High-speed Counter-current Chromatography Separation Research Articles

Efficient discovery of active isolates from Dioscorea spongiosa by the combination of bioassay-guided macroporous resin column chromatography and high-speed counter-current chromatography.

In the present study, twelve compounds from Dioscorea spongiosa were successfully purified by an efficient technique combined bioassay-guided fractionation macroporous resin column chromatography (MRCC) pretreatment and high-speed counter-current chromatography (HSCCC) separation for the first time. Then, D101 MRCC was used to fractionate the crude extract into five parts, which further applied the bioassay-guided fractionation strategy to screen the active fractions of 2 and 4. As for the separation, 200mg Fr.2 was purified by HSCCC using EtOAc/n-BuOH/H2 O (2:2:3, v/v), leading to annulatomarin (1), dioscoresides C (2), diosniponol C (3), methyl protodioscin (4), pseudoprotodioscin (5), protogracillin (6), as well as 200mg Fr.4 yielding montroumarin (7), dioscorone A (8), diosniponol D (9), protodioscin (10), gracillin (11), and dioscin (12) using CH2 Cl2 /MeOH/H2 O (3:3:2, v/v) with the purities over 95.0%. Finally, the isolates were assayed for their anti-inflammatory, urico-lowering, and anti-diabetic activities in vitro, which indicated that the steroidal saponins of 5, 6, and 11 showed all these three activities.

Iridoid-glycoside isolation and purification from Premna fulva leaves.

Premna fulva Craib, rich in iridoid glycosides, is widely used to treat periarthritis, osteoproliferation, pain, and other diseases. However, no studies have reported effective purification methods for obtaining iridoid glycosides as active materials. This paper describes an efficient strategy for separating iridoid glycosides from Premna fulva leaves using high-speed counter-current chromatography and preparative high-performance liquid chromatography. A two-phase solvent system, ethyl acetate/n-butanol/water (7.5:2.5:10, v/v), was selected for high-speed counter-current chromatography separation. The proposed method effectively separated and purified four iridoid glycosides and four lignans, including three new iridoid glycosides (4-6) and five known compounds (1-3, 7, 8), from Premna fulva leaves, indicating that high-speed counter-current chromatography combined with prep-HPLC can efficiently isolate catalpol derivatives from the genus Premna. Additionally, the in vitro anti-inflammatory activities of all isolated compounds were analyzed using lipopolysaccharide-stimulated RAW 264.7 cells, and the results indicated that six compounds (1 and 3-7) exhibited potential anti-inflammatory activities.

Large-scale preparation of five polar polyphenols including three isomers from Phyllanthus emblica Linn. by preparative high-speed counter-current chromatography.

The separation of polar compounds is challenging work due to poor retention and insufficient selectivity. In the present study, an efficient strategy for large-scale preparation of five polar polyphenols including three isomers from Phyllanthus emblica Linn has been established by preparative high-speed counter-current chromatography. Macroporous resin column chromatography was used for the enrichment of the polar polyphenols. However, sugar and other ultra-polar impurities were co-washed out with the targets. Liquid-liquid extraction with ethyl acetate/water (1/1, v/v) solvent system was developed to remove the ultra-polar impurities with a clearance rate of 95%. Finally, the targets were introduced to preparative high-speed counter-current chromatography for separation using ethyl acetate/n-butanol/acetic acid/water (2/7/1/10, v/v/v/v) solvent system. As a result, 191 mg of Mucic acid 1,4-lactone 5-O-gallate, 370 mg of β-Glucogallin, 301 mg of Gallic acid, 195 mg of Mucic acid 1,4-lactone 3-O-gallate and 176 mg of Mucic acid 1,4-lactone 2-O-gallate with purity higher than 98% were obtained from 1.5 g of sample. Mucic acid 1,4-lactone 3-O-gallate, Mucic acid 1,4-lactone 3-O-gallate, and Mucic acid 1,4-lactone 2-O-gallate are isomers. The results showed that high-speed counter-current chromatography could be well developed for the separation of polar compounds from natural products.

Large-scale preparation of two minor polar polyphenols from Phyllanthus emblica Linn. by macroporous resin column chromatography and high-temperature preparative high-speed counter-current chromatography.

