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.

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.

Effect of Extract and Synthesized Derivatives of Isolated Compound from Symplocos chinensis f. Pilosa Ohwi on Neuropathic Pain in Mice.

Neuropathic pain is described as the "most terrible of all tortures that a nerve wound may inflict." The aim of the present study was to demonstrate the antinociceptive effect of Symplocos chinensis f. pilosa Ohwi water extract (SCW) and synthesized derivatives of the isolated compound. The antinociceptive effect was tested using the acetic acid-induced writhing and 5% formalin tests. Antinociceptive effects on neuropathic pain were evaluated using the von Frey test with chronic constriction injury (CCI) and surgical nerve injury (SNI) models and tail-flick test with a vincristine-induced pain model. An Ames test was also conducted. 5-hydroxymethylfurfural (5-HMF) was isolated and derivatives were synthesized with various acid groups. Among the plant water extracts, SCW showed significantly effective activity. Additionally, SCW presented antinociceptive effects in the neuropathic pain models. The SCW water fraction resulted in fewer writhes than the other fractions, and isolated 5-HMF was identified as an effective compound. Because 5-HMF revealed a positive response in the Ames test, derivatives were synthesized. Among the synthesized derivations, 5-succinoxymethylfurfural (5-SMF) showed the best effect in the neuropathic pain model. Our data suggest that SCW and the synthesized compound, 5-SMF, possess effective antinociceptive activity against neuropathic pain.

Efficient Separation of Phytochemicals from Muehlenbeckia volcanica (Benth.) Endl. by Polarity-Stepwise Elution Counter-Current Chromatography and Their Antioxidant, Antiglycation, and Aldose Reductase Inhibition Potentials.

Muehlenbeckia volcanica (Benth.) Endl. (M. volcanica), native to South America, is a traditional Peruvian medicinal plant that has multi-therapeutic properties; however, no phytochemicals have been identified from it yet. In this study, a five-step polarity-stepwise elution counter-current chromatography (CCC) was developed using methanol/water (1:5, v/v) as the stationary phase and different ratios of n-hexane, ethyl acetate, and n-butanol as mobile phases to separate the compounds from the 70% methanol extract of M. volcanica, by which six compounds with a wide range of polarities were separated in a single run of CCC and were identified as gallic acid, protocatechuic acid, 4,4'-dihydroxy-3,3'-imino-di-benzoic acid, rutin, quercitrin, and quercetin. Then, two compounds from the fractions of stepwise elution CCC were separated using conventional high-speed CCC, pH-zone-refining CCC, and preparative high-performance liquid chromatography, and identified as shikimic acid and miquelianin. These compounds are reported from M. volcanica for the first time. Notably, except for shikimic acid, all other compounds showed anti-diabetic potentials via antioxidant, antiglycation, and aldose reductase inhibition. The results suggest that the polarity-stepwise elution CCC can be used to efficiently separate or fractionate compounds with a wide range of polarities from natural products. Moreover, M. volcanica and its bioactive compounds are potent anti-diabetic agents.

Orthogonality in the selection of biphasic solvent systems for off-line two-dimensional countercurrent chromatography from Polygonum cuspidatum Sieb. et Zucc.

Off-line two-dimensional countercurrent chromatography has been widely applied to the isolation of complex samples, but little research on the investigation of orthogonality in the selection of biphasic solvent systems is available. In the present work, the orthogonality in the selection of a biphasic solvent system for liquid-liquid chromatographic separation of aqueous extract and ether extract from the traditional Chinese medicinal plant Polygonum cuspidatum Sieb. et Zucc was evaluated by the correlation coefficient and space occupancy rate. In total, 25 different biphasic solvent systems were tested, and 313 system combinations were analysed. A convex hull methodology was used to determine the separation space and to optimize separation conditions. The correlation coefficient matrix was transformed into dendrograms and a colour map to visualize the dissimilarity between, and orthogonality for, all solvent systems. The aqueous extracts from Polygonum cuspidatum were separated using selected biphasic solvent systems with high orthogonality: ethyl acetate-ethanol-water (70:1:70, v/v) and petroleum ether-ethyl acetate-water (1:5:5, v/v). The ether extracts from Polygonum cuspidatum were also separated using selected biphasic solvent systems with high orthogonality: petroleum-ethyl acetate-methanol-aqueous 0.25 M NH3•H2O (5:5:5:5, v/v) and petroleum-ethyl acetate-methanol-water (5:5:5:5, v/v). Thirteen compounds were successfully obtained. The experimental results demonstrated that the evaluation of orthogonality provided an alternative strategy to select an applicable solvent system for the separation of complex samples using off-line two-dimensional countercurrent chromatography.

