Application of two-dimensional reversed phase countercurrent chromatography × high-performance liquid chromatography to bioactivity-guided screening and isolation of α-glucosidase inhibitors from Rheum palmatum L.

In the present work, comprehensive two-dimensional reversed-phase countercurrent chromatography × reversed-phase liquid chromatography combined (2D RPCCC × RPLC) with 2D microfraction bioactive evaluation was employed to screen and isolate α-glucosidase inhibitors from Rheum palmatum L. Countercurrent chromatography was employed to improve 2D analysis and preparative separation. A selected biphasic solvent system composed of petroleum ether/ethyl acetate/methanol/water with gradient elution mode was used for the first dimension RPCCC separation (1D RPCCC). Solid-phase extraction was applied to eliminate interfering polar compounds before the second dimension analysis (2D RPLC). 76 components were shown in 2D contour plot in UV 280 nm. 11 Candidates were separated by a scaled-up CCC and identified by 1H NMR and 13C NMR, including anthraquinones, flavonoids, stilbenes, phenols, and glucoside derivatives. In addition, it was found that two components, resveratrol-4'-O-(6″-galloyl)glucoside (36) and lyciumaside (43) were identified as natural α-glucosidase inhibitors in Rheum palmatum L. for the first time.

Screening and identification of antibacterial components in Artemisia argyi essential oil by TLC-direct bioautography combined with comprehensive 2D GC × GC-TOFMS.

One of the primary components that contribute to Artemisia argyi 's effectiveness is essential oil, which has an exceptional antibacterial effect that has been well documented. The actual cause of its antibacterial activity and associated mechanism, however, are still not completely understood. For the first time, comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (2D GC × GC-TOFMS) and thin-layer chromatography-direct bioautography (TLC-DB) were employed to investigate its antibacterial components. The antibacterial properties of A. argyi essential oil were investigated, and the antibacterial activity of six compounds was evaluated, using Staphylococcus aureus (S. aureus) and Escherichia coli (E. coil) as test microorganisms. TLC-direct bioautography was used to screen two bioactive clusters. Following 2D GC × GC-TOFMS identification of bioactive clusters, six compounds were evaluated for in vitro antibacterial activity verification. All the components tested displayed antibacterial action. Results showed that α-terpineol and eugenol had high potent antibacterial activity (MIC＜0.62 mg/mL, IC50＜2.00 mg/mL). For complex essential oils from traditional Chinese medicine, this method is efficient for quick screening and identifying antibacterial compounds.

[Off-line comprehensive two-dimensional countercurrent chromatography-liquid chromatography separation of Curcuma volatile oil].

The chemical constituents of volatile oils used in traditional Chinese medicine are highly complex. Thus, achieving the complete separation of volatile oils by one-dimensional chromatography is difficult owing to the low peak capacity of the technique. Although comprehensive two-dimensional gas chromatography provides an efficient means for separating volatile oils, it cannot be used to screen bioactive components because of its limitations. Therefore, developing a new method to separate volatile oils based on liquid chromatography is of great significance in efforts to obtain new approaches to screen bioactive components in volatile oil. The objectives of the present study are to establish an efficient method for separating the chemical constituents of Curcuma volatile oil using off-line comprehensive two-dimensional countercurrent chromatography-liquid chromatography (CCC-LC) and to investigate the two-dimensional peak capacity, orthogonality, and spatial coverage of this method. Both CCC and LC conditions were optimized. A biphasic n-hexane-methanol-water solvent system was selected via the colorimetric method, and the lower phase was used as the mobile phase in gradient-elution mode: 0-55 min, n-hexane-methanol-water (5∶2∶3 v/v/v); 55-170 min, n-hexane-methanol-water (5∶3∶2, v/v/v); 170-290 min, n-hexane-methanol-water (5∶4∶1, v/v/v). After gradient elution, elution-extrusion elution mode was applied within 290-375 min. Good resolution was achieved by the CCC separation process. The HPLC separation process was carried out with gradient elution using a mobile phase composed of acetonitrile (A)-water (B): 0-10 min, 50%A-65%A; 10-14 min, 65%A; 14-21 min, 65%A-85%A; 21-25 min, 85%A-95%A; 25-30 min, 95%A-55%A; 30-40 min, 55%A. Curcuma volatile oil was separated under the above optimized two-dimensional separation conditions, and the data obtained were drawn into a two-dimensional contour map using Matlab software. The calculated total peak capacity exceeded 954, which was 10 times more than that of one-dimensional chromatography. High orthogonality (r=0.17) and spatial coverage factor (68.1%) were also obtained. Our research provides a new methodology for separating volatile oils used in traditional Chinese medicine as well as an approach for evaluating the quality of traditional Chinese medicinal herbs using two-dimensional chromatographic fingerprints.

