elution solvents and different elution flow rates. The resveratrol separation effect was best at 0.5 ml/min. The purity of resveratrol reached 80.34% and the recovery reached 86.16% by medium pressure liquid chromatography. Membrane separation
Membrane separation technology is a high-tech of the century， including microfiltration， ultrafiltration， nanofiltration， electrodialysis， reverse osmosis， membrane electrolysis， membrane distillation， membrane extraction and other techniques. Because it has the functions of separation， concentration and purification， and has the advantages of high efficiency， energy saving， environmental protection， and molecular filtration， it is widely used in the field of natural product separation[15].
Jiang et al.[16]firstly removed emodin from the alcohol extract of P. cuspidatum by low alcohol and then removed tannin and some impurities by ethyl acetate countercurrent extraction， and the resveratrol purity reached 20%. Then an ultrafiltration membrane with a relative molecular weight of 4 000 to be intercepted was used to perform further purification， and the purity of resveratrol after the purification was more than 95%. Liu et al.[17]used membrane separation technology to purify resveratrol from P. cuspidatum which increased the purity of resveratrol in the crude extract of P. cuspidatum from 8.7% to 30.5%， and concentrated and separated the filtrate using an ultrafiltration membrane， obtaining resveratrol with a purity reaching 55.8%. Membrane separation is a physical separation process， which produces no waste， and is green and environmentally friendly， but the cost of the membrane is relatively high， so membrane separation is suitable for the separation and purification of end products.
Macroporous resin method
The macroporous adsorption resin is a kind of synthetic polymer adsorbent with a multi-hollow three-dimensional structure. It can selectively adsorb components through physical adsorption， and its adsorption is mainly achieved through surface adsorption， surface electrical properties， or morphological hydrogen bond[18].
Li et al.[19]used 18 adsorption resins to purify resveratrol in P. cuspidatum， and found that the adsorption capacity and resolution of HPD-722 resin are suitable for the purification of resveratrol. Yang et al.[20]studied the method of extracting and purifying resveratrol from P. cuspidatum with macroporous adsorption resin combined with enzymolysis， and found that H1020 resin not only has high adsorption capacity， easy desorption， but also can make the content of resveratrol in crude extract increase from 8.7% to 71.5%. Liu[21]used D101 macroporous resin to separate and purify the effective parts of P. cuspidatum. The adsorption flow rate was 1.0 ml/min， and the eluent was 50% ethanol. After eluting with 6BV eluent， the resveratrol was almost completely desorbed. It was determined that the content of resveratrol increased by more than 10% after purification. Molecular imprinting
Molecularly imprinted polymers （MIPs） polymerize functional monomers in the presence of template molecules （imprinted molecules） by a cross-linking agent. MIPs have spatial structures that can specifically bind to target molecules， and thus have high selectivity for the target molecules. They mainly have two different forms： non-covalent and covalent[22].
Zhuang et al.[23]studied the separation of resveratrol and emodin from P. cuspidatum. They prepared resveratrol imprinted polymer using resveratrol as a template， 4-vinylpyridine as a functional monomer， ethylene glycol dimethacrylate as a cross-linking agent， acetone as a porogen and 2，2-azobisisobutyronitrile as a directing agent， performed online high-performance liquid extraction with methanol-water （80∶20） as a mobile phase and detected the extract by reverse-phase high-performance liquid chromatography， and the recovery and purity of resveratrol were both higher than 80%. Feng et al.[24]separated resveratrol from P. cuspidatum， and prepared a molecularly imprinted polymer with resveratrol as a template， acrylamide as a functional monomer， and chloroform and tetrahydrofuran mixture as a cross-linking agent of the imprinted polymer. With the adsorption performance， selection performance and solid-phase extraction performance of the molecularly imprinted polymer as the investigation indicators， the molecularly imprinted polymer was subjected to solid phase extraction and HPLC determination with 40% ethanol solution and 80% ethanol solution as eluents， respectively， at a flow rate of 1 ml/min. The purity of resveratrol in the eluate was 89.2%， and the yield of resveratrol was 73.6%.
High-speed countercurrent chromatography
High-speed countercurrent chromatography technology is a liquid-liquid chromatography technology that does not use any solid support or carrier. Compared with the traditional solid-liquid column chromatography technology， it does not use a solid phase carrier as a stationary phase， and the separated substances are distributed and separated in two phases which are incompatible with each other. Therefore， this technology overcomes the disadvantages of sample adsorption， loss and pollution brought by the solid phase carrier.