Constituents from Solidago virgaurea var. gigantea and their inhibitory effect on lipid accumulation.

In this study, the anti-adipogenic activities of compounds isolated from Solidago viraurea var. gigantea (SG) extracts were investigated using Oil Red O staining in the 3T3-L1 cell line. Four known compounds including 3,5-di-O-caffeoylquinic acid (5), protocatechuic acid (6), chlorogenic acid (7), and kaempferol-3-O-rutinoside (8), and four undescribed compounds including (1R,2S,3S,5R,7S)-methyl 7-((cinnamoyloxy)methyl)-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]octane-5-carboxylate (1), (1R,2S,3S,5R,7S)-methyl 2,3-dihydroxy-7-((((Z)-3-phenylacryloyl)oxy)methyl)-6,8-dioxabicyclo[3.2.1]octane-5-carboxylate (2), (1R,2S,3S,5R,7S)-2,3-dihydroxy-7-((((Z)-3-phenylacryloyl)oxy)methyl)-6,8-dioxabicyclo[3.2.1]octane-5-carboxylic acid (3), and (1R,2S,3S,5R,7S)-7-((cinnamoyloxy)methyl)-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]octane-5-carboxylic acid (4) were isolated from S. viraurea var. gigantea. The structures of the compounds were first identified by comparing their 1H NMR spectra with spectral data from the literature and a more detailed identification was then performed using 2D NMR (Correlated spectroscopy (COSY), heteronuclear single quantum correlation (HSQC), heteronuclear multiple bond correlation (HMBC), and nuclear overhauser spectroscopy (NOESY)), and X-ray crystallography analyses. The anti-adipogenic activities of all compounds were evaluated by MTT assay and Oil Red O staining in 3T3-L1 cells. 3,5-di-O-caffeoylquinic acid was found to inhibit lipid accumulation more potently than the other tested compounds.

Retention mechanism of pH-peak-focusing in countercurrent chromatographic separation of baicalin and wogonoside from Scutellaria baicalensis Georgi.

An efficient and target-oriented pH-peak-focusing countercurrent chromatographic method was established for large-scale separation of baicalin and wogonoside from the crude exact of traditional Chinese medicinal herb Scutellaria baicalensis Georgi. An optimized two-phase solvent system composed of n-butanol-ethyl acetate-methanol-water (1:4:0.5:5, v/v) was selected. Trifluoroacetic acid (10 mmol/L) was added to the upper organic phase, used as the stationary phase. One liter of the aqueous lower phase was used as the mobile phase for 0-350 min, and then 10 mmol/L ammonia was added to remaining 1 L of the aqueous lower phase and used as the mobile phase for 350-600 min. In total, 493.2 mg of baicalin with 98.6% purity and 88.6 mg of wogonoside with 98.9% purity were obtained from 1.0 g of crude exact of S. baicalensis by countercurrent chromatography in a single run. The acid dissociation constant (pKa) and oil-water partition coefficient values of two components were measured to better understand the mechanism of separation. Results showed that pH-peak-focusing countercurrent chromatography with a polar solvent system added with trifluoroacetic acid could be an efficient method for large-scale isolation of organic acids, which are difficult to separate with conventional countercurrent chromatography due to their poor solubility in non-polar solvents.