Coumarins from Jinhua Finger Citron: Separation by Liquid-Liquid Chromatography and Potential Antitumor Activity.

In this paper, liquid-liquid chromatography was introduced for the first time for the separation of fingered citron (Citrus medica L. var. sarcodactylis Swingle). The fingered citron cultivated in Jinhua is of significant industrial and medicinal value, with several major coumarin compounds detected in its extract. Therefore, further separation for higher purity was of necessity. A preparative liquid-liquid chromatographic method was developed by combining two elution modes (isocratic and step-gradient) with selection according to different polarities of the target sample. Five coumarin derivatives-5,7-dimethoxycoumarin (52.6 mg, 99.6%), phellopterin (4.9 mg, 97.1%), 5-prenyloxy-7-methoxycoumarin (6.7 mg, 98.7%), 6-hydroxy-7-methoxycoumarin (7.1 mg, 82.2%), and byakangelicol (10.5 mg, 90.1%)-with similar structures and properties were isolated on a large scale from 100 mg of petroleum ether (PE) extract and 100 mg of ethyl acetate (EA) extract in Jinhua fingered citron. The productivity was much improved. The anti-growth activity of the isolated coumarins was evaluated against three cancer cell lines (HeLa, A549, and MCF7) with an MTT assay. The coumarins demonstrated potential anti-tumor activity on the HeLa cell line, with 5,7-dimethoxycoumarin in particular exhibiting the best anti-growth activity (IC50 = 10.57 ± 0.24 µM) by inhibiting proliferation. It inhibited colony formation and reduced the size of the tumor sphere in a concentration-dependent manner. The main mechanism was confirmed as inducing apoptosis. This work was informative for further studies aimed at exploring new natural-product-based antitumor agents.

Matrix solid-phase dispersion combined with micro-fractionation bioactivity evaluation screening polymethoxylated flavones from Citrus peels.

Polymethoxyflavones were a unique class of natural and safe flavonoids containing two or more methoxy groups, which were also the most abundant edible part in Citrus peel. The optimum condition in the process of selective extraction of polymethoxylated flavones from Citrus peel by matrix solid-phase dispersion (MSPD) was as follows: SBA-15 as adsorbent, ethyl acetate as eluent, the mass ratio of adsorbent to sample 1:1, and the mixture of sample and adsorbent was ground for 3 min. Twelve antioxidants were successfully screened by micro-fractionation bioactivity evaluation assay, in which four of them were flavonoid glycosides, seven of them were polymethoxylated flavones, and one was phenylpropanoid. 1-sinapoly-ß-D-glucopyranoside (1) was reported for the first time in Citrus peel. And antioxidant capacity of 1-sinapoly-ß-D-glucopyranoside, 5, 7, 8, 3', 4', 5'-hexamethoxyflavone (6), hexamethoxyflavone (11), and 5, 6, 7, 4'-tetramethoxyflavone (7) were reported for the first time. Nobiletin (compound 8), 3, 5, 6, 7, 8, 3', 4'-heptamethoxyflavone (9) and tangeretin (10) were isolated and purified by countercurrent chromatography combined with preparative liquid chromatography. Antioxidant activity evaluation indicated that the three isolated polymethoxylated flavones owned similar antioxidant activity. This study indicated that MSPD combined with micro-fractionation bioactive evaluation was efficient in screening bioactive compounds for rapid extraction and effective pinpointing bioactive substances in natural products.

A comprehensive strategy for prediction and quality evaluation of standardized planting herbs based on plant metabolomics coupled with extreme learning machine: Astragali Radix as an example.

