Separation of five flavone glycosides including two groups with similar polarities from Dracocephalum tanguticum by a combination of three high-speed counter-current chromatography modes

The separation of compounds with similar polarities is challenging. In the present study, five flavone glycosides, including two groups with similar polarities, were obtained from Dracocephalum tanguticum by three high-speed counter-current chromatography modes, including flow rate conversion mode, recycling mode, and heart-cut mode. With flow rate conversion mode, compounds 3 and 4 with similar polarities and compound 5 were separated by high-speed counter-current chromatography with ethyl acetate/methanol/water (5.0% acetic acid) (8:2:10, v/v) system. The flow rate was controlled as: 1.8 mL/min for 0–160 min, 2.2 mL/min for 160–200 min, and 2.5 mL/min for 200–400 min. However, compounds 1 and 2 with similar polarities were not separated due to the similar distributive properties. Then, a recycling and heart-cut mode were introduced to improve the separation efficiency. The heart-cut mode was introduced in the second and third cycles, and compounds 1 and 2 were well separated in the fourth cycle. Consequently, five flavone glycosides, including two groups with similar polarities were obtained and identified as cosmosiin (1), pedaliin (2), quercetin-3-O-rutinoside (3), pedaliin-6''-acetate (4), and sorbifolin-6-O-β-glucopyranoside (5). The current strategy provides a reference for separating compounds with similar polarities from a crude sample.     

High-speed counter-current chromatography assisted preparative isolation of phenolic compounds from the flowers of Chrysanthemum morifolium cv. Fubaiju

Chrysanthemum morifolium cv. Fubaiju is rich in phenolic compounds with various benefits such as anti-inflammatory, antioxidant, and cardiovascular protection. In this study, 12 phenolic compounds, including five flavonoid glycosides and seven quinic acid derivatives, were successfully separated from the flowers of Chrysanthemum morifolium cv. Fubaiju by high-speed counter-current chromatography and preparative high-performance liquid chromatography. Ethyl acetate-n-butanol–acetonitrile–water–acetic acid (5:0.5:2.5:5:0.25, v/v/v/v/v) was selected as solvent system to separate six fractions from the flowers of Chrysanthemum morifolium cv. Fubaiju, and 20% aqueous acetonitrile (containing 0.1% formic acid) was chosen to be the elution solvent in preparative high-performance liquid chromatography for purifying the fractions above. Luteolin-7-O-β-D-glucoside ( 1 ), luteolin-7-O-β-D-glucuronide ( 2 ), apigenin-7-O-β-D-glucoside ( 3 ), luteolin-7-O-β-D-rutinoside ( 4 ), diosmetin-7-O-β-D-glucoside ( 5 ), chlorogenic acid ( 6 ), 1,5-dicaffeoylquinic acid ( 7 ), 1,4-dicaffeoylquinic acid ( 8 ), 3,4-dicaffeoylquinic acid ( 9 ), 3,4-dicaffeoyl-epi-quinic acid ( 10 ), 3,5-dicaffeoylquinic acid ( 11 ), and 4,5-dicaffeoylquinic acid ( 12 ) were isolated with purities all above 95%, respectively. In addition, all isolates were evaluated for their protective effects on H₂O₂-induced oxidative damage in adult retinal pigment epithelial cells.     

Isolation of Anti-HIV Components from Canarium album Fruits by High-Speed Counter-Current Chromatography

The fruit of Canarium album, also called Chinese olive, is a popular food and traditional Chinese herb. The ethyl acetate extract of Canarium album fruits was found to exhibit an inhibitory effect on six-helix bundle formation of the human immune deficiency virus (HIV) glycoprotein transmembrane subunit gp41 in our previous study. In this paper, a preparative separation of anti-HIV components from an ethyl acetate extract of Canarium album fruits was performed by high-speed counter-current chromatography, using a solvent system of n-hexane–ethyl acetate–ethanol–0.5% acetic acid aqueous (0.5:9.5:2:8, v/v/v/v). Among the six fractions obtained from a single separation process, three exhibited over 90% purity as determined by HPLC, two compounds were identified as 5-hydroxymethylfurfural and 3,4,5-trihydroxybenzoic acid, and four fractions displayed significant inhibitory effects on HIV-1 IIIB infection. These findings suggest that the high-speed counter-current chromatography is an effective and reliable technique for separating bioactive components from medicinal herbs. Further identification of the active compounds in Canarium album fruits and studying their mechanism of action are warranted.     