Liquid chromatographic and liquid-liquid chromatographic separation of structural isomeric oleanolic acid and ursolic acid using hydroxypropyl-ß-cyclodextrin as additive.

Two structural isomeric pentacyclic triterpenes, oleanolic acid and ursolic acid, were considered as the models for the quality control of many traditional Chinese herbal medicines and they have been proved to own important pharmacological activities. In the present work, liquid chromatographic and liquid-liquid chromatographic separation with high peak resolution of structural isomeric oleanolic acid and ursolic acid using hydroxypropyl-ß-cyclodextrin as mobile phase additive was successfully achieved, respectively. A high peak resolution, RS=8.143, was achieved for the two structural isomeric compounds by conventional reverse phase high performance liquid chromatography, which was greatly improved compared with the published values. Meanwhile, a biphasic solvent system composed of n-hexane-ethyl acetate-0.1 mol/L hydroxypropyl-ß-cyclodextrin (9:1:10, v/v) was selected for liquid-liquid chromatography, which provided a high peak resolution, RS = 6.573, for analytical apparatus and Rs = 8.500 for semi-preparative apparatus after optimization by liquid-liquid extractions. Two elution modes including reverse phase mode and normal phase mode were investigated for preparative separation of two acids from crude exact of Eriobotrya japonica Thunb. Furthermore, the inclusion complex between each of the two structural isomers and hydroxypropyl-ß-cyclodextrin were also investigated for high performance liquid chromatography and liquid-liquid chromatography, respectively, in which formation constants were determined for oleanolic acid and ursolic acid.

Acute and subchronic toxicity study of nonpolar extract of licorice roots in mice.

Licorice is used as a medicinal plant, and several studies have shown that licorice has beneficial effects. The objective of this study was to evaluate the safety of nonpolar licorice extract using toxicity experiments. Nonpolar extract from the root of Glycyrrhiza uralensis (NERG) was analyzed by high-performance liquid chromatography (HPLC). Antioxidant ability was determined by method of TPC and DPPH. Blood pressure was monitored by using blood pressure meter. In the acute study, a single dose (2,000 mg/kg) was orally administered to mice. In the subchronic study, mice were treated with extract at doses (50, 100, 500, and 1,000 mg/kg) for 120 days. Significantly difference was not shown at blood pressure, hematological, and biochemical parameters, and histopathology on mice. The results suggested that at acute and subchronic toxicity, each levels of nonpolar licorice extract administration in experiments did not cause toxicity effects or death on mice.

Affinity measurement of ligands in Perilla frutescens extract towards α-glucosidase using affinity-based ultrafiltration-high-performance liquid chromatography.

It is a time-consuming and challenging task for affinity measurement of drug lead compounds from a plant extract because of its chemical complexity. In this research, a strategy of ultrafiltration-high-performance liquid chromatography (HPLC) was developed to directly measure dissociation constant (Kd) of compounds from natural product extract to target protein, and the Kd measurement of α-glucosidase ligands from the ethyl acetate fraction of Perilla frutescens (L.) Britt. (PFEA) was performed. The recovery value, binding degree, and signal-to-noise ratio of α-glucosidase ligands from PFEA were first determined according to the ultrafiltration-HPLC results; the Kd values were then calculated using proposed equilibrium. Finally, oleanolic acid (4) and apigenin (8) from PFEA were determined as the high affinity ligands for α-glucosidase, and their Kds were calculated as 44.9 µM and 88.5 µM, respectively, which agreed with the isothermal titration calorimetry analysis, kinetic analysis, and computer simulation of molecular docking. These results suggested that the proposed strategy is a simple and convenient method for the direct Kd determination of compounds from natural product extract without using any internal calibrants or internal standards.

Aldose Reductase, Protein Glycation Inhibitory and Antioxidant of Peruvian Medicinal Plants: the Case of Tanacetum parthenium L. and Its Constituents.