INTRODUCTION: Standardizing the planting process is an effective way to control the quality stability of herbal resources, which are susceptible to external environmental factors (e.g., moisture, soil, etc.). However, how to scientifically and comprehensively assess the effects of standardized planting on plant quality and quickly test unknown samples has not been addressed. OBJECTIVE: The aim of this study was to determine and compare the metabolite levels of herbs before and after standardized planting, to quickly distinguish their sources, and to evaluate their quality, using the typical herb Astragali Radix (AR) as an example. METHODS: In this study, an efficient strategy using liquid chromatography-mass spectrometry (LC-MS) based on plant metabolomics combined with extreme learning machine (ELM) has been developed to efficiently distinguish and predict AR after standardized planting. Moreover, a comprehensive multi-index scoring method has been developed for the comprehensive evaluation of the quality of AR. RESULTS: The results confirmed that AR after standardized planting was significantly differentiated, with a relatively stable content of 43 differential metabolites, mainly including flavonoids. An ELM model was established based on LC-MS data, and the accuracy in predicting unknown samples could reach more than 90%. As expected, higher total scores were obtained for AR after standardized planting, indicating much better quality. CONCLUSION: A dual system for evaluating the impact of standardized planting on the quality of plant resources has been established, which will significantly contribute to innovation in the quality evaluation of medicinal herbs and support the selection of optimal planting conditions.

High-resolution monophenolase/diphenolase/radical scavenging profiling for rapid screening of natural whitening candidates from Rosa rugosa cv. 'Plena'.

Antioxidants and tyrosinase inhibitory components were successfully screened and separated from Rosa rugosa cv. 'Plena' by high-performance liquid chromatography microfractionation bioactive screening combined with several separation and purification methods. Ethyl acetate extract of Rosa rugosa cv. 'Plena' showed high antioxidant activity and tyrosinase inhibitory activity. High-speed countercurrent chromatography, silica gel column chromatography, and semi-preparative high-performance liquid chromatography were used for the preparative separation of four bioactive components from ethyl acetate extract. Two tyrosinase-inhibiting active substances, flavogallonic acid, and N1 -N5 -N10 -tri-4-p-coumaroylspermidine, were isolated from Rosa rugosa cv. 'Plena', and they showed great monophenolase inhibition activity (half-maximal inhibitory concentration: 664.60 and 23.77 µg/ml, respectively) and excellent diphenolase inhibition activity (half-maximal inhibitory concentration: 23 614.61 and 16.80 µg/ml, respectively). Meanwhile, gallic acid, flavogallonic acid, and ellagic acid were shown to have excellent 1,1-diphenyl-2-picryl-hydrazyl antioxidant activity (half maximal inhibitory concentration: 6.66, 20.17, and 13.45 µg/ml), and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) antioxidant activity (half maximal inhibitory concentration: 3.53, 3.83, and 2.78 µg/ml). Molecular docking revealed that flavogallonic acid and N1 -N5 -N10 -tri-4-p-coumaroylspermidine had a strong binding affinity (-9.3 and -10 kcal/mol, respectively) to tyrosinase through hydrogen bonding and hydrophobic interactions.

High-performance liquid chromatography micro-fraction bioactive evaluation combined with countercurrent chromatographic separation of antioxidants from Citrus peel and their tyrosinase inhibition activities.

In the present study, high-performance liquid chromatography micro-fraction bioactive evaluation and high speed countercurrent chromatography were performed on screening, identification and isolation of antioxidants from Citrus peel. Three compounds were screened as antioxidants and tyrosinase inhibitors using 2,2'-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) radical cation scavenging assay and tyrosinase activity test, then they were identified as eriocitrin, narirutin and hesperidin. Moreover, the solvent system ethyl acetate-n-butanol-water (6:4:10, v/v/v) was used for separation of ethyl acetate extract of Citrus peel by high speed countercurrent chromatography. In total, 0.45 mg of eriocitrin with 87.10% purity, 2.04 mg of narirutin with 95.19% purity and 1.35 mg of hesperidin with 95.19% purity were obtained from 20 mg of ethyl acetate extract of Citrus peel in a single run and then each component was subjected to 2,2'-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) radical cation scavenging assay and tyrosinase inhibition assay. Eriocitrin showed great antioxidant activity (the half-maximum concentration: 3.65 µM) and tyrosinase inhibition activity (the half-maximum concentration: 115.67 µM), while narirutin and hesperidin exhibited moderate activity. Tyrosinase inhibition activity for eriocitrin in vitro was reported for the first time. Furthermore, molecular docking between eriocitrin and mushroom tyrosinase was also studied.