In the present study, an efficient strategy for the large-scale preparation of two minor polar polyphenols from Phyllanthus emblica L. has been established by macroporous resin column chromatography and high-temperature preparative high-speed counter-current chromatography. Macroporous resin column chromatography was used for the enrichment of the target polyphenols. The target polyphenols could be well enriched in the 10% ethanol fraction and the peak area proportion increased from 2.2% in the crude extract to 85.3% in the 10% ethanol fraction. Then, the 10% ethanol fraction was introduced to preparative high-speed counter-current chromatography for separation using an n-butanol-ethyl acetate-water (17:3:20, v/v/v) solvent system. In order to improve the retention of the stationary phase, a high-temperature preparative high-speed counter-current chromatography had been developed by using a 316 L stainless steel pipe as a column. A 50°C of column temperature was used and the retention rate of the stationary phase could reach 75%. Finally, 871mg of compound Ⅰ and 534mg of compound Ⅱ with purities higher than 98% were obtained from 4g of the sample. The chemical structures were identified as corilagin and 1, 6-di-O-galloyl-β-D-glucoside by 1 H-NMR and 13 C-NMR.

Review of High-Speed Countercurrent Chromatography in Separation of Lanthanides and Actinides

Review of High-Speed Countercurrent Chromatography in Separation of Lanthanides and Actinides

Characterization, Large-Scale HSCCC Separation and Neuroprotective Effects of Polyphenols from Moringa oleifera Leaves

Moringa oleifera leaves have been widely used for the treatment of inflammation, diabetes, high blood pressure, and other diseases, due to being rich in polyphenols. The main objective of this work was to largely separate the main polyphenols from Moringa oleifera leaves using the technique of high-speed counter-current chromatography (HSCCC). The phenolic composition in Moringa oleifera leaves was first analyzed qualitatively and quantitatively by UPLC-Q-Exactive Orbitrap/MS and UPLC-QqQ/MS, respectively, indicating that quercetin and kaempferol derivatives, phenolic acid and apigenin are the main polyphenols in Moringa oleifera leaves, with quercetin and kaempferol derivatives predominating. Furthermore, the conditions of HSCCC for large-scale separation of polyphenols from Moringa oleifera leaves were optimized, which included the selection of the solvent system, flow rate and the sample load. Only by one-step HSCCC separation (within 120 min) under the optimized conditions, six quercetin and kaempferol derivatives, a phenolic acid and an apigenin could be individually isolated at a large scale (yield from 10% to 98%), each of which possessed high purity. Finally, the isolated polyphenols and phenolic extract from Moringa oleifera leaves (MLPE) were verified to have strong neuroprotective activities against H2O2-induced oxidative stress in PC-12 cells, suggesting that these compounds would contribute to the main beneficial effects of Moringa oleifera leaves.

Study on the Synthesis of Methylated Reference and Their Application in the Quantity of Curcuminoids Using Single Reference Liquid Chromatography Based on Relative Molar Sensitivity.

We report on the recommendation of the simple and versatility of methylated reference (MR) to improve applications in the single reference (SR)-LC based on relative molar sensitivity (RMS). Three curcuminoids (Curs) such as curcumin, demethoxycurcumin and bisdemethoxycurcumin in turmeric products were determined using authentic standards and methylated curcumin. In addition, high-speed countercurrent chromatography (HSCCC) purification is necessary to separate Curs for indicating the RMS. For HSCCC separation, a biphasic solvent system was used to obtain these fractions, which were then subjected to 1H quantitative NMR to determine their contents in each test solution. Using these solutions, the RMS of Curs are calculated from slopes ratios of calibration curves (three ranges from 0-100 µmol/L, r2 > 0.998). The averaged RMS of Curs were 8.92 (relative standard deviation (RSD), 1.17%), 8.97 (2.18%), and 9.61 (0.77%), respectively. Cur concentrations in turmeric products can be determined using RMS, peak area, and MR content added in these samples. This proposed method, which is based on chemical methylation and the SR-LC assay has been successfully applied for the simple and reliable estimation of Curs in turmeric products.

Separation and Identification of Antioxidants and Aldose Reductase Inhibitors in Lepechinia meyenii (Walp.) Epling.

