Separation and purification of α-glucosidase inhibitors from Polygonatum odoratum by stepwise high-speed counter-current chromatography combined with Sephadex LH-20 chromatography target-guided by ultrafiltration-HPLC screening.

Although Polygonatum odoratum has been widely used as medicinal plant and food supplement for treating diabetes, little is known regarding its bioactive components. In this study, ultrafiltration-HPLC based ligand screening was developed to screen α-glucosidase inhibitors from P. odoratum for the first time. Then bioactive components were target-guided separated by combining stepwise high-speed counter-current chromatography (HSCCC) using petroleum ether-ethyl acetate-methanol-water (1:4:0.8:4.2, v/v/v/v), (1:4:1.8:3.2, v/v/v/v) and (1:4:2.3:2.7, v/v/v/v) as solvent systems with Sephadex LH-20 chromatography eluted by MeCN-MeOH (1:1, v/v). Five phenethyl cinnamides, N-cis-feruloyloctopamine (1); N-trans-p-coumaroyloctopamine (2), N-trans-feruloyloctopamine (3), N-trans-p-coumaroyltyramine (4) and N-trans-feruloyltyramine (5), and four homoisoflavanones, (3R)-5,7-dihydroxyl-3-(2',4'-dihydroxylbenzyl)-chroman-4-one (6), (3R)-5,7-dihydroxyl-6-methyl-3-(4'-hydroxylbenzyl)-chroman-4-one (7), (3R)-5,7-dihydroxyl-6-methyl-8-methoxyl-3-(4'-hydroxylbenzyl)-chroman-4-one (8); and (3R)-5,7-dihydroxyl-6,8-dimethyl-3-(4'-hydroxylbenzyl)-chroman-4-one) (9), with purity over 98.5% were purified, and their structures were identified by UV, MS, and (1)H NMR. Notably, compounds 2 and 4 were first reported in genus Polygonatum, while compound 1 was first obtained from family Liliaceae. In addition, α-glucosidase inhibitory activities of compounds 1-9 were evaluated, and compounds 2 and 4 exhibited stronger α-glucosidase inhibitory activity with IC50 values of 2.3 and 2.7μM. The results suggested the potential medicinal use of P. odoratum, and the technology could be widely applied for rapid screening and preparative separation of a group of bioactive compounds from complex matrix.

Zhou X ，Liang J ，Zhang Y ，Zhao H ，Guo Y ，Shi S ... -  《-》

Separation of α-glucosidase inhibitors from Potentilla kleiniana Wight et Arn using solvent and flow-rate gradient high-speed counter-current chromatography target-guided by ultrafiltration HPLC-MS screening.

Potentilla kleiniana Wight et Arn is widely used as a herbal medicine to treat type 2 diabetes. However, detailed information about its active compounds is lacking. To develop an efficient method for the rapid screening and separation of α-glucosidase inhibitors from Potentilla kleiniana Wight et Arn. Potential α-glucosidase inhibitors from Potentilla kleiniana Wight et Arn were rapidly screened out through ultrafiltration high-performance liquid chromatography mass spectrometry (HPLC-MS), and then followed by a target-guided high-speed counter-current chromatography (HSCCC) separation using two-phase solvent systems composed of n-hexane/ethyl acetate/methanol/water (1:10:1:10, v/v/v/v and 1:10:5:6, v/v/v/v), and adopting increasing flow-rate from 1.5 to 3.0 mL/min after 200 min. Their structures were identified by ultraviolet (UV), MS, proton nuclear magnetic resonance (1 H-NMR) and carbon-13 (13 C)-NMR, and their α-glucosidase inhibitory activities were assessed by in vitro assay. Five α-glucosidase inhibitors including gallic acid (25.7 mg, 98.2%, 1), brevifolincarboxylic acid (9.86 mg, 95.3%, 2), ethyl evifolincarboxylate (13.26 mg, 97.6%, 3), 3,3'-di-O-methylellagic acid-4'-O-β-d-glucopyranoside (16.26 mg, 95.1%, 4), and 3,3'-di-O-methylellagic acid (10.54 mg, 96.8%, 5) were successfully purified from 250 mg n-butanol extract in a single run. Compounds 1, 2, 4 and 5 exhibited stronger α-glucosidase inhibitory activities[half maximal inhibition concentration (IC50 ) values at 173.41 ± 6.35, 323.46 ± 8.08, 44.63 ± 2.50, and 20.73 ± 2.56 μM, respectively] than acarbose (IC50 value at 332.12 ± 5.52 μM, reference compound). Notably, compounds 2-5 were reported in the Potentilla kleiniana Wight et Arn for the first time. The results indicated that the proposed method could be applied for the rapid screening and preparative separation of α-glucosidase inhibitors from a complex matrix.