Cucurbitane-type triterpenoids from the fruits of Citrullus colocynthis.

A phytochemical investigation of the fruits of Citrullus colocynthis resulted in the isolation of 21 structurally diverse cucurbitane triterpenoids, including 9 previously undescribed ones, colocynins A-I (1-9). Their absolute configurations were elucidated by means of quantum chemical electronic circular dichroism (ECD) calculations, CD exciton chirality method, and single-crystal X-ray crystallography. Colocynins A-C (1-3) represent the first examples of nonanorcucurbitane-type triterpenoids. An anti-acetylcholinesterase activity assay showed that 6, 10, 13, 18, and 20 exhibited inhibitory activities, with IC50 values ranging from 5.0 to 21.7 µM. In addition, 18 and 21 showed significant cytotoxicity against PACA, A431, and HepG2 cells, with IC50 values ranging from 0.042 to 0.60 and 3.6-14.4 µM, respectively.

Comprehensive two-dimensional countercurrent chromatography × gas chromatography characterization of Artemisia argyi essential oil.

A comprehensive two-dimensional (2D) countercurrent chromatography (CCC) × gas chromatography (GC) was investigated for characterization of chemical constituents of Artemisia argyi essential oil, and orthogonality for the 2D chromatographic system was evaluated. A solvent system composed of n-hexane/acetonitrile/methanol (2:2:1, v/v/v) was selected for first dimensional separation of Artemisia argyi essential oil. Then all CCC fractions were analyzed by GC, which provided a wealth of information regarding the composition of the essential oil. Visualization of chemical compositions obtained from the comprehensive 2D CCC × GC separation was achieved by creation of a 2D contour plot map. Total peak capacity was evaluated and approximately 1392 peaks were obtained through a comprehensive 2D CCC × GC separation. A high spatial coverage and a low linear correlation coefficient were achieved. Meanwhile, all compounds were identified by GC-MS. The obtained 2D contour plot could be divided into six zones to show the characteristic chemical compositions. Six zones could be divided into different component groups, including monoterpenes, sesquiterpenes, monoterpene alcohols, phenols, aldehydes, ketones and esters, which could be used to identify compounds that have not been reported, and to predict the structure of unknown compounds in Artemisia argyi essential oil and comprehensively characterize fingerprint peak.

Discovery of potential hypoglycemic metabolites in Cassiae Semen by coupling UHPLC-QTOF-MS/MS combined plant metabolomics and spectrum-effect relationship analyses.

Cassiae Semen (CS) is consumed as fried tea or medicinal food in Asian areas. Its two commercial forms, namely raw and fried CS, exert different clinical applications, in which fried CS is commonly applied as a functional tea for losing weight. To prevent confusion in the use of the two forms of CS, a comprehensive strategy by combining plant metabolomics and spectrum-effect relationship analyses was developed for the fast and efficient discrimination of raw and fried CS, and further for the discovery of the potential hypoglycemic metabolites of CS to control its quality. First, the plant metabolic profiling of raw and processed samples was performed by UHPLC-QTOF-MS/MS. A total of 1111 differential metabolites were found to well distinguish the raw and fried CS after analyzing by MPP software. Subsequently, α-glucosidase inhibition of raw and fried CS was investigated. As a result, fried CS demonstrated much stronger α-glucosidase inhibition activity than the raw sample. By analyzing the spectrum-effect relationship with GRA, BCA, and PLSR, 14 potential hypoglycemic-related compounds were discovered. As anticipated, they were also found as the differential metabolites of the raw and fried samples with a potential hypoglycemic effect, which might be beneficial for the quality control of CS tea. Additionally, molecular docking analysis was conducted to reveal the underlying inhibition mechanisms of the four most critical constituents, including physcion, chrysoobtusin, aurantio-obtusin, and obtusifolin. This study provides a powerful tool for the discrimination of processed samples and fast screening of the active constituents in complex natural products on a high-throughput basis.

