Five New Diarylbutyrolactones and Sesquilignans from Saussurea medusa and Their Inhibitory Effects on LPS-induced NO Production.

Five new diarylbutyrolactones and sesquilignans (1A/1B:  - 4: ), including one pair of enantiomers (1A/1B: ), together with 10 known analogues (5:  - 14: ), were isolated from the whole plants of Saussurea medusa. Compound 1: was found to possess an unusual 7,8'-diarylbutyrolactone lignan structure. Separation by chiral HPLC analysis led to the isolation of one pair of enantiomers, (+)-1A: and (-)-1B: . The structures of the new compounds were elucidated by extensive spectroscopic data. All compounds, except compounds 5, 7: and 9: , were isolated from S. medusa for the first time. Moreover, compounds 1:  -  4, 8: and 10:  - 14: had never been obtained from the genus Saussurea previously. Compounds (+)- 1A, 2, 5, 7: , and 9:  - 11: were found to inhibit the lipopolysaccharide (LPS)-induced release of NO by RAW264.7 cells with IC50 values ranging from 10.1 ± 1.8 to 41.7 ± 2.1 µM. Molecular docking and iNOS expression experiments were performed to examine the interactions between the active compounds and the iNOS enzyme.

Three Pairs of Novel Enantiomeric 8-O-4' Type Neolignans from Saussurea medusa and Their Anti-inflammatory Effects In Vitro.

Three pairs of novel enantiomeric 8-O-4' type neolignans (1a/1b−3a/3b), together with seven known analogues (4−10), were isolated from the whole plants of Saussurea medusa. Their structures were elucidated by extensive spectroscopic data analysis and electric circular dichroism (ECD) calculations after chiral separations. All compounds were obtained from S. medusa for the first time, and compounds 1−3 and 5−10 had never been obtained from the genus Saussurea previously. The anti-inflammatory activities of the compounds were evaluated by determining their inhibitory activities on the production of NO and inducible nitric oxide synthase (iNOS) expression in LPS-stimulated RAW 264.7 cells. Compounds (+)-1a, (−)-1b and 5−7 inhibited NO production and had IC50 values ranging from 14.3 ± 1.6 to 41.4 ± 3.1 µM. Compound 7 induced a dose-dependent reduction in the expression of iNOS in LPS-treated RAW 264.7 cells. Molecular docking experiments showed that all active compounds exhibited excellent docking scores (<−7.0 kcal/mol) with iNOS. Therefore, compounds (+)-1a, (−)-1b and 5−7 isolated from the whole plants of S. medusa may have therapeutic potential in inflammatory diseases.

The Antibacterial Activity Mode of Action of Plantaricin YKX against Staphylococcus aureus.

We aimed to evaluate the inhibitory effect and mechanism of plantaricin YKX on S. aureus. The mode of action of plantaricin YKX against the cells of S. aureus indicated that plantaricin YKX was able to cause the leakage of cellular content and damage the structure of the cell membranes. Additionally, plantaricin YKX was also able to inhibit the formation of S. aureus biofilms. As the concentration of plantaricin YKX reached 3/4 MIC, the percentage of biofilm formation inhibition was over 50%. Fluorescent dye labeling combined with fluorescence microscopy confirmed the results. Finally, the effect of plantaricin YKX on the AI-2/LuxS QS system was investigated. Molecular docking predicted that the binding energy of AI-2 and plantaricin YKX was -4.7 kcal/mol and the binding energy of bacteriocin and luxS protein was -183.701 kcal/mol. The expression of the luxS gene increased significantly after being cocultured with plantaricin YKX, suggesting that plantaricin YKX can affect the QS system of S. aureus.

Enrichment and separation of high-polar compounds from Saussurea obvallata using solid-phase extraction combining with offline two-dimensional liquid chromatography.

The high-polar compounds from natural products are often used as medicines due to their good bioactivities. However, owing to the complexity and diversity of their structure, the separation of high-polar compounds is still a challenging work. For this, an efficient method for enrichment and separation of the high-polar compounds from Saussurea obvallata was developed. First, the target compounds were enriched from the total extract using a solid-phase extraction method. An offline two-dimensional liquid chromatography method was used for the separation of pure compounds from the enriched sample. After optimization of chromatographic conditions, high separation selectivity of target compounds was obtained on a polar-modified C18 column and a HILIC XAmide column. Hence, a two-dimensional reversed-phase × hydrophilic interaction liquid chromatography system was constructed and enlarged from the analytical level to the preparative level. In the first dimension, four fractions were obtained on the XCharge C18 column with a recovery rate of 71.2%. In the second-dimension preparation on the XAmide column, eight high-polar compounds with more than 96% purity were isolated. All compounds were isolated from Saussurea obvallata for the first time. The results demonstrated that this developed strategy is effective for preparative-scale isolation of high-polar compounds from natural products.

Preparative isolation of 1,1-diphenyl-2-picrylhydrazyl inhibitors from Ribes himalense using medium-pressure and two-dimensional reversed-phase/reversed-phase liquid chromatography guided by an online HPLC-1, 1-diphenyl-2-picrylhydrazyl assay.

