Target-guided isolation and purification of cyclooxygenase-2 inhibitors from Meconopsis integrifolia (Maxim.) Franch. by high-speed counter-current chromatography combined with ultrafiltration liquid chromatography.

Meconopsis integrifolia (Maxim.) Franch. is used extensively in traditional Tibetan medicine for its potent anti-inflammatory properties. In this study, six cyclooxygenase-2 (COX-2) inhibitors were purified from M. integrifolia using high-speed counter-current chromatography guided by ultrafiltration liquid chromatography (ultrafiltration-LC). First, ultrafiltration-LC was performed to profile the COX-2 inhibitors in M. integrifolia. The reflux extraction conditions were further optimized using response surface methodology, and the results showed that the targeted COX-2 inhibitors could be well enriched under the optimized extraction conditions. Then the six target COX-2 inhibitors were separated by high-speed countercurrent chromatography with a solvent system composed of ethyl acetate/n-butanol/water (4:1:4, v/v/v. Finally, the six COX-2 inhibitors, including 21.2 mg of 8-hydroxyluteolin 7-sophoroside, 29.6 mg of 8-hydroxyluteolin 7-[6'''-acetylallosyl-(1→2)-glucoside], 42.5 mg of Sinocrassoside D3, 54.1 mg of Hypolaetin 7-[6'''-acetylallosyll-(l→2)-3''-acetylglucoside, 30.6 mg of Hypolaetin 7-[6'''-acetylallosyll-(l→2)-6''-acetylglucoside and 17.8 mg of Hypolaetin were obtained from 500 mg of sample. Their structures were elucidated by 1 H-NMR spectroscopy. This study reveals that ultrafiltration-LC combined with high-speed counter-current chromatography is a robust and efficient strategy for target-guided isolation and purification of bioactive molecules. It also enhances the scientific understanding of the anti-inflammatory properties of M. integrifolia but also paves the way for its further medicinal applications.

Target-Guided Isolation and Purification of Antioxidants from Urtica laetevirens Maxim. by HSCCC Combined with Online DPPH-HPLC Analysis.

Urtica laetevirens Maxim. is used extensively in traditional Chinese medicine (TCM) for its potent antioxidative properties. In this study, three antioxidants were purified from U. laetevirens. using HSCCC guided by online DPPH-HPLC analysis. Firstly, the online DPPH-HPLC analysis was performed to profile out the antioxidant active molecules in U. laetevirens. The ultrasonic-assisted extraction conditions were optimized by response surface methodology and the results showed the targeted antioxidant active molecules could be well enriched under the optimized extraction conditions. Then, the antioxidant active molecules were separated by high-speed countercurrent chromatography ethyl acetate/n-butanol/water (2:3:5, v/v/v) as the solvent system. Finally, the three targets including 16.8 mg of Isovitexin, 9.8 mg of Isoorientin, and 26.7 mg of Apigenin-6,8-di-C-ß-d-glucopyranoside were obtained from 100 mg of sample. Their structures were identified by 1H NMR spectroscopy.

An efficient strategy for large-scale preparation of low polarity gingerols directly from ginger crude extract by high-speed countercurrent chromatography with different rotation mode.

This study presents an efficient strategy for large-scale preparation of low polarity gingerols directly from ginger crude extract by high-speed countercurrent chromatography with different rotation mode. The ultrasonic-assisted extraction conditions were optimized by response surface methodology and the results showed the major low polarity gingerols could be well enriched under the optimized extraction conditions. Then the crude extract without any pretreatment was directly separated by high-speed countercurrent chromatography with different rotation mode using n-hexane/ethyl acetate/methanol/water (6:4:6:4, v/v/v/v) as the solvent system. In about 400 min, five major gingerols including 150 mg of [6]-gingerol, 50 mg of [8]-gingerol, 20 mg of [6]-shogaol, 43 mg of [6]-dehydrogingerdione, and 40 mg of [10]-gingerol were obtained from 1.2 g of crude extract in a single run with repeated injection. Their structures were identified by 1 H-NMR spectroscopy.

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.

Activity labelled molecular networking fuels the antioxidation active molecules profile of Ginger.

Ginger has been used as consumed food spice and folk medicine in daily life for thousands of years in various regions of the world. Considerable antioxidation is one of the major activities for Ginger to exhibit health-promoting effects. In this study, a bioinformatic workflow was developed to generate activity labelled molecular networking (ALMN) to fuel the antioxidation active molecules profile of Ginger. In ALMN, antioxidation activity data, which was defined as correlation (r and p value) between the relative abundance of a molecule in fractions and the activity level of each fraction, was labelled to feature-based molecular network to profile out antioxidation active molecules visually. Fragmentation tree was further computed as a complementary way to conduct high confidence structure annotations of antioxidation active molecules. Consequently, 48 molecules were prioritized as antioxidation active molecules from 11,720 metabolite molecules of Ginger in a systematical way.