We previously reported that Lepechinia meyenii (Walp.) Epling has antioxidant and aldose reductase (AR) inhibitory activities. In this study, L. meyenii was extracted in a 50% MeOH and CH2Cl2/MeOH system. The active extracts of MeOH and 50% MeOH were subjected to fractionation, followed by separation using high-speed counter-current chromatography (HSCCC) and preparative HPLC. Separation and identification revealed the presence of caffeic acid, hesperidin, rosmarinic acid, diosmin, methyl rosmarinate, diosmetin, and butyl rosmarinate. Of these, rosmarinic acid, methyl rosmarinate, and butyl rosmarinate possessed remarkable antioxidant and AR inhibitory activities. The other compounds were less active. In particular, rosmarinic acid is the key contributor to the antioxidant and AR inhibitory activities of L. meyenii; it is rich in the MeOH extract (333.84 mg/g) and 50% MeOH extract (135.41 mg/g) of L. meyenii and is especially abundant in the EtOAc and n-BuOH fractions (373.71–804.07 mg/g) of the MeOH and 50% MeOH extracts. The results clarified the basis of antioxidant and AR inhibitory activity of L. meyenii, adding scientific evidence supporting its traditional use as an anti-diabetic herbal medicine. The HSCCC separation method established in this study can be used for the preparative separation of rosmarinic acid from natural products.

A strategy to process hundred-gram level complex sample using liquid-liquid-refining extraction and consecutive counter-current chromatography: Toona sinensis case study

Large-scale preparation of target compounds from complex samples is facing great challenges. In the present study, an efficient strategy for large-scale preparation of target compound was proposed and successfully applied in the separation of active components from Toona sinensis. The pretreatment technology of liquid-liquid refining extraction (LLRE) combined with consecutive high-speed counter-current chromatography (HSCCC) was used to process hundred grams of extractions. Firstly, two phase solvent systems composed of n-hexane–ethyl acetate–methanol–water (5:5:5:5, v/v) and (2:5:2:5, v/v) were used to remove low polar and high polar impurities from 100 g crude extracts of T. sinensis, respectively, and 9.25 g of crude sample was obtained. And then, n-hexane–ethyl acetate–methanol–water (2.5:5:2.5:5, v/v) was used as the solvent system for HSCCC separation. The isocratic elution mode with max loading and consecutive injections mode were investigated to obtain more target compound. As a result, ethyl gallate with purity of 97% was successfully separated by 5 times consecutive counter-current chromatography. The separation was repeated once. Finally, ethyl gallate (3.73 g) was isolated from 9.25 g of crude sample (100 g crude extracts). The results demonstrated that the yield increased from 0.26 g/h/L of untreated crude extract to 0.93 g/h/L of LLRE pre-treated sample for single injection, and further increased to 1.62 g/h/L for 5 consecutive injections mode with the present method.

Separation of eight phenolic compounds from the over-ground parts of Aconitum pendulum Busch by repeated injection high-speed counter-current chromatography

ABSTRACT In this study, eight phenolic compounds were obtained from the over-ground parts of the Aconitum pendulum (A. pendulum) Busch through an efficient high-speed counter-current chromatography （HSCCC）method. First, the column chromatography for macroporous resin was used for the treatment of crude extract of over-ground parts of A. pendulum Busch. And then, 30% ethanol elution fraction was transferred to HSCCC for separation using the classic ethyl acetate, n-butanol and water system with the solvent ratio of 7:3:10. To improve parting efficiency, a mode of repeated injection was developed. Two groups of compounds existing in one HPLC peak were effectively separated by HSCCC. The results revealed that HSCCC could effectively isolate compounds with the same or similar polarity. Preparative HPLC was used to perform further purification, and then eight phenolic compounds were separated from the over-ground parts of the A. pendulum Busch for the first time. The current investigation successfully provides a reference for phytochemical research and, comprehensive development and utilization of A. pendulum Busch in the future. Furthermore, HSCCC efficiently separated compounds with similar polarity and its combination with preparative HPLC could play a great role in the separation of natural materials.