He et al.[25]used countercurrent chromatography to separate the cis-trans isomers of resveratrol in Parthenocissus laetevirens Rehd. roots using n-hexane-ethyl acetate-methanol-water （1∶2∶1∶2） as the solvent system. After purification， the purity was 95.4% and 97.6% by HPLC. In the experiment of purifying resveratrol from P. cuspidatum by high-speed countercurrent chromatography， Liu et al.[26]selected a solvent system of chloroform∶methanol∶water = 4∶3∶2， a rotation speed at 850 r/min， and a flow rate at 2 ml/min， the purity of resveratrol obtained after purification was greater than 96%， and a good separation effect was achieved. Two-phase water extraction
Li et al.[27]used two-aqueous phase extraction technology to purify resveratrol from P. cuspidatum， and compared with chloroform organic extraction. It was found that when using ethanol-ammonium sulfate solution as a two-aqueous phase system to separate and enrich the extract of P. cuspidatum， the content of resveratrol in the liquid was 34.29%， which was 5.81% higher than that of organic extraction. He et al.[28]found that in the experiment of purifying resveratrol from wine， compared with organic solvent extraction， ethanol-ammonium sulfate-water extraction as a two-aqueous system had a better separation effect， and the resveratrol content was 5.68 times higher than organic solvent extraction. Jiang et al.[29]used ultrasonic-assisted two-phase aqueous extraction to purify resveratrol. The enzymatic hydrolysate of P. cuspidatum was extracted with ammonium sulfate-absolute ethanol as a two-phase aqueous system. The recovery of resveratrol was more than 98%. A two-phase system is a non-miscible two-phase or multi-phase water system formed by dissolving certain organic compounds or organic matter and inorganic salts in appropriate concentrations in water， and the separation principle of the system is based on the selective distribution of substances in the aqueous phase system.
Prospect
Resveratrol has extremely unstable chemical properties， and is prone to deteriorate after long time storage due to phenolic hydroxyl oxidation and double bond addition. This property affects the production of high-purity resveratrol. The stability of this compound can be enhanced after a certain structural modification， and the addition of the purification technology of resveratrol comprising its structural modification during the production process is currently less reported， so the new purification technology needs further study.
References
[1]CHIN YT， HSIEH MT， YANG SH， et al. Anti-proliferative and gene expression actions of resveratrol in breast cells in vitro[J]. Oncotarget， 2014， 5（24）： 12891-12907.
[2]LI C， XU XF， TAO ZH， et al. Resveratrol dimers， nutritional components in grape wine， are selective ROS scavengers and weak Nrf2 activators[J]. Food Chemistry， 2015， （173）： 218-223.
[3]KURITA S， KASHIWAGI T， EGISU T， et al. Content of resveratrol and glycoside and its contribution to the antioxidative capacity of Polygonum cuspidatum （Itadori） harvested in Kochi[J]. Biosci Biotechnol Biochem， 2014， 78（3）： 499-502. [4]HUANG FC， KUO HC， HUANG YH， et al. Anti-inflammtory effect of resveratrol in human coronary arterial endothelial cells via induction of autophagy： implication of the treatment of Kawasaki disease[J]. Circulation， 2017， 18（1）： 1-8.
[5]SHIN JA， OH S， AHN JH， et al. Estrogen receptormediated resveratrol actions on blood-brain barrier of ovariecto-mized mice[J]. Neurobiology of Aging， 2015， 36（2）： 993-1006.
[6]ALI MH， MESSIHA BA， ABDEL-LATIF HA. Protective effect of ursodeoxycholic acid， resveratrol， and N-acetylcysteiine on nonalcoholic fatty liver disease in rats[J]. Pharmaceutical Biology， 2016， 54（7）： 1198-1208.
[7]ZHANG QH， BIAN YH， SHI YY， et al. An economical and efficient technology for the extraction of resveratrol from peanut （Arachis hypogaea） sprouts by multi-stage countercurrent extraction[J]. Food Chemistry， 2015， （179）： 15-25.
[8]FAN XW， GAO MB， CAO XJ， et al. response surface optimization in extraction of resveratrol from grape seeds[J]. Guangzhou Chemical Industry， 2016， 44（15）： 74-79. （In Chinese）
[9]ROUPE KA， REMSBERG CM， YANEZ JA， et al. Pharma-cometrics of stilbenes： segueing towards the clinic[J]. Curr Clin Pharmacol， 2006， 1（1）： 81-101.