A Strategy for Pinpointing Natural Bioactive Components Using Two-Dimensional Bioassay Profilings Combined with Comprehensive Two-Dimensional Countercurrent Chromatography × High-Performance Liquid Chromatography.

Inspired by the interpretation of two-dimensional (2D) nuclear magnetic resonance spectra, an efficient strategy was proposed for pinpointing bioactive components from complex natural products. An off-line comprehensive countercurrent chromatography (CCC) × high-performance liquid chromatography (HPLC) was employed to achieve a 2D chemical chromatogram, and 2D bioassay profilings were obtained from bioassays of the eluent of the first dimension (1D) CCC and the eluent of the second dimension (2D) HPLC. Then 2D chemical chromatograms and 2D bioassay profilings were matched for pinpointing bioactive natural components from complex matrices. Thus, bioactive components in a complex matrix could be efficiently analyzed, separated, and bioactivity-determined. This experimental scheme was successfully demonstrated with a traditional medicinal herb Polygonum cuspidatum Sieb. et Zucc. The feasibility of this 2D strategy was verified with tyrosinase inhibition assay, α-glucosidase inhibition assay, DPPH radical scavenging assay, and ABTS•+ decolorization assay. Eight natural inhibitors were successfully pinpointed and identified from P. cuspidatum. Both pieceid-2″-O-gallate (10) and vanicoside B (20) were screened and identified as natural tyrosinase inhibitors for the first time. Meanwhile, vanicoside B (20) was also found as the strongest α-glucosidase inhibitor among all the isolated components. Most of the compounds exhibited much higher radical scavenging activities. Compared with traditional methodology based on one-dimensional chromatographic separation, the present 2D strategy would be more precise, efficient, and convenient to screen and separate bioactive compounds from complex matrices.

In silico screening of off-line comprehensive two-dimensional counter-current chromatography with liquid chromatography for four saponins isolation.

Being restrained by the limited peak capacity, one-dimensional chromatography usually leads to an unsatisfactory separation with low purity of compounds in a complex mixture. To obtain more highly pure targets for standard reference and to discover new substances for structural elucidation, two-dimensional chromatography is more and more prevalent in many fields. As few metrics on assessment of the preparative capability of two-dimensional chromatographic separations are reported, a methodology of in silico screening of various two-dimensional chromatographic separations with a minimal number of experiments was demonstrated in this work, which was based on three descriptors including the occupation rate of peaks and system homogeneity of a two-dimensional separation space, and the minimal distance of all nearest-neighbor distances of peaks. Combining the advantages of counter-current chromatography and liquid chromatography, we elaborated the methodology by employing off-line comprehensive two-dimensional counter-current chromatography with liquid chromatography to be in silico screened for separation of four saponins from Panax notoginseng at an analytical scale to simulate the case of preparative scale transfer. The predictive results were presented by two-dimensional contour plots and verified by experiments. The result showed that the experimental results were in general accord with the predictive results.

Solvent strength of aqueous phase for two typical biphasic solvent systems in high-speed countercurrent chromatography.

In the present work, influence of solvent strength of aqueous phase for two frequently-used biphasic solvent system in partition coefficient (K) of selected solutes were mainly studied, and a new method for selection of biphasic solvent system was proposed for high-speed countercurrent chromatographic separations. Solvent strength was referred to the typical theory that was deeply investigated in conventional reversed-phase liquid chromatography. Experimental results showed that a linear relationship between log(K) of solutes and apparent content of methanol in biphasic solvent system was found for the biphasic solvent system hexane-ethyl acetate-methanol-water (HEMWat), which was consistent with the relationship between real content and apparent content of methanol in this system. Meanwhile, a quadratic relationship was found between log(K) of solutes and apparent content of methanol in biphasic solvent system chloroform-methanol-water (ChMWat), in which it was found that the relationship between real content and apparent content of methanol in this system was also quadratic. In addition, a visual and simple method was proposed to select a suitable biphasic solvent system for separation of target compounds by high-speed countercurrent chromatography with isocratic elution, which saves a lot of manpower and material resources in order to find a suitable two-solvent system. An optimal biphasic solvent system for isolation of several tested compounds by high-speed countercurrent chromatography was easily obtained using our proposed method.