A rational route based on liquid-liquid-refining extraction and high-speed counter-current chromatography for separation of target compound from Toona sinensis

Gallotannin, 1,2,3,4,6-penta-O-galloyl-β-d-glucose (PGG), exhibited a wide range of biological activities. In the present study, a method combined liquid-liquid refining extraction and high-speed counter-current chromatography was established for improving the separation yield of PGG from Toona sinensis. Liquid-liquid refining extraction was used to enrich the target compound, the impurities with high polarity and low polarity can be removed with appropriate two-phase solvent systems, and a series of equations were developed to determine the times of extraction and loss rate of target compound. As a result, the two solvent systems composed of n-hexane–ethyl acetate–methanol–water (1:2:1:2, v/v) and ethyl acetate–water (1:1, v/v) were selected to remove the low polarity impurities and high polarity impurities, respectively, and 78.08 % by weight of the crude extract was removed, while the loss rate of PGG was only 12.09 % after extraction. Then, the PGG enriched samples were further separated by high-speed counter-current chromatography with a two-phase solvent system n-hexane–ethyl acetate–methanol–water (0.4:5:0.4:5, v/v). PGG (75 mg) with purity of 97.33 % was isolated from 350 mg of the PGG enriched samples. This study proved that the liquid-liquid refining extraction was a very effective method to enrich the target compound from complex mixture and a good assistant for large-scale preparation of natural products.

Preparative separation of three terpenoids from edible brown algae Sargassum fusiforme by high-speed countercurrent chromatography combined with preparative high-performance liquid chromatography

Edible brown algae Sargassum fusiforme is an economic seaweed with huge yield in Asia. Terpenoids are an important class of active substances in Sargassum fusiforme, which have replaced some synthetic drugs in clinical treatment. In this work, the preparative separation protocol of three terpenoids, loliolide (1), isololiolide (2), and (3R)-4-[(2R,4S)-4-acetoxy-2-hydroxy-2,6,6-trimethylcyclohexylidene]-3-buten-2-one (3), from Sargassum fusiforme by high-speed countercurrent chromatography combined with preparative high-performance liquid chromatography was presented. n-Hexane-ethyl acetate-methanol-water (1:2:1:2, v/v) was selected as biphasic solvent system through optimization. Under the optimal high-speed countercurrent chromatography separation conditions, 35.87 mg mixture of isomers 1 and 2, and 8.87 mg compound 3 were obtained from 300 mg of the crude extract of Sargassum fusiforme. The isomer mixture was further separated by preparative high-performance liquid chromatography to obtain 11.56 mg compound 1 and 12.82 mg compound 2. Their purities were all above 95%. The chemical structures of three isolated terpenoids were identified by 1H and 13C nuclear magnetic resonance spectroscopy.

Combined Ultrahigh Pressure Extraction and High-Speed Counter-Current Chromatography for Separation and Purification of Three Glycoside Compounds from Dendrobium officinale Protocorm.

As an alternative to Dendrobium candidum, protocorm-like bodies (PLBs) of Dendrobium candidum are of great value due to their high yield and low cost. In this work, three glycoside compounds, β-D-glucopyranose 1-[(E)-3-(4-hydroxyphenyl)-2-propenoat] (I), β-D-glucopyranose 1-[(E)-3-(3, 4-dihydroxyphenyl)-2-propenoat] (II), and 1-O-sinapoyl glucopyranoside (III), were extracted and isolated by ultrahigh pressure extraction (UPE) coupled with high-speed counter-current chromatography (HSCCC) from PLBs of D. officinale. First, the target compounds were optimized and prepared with 50% ethanol solution at a 1:30 (g/mL) solid/liquid ratio in 2 min under 300 MPa by UPE. Then, the crude extract was chromatographed with a silica gel column, and primary separation products were obtained. In addition, the products (150 mg) were separated by HSCCC under the solvent system of MTBE-n-butyl alcohol-acetonitrile-water (5:1:2:6, v/v/v/v), yielding 31.43 mg of compound I, 10.21 mg of compound II, and 24.75 mg of compound III. Their structures were further identified by ESI-MS, 1H NMR, and 13C NMR. The antioxidant results showed that the three compounds expressed moderate effects on the DPPH· scavenging effect. Compound II had the best antioxidant capacity and its IC50 value was 0.0497 mg/mL.

Preparative separation of specific triterpenoids from Inonotus obliquus based on negative-pressure cavitation extraction and high-speed counter-current chromatography