UV-based solvent system screening for high-speed counter-current chromatography fractionation of compounds with similar UV absorption from complex samples followed by preparative HPLC purification: Flavonoids from barley seedlings as sample

This article proposes a solvent system screening strategy for compounds with similar UV absorption in complex samples by UV spectrophotometer. There is no need to calculate the partition coefficient value of each compound, only the partition coefficient of the whole sample. The partition coefficient value should be close to 1 in order to obtain as many high-speed counter-current chromatography fractions as possible. Then, preparative HPLC was used to purify the high-speed counter-current chromatography fractions. Based on the above strategy, seven c-glycosyl flavonoids and an amino acid were successfully obtained from barley seedlings through high-speed counter-current chromatography fractionation with ethyl acetate/n-butanol/water (8:2:10, v:v:v) system followed by preparative HPLC purification. The research shows that high-speed counter-current chromatography could be well developed as a tool for fractionation before purification, and greatly improves the separation efficiency.     

Preparative isolation of diterpenoids from Salvia bowleyana Dunn roots by high-speed countercurrent chromatography combined with high-performance liquid chromatography

The root of Salvia bowleyana Dunn (Lamiaceae) is used as a traditional Chinese medicine that has multiple therapeutic effects. In this study, an efficient strategy was developed to separate diterpenoid compounds, which are the main active ingredients in Salvia bowleyana Dunn roots, from complex crude extracts by high-speed countercurrent chromatography combined with preparative high-performance liquid chromatography. A two-phase solvent system comprising n-hexane–ethyl acetate–methanol–water (7:3:7:3, v/v/v/v) was selected for high-speed countercurrent chromatographic separation. Three major diterpenoids, 6α-hydroxysugiol ( 7 ), sugiol ( 8 ), and 6, 12-dihydroxyabieta-5,8,11,13-tetraen-7-one ( 9 ) were obtained at purities of 98.9, 95.4, and 96.2%, respectively, and minor diterpenoids were enriched via one-step separation. The enriched minor diterpenoids were further purified by continuous preparative high-performance liquid chromatography to yield two new norabietanoids ( 1 , 6 ) and four known compounds ( 2 – 5 ). The structures of these new compounds were determined using NMR spectroscopy, high-resolution electrospray ionization mass spectrometry, and electronic circular dichroism spectroscopy. The results suggest that high-speed countercurrent chromatography combined with preparative high-performance liquid chromatography efficiently isolates diterpenoids, including minor components, from complex natural products.     

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.     

Isolation and purification of ginkgo flavonol glycosides from Ginkgo biloba leaves by high-speed counter-current chromatography

A high-speed counter-current chromatography method was developed for the separation and purification of bioactive flavonol glycosides from a crude ethanol extract of Ginkgo biloba leaves. The separation was performed with a two-phase solvent system composed of n-hexane-butanol-ethyl acetate-methanol-0.5% acetic acid (1:0.5:3.5:1:4, v/v) and three pure compounds were eluted in high purities in a one-step separation. Their purities were determined by HPLC and identified by MS,(1)H-NMR, and(13)C-NMR.     

Separation and purification of alkaloids and phenolic acids from Phellodendron chinense by pH-zone refining and online-storage inner-recycling counter-current chromatography

In this work, eight compounds from Phellodendron chinense were separated and purified by pH-zone refining counter-current chromatography and traditional counter-current chromatography coupled with online-storage inner-recycling counter-current chromatography (IRCCC). The pH-zone-refining mode was adopted for separating 2.0 g of crude extract with the solvent system of chloroform–methanol–water (4:3:3, v/v), in which 10 mM hydrochloric acid and 10 mM triethylamine were added in the stationary and mobile phases, respectively. Meanwhile, traditional counter-current chromatography coupled with online-storage IRCCC separation was performed by the solvent system of n-hexane-ethyl acetate-methanol-water (5:5:2:8, v/v). Finally, eight compounds, including six alkaloids as 6-methylpiperidin-2-one( 1 ), isoplatydesmine( 4 ), berlambine( 5 ), epiberberine( 6 ), palmatine( 7 ), berberine( 8 ) and two phenolic acids as ferulic acid( 2 ), isoferulic acid( 3 ), were successfully obtained using these three different CCC modes with the purities over 95.0%.     