Bufadienolides originated from toad source and their anti-inflammatory activity.

Bufadienolide, an essential member of the C-24 steroid family, is characterized by an α-pyrone positioned at C-17. As the predominantly active constituent in traditional Chinese medicine of Chansu, bufadienolide has been prescribed in the treatment of numerous ailments. It is a specifically potent inhibitor of Na+/K+ ATPase with excellent anti-inflammatory activity. However, the severe side effects triggered by unbiased inhibition of the whole-body cells distributed α1-subtype of Na+/K+ ATPase, restrict its future applicability. Thus, researchers have paved the road for the structural alteration of desirable bufadienolide derivatives with minimal adverse effects via biotransformation. In this review, we give priority to the present evidence for structural diversity, MS fragmentation principles, anti-inflammatory efficacy, and structure modification of bufadienolides derived from toads to offer a scientific foundation for future in-depth investigations and views.

The applicability of pH-zone-refining counter-current chromatography for preparative separation of biosynthesis products: Glycosylation products as example.

Biosynthesis is a research hot-spot in recent years, however, the purification of its final products is a tough work. Liquid stationary phase and large-scale separation ability of PZRCCC could easily avoid the commonly disadvantages occurred in traditional column chromatography. These characteristics makes PZRCCC particularly applicable for final products separation in biosynthesis. In this study, the glycosylation products of ellagic acid by one-pot glycosylation were successfully purified by PZRCCC to show the applicability of PZRCCC for preparative separation of biosynthesis products. An optimized ethyl acetate/n-buthanol/water (3:3:5, v/v/v) system was applied in this study, where 5 mM trifluoroacetic acid (TFA) as the retainer and 30 mM triethylamine (TEA) as the eluter were added. As a result, four ellagic acid glycosylation products, including 51 mg of ellagic acid-4, 3'-O-ß-D-diglucoside (EG-1), 24 mg of ellagic acid-4, 4'-O-ß-D-diglucoside (EG-2), 11 mg of ellagic acid-4-O-ß-D-glucosyl (1→2)-ß-D-glucoside (EG-3) and 64 mg of ellagic acid-4-O-ß-D-glucoside (EG-4) were simultaneously separated from 500 mg of glycosylation crude products, with the purity of 93.3%, 91.2%, 89.4% and 95.5%, respectively. Their structures were identified by spectroscopic analysis.

Targeted isolation of 1,1-diphenyl-2-picrylhydrazyl inhibitors from Saxifraga atrata using medium- and high- pressure liquid chromatography combined with online high performance liquid chromatography-1,1-diphenyl-2- picrylhydrazyl detection.

Traditional Tibetan medicine (TTM) is a valuable source of novel therapeutic lead molecules inspired by natural products (NPs). The health benefits of Saxifraga atrata are well documented in TTM, but reports on its chemical composition are limited, most likely due to the complicated purification process. Herein, target separation and identification of 4 main radical scavenging compounds from the methanolic extract of S. atrata was were performed using medium- and high-pressure liquid chromatography coupled with online HPLC-DPPH detection. The sample was pretreated using medium pressure liquid chromatography with MCI GELⓇ CHP20P styrene-divinylbenzene beads as a stationary phase, yielding 1.4 g of the target DPPH inhibitors (Fr4, 11.9% recovery). The compounds were further purified and isolated using HPLC on RP-C18 (ReproSil-Pur C18 AQ) followed by HILIC (Click XIon) column separation, resulting in 2.8 mg of fraction Fr4-1-1, 6.8 mg of fraction Fr4-2, 244.9 mg of the Fr4-3-1 sample, and 38.3 mg of Fr4-4-1. The structure and purity of the target compounds were determined, and four compounds (ethyl gallate, 11-O-galloylbergenin, rutin and isoquercitrin) were isolated with >95% purity. The developed methodology is efficient for targeted isolation of high-purity radical scavengers from NP extracts and could be used for rapid identification and isolation of DPPH inhibitors from various NPs.

Separation of three anthraquinone glycosides including two isomers by preparative high-performance liquid chromatography and high-speed countercurrent chromatography from Rheum tanguticum Maxim. ex Balf.