BackgroundInonotus obliquus, a famous dark brown white-rot fungus on birch trees, has remarkable anti-cancer activity due to its special triterpenpoid components. The aims of this study were to develop an efficient and integrated strategy for preparative separation of triterpenoids from Inonotus obliquus and to provide ideas for the preparation of bioactive ingredients in other complex materials. MethodsIn the present study, an integrated technology of negative pressure cavitation extraction (NPCE) and high-speed counter-current chromatography (HSCCC) separation of four main triterpenoids in Inonotus obliquus was developed for the first time. Response surface methodology was used to optimize the negative pressure cavitation extraction parameters of triterpenoids from Inonotus obliquus. For the rapid and effective preparative separation of triterpenoids, the HSCCC method with Hex-ACN (1:1; v/v) system was established. Significant FindingsThe extraction yield of total triterpenoids reached 17.41 mg/g under the optimal NPCE extraction conditions. The specific four triterpenoids including 3β-hydroxy-lanosta-8, 24-diene-21-al (0.84 mg/g), Inoterpene F (1.09 mg/g), Inotodiol (2.93 mg/g), Lanosterol (1.42 mg/g) were obtained by one step separation with the purities of more than 95%. This is the first report on the preparative separation of triterpenoids from Inonotus obliquus by NPCE-HSCCC.

A hydrophobic deep eutectic solvents-based integrated method for efficient and green extraction and recovery of natural products from Rosmarinus officinalis leaves, Ginkgo biloba leaves and Salvia miltiorrhiza roots

Green extraction and separation of natural products is crucial for food industry, and has been widely concerned. Compared with hydrophilic natural products, the green extraction and separation of hydrophobic natural products is more challenging. In this work, a novel method which coupled the ultrasound-assisted extraction with high-speed countercurrent chromatography (HSCCC) separation based on hydrophobic deep eutectic solvent (DES) was proposed for hydrophobic compounds extraction and recovery. By using the developed method, the hydrophobic compounds in Rosmarinus officinalis leaves, Ginkgo biloba leaves and Salvia miltiorrhiza roots have been successfully extracted and recovered. The results indicated that hydrophobic DESs are more effective for extraction hydrophobic compounds than hydrophilic DESs and traditional solvents. And hydrophobic DESs can be used as HSCCC separation solvents to separate and recover hydrophobic compounds effectively. The infinite possibilities of DESs components and their combinations can provide inexhaustible selective space for hydrophobic compounds in the HSCCC separation process.

Separation and anti-inflammatory evaluation of phytochemical constituents from Pleurospermum candollei (Apiaceae) by high-speed countercurrent chromatography with continuous sample load.

Pleurospermum (Apiaceae) species possess a wide range of biological properties viz. analgesic, anti-inflammatory, antimalarial, and so on. Pleurospermum candollei (DC.) Benth. Ex C. B. Clark. is reported to cure diarrhea, gastric, respiratory, stomach, abdominal, joint, and back pain problems. In addition, it is also used for both male and female infertility. The present study deals with an efficient technique using high-speed countercurrent chromatography for separation of chemical components from the methanol extract of P. candollei. Notably, nine main compounds namely luteolin 7-O-glucoside (1), oxypeucedanin hydrate (2), pabulenol (3), bergapten (4), heptadecanoic acid (5), (E)-isoelemicin (6), trans-asarone (7), α-linolenic acid (8), and isoimperatorin (9) were very efficiently separated and isolated in pure form. Multiple injections were applied followed by two off-line recycling high-speed countercurrent chromatography. The inhibitory effect of nitric oxide production of all compounds was tested in the presence of 200ng/mL lipopolysaccharide in RAW264.7 mice macrophage cells. The results demonstrated that compounds 7 and 8 effectively inhibited nitric oxide production, with IC50 values of 28.44 and 53.18μM, respectively. This study thus validates the traditional claim of using P. candollei. Taken together, these findings will be useful in future research to find a potential candidate with anti-inflammatory properties.

Valeriana rigida Ruiz & Pav. Root Extract: A New Source of Caffeoylquinic Acids with Antioxidant and Aldose Reductase Inhibitory Activities.

Valeriana rigida Ruiz & Pav. (V. rigida) has long been used as a herbal medicine in Peru; however, its phytochemicals and pharmacology need to be scientifically explored. In this study, we combined the offline 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH)-/ultrafiltration-high-performance liquid chromatography (HPLC) and high-speed counter-current chromatography (HSCCC)/pH-zone-refining counter-current chromatography (pH-zone-refining CCC) to screen and separate the antioxidants and aldose reductase (AR) inhibitors from the 70% MeOH extract of V. rigida, which exhibited remarkable antioxidant and AR inhibitory activities. Seven compounds were initially screened as target compounds exhibiting dual antioxidant and AR inhibitory activities using DPPH-/ultrafiltration-HPLC, which guided the subsequent pH-zone-refining CCC and HSCCC separations of these target compounds, namely 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, 3,4-O-di-caffeoylquinic acid, 3,5-O-di-caffeoylquinic acid, 4,5-O-di-caffeoylquinic acid, and 3,4,5-O-tri-caffeoylquinic acid. These compounds are identified for the first time in V. rigida and exhibited remarkable antioxidant and AR inhibitory activities. The results demonstrate that the method established in this study can be used to efficiently screen and separate the antioxidants and AR inhibitors from natural products and, particularly, the root extract of V. rigida is a new source of caffeoylquinic acids with antioxidant and AR inhibitory activities, and it can be used as a potential functional food ingredient for diabetes.