Preparative separation of six terpenoids from Wedelia prostrata Hemsl. by two-step high-speed counter-current chromatography

Terpenoids are principal chemical compounds of Wedelia prostrata Hemsl. and have different biological activities, thus the study on separation and purification of terpenoids from W. prostrata Hemsl. is necessary. In this paper, high-speed counter-current chromatography (HSCCC) was successfully established for preparative isolation and purification of terpenoids from extracts of petroleum ether fraction which extracted from whole herbs of W. prostrata Hemsl. In the process, a total of 750?mg of sample was prepared for HSCCC isolation. Terpenoids were separated and purified with the two-phase solvent system n-hexane–ethyl acetate–methanol–water (9:1:9:1, 19:1:19:1, v/v/v/v). Therefore, 5α-hydroxy-2-oxo-p-menth-6(1)-ene (4.4?mg), 3α-angeloyloxy-ent-kaur-16-en-oic acid (5.6?mg), 3α-tigloyloxy-ent-kaur-16-en-oic acid (5.7?mg), 3α-phenylpropionoyloxy-ent-kaur-16-en-oic acid (7.3?mg), 3α-senecioyloxy-ent-kaur-16-en-oic acid (11.4?mg), and kaurenoic acid (12.3?mg) were obtained from W. prostrata Hemsl. and their purities reached standard determined by HPLC. Among them, 3α-phenylpropionoyloxy-ent-kaur-16-en-oic acid and 5α-hydroxy-2-oxo-p-menth-6(1)-ene were first isolated with high quantity as a useful chemical resource. The structures of these compounds were identified by ESI-MS, ¹H-NMR, and ¹³C-NMR. The present results demonstrated that high-speed counter-current chromatography was a fast and efficient technique for preparative separation of six terpenoids from W. prostrata Hemsl. which provided a useful reference to solve the problem of their sample availability for drug development.     

Extraction and preparation of 5-lipoxygenase and acetylcholinesterase inhibitors from Astragalus membranaceus stems and leaves

In this study, an efficient method that employs 5-lipoxygenase and acetylcholinesterase as biological target molecules in receptor–ligand affinity ultrafiltration–liquid chromatography was developed for the screening of enzyme inhibitors derived from the Astragalus membranaceus stems and leaves. The effects of the extraction time, number of extraction cycles, ethanol concentration, and liquid–solid ratio on the total yield of the target compounds were investigated using response surface methodology, and the bioactive components were isolated using a combination of semi-preparative high-performance liquid chromatography and high-speed countercurrent chromatography via a two-phase solvent system consisting of n-hexane–ethyl acetate–methanol–water (1:6:2:6, v/v/v/v). Subsequently, 10 naturally-occurring bioactive components in the Astragalus membranaceus stems and leaves, including wogonin, ononin, isoquercitrin, calycosin-7-glucoside, 3-hydroxy-9,10-dimethoxyptercarpan, hyperoside, 7,2′-dihydroxy-3′,4′-dimethoxyisoflavan, baicalein, calycosin, and soyasaponin, were screened using affinity ultrafiltration to determine their potential effects against Alzheimer's disease. Consequently, all target compounds had purities higher than 95.0%, and the potential anti-Alzheimer's disease effect of the obtained bioactive compounds was verified using molecular docking analysis. Based on the results, the back-to-back screening of complex enzyme inhibitors and separation of the target bioactive compounds using complex chromatography could provide a new approach to the discovery and preparation of natural active ingredients.     