[10]LI BY， ZHU XQ， QIU XL， et al. Study on the stability and reaction performance of resveratrol by ultraviolet-visible spectroscopy[J]. Jiangsu Agricultural Sciences， 2016， 44（11）： 302-305. （In Chinese）
[11]XU Z， ZHAO ZQ. Application of column chromatography in separation and purification of effective components of Chinese medicinal materials[J]. Chinese Journal of Information on Traditional Chinese Medicine， 2013， 20（12）： 109-110. （In Chinese）
[12]JIANG RQ， ZHOU L， LI JL， et al. Study on purification of resveratrol from peanut root through the silica gel column chromatography[J]. Food Science and Technology， 2010， 35（1）： 199-202. （In Chinese）
[13]CHEN ZG. Analysis on extraction process of resveratrol from peanut roots[J]. China Chemical Trade， 2014， （31）： 158. （In Chinese）
[14]ZHANG LY， GUO JL， YE BY， et al. Study on the isolation method of resveratrol from Polygonum cuspidatum by Sephadex LH-20[J]. Natural Product Research and Development， 2009， 21（1）， 104-107. （In Chinese）
[15]TONG X， ZHANG H. Application of membrane separation technology in natural products[J]. Biotech World， 2015（3）： 98. （In Chinese）
[16]JIANG ML， LI ZF. Study on process of separating resveratrol from GentrinKentrin with membrance separation technique and the determination[J]. Acta Medicinae Sinica， 2008， 21（4）， 628-629. （In Chinese） [17]LIU ZC， XIA Y， ZHANG Y， et al. Study on membrane separation technology used in the purification of resveratrol[J]. Lishizhen Medicine and Materia Medica Research， 2009， 20（1）： 203-204. （In Chinese）
[18]ZHENG J， WU Q， LI XM， et al. Application of high speed countercurrent chromatography in separation of plant active components[J]. Science and Technology of Food Industry， 2009， 3（30）： 351-354. （In Chinese）
[19]LI Y， LIU JH. Study on the static adsorption kinetics of resveratrol from Polygonum cuspidatum sieb. et zucc by macroporous resin[J]. Food & Machinery， 2011， 27（5）： 82-86. （In Chinese）
[20]YANG JH， WANG N， SUN YH. Study on separation and purification of resveratrol from Polygonum cuspidatum by macroporous adsorption resin and enzymatic hydrolysis[J]. Ion Exchange and Adsorption， 2009， 25（3）： 273-275. （In Chinese）
[21]LIU LM. Study on purification technology of resveratrol in Polygonum cuspidatum[J]. Agricultural products processing， 2014， （11）： 29-35. （In Chinese）
[22]LANZA F， SELLERGEN B. The application of molecular imprinting technology to solid phase extraction[J]. Chromatographi， 2001， 53（11-12）： 599-611.
[23]ZHUANG XL， DONG XC， MA SJ. Selective on-line extraction of trans-resveratrol and emodin from Polygonum cuspidatum using molecularly imprinted polymer[J]. Journal of Chromatographic Science， 2008， 46（8）： 739-742.
[24]FENG T， LIU P， LIU HY， et al. Application and performance study of molecular imprinted polymer of resveratrol[J]. Food Research and Development， 2016， 37（16）： 37-41. （In Chinese）
[25]HE S， LU YB， WU B， et al. Isolation and purification of antioxidative isomeric polyphenols from the roots of parthenocissus laetecvirens by counter-current chromatography[J]. Journal of Chromatography A， 2007， 1151（1-2）： 175-179.
[26]LIU SX， CHENG LY， GENG W， et al. Study on the purification of resveratrol by HSCCC[J]. Agricultural products processing， 2005（9）： 121-123. （In Chinese）
[27]LI MQ， GENG YH， LIU GM， et al. Application of two-aqueous phase extraction technology in purification process of resveratrol[J]. Natural Product Research and Development， 2006， 18（4）：647-649. （In Chinese）
[28]HE YZ， WANG B， ZHUANG Y， et al. Study on separation and purification of resveratrol in wine grape residue with aqueous two phase extraction method[J]. Advanced Materials Research， 2012， （550-553）： 1743-1746.