A novel high-resolution monophenolase/diphenolase/radical scavenging profiling for the rapid screening of natural whitening candidates from Peaonia lactiflora root and their mechanism study with molecular docking.

ETHNOPHARMACOLOGICAL RELEVANCE: The root of Paeonia lactiflora is a traditionally-used whitening medicine in China for thousands of years. Although some tyrosinase inhibitors and/or antioxidants such as 1,2,3,4,6-pentagalloylglucose, gallic acid, have been isolated and identified, their tyrosinase inhibition pathway (monophenolase or diphenolase inhibition, or both two) have not been systematically studied and the underlying tyrosinase inhibition mechanism has not been revealed yet. Moreover, the exploring of new natural tyrosinase inhibitors and antioxidants is urgently needed. AIM OF THE STUDY: This review aimed to develop a new microplate-based high-resolution tyrosinase inhibition profiling assay and establish a furthermore triple high-resolution monophenolase/diphenolase/radical scavenging profiling for accelerating identification bioactive compounds from complicated plant extract. MATERIALS AND METHODS: The targeted isolation and structure elucidation were performed with high-performance liquid chromatography-high-resolution mass spectrometry and preparative high-performance liquid chromatography. It allows to be a proof of concept with the root of Paeonia lactiflora crude extract as a natural whitening herbal drug. RESULTS: The result showed that galloylpaeoniflorin specifically inhibited monophenolase activity. While 1,2,3,4,6-pentagalloylglucose, gallic acid and catechin demonstrated the inhibition towards both monophenolase and diphenolase. Among them, 1,2,3,4,6-pentagalloylglucose can inhibit monophenolase activity was reported for the first time. In addition, antioxidant properties were attributed to catechin, 1,2,3,4,6-pentagalloylglucose and gallic acid. Due to its low content and complicated configuration in the root of Paeonia lactiflora, a new potential tyrosinase inhibitor and radical scavenger which tentatively identified as hexagalloylglucose by high-resolution MS was still need further verification. What's more, the molecular docking unveiled that bioactive enzymatic inhibitors mainly interacted with amino acid catalytic residues of tyrosinase via H-bonds and van der wals, which may be helpful to understand their inhibition mechanisms with tyrosinase in the skin whitening. CONCLUSIONS: The platform provided a promising and efficient strategy for the rapid screening of whitening active components from natural sources.

[Definitive screening design combined with design space for optimizing purification process of Scutellariae Radix extract].

In the pharmacopoeia, many process parameters for the purification process of Scutellariae Radix are unclear. In this study, deterministic screening design combined with design space method was used to optimize the purification process of Scutellariae Radix extract. Nine method parameters such as mass fraction of solution(X_1), first acid precipitation pH(X_2) and first holding time(X_3) in the purification process were firstly studied by definitive screening design. The yield of baicalin was defined as the evaluation index. A stepwise regression method was used then to build quantitative models between evaluation index and method parameters and the three most critical impact parameters were determined. Probability-based design space was calculated and successfully verified with the experimental error simulation method. Finally, the second standing temperature, the first standing temperature and the pH value of the second acid precipitation were determined as the three most critical method parameters. The recommended operating space was as follows: the second standing temperature 5-7 ℃, the first standing temperature 13-15 ℃, and the pH of the second acid precipitation 1.5-1.7. Within this operating space, the baicalin yield in the purification process was over 80%, and the probability of reaching the standard was over 0.96. In this study, we optimized the effect of various parameters for the purification process of the Scutellariae Radix extract in the pharmacopoeia on the yield of baicalin and provided a reference for industrial production of the exact of Scutellariae Radix.

An efficient high-speed countercurrent chromatography method for preparative isolation of highly potent anti-cancer compound antroquinonol from Antrodia camphorata after experimental design optimized extraction.