Preparative isolation and purification of three flavonoid glycosides from Taraxacum mongolicum by high-speed counter-current chromatography

A preparative high-speed counter-current chromatography (HSCCC) was successively applied to purify three flavonoid glycosides from the aerial part of Taraxacum mongolicum, a traditional Chinese medicine. Subsequent UV, MS, and NMR analyses have led to the characterization of three flavonoid glycosides including two new compounds isoetin-7-O-beta-D-glucopyranosyl-2'-O-alpha-L-arabinopyranoside and isoetin-7-O-beta-D-glucopyranosyl-2'-O-alpha-D-glucopyranoside, and a known compound, isoetin-7-O-beta-D-glucopyranosyl-2'-O-alpha-D-xyloypyranoside, which were first isolated from T. mongolicum. The two-phase solvent system composed of ethyl acetate/n-butanol/water (2:1:3, v/v/v) was performed in HSCCC. Consequently, a total of 25.7 mg isoetin-7-O-beta-D-glucopyranosyl-2'-O-alpha-L-arabinopyranoside, 19.1 mg isoetin-7-O-beta-D-glucopyranosyl-2'-O-alpha-D-glucopyranoside, and 10.6 mg isoetin-7-O-beta-D-glucopyranosyl-2'-O-alpha-D-xyloypyranoside were obtained with purity of 98.7, 98.3, and 99.1%, respectively, as determined by HPLC from 500 mg enriched extract after cleaning-up by polyamide resin.     

Preparative separation of flavonoid glycosides in leaves extract of Ampelopsis grossedentata using high-speed counter-current chromatography

Preparative separation of flavonoid glycosides in leaves extract of Ampelopsis grossedentata was conducted using high-speed counter-current chromatograph (HSCCC) with a solvent system composed of n-hexane-ethyl acetate-methanol-water (1:6:1.5:7.5, v/v). In a single operation, 28 mg of 5,7-dihydroxy-3',4'-trihydroxyflavone-3-O-6'-rhamnose and 18 mg of 5,7-dihydroxy-3',4'-dihydroxyflavone-3-O-6'-rhamnose was obtained from 150 mg of the extract. The chemical structure of the two compounds was elucidated by electrospray ionization (EIS) MS and NMR.     

Isolation and structure determination of a new flavone glycoside from seed residues of seabuckthorn (Hippophae rhamnoides L.)

In this study, a valid method was established for the isolation and purification of flavone glycosides from Hippophae rhamnoides L. seed residues using high-speed counter-current chromatography in one step, with a solvent system of ethyl acetate-methanol-n-butyl alcohol-water (9:1:0.5:9, v/v/v/v). A total of 28.8 mg compound I and 57.3 mg compound II were obtained from 200 mg of flavone H-glycosides rich extract, with purities of 98.3 and 96.4%, respectively. The structures of two compounds were identified by MS and NMR. 3-O-β-D-Sophorosylkaempferol-7-O-{3-O-[2(E)-2,6-dimethyl-6-hydroxyocta-2,7-dienoyl]}-α-L-rhamnoside is compound I and compound II named hippophanone is a new compound were identified by MS and NMR. The method was efficient and convenient, which could be used for the preparative separation of flavone glycosides from H. rhamnoides L. seed residues.     

Efficient strategies for preparative separation of iridoid glycosides and flavonoid glycosides from Hedyotis diffusa

Efficient strategies for the preparative separation of iridoid glycosides and flavonoid glycosides from Hedyotis diffusa using preparative high-performance liquid chromatography combined with appropriate pretreatment technologies were developed. Four fractions (Fr.1-1, Fr.1-2, Fr.1-3, and Fr.2-1) were firstly isolated from the crude extract of Hedyotis diffusa by column chromatography with C18, resin, and silica gel materials, respectively. Then, corresponding separation strategies were developed according to the polarity and chemical constituents. High-polar compounds of Fr.1-1 were purified by hydrophilic reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography mode. The combination of C18 and phenyl columns realized the complementary separation of iridoid glycosides in Fr.1-2. Meanwhile, the improved selectivity caused by the change of organic solvent in the mobile phase was utilized to realize the purification of flavonoid glycosides in Fr.1-3 and Fr. 2-1. Finally, 27 compounds (purity > 95%) mainly involving nine iridoid glycosides and five flavonoid glycosides were obtained. A complete strategy was established for the separation of a complex sample with a wide polarity range, to jointly solve the problems of enrichment of target components and separation of structural analogs.     