[29]JIANG B， ZHANG T， ZHOU LF， et al. An ultrasonic-assisted two-aqueous phase method for extracting resveratrol[P]. China， CN10503039426A， 2015. （In Chinese）
Key words Resveratrol; Physiology activity; Purification
Received： February 25， 2020 Accepted： April 27， 2020
Supported by Hebei Provincial Phase II Modern Agricultural Industry Technology System Innovation Team Construction Project （HBCT2018120207）; Tangshan Science and Technology Planning Project （19150204E）; Hebei Province Innovation Ability Promotion Plan Project （20567673H）.
Lei WANG （1982-）， male， P. R. China， associate researcher， devoted to functional ingredients and food additives.
Di WANG （1989-）， female， P. R. China， associate researcher， devoted to functional ingredients and food additives. E-mail： [email protected].
#These authors contributed equally to this work.
Corresponding author. E-mail： [email protected]; [email protected].
Resveratrol is an antitoxin produced by plants under fungal infection， ultraviolet radiation or pathological conditions， and also acts as an antioxidant and anti-mutagenic agent[1]. Resveratrol has anti-tumor， anti-atherogenesis， anti-free radical， anti-oxidation， anti-viral， anti-inflammatory， estrogen-like， liver protection and other effects[2-4]. Resveratrol is rich in plant sources and widely exists in Polygonum cuspidatum， peanut， grape， mulberry， cassia and other plants. It has been found in at least 72 plants of 31 genera in 21 families[5-8]. The resveratrol circulating on the market is mainly derived from the extraction and purification of plants， and the highest purity of resveratrol has reached 99%. However， there are few finished products of high-purity resveratrol on the market， and the price is high. Resveratrol with a purity of 99% in China has been sold for 32 000 yuan/kg.
In recent years， the industry has made great efforts to develop resveratrol， but the current processes and methods have problems of low extraction rate and relatively high production cost. Therefore， improving the extraction efficiency of resveratrol is one of the problems to be solved in the pharmaceutical and food industries. This paper reviewed the current status of resveratrol separation and purification technology， providing a reference for its development and utilization value. Physical and Chemical Properties of Resveratrol
Resveratrol is a non-flavonoid polyphenolic compound with a molecular formula of C14H12O3 and a relative molecular weight of 228.25. It is white needle-like crystals， which is difficult to dissolve in water and soluble in organic solvents. It has a melting point of 256-257 ℃ and sublimates at 261 ℃. It can produce fluorescence when irradiated by ultraviolet light with a wavelength of 365 nm， and can react with ferric chloride-potassium ferricyanide[9]. Resveratrol exists in cis and trans forms in plants. Resveratrol forms with sugar， a glycoside， which releases resveratrol under the action of glycosidase in the intestine. Resveratrol mainly exists in trans in plants， and its physiological activity is stronger than its cis isomer[10].
Separation and Purification of Resveratrol
At present， there are many methods for the separation and purification of resveratrol， and the specific method should depend on the extraction conditions and the purpose of purification.
Column chromatography
Column chromatography is a chromatography method that uses a solid adsorbent as the stationary phase and an organic solvent or buffer solution as the mobile phase. This technique gradually separate components in a mixture depending on the differences in physical and chemical properties of substances such as the molecular shape， size， charged state， solubility， adsorption capacity， molecular polarity and affinity. According to different substance separation mechanisms， column chromatography can be divided into adsorption column chromatography， ion exchange column chromatography， gel column chromatography， distribution column chromatography， and electrofocusing column chromatography[11].
Jiang et al.[12]used silica gel column chromatography to purify resveratrol in peanut roots， with chloroform-methanol （9∶1） as the eluting solvent and a ratio of silica gel adsorbent to sample volume at 50∶1. The purity of resveratrol increased from 39% to more than 99%. Chen[13]used silica gel column chromatography to purify resveratrol in peanut roots. The thin layer chromatography and silica gel column plate and HPLC purity detection were used. When the ratio of chloroform to methanol was 9∶1， the purity of resveratrol in the eluate of silica gel column chromatography increased to more than 99%. Zhang et al.[14]investigated the separation effect of resveratrol samples on Sephadex LH-20 gel chromatography column under different elution solvents and different elution flow rates. The resveratrol separation effect was best at 0.5 ml/min. The purity of resveratrol reached 80.34% and the recovery reached 86.16% by medium pressure liquid chromatography. Membrane separation
Membrane separation technology is a high-tech of the century， including microfiltration， ultrafiltration， nanofiltration， electrodialysis， reverse osmosis， membrane electrolysis， membrane distillation， membrane extraction and other techniques. Because it has the functions of separation， concentration and purification， and has the advantages of high efficiency， energy saving， environmental protection， and molecular filtration， it is widely used in the field of natural product separation[15].