Diabetes complications, including peripheral neuropathy, cataracts, impaired wound healing, vascular damage, arterial wall stiffening and retinopathy diseases, are among the most predominant health problems facing the world's population today. The 22 Peruvian plant extracts were screened for their potential inhibitory activity against rat lens aldose reductase (RLAR) and DPPH radical scavenging. Among them, we have found that Tanacetum parthenium L. (TP) has the RLAR, AGEs and DPPH radical scavenging activities. We used for screening of active components in TP against RLAR and DPPH for the first time by ultrafiltration (UF) and DPPH. Compounds in TP were isolated by Sephadex column chromatography and their structures were established by MS and NMR spectroscopic analyses. Among the isolated compounds, ferulic acid, apigenin, luteolin-7-O-glucoside, luteolin, chrysosplenol, and kaempferol showed potent inhibition with IC50 values of 1.11-3.20 and 6.44-16.23 µM for RLAR and DPPH radical scavenging. Furthermore, these compounds suppressed sorbitol accumulation in rat lenses and ferulic acid, luteolin-7-O-glucoside, and luteolin have AGEs inhibitory activities with IC50 values of 3.43-6.73 µM. In summary, our study provides interesting plants for further study with respect to the treatment and prevention of diabetic complication of Peruvian plant and can provide the scientific base of the traditional uses.

An in vitro affinity-based method for studying herb-drug interactions for direct identification of cytochrome P450 1A2, 3A4, and 2C9 specific ligands from herbal extracts using ultrafiltration-high performance liquid chromatography.

A novel in vitro strategy for affinity-based ultrafiltration-high performance liquid chromatography (HPLC) was developed for the direct identification of cytochrome P450 (CYP) 1A2, 3A4, and 2C9 specific ligands from Danshen extracts, in which human liver microsome (HLM) was used as the source of CYP enzymes. The Danshen extract was incubated without HLM, with HLM, or with HLM where the active site of the target enzyme was blocked with a specific competitive probe before ultrafiltration to isolate ligand-enzyme complexes from unbound compounds. Subsequently, HPLC analysis was performed on the released ligands from the complexes. α-Naphthoflavone, ketoconazole, and sulfaphenazole were used as specific competitive probes for CYP1A2, 3A4, and 2C9, respectively. The signal-to-noise ratio (S/N) and specific-signal-to-noise ratio (S-S/N) of each compound were calculated using the obtained peak areas. Finally, two criteria were applied to select putative ligands for each specific target: (1) S/N > 1; (2) S-S/N > 0. Finally, dihydrotanshinone was identified as a specific ligand for CYP1A2 and tanshinone I, cryptotanshinone, and tanshinone IIA were identified as specific ligands for CYP1A2, 2C9, and 3A4. It was demonstrated that the newly developed method can be used to rapidly and directly detect specific ligands from natural product extracts in multi-target systems.

Screening In Vitro Targets Related to Diabetes in Herbal Extracts from Peru: Identification of Active Compounds in Hypericum laricifolium Juss. by Offline High-Performance Liquid Chromatography.

This study investigates in vitro targets related to diabetes in 30 herbal extracts from Peru, for the first time, using α-glucosidase, aldose reductase (AR) inhibitory assays and 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) scavenging assays. Among the 30 herbal extracts, Hypericum laricifolium Juss. (HL) was the herb which showed more than 50% inhibition in all assays, presenting 97.2 ± 2.0%, 56.9 ± 5.6%, 81.9 ± 2.5%, and 58.8 ± 4.6% inhibition for the α-glucosidase, AR, DPPH, and ABTS assays, respectively. Finally, six bioactive compounds, namely, protocatechuic acid, chlorogenic acid, caffeic acid, kaempferol 3-O-glucuronide, quercetin, and kaempferol were identified in HL by offline high-performance liquid chromatography (HPLC). Quercetin exhibited the strongest inhibition in all enzyme assays and the strongest antioxidant activity. The results suggest that HL shows great potential for the complementary treatment of diabetes and its complications.