Nuclear magnetic resonance-based solvent system selection for counter-current chromatography separation of compounds present in the same high-performance liquid chromatography peak: Flavonoids in barley seedlings as an example

Partition coefficient is a key parameter for counter-current chromatography separation. High-performance liquid chromatography (HPLC) is the most commonly used tool for the screening of partition coefficients. However, HPLC technology is not applicable to the compounds present in the same chromatographic peak. Nuclear magnetic resonance (NMR) technology could easily distinguish compounds according to their characteristic absorption even if they exist in the same HPLC peak. In this study, two flavonoids present in the same HPLC peak were successfully purified by counter-current chromatography with a solvent system screened by NMR to show the great potential of NMR technology in the screening of the partition coefficient of co-efflux compounds. Through NMR screening, an optimized ethyl acetate/n-buthanol/water (7:3:10, v/v/v) system was applied in this study. As a result, two flavonoids, including 4.8 mg of 3′-methoxyl-6′’’-O-feruloylsaponarin and 9.8 mg of 6′’’-O-feruloylsaponarin were separated from 15 mg of the mixture. There is only one methoxy group difference between the two flavonoids. This study provides a new strategy for the screening of counter-current chromatography solvent systems and broadens the application scope of counter-current chromatography.     

Systematic characterization of flavonoids from Siraitia grosvenorii leaf extract using an integrated strategy of high‐speed counter‐current chromatography combined with ultra high performance liquid chromatography and electrospray ionization quadrupole time‐of‐flight mass spectrometry

The chemical constituents of the Siraitia grosvenorii leaf extract were studied. Firstly, high‐speed counter‐current chromatography was applied to the one‐step separation of four compounds from S. grosvenorii leaf extract with the solvent system composed of 0.01% acetic acid water/n‐butanol/n‐hexane/methanol (5:3:1:1, v/v/v/v). In this work, 270 mg of crude sample yielded four compounds, a new kaempferol O‐glycoside derivative, kaempferol 3‐O‐α‐L‐[4‐O‐(4‐carboxy‐3‐hydroxy‐3‐methylbutanoyl)]‐rhamnopyranoside‐7‐O‐α‐L‐rhamnopyranoside, named kaempferitrin A (2.1 mg, 90%), and three known compounds, grosvenorine (3.4 mg, 93%), kaempferitrin (14.4 mg, 99%) and afzelin (4 mg, 98%), and the structures of these compounds were identified by NMR spectroscopy and mass spectrometry. Then, ultra high performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight mass spectrometry was used to illustrate the dominant flavonoids in S. grosvenorii leaf extract. 34 flavonoids including 19 kaempferol O‐glycosides, 4 quercetin O‐glycosides, 6 flavanone derivatives, and 5 polymethoxyflavones, were accurately or tentatively identified by carefully comparing their retention times, UV data, precise masses, the typical fragments of the standards and literature data. Most of these compounds were reported for the first time. This study establishes a foundation for the further development and utilization of S. grosvenorii leaves in future.     

Preparation of five high‐purity iridoid glycosides from Gardenia jasminoides Eills by molecularly imprinted solid‐phase extraction integrated with preparative liquid chromatography

Five iridoid glycosides were prepared using molecularly imprinted solid‐phase extraction combined with preparative high‐performance liquid chromatography. Hydrophilic molecularly imprinted polymers were synthesized using α‐1‐allyl‐2‐N‐acetyl glucosamine, which introduced an abundance of hydrophilic groups into the polymers. Using molecularly imprinted solid‐phase extraction as the sample pretreatment procedure, five iridoid glycosides, gardenoside, geniposide, shanzhiside, geniposidic acid, and genipin‐1‐O‐gentiobioside, were selectively enriched from Gardenia fructus extracts. Preparative high‐performance liquid chromatography then provided iridoid glycosides with a purity >98%. The structures were elucidated by using nuclear magnetic resonance spectroscopy, optical rotation and melting point measurements, and mass spectrometry. The results demonstrate that molecularly imprinted solid‐phase extraction combined with preparative high‐performance liquid chromatography was an efficient, rapid, and economical method for the preparation of bioactive compounds from natural products.     