Jiang et al.[16]firstly removed emodin from the alcohol extract of P. cuspidatum by low alcohol and then removed tannin and some impurities by ethyl acetate countercurrent extraction， and the resveratrol purity reached 20%. Then an ultrafiltration membrane with a relative molecular weight of 4 000 to be intercepted was used to perform further purification， and the purity of resveratrol after the purification was more than 95%. Liu et al.[17]used membrane separation technology to purify resveratrol from P. cuspidatum which increased the purity of resveratrol in the crude extract of P. cuspidatum from 8.7% to 30.5%， and concentrated and separated the filtrate using an ultrafiltration membrane， obtaining resveratrol with a purity reaching 55.8%. Membrane separation is a physical separation process， which produces no waste， and is green and environmentally friendly， but the cost of the membrane is relatively high， so membrane separation is suitable for the separation and purification of end products.
Macroporous resin method
The macroporous adsorption resin is a kind of synthetic polymer adsorbent with a multi-hollow three-dimensional structure. It can selectively adsorb components through physical adsorption， and its adsorption is mainly achieved through surface adsorption， surface electrical properties， or morphological hydrogen bond[18].
Li et al.[19]used 18 adsorption resins to purify resveratrol in P. cuspidatum， and found that the adsorption capacity and resolution of HPD-722 resin are suitable for the purification of resveratrol. Yang et al.[20]studied the method of extracting and purifying resveratrol from P. cuspidatum with macroporous adsorption resin combined with enzymolysis， and found that H1020 resin not only has high adsorption capacity， easy desorption， but also can make the content of resveratrol in crude extract increase from 8.7% to 71.5%. Liu[21]used D101 macroporous resin to separate and purify the effective parts of P. cuspidatum. The adsorption flow rate was 1.0 ml/min， and the eluent was 50% ethanol. After eluting with 6BV eluent， the resveratrol was almost completely desorbed. It was determined that the content of resveratrol increased by more than 10% after purification. Molecular imprinting
Molecularly imprinted polymers （MIPs） polymerize functional monomers in the presence of template molecules （imprinted molecules） by a cross-linking agent. MIPs have spatial structures that can specifically bind to target molecules， and thus have high selectivity for the target molecules. They mainly have two different forms： non-covalent and covalent[22].
Zhuang et al.[23]studied the separation of resveratrol and emodin from P. cuspidatum. They prepared resveratrol imprinted polymer using resveratrol as a template， 4-vinylpyridine as a functional monomer， ethylene glycol dimethacrylate as a cross-linking agent， acetone as a porogen and 2，2-azobisisobutyronitrile as a directing agent， performed online high-performance liquid extraction with methanol-water （80∶20） as a mobile phase and detected the extract by reverse-phase high-performance liquid chromatography， and the recovery and purity of resveratrol were both higher than 80%. Feng et al.[24]separated resveratrol from P. cuspidatum， and prepared a molecularly imprinted polymer with resveratrol as a template， acrylamide as a functional monomer， and chloroform and tetrahydrofuran mixture as a cross-linking agent of the imprinted polymer. With the adsorption performance， selection performance and solid-phase extraction performance of the molecularly imprinted polymer as the investigation indicators， the molecularly imprinted polymer was subjected to solid phase extraction and HPLC determination with 40% ethanol solution and 80% ethanol solution as eluents， respectively， at a flow rate of 1 ml/min. The purity of resveratrol in the eluate was 89.2%， and the yield of resveratrol was 73.6%.
High-speed countercurrent chromatography
High-speed countercurrent chromatography technology is a liquid-liquid chromatography technology that does not use any solid support or carrier. Compared with the traditional solid-liquid column chromatography technology， it does not use a solid phase carrier as a stationary phase， and the separated substances are distributed and separated in two phases which are incompatible with each other. Therefore， this technology overcomes the disadvantages of sample adsorption， loss and pollution brought by the solid phase carrier.