An approach based on consecutive high-speed counter-current chromatography for preparation of an active compound rutin from Apocynum venetum L

Apocynum venetum is a kind of plant with high medicinal value. Its main component is rutin, which is widely used. In the present study, an efficient method based on high-speed counter-current chromatography (HSCCC) was established to separate rutin from A. venetum. The optimized solvent system composed of ethyl acetate-n-butanol-water (4:1:5, v/v) was selected for HSCCC separation. It was found that the maximum loading amount was 1 g, but the loading amount did not affect the two-phase equilibrium of solvent system. Therefore, consecutive high-speed counter-current chromatography was introduced to further improve the yield in the present study. As a result, 292 mg of rutin with 95% purity was obtained from 2.4 g crude sample. The retention of the stationary phase was 27%, which was almost unchanged. The current strategy can not only be used for the scale-up preparation of rutin, but also provide an example for the preparation of other natural active compounds.

Purification of Four Caffeoylquinic Acid Derivatives from the Flowers of Gynura Procumbens by HSCCC.

Four caffeoylquinic acid derivatives from the Gunura procumbens flowers (GPF) were successfully isolated and purified by high-speed counter-current chromatography (HSCCC). Ethyl acetate-methanol-water (3:1:3, v/v/v) was the optimum biphasic solvent system, which was selected by high-performance liquid chromatography (HPLC) and run on a preparative scale where the lower aqueous phase was used as the mobile phase with a head-to-tail elution mode. Chlorogenic acid (3.83 mg), Isochlorogenic acid A (6.51 mg), Isochlorogenic acid B (4.38 mg) and Isochlorogenic acid C (4.47 mg) were obtained for the first time in an one-step HSCCC separation from 800 mg of the crude extracts. The purities of four compounds were determined to be >95% by HPLC. Chemical structures of each isolated compounds were identified by nuclear magnetic resonance and electrospray ionization mass spectrometry methods. It is worth noting that all the four compounds were isolated here for the first time from GPF and this work confirms the effectiveness of HSCCC for the separation of compounds contained in complex samples, and provides a foundation for further exploitation of G. procumbens.

Rapid enrichment and separation of two novel minor phenols from Malus hupehensis utilizing liquid-liquid extraction with three-phase solvent system and high-speed counter-current chromatography based on the polarity parameter.

For a rapid enrichment and separation of minor components from Malus hupehensis, the selection of suitable solvent system is the great challenge for liquid-liquid extraction with a three-phase solvent system and high-speed counter-current chromatography. According to the concept of "like dissolves like," the similarity of the average polarity between solvent system and target compounds was the significant characteristic of liquid-liquid extraction with a three-phase solvent system and high-speed counter-current chromatography separation. The polarity parameter model provides a way to calculate the polarity of unknown compounds. Under the guidance of the polarity, an efficient enrichment and separation approach was established through liquid-liquid extraction and high-speed counter-current chromatography with solvent systems composed of n-hexane-ethyl acetate-acetonitrile-water (5:3:5:7, v/v), n-hexane-ethyl acetate-methanol-water (1:2:1:2, v/v), respectively. Thus, the total content of minor compounds was increased from 2.6% to 17.2%, and two novel compounds (6´´-O-coumaroyl-2´-O-glucopyranosylphloretin and 3´´´-methoxy-6´´-O-feruloy-2´-glucopyranosylphloretin) were obtained. The discovery of the new dihydrochalcones expanded the structural diversity of compounds produced by the genus Malus. The experimental results demonstrated that compound polarity can be described by the polarity parameter model and is an important reference for investigating optimum solvent systems for liquid-liquid extraction with a three-phase solvent system and high-speed counter-current chromatography.