The lack of suitable chromatographic purification methods makes it a challenge to effectively isolate the chemical components of traditional Tibetan medicines. Ribes himalense is a rarely studied Tibetan medicine, reputed to have free radical-scavenging effects. In the present work, we utilized it as a model herb to highlight an approach for the separation of 1,1-diphenyl-2-picrylhydrazyl inhibitors via medium-pressure chromatography and two-dimensional reversed-phase/reversed-phase interaction liquid chromatography under the guidance of an online high-performance liquid chromatography-1,1-diphenyl-2-picrylhydrazyl assay. Finally, we obtained two free radical inhibitors (>95% purity) from the R. himalense extract. This is the first report of the rapid isolation of these free radical inhibitors from R. himalense. This method can be useful in quality standard assessment and further pharmacological activity research, and may be used as a reference for the composition research of various natural products.

Arenaria kansuensis attenuates pulmonary fibrosis in mice via the activation of Nrf2 pathway and the inhibition of NF-kB/TGF-beta1/Smad2/3 pathway.

Pulmonary fibrosis is a key feature of COVID-19, Chinese herbal medicine Arenaria kansuensis has been used for curing pulmonary disease and antivirus for a long time and it has the potential against COVID-19. In this work, protective effect of A. kansuensis ethanol extract (AE) on pulmonary fibrosis was evaluated through paraquat (PQ)-induced pulmonary fibrosis animal model. Results showed that AE could significantly improve the survival rate, increase the body weight and reduce the lung index of mice at the raw drug doses of 700 and 350 mg/kg. Histopathological observation results showed that the destruction degree of lung tissue structure in mice was significantly improved with the increase of AE dosage. Collagen deposition in lung interstitium was significantly reduced. The marker protein alpha-SMA involved in PF were significantly inhibited through repressing TGF-beta1/Smads pathway. The degree of inflammatory infiltration was significantly reduced and inflammatory cytokines were significantly inhibited in mice through inhibiting the NF-kB-p65. Besides, oxidant stress level including upregulated ROS and down-regulated SOD and GSH was efficiently improved by AE through upregulation of Nrf2 and downregulation of NOX4. In summary, this study firstly showed that the protective effect of AE on pulmonary fibrosis was partly due to activation of Nrf2 pathway and the inhibition of NF-kB/TGF-beta1/Smad2/3 pathway.

Multivariate statistical and comparison analysis of chemical constituents in Arenaria kansuensis Maxim. from different regions in Qinghai-Tibet Plateau.

INTRODUCTION: Arenaria kansuensis Maxim. (AKM) is one of the most valued medicinal and edible herbs widely used in Qinghai-Tibet Plateau and there is also a large number of AKM bioactive constituents for health benefits of human beings. However, few works have referred to phytochemical content, fingerprint analysis and quality control of AKM. Therefore, the establishment of validated analytical methods is urgently needed for fingerprint comparison and quantitative analysis of AKM multicomponent. OBJECTIVES: To determine quantitatively and compare the phytochemical constituents of AKM located at different areas. METHODOLOGY: The chemical constituents in AKM samples were separated, identified, and quantified by high-performance liquid chromatography (HPLC) with a diode array detector. The discrimination and separation models for the chemical constituents were developed by chemometric analysis. RESULTS: The flavones and ß-carboline alkaloids were rich in AKM herbs, and the overall pattern of phytochemical profiles was the same, while the significant differences were detected in the total flavonoids, total ß-carboline alkaloids and individual contents, especially the predominant compounds such as tricin and arenarine B. This demonstrated that ecogeographical origin gave an important impact on phytochemical compositions which could be considered as reliable parameters for classifying the AKM resources. Moreover, the contents of AKM constituents were higher in July and/or August than other months of the year, and there were no significant differences in the main phytochemical contents between cultivated and wild AKM herbs. CONCLUSION: This study could provide credible data and method for geographical origin trace, comprehensive evaluation and further utilization of AKM resources.

Two new polycyclic polyprenylated acylphloroglucinols derivatives from Hypericum acmosepalum.

Polycyclic polyprenylated acylphloroglucinols (PPAPs) were mainly obtained from the plants of Hypericum genus of Guttiferae family, and possessed intriguing chemical structures and appealing biological activities. Two new PPAPs derivatives, hyperacmosin C (1) and hyperacmosin D (2) were isolated from H. acmosepalum. Their structures were established by NMR, HREIMS, and experimental electronic circular dichroism spectra. Besides, compound 1 showed significant hepatoprotective activity at 10 µM against paracetamol-induced HepG2 cell damage and compound 2 could moderately increase the relative glucose consumption.

Targeted Separation of COX-2 Inhibitor from Pterocephalus hookeri Using Preparative High-Performance Liquid Chromatography Directed by the Affinity Solid-Phase Extraction HPLC System.

Pterocephalus hookeri, as a kind of popular traditional Tibetan medicine, is reputed to treat inflammatory related diseases. In the present work, a cyclooxygenase-2 functionalized affinity solid-phase extraction HPLC system was developed and combined with preparative-HPLC for rapidly screening and separating cyclooxygenase-2 ligand from P. hookeri extracts. Firstly, ligands of cyclooxygenase-2 were screened from extracts by affinity solid-phase extraction HPLC system. Then directed by the screening results, the recognized potential active compounds were targeted separated. As a result, the major cyclooxygenase-2 inhibitor of P. hookeri was obtained with a purity of >95%, which was identified as sylvestroside I. To test the accuracy of this method, the anti-inflammatory activity of sylvestroside I was inspected in lipopolysaccharide-induced RAW 264.7 cells. The results show that sylvestroside I significantly suppressed the release of prostaglandin E2 with dose-dependent, which was in good agreement with the screening result of the affinity solid-phase method. This method of integration of screening and targeted separation proved to be very efficient for the recognition and isolation of cyclooxygenase-2 inhibitors from natural products.

A novel chromatographic separation method for rapid enrichment and isolation of novel flavonoid glycosides from Sphaerophysa salsula.

Flavonoid glycosides exist widely in medicine herbs and often used as nutraceuticals because of their excellent bioactivity and low toxicity. For accurate quality control and bioactivity assessment of Sphaerophysa salsula, a rapid and productive method to isolate flavonoid glycosides is needed. Therefore, this work reports the development of a novel comprehensive strategy based on an online middle-pressure chromatography and preparative high-performance liquid chromatography for rapid enrichment and separation of flavonoid glycosides from S. salsula. First, the flavonoid glycosides were enriched using an online middle-pressure chromatographic column containing stationary middle chromatogram isolated phase. During this process, the high-volume injection of the extracting solution was realized by an empty precolumn positioned before the main chromatographic tower. Then, the compounds were separated through preparative high-performance liquid chromatography with Megress C18. As a result, one new flavonol 3-O-glycoside (2) and two known flavonol 3-O-glycosides (1, 3) were targetedly isolated from S. salsula. The content of compounds 1-3 in S. salsula was 0.09, 0.11, and 0.18 wt%, respectively. Comparing to traditional enrichment and separation methods, our technique offers significantly shorter sample pretreatment time as well as high reproducibility. We believe that our separation method has a strong potential to be used for the processing of other medicinal plants.

Preparative separation of isoquinoline alkaloids from Corydalis impatiens using middle chromatogram isolated gel column coupled with positively charged reversed-phase liquid chromatography.

Positively charged reversed-phase liquid chromatography was employed for the efficient preparative separation of isoquinoline alkaloids from Corydalis impatiens. Ten commercially available columns were compared for isoquinoline alkaloids analysis. While tailing, overloading, lower resolution, and buffer salts limited the application in purification of isoquinoline compounds of many of these columns, one positively charged reversed-phase C18 column (XCharge C18) overcame these drawbacks, allowing for favorable separation resolution, even when loading isoquinoline compounds on a larger, preparative scale. The general separation process is as follows. First, isoquinoline alkaloids are enriched with Corydalis impatiens extract via a middle chromatogram isolated gel column. After column selection, separation is performed on an XCharge C18 analytical column, from which two evident chromatographic peaks are readily obtained. Finally, two isoquinoline alkaloids (protopine and corydamine) are selectively purified on the XCharge C18 preparative column. These results demonstrate that a middle chromatogram isolated gel column coupled with positively charged reversed-phase liquid chromatography is effective for the preparative separation of isoquinoline alkaloids from Corydalis impatiens.

Preparative isolation of arylbutanoid-type phenol [(-)-rhododendrin] with peak tailing on conventional C18 column using middle chromatogram isolated gel column coupled with reversed-phase liquid chromatography.

Reversed-phase liquid chromatography coupled with middle chromatogram isolated gel column was employed for the efficient preparative separation of the arylbutanoid-type phenol [(-)-rhododendrin] from Saxifraga tangutica. Universal C18 (XTerra C18) and XCharge C18 columns were compared for (-)-rhododendrin fraction analysis and preparation. Although tailing and overloading occurred on the XTerra C18 column, the positively charged reversed-phase C18 column (XCharge C18) overcame these drawbacks, allowing for favorable separation resolution, even when loading at a on a preparative scale (3.69 mg per injection). The general separation process was as follows. First, 365.0 mg of crude (-)-rhododendrin was enriched from 165 g Saxifraga tangutica extract via a middle chromatogram isolated gel column. Second, separation was performed on an XTerra C18 preparative column, from which 73.8 mg of the target fraction was easily obtained. Finally, the 24.0 mg tailing peak of (-)-rhododendrin on XTerra C18 column was selectively purified on the XCharge C18 analytical column. These results demonstrate that the tailing nonalkaloid peaks can be effectively used for preparative isolation on XCharge C18 columns.

Two New Prenylated Indole Diterpenoids from Tolypocladium sp. and Their Antimicrobial Activities.

Two new prenylated indole diterpenoids, tolypocladins K and L (