Liu M ，Huang X ，Liu Q ，Li X ，Chen M ，Zhu Y ，Chen X ... -  《-》

Antioxidant homoisoflavonoids from Polygonatum odoratum.

Polygonatum odoratum is widely used as a traditional food supplement and herbal medicine with strong antioxidant activity. However, systematic investigation of its antioxidants was limited. Ethanol extract of P. odoratum was fractioned on macroporous absorptive resin (D101) column. A bioassay-guided purification of flavonoid-rich fraction (IC50 value at 74.1 ± 11.9 μg/mL for DPPH scavenging) was realised via high-speed counter-current chromatography (HSCCC) using petroleum ether-ethyl acetate-methanol-water (2:3:3:2, v/v/v/v) as the solvent system combination with Sephadex LH-20 column chromatography (CC) eluting with MeCN-MeOH (1:1, v/v). Three novel homoisoflavonoids (1-3), along with eight homoisoflavonoids (4-11), were isolated. Their structures were elucidated by interpretating various spectroscopic data. All the isolated homoisoflavonoids showed potent antioxidant activities, while compounds 1, 4, and 6 with dihydroxylated B-rings exhibited stronger antioxidant activities (IC50 values at 3.8 ± 0.5, 4.9 ± 0.3 and 3.9 ± 0.4 μg/mL) than ascorbic acid (IC50 value at 5.3 ± 0.6 μg/mL).

Zhou X ，Yuping Z ，Zhao H ，Liang J ，Zhang Y ，Shi S ... -  《-》

At-line hyphenation of high-speed countercurrent chromatography with Sephadex LH-20 column chromatography for bioassay-guided separation of antioxidants from vine tea (Ampelopsis grossedentata).

Vine tea (Ampelopsis grossedentata), a widely used healthy tea, beverage and herbal medicine, exhibited strong antioxidant activity. However, systematic purification of antioxidants, especially for those with similar structures or polarities, is a challenging work. Here, we present a novel at-line hyphenation of high-speed countercurrent chromatography with Sephadex LH-20 column chromatography (HSCCC-Sephadex LH-20 CC) for rapid and efficient separation of antioxidants from vine tea target-guided by 1,1-diphenyl-2-picryl-hydrazyl radical-high performance liquid chromatography (DPPH-HPLC) experiment. A makeup pump, a six-port switching valve and a trapping column were served as interface. The configuration had no operational time and mobile phase limitations between two dimensional chromatography and showed great flexibility without tedious sample-handling procedure. Seven targeted antioxidants were firstly separated by stepwise HSCCC using petroleum ether-ethyl acetate-methanol-water (4:9:4:9, v/v/v/v) and (4:9:5:8, v/v/v/v) as solvent systems, and then co-eluted antioxidants were on-line trapped, concentrated and desorbed to Sephadex LH-20 column for further off-line purification by methanol. It is noted that six elucidated antioxidants with purity over 95% exhibited stronger activity than ascorbic acid (VC). More importantly, this at-line hyphenated strategy could sever as a rapid and efficient pathway for systematic purification of bioactive components from complex matrix.

Ma R ，Zhou R ，Tong R ，Shi S ，Chen X ... -  《-》

Ultrafiltration liquid chromatography combined with high-speed countercurrent chromatography for screening and isolating potential α-glucosidase and xanthine oxidase inhibitors from Cortex Phellodendri.

Cortex Phellodendri is a typical Chinese herb with a large number of alkaloids existing in all parts of it. The most common methods for screening and isolating alkaloids are mostly labor intensive and time consuming. In this study, a new assay based upon ultrafiltration liquid chromatography was developed for the rapid screening of ligands for α-glucosidase and xanthine oxidase. The C. Phellodendri extract was found to contain two alkaloids with both α-glucosidase- and xanthine oxidase binding activities and one lactone with α-glucosidase-binding activity. Subsequently, with the help of high-speed countercurrent chromatography, the specific binding ligands including palmatine, berberine, and obaculactone with purities of 97.38, 96.12, and 96.08%, respectively, were successfully separated. An optimized low-toxicity two-phase solvent system composed of ethyl acetate/n-butanol/ethanol/water (3.5:1.7:0.5:5, v/v/v/v) was used to isolate the three compounds mentioned above from C. Phellodendri. The targeted compounds were identified by liquid chromatography coupled with mass spectrometry and NMR spectroscopy. Therefore, ultrafiltration liquid chromatography combined with high-speed countercurrent chromatography is not only a powerful tool for screening and isolating α-glucosidase and xanthine oxidase inhibitors in complex samples but is also a useful platform for discovering bioactive compounds for the prevention and treatment of diabetes mellitus and gout.

Li S ，Liu C ，Guo L ，Zhang Y ，Wang J ，Ma B ，Wang Y ，Wang Y ，Ren J ，Yang X ，Qin Y ，Tang Y ... -  《-》