He et al.[25]used countercurrent chromatography to separate the cis-trans isomers of resveratrol in Parthenocissus laetevirens Rehd. roots using n-hexane-ethyl acetate-methanol-water （1∶2∶1∶2） as the solvent system. After purification， the purity was 95.4% and 97.6% by HPLC. In the experiment of purifying resveratrol from P. cuspidatum by high-speed countercurrent chromatography， Liu et al.[26]selected a solvent system of chloroform∶methanol∶water = 4∶3∶2， a rotation speed at 850 r/min， and a flow rate at 2 ml/min， the purity of resveratrol obtained after purification was greater than 96%， and a good separation effect was achieved. Two-phase water extraction
Li et al.[27]used two-aqueous phase extraction technology to purify resveratrol from P. cuspidatum， and compared with chloroform organic extraction. It was found that when using ethanol-ammonium sulfate solution as a two-aqueous phase system to separate and enrich the extract of P. cuspidatum， the content of resveratrol in the liquid was 34.29%， which was 5.81% higher than that of organic extraction. He et al.[28]found that in the experiment of purifying resveratrol from wine， compared with organic solvent extraction， ethanol-ammonium sulfate-water extraction as a two-aqueous system had a better separation effect， and the resveratrol content was 5.68 times higher than organic solvent extraction. Jiang et al.[29]used ultrasonic-assisted two-phase aqueous extraction to purify resveratrol. The enzymatic hydrolysate of P. cuspidatum was extracted with ammonium sulfate-absolute ethanol as a two-phase aqueous system. The recovery of resveratrol was more than 98%. A two-phase system is a non-miscible two-phase or multi-phase water system formed by dissolving certain organic compounds or organic matter and inorganic salts in appropriate concentrations in water， and the separation principle of the system is based on the selective distribution of substances in the aqueous phase system.
Prospect
Resveratrol has extremely unstable chemical properties， and is prone to deteriorate after long time storage due to phenolic hydroxyl oxidation and double bond addition. This property affects the production of high-purity resveratrol. The stability of this compound can be enhanced after a certain structural modification， and the addition of the purification technology of resveratrol comprising its structural modification during the production process is currently less reported， so the new purification technology needs further study.
References
[1]CHIN YT， HSIEH MT， YANG SH， et al. Anti-proliferative and gene expression actions of resveratrol in breast cells in vitro[J]. Oncotarget， 2014， 5（24）： 12891-12907.
[2]LI C， XU XF， TAO ZH， et al. Resveratrol dimers， nutritional components in grape wine， are selective ROS scavengers and weak Nrf2 activators[J]. Food Chemistry， 2015， （173）： 218-223.
[3]KURITA S， KASHIWAGI T， EGISU T， et al. Content of resveratrol and glycoside and its contribution to the antioxidative capacity of Polygonum cuspidatum （Itadori） harvested in Kochi[J]. Biosci Biotechnol Biochem， 2014， 78（3）： 499-502. [4]HUANG FC， KUO HC， HUANG YH， et al. Anti-inflammtory effect of resveratrol in human coronary arterial endothelial cells via induction of autophagy： implication of the treatment of Kawasaki disease[J]. Circulation， 2017， 18（1）： 1-8.
[5]SHIN JA， OH S， AHN JH， et al. Estrogen receptormediated resveratrol actions on blood-brain barrier of ovariecto-mized mice[J]. Neurobiology of Aging， 2015， 36（2）： 993-1006.
[6]ALI MH， MESSIHA BA， ABDEL-LATIF HA. Protective effect of ursodeoxycholic acid， resveratrol， and N-acetylcysteiine on nonalcoholic fatty liver disease in rats[J]. Pharmaceutical Biology， 2016， 54（7）： 1198-1208.
[7]ZHANG QH， BIAN YH， SHI YY， et al. An economical and efficient technology for the extraction of resveratrol from peanut （Arachis hypogaea） sprouts by multi-stage countercurrent extraction[J]. Food Chemistry， 2015， （179）： 15-25.
[8]FAN XW， GAO MB， CAO XJ， et al. response surface optimization in extraction of resveratrol from grape seeds[J]. Guangzhou Chemical Industry， 2016， 44（15）： 74-79. （In Chinese）
[9]ROUPE KA， REMSBERG CM， YANEZ JA， et al. Pharma-cometrics of stilbenes： segueing towards the clinic[J]. Curr Clin Pharmacol， 2006， 1（1）： 81-101.