To avoid irreversible stationary phase adsorption and tedious and time-consuming separation steps, high-speed countercurrent chromatography was employed for the preparative separation of anti-tumor compound antroquinonol from solid fermentation culture of Antrodia camphorata for the first time. A Box-Behnken experimental design, based on three parameters including liquid-to-solid ratio, extraction time, and extraction temperature, was applied to optimize the ultrasonic extraction procedure. The optimal extraction condition was set as follows: liquid-to-solid ratio: 49.57:1; extraction time: 55.76 min; extraction temperature was arranged as 44.21°C. Meanwhile, an optimized solvent system containing petroleum ether, ethyl acetate, methanol, and water (4:1:4:1, v/v/v/v) was selected for the preparative separation of antroquinonol at a flow rate of 2.0 mL/min. The yield of isolated antroquinonol was determined to be 6.0 mg from 0.67 g of ethyl acetate extracts. The isolated antroquinonol was elucidated by ultra-high-performance liquid chromatography-tandem mass spectrometry, and NMR spectroscopy, and by comparison with literature data. The purity of isolated antroquinonol was determined to be 97.12%. This study confirmed that high-speed countercurrent chromatography was powerful and cost-effective for the preparative separation of the high-potently anti-tumor compound antroquinonol from solid fermentation culture of A. camphorata.

Screening and characterization of aldose reductase inhibitors from Traditional Chinese medicine based on ultrafiltration-liquid chromatography mass spectrometry and in silico molecular docking.

ETHNOPHARMACOLOGICAL RELEVANCE: Shenqi Jiangtang granule (SJG) is an ancient Chinese herbal formula used for treatment of Diabetes mellitus and its complications. AIM OF THE STUDY: To establish an integrated approach for discovery of effective Aldose reductase inhibitors (ARIs) from SJG. MATERIALS AND METHODS: An integrated approach combining ultrafiltration-liquid chromatography-mass spectrometry (UF-LC-MS) with in silico molecular docking was established for development of ARIs. AR enzyme was separated from the rabbit's crystalline lens. The inhibitory activities of these compounds were detected by UV spectrophotometry with DL-glyceraldehyde as a substrate. Furthermore, molecular docking was used to understand the binding mechanism of these screened compounds interacting with AR. RESULTS: After optimization of AR reaction system and ultrafiltration incubation system, 17 active ingredients were screened from SJG by UF-LC-MS technique. Among these potential AR inhibitors, ginsenoside Rd exhibited the strongest activity with IC50 value of 45.77 µM. Three of them, calycosin, gomisin J and schisandrin A were demonstrated to be potential inhibitors for the first time, with IC50 at 447.34 µM, 181.73 µM, and 429.00 µM, respectively. Most of the active compounds exhibited competitive inhibition against AR. The docking scores of saponins were higher than that of lignans, which was consistent with the verification results. CONCLUSION: The results indicated that TCM formula with clinical efficacy was indeed hopeful source for screening active ingredients, and the combination of UF-LC-MS and in silico molecular docking was a universal and promising approach for development of effective enzyme inhibitors.

Isolation and Purification of Five Phloroglucinol Derivatives From Agrimonia Pilosa Ledeb by Counter-Current Chromatography Using Non-Aqueous Solvent Systems.

Five non-polar phloroglucinol derivatives, viz. pseudo-aspidin, α-kosin and agripinol A-C were isolated and purified from Agrimonia pilosa Ledeb by semi-preparative counter-current chromatography. The separation was performed by a two-step elution with non-aqueous solvent systems. In the first step, an elution mode of a two-phase solvent system consisting of n-hexane-acetonitrile-dichloromethane-methanol (6:6:0.5:0.5, v/v/v/v) was used. We obtained sample Ι containing three components (47.0 mg) and sample ΙΙ containing two components (24.8 mg) from crude extract (371.0 mg). In the second step, sample Ι was successfully separated by closed-loop recycling mode with a solvent system consisting of n-hexane-acetonitrile-dichloromethane (10:7:3, v/v/v), yielding 17.8 mg of pseudo-aspidin, 18.5 mg of α-kosin and 6.4 mg of agripinol A. The other two compounds-8.7 mg of agripinol B and 13.6 mg of agripinol C-were obtained from sample ΙΙ in the same manner. All the isolated compounds had a high purity exceeding 95%.

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.