Anthraquinone glycosides, such as chrysophanol 1-O-ß-d-glucoside, chrysophanol 8-O-ß-d-glucoside, and physion 8-O-ß-d-glucoside, are the accepted important active components of Rheum tanguticum Maxim. ex Balf. due to their pharmacological properties: antifungal, antimicrobial, cytotoxic, and antioxidant activities. However, an effective method for the separation of the above-mentioned anthraquinone glycosides from this herb is not currently available. Especially, greater difficulty existed in the separation of the two isomers chrysophanol 1-O-ß-d-glucoside and chrysophanol 8-O-ß-d-glucoside. This study demonstrated an efficient strategy based on preparative high-performance liquid chromatography and high-speed countercurrent chromatography for the separation of the above-mentioned anthraquinone glycosides from Rheum tanguticum Maxim.ex Balf.

An Efficient Protocol for Preparation of Gallic Acid from Terminalia bellirica (Gaertn.) Roxb by Combination of Macroporous Resin and Preparative High-Performance Liquid Chromatography.

In this article, macroporous resin column chromatography and preparative high-performance liquid chromatography were applied for preparation of gallic acid from Terminalia bellirica (Gaertn.) Roxb. In the first step, six kinds of resins were investigated by adsorption and desorption tests and AB-8 macroporous resin was selected for the enrichment of gallic acid. As a result, 20 g of gallic acid at a purity of 71% could be separated from 100 g of crude extract in which the content of gallic acid was 16.7% and the recovery of gallic acid reached 85.0%. In the second step, preparative high-performance liquid chromatography was selected to purify gallic acid. As a result, 640 mg of gallic acid at a purity of 99.1% was obtained from 1 g of sample in 35 min. The results demonstrated that macroporous resin coupled with preparative high-performance liquid chromatography was suitable for preparation of gallic acid from T. bellirica (Gaertn.) Roxb.

Separation of three phenolic high-molecular-weight compounds from the crude extract of Terminalia Chebula Retz. by ultrasound-assisted extraction and high-speed counter-current chromatography.

This study presents an efficient strategy for separation of three phenolic compounds with high molecular weight from the crude extract of Terminalia chebula Retz. by ultrasound-assisted extraction and high-speed counter-current chromatography. The ultrasound-assisted extraction conditions were optimized by response surface methodology and the results showed the target compounds could be well enriched under the optimized extraction conditions. Then the crude extract was directly separated by high-speed counter-current chromatography without any pretreatment using n-hexane/ethyl acetate/methanol/water (1:7:0.5:3, v/v/v/v) as the solvent system. In 180 min, 13 mg of A, 18 mg of B, and 9 mg of C were obtained from 200 mg of crude sample. Their structures were identified as Chebulagic acid (A, 954 Da), Chebulinic acid (B, 956 Da), and Ellagic acid (C) by (1) H NMR spectroscopy.

Application of high-speed counter-current chromatography combined with macroporous resin for rapid enrichment and separation of three anthraquinone glycosides and one stilbene glycoside from Rheum tanguticum.

In this paper, an efficient method was successfully established by the combination of macroporous resin (MR) and high-speed counter-current chromatography (HSCCC) for rapid enrichment and separation of aloe-emodin 8-O-ß-D-glucoside, emodin 1-O-ß-D-glucoside, emodin 8-O-ß-D-glucoside and piceatannol 4'-O-ß-D-(6″-O-gallate)-glucoside. Six kinds of macroporous resins were investigated in the first step and X-5 macroporous resin was selected for the enrichment of the target compounds. The recoveries of the target compounds reached 89.0, 85.9, 82.3 and 84.9% respectively after 40% ethanol elution. In the second step, the target compounds were separated by HSCCC with a two-phase solvent system composed of chloroform/ethyl acetate/methanol/water (8:1:6:5, v/v). The established method will be helpful for further characterization and utilization of Rheum tanguticum. The results demonstrate that MR coupled with HSCCC is a powerful technique for separation of bioactive compounds from natural products.

Application of an efficient strategy based on liquid-liquid extraction, high-speed counter-current chromatography, and preparative HPLC for the rapid enrichment, separation, and purification of four anthraquinones from Rheum tanguticum.

This study presents an efficient strategy based on liquid-liquid extraction, high-speed counter-current chromatography, and preparative HPLC for the rapid enrichment, separation, and purification of four anthraquinones from Rheum tanguticum. A new solvent system composed of petroleum ether/ethyl acetate/water (4:2:1, v/v/v) was developed for the liquid-liquid extraction of the crude extract from R. tanguticum. As a result, emodin, aloe-emodin, physcion, and chrysophanol were greatly enriched in the organic layer. In addition, an efficient method was successfully established to separate and purify the above anthraquinones by high-speed counter-current chromatography and preparative HPLC. This study supplies a new alternative method for the rapid enrichment, separation, and purification of emodin, aloe-emodin, physcione, and chrysophanol.