[10]LI BY， ZHU XQ， QIU XL， et al. Study on the stability and reaction performance of resveratrol by ultraviolet-visible spectroscopy[J]. Jiangsu Agricultural Sciences， 2016， 44（11）： 302-305. （In Chinese）
[11]XU Z， ZHAO ZQ. Application of column chromatography in separation and purification of effective components of Chinese medicinal materials[J]. Chinese Journal of Information on Traditional Chinese Medicine， 2013， 20（12）： 109-110. （In Chinese）
[12]JIANG RQ， ZHOU L， LI JL， et al. Study on purification of resveratrol from peanut root through the silica gel column chromatography[J]. Food Science and Technology， 2010， 35（1）： 199-202. （In Chinese）
[13]CHEN ZG. Analysis on extraction process of resveratrol from peanut roots[J]. China Chemical Trade， 2014， （31）： 158. （In Chinese）
[14]ZHANG LY， GUO JL， YE BY， et al. Study on the isolation method of resveratrol from Polygonum cuspidatum by Sephadex LH-20[J]. Natural Product Research and Development， 2009， 21（1）， 104-107. （In Chinese）
[15]TONG X， ZHANG H. Application of membrane separation technology in natural products[J]. Biotech World， 2015（3）： 98. （In Chinese）
[16]JIANG ML， LI ZF. Study on process of separating resveratrol from GentrinKentrin with membrance separation technique and the determination[J]. Acta Medicinae Sinica， 2008， 21（4）， 628-629. （In Chinese） [17]LIU ZC， XIA Y， ZHANG Y， et al. Study on membrane separation technology used in the purification of resveratrol[J]. Lishizhen Medicine and Materia Medica Research， 2009， 20（1）： 203-204. （In Chinese）
[18]ZHENG J， WU Q， LI XM， et al. Application of high speed countercurrent chromatography in separation of plant active components[J]. Science and Technology of Food Industry， 2009， 3（30）： 351-354. （In Chinese）
[19]LI Y， LIU JH. Study on the static adsorption kinetics of resveratrol from Polygonum cuspidatum sieb. et zucc by macroporous resin[J]. Food & Machinery， 2011， 27（5）： 82-86. （In Chinese）
[20]YANG JH， WANG N， SUN YH. Study on separation and purification of resveratrol from Polygonum cuspidatum by macroporous adsorption resin and enzymatic hydrolysis[J]. Ion Exchange and Adsorption， 2009， 25（3）： 273-275. （In Chinese）
[21]LIU LM. Study on purification technology of resveratrol in Polygonum cuspidatum[J]. Agricultural products processing， 2014， （11）： 29-35. （In Chinese）
[22]LANZA F， SELLERGEN B. The application of molecular imprinting technology to solid phase extraction[J]. Chromatographi， 2001， 53（11-12）： 599-611.
[23]ZHUANG XL， DONG XC， MA SJ. Selective on-line extraction of trans-resveratrol and emodin from Polygonum cuspidatum using molecularly imprinted polymer[J]. Journal of Chromatographic Science， 2008， 46（8）： 739-742.
[24]FENG T， LIU P， LIU HY， et al. Application and performance study of molecular imprinted polymer of resveratrol[J]. Food Research and Development， 2016， 37（16）： 37-41. （In Chinese）
[25]HE S， LU YB， WU B， et al. Isolation and purification of antioxidative isomeric polyphenols from the roots of parthenocissus laetecvirens by counter-current chromatography[J]. Journal of Chromatography A， 2007， 1151（1-2）： 175-179.
[26]LIU SX， CHENG LY， GENG W， et al. Study on the purification of resveratrol by HSCCC[J]. Agricultural products processing， 2005（9）： 121-123. （In Chinese）
[27]LI MQ， GENG YH， LIU GM， et al. Application of two-aqueous phase extraction technology in purification process of resveratrol[J]. Natural Product Research and Development， 2006， 18（4）：647-649. （In Chinese）
[28]HE YZ， WANG B， ZHUANG Y， et al. Study on separation and purification of resveratrol in wine grape residue with aqueous two phase extraction method[J]. Advanced Materials Research， 2012， （550-553）： 1743-1746.
[29]JIANG B， ZHANG T， ZHOU LF， et al. An ultrasonic-assisted two-aqueous phase method for extracting resveratrol[P]. China， CN10503039426A， 2015. （In Chinese）