Comprehensive separation of iridoid glycosides and triterpenoid saponins from Dipsacus asper with salt‐containing solvent by high‐speed countercurrent chromatography coupled with recycling mode

The roots of Dipsacus asper Wall as a commonly used traditional Chinese medicine are used for tonifying liver and kidney and strengthening bones and muscles. However, an effective separation strategy for comprehensive and rapid separation of the main active compounds from the roots of D. asper is nonexistent. This investigation provided an effective separation method based on AB‐8 macroporous resin column chromatography using different ratios of ethanol in water and two different modes of high‐speed countercurrent chromatography with salt‐containing solvent system for rapid enrichment and separation from the roots of D. asper. The macroporous resin column chromatography was performed on AB‐8 resin using ethanol in water ratios of 10, 30, 40, 50, and 80% as the optimized enrichment conditions for iridoid glycosides and triterpenoid saponins with different polarities. For high‐speed countercurrent chromatography separation, the conventional and recycling modes were combined together to develop a strategy for 12 compounds ( 1 – 12 ) from the enriched parts of 30, 40, and 80% ethanol, including six high‐polarity iridoid glycosides ( 1 – 6 ) using inorganic salt‐containing solvent system and six triterpenoid saponins ( 7 – 12 ). Recycling high‐speed countercurrent chromatography separation was successfully applied to separate two isomers ( 9 and 10 ) after 11 cycles.     

Isolation of isomangiferin from honeybush (Cyclopia subternata) using high-speed counter-current chromatography and high-performance liquid chromatography

Isomangiferin was isolated from Cyclopia subternata using a multi-step process including extraction, liquid–liquid partitioning, high-speed counter-current chromatography (HSCCC) and semi-preparative reversed-phase high-performance liquid chromatography (HPLC). Enrichment of phenolic compounds in a methanol extract of C. subternata leaves was conducted using liquid–liquid partitioning with ethyl acetate–methanol–water (1:1:2, v/v). The enriched fraction was further fractionated using HSCCC with a ternary solvent system consisting of tert-butyl methyl ether–n-butanol–acetonitrile–water (3:1:1:5, v/v). Isomangiferin was isolated by semi-preparative reversed-phase HPLC from a fraction containing mostly mangiferin and isomangiferin. The chemical structure of isomangiferin was confirmed by LC–high-resolution electrospray ionization MS, as well as one- and two-dimensional NMR spectroscopy.     

Isolation and purification of high polar glycosides from aerial parts of Gynostemma pentaphyllum (Thunb.) Makino by linear gradient counter-current chromatography coupled with inner-recycling mode

Gynostemma pentaphyllum (Thunb.) Makino represents the popular health food and supplemental product with broad pharmacological activities. The highly polar glycosides, including flavonoids and saponins, are major effective active components that contain diverse sugar positions and quantities, which result in diverse chemical polarities, making it challenging to separate and isolate these components. The present work described the rapid and efficient linear gradient counter-current chromatography to preparatively separate glycosides from aboveground parts of G. pentaphyllum. Besides, the ethyl acetate and n-butanol binary mobile phases were achieved through adjusting associated proportions. Six glycosides, including quercetin-3-O-neohesperidoside ( 1 ), kaempferol-3-O-robinobioside ( 2 ), kaempferol-3-O-neohesperidoside ( 3 ), gypenoside LVI ( 4 ), ginsenoside Rb3 ( 5 ), and gypenoside XLVI ( 6 ), were isolated at the purities greater than 98%. Moreover, electrospray ionization mass spectrometry and nuclear magnetic resonance tandem mass spectrometry were conducted for structural identification. According to our findings, the established linear gradient counter-current chromatography was an efficient approach to separate the highly polar glycosides from aboveground parts of G. pentaphyllum. Our proposed strategy can be used to separate active compounds from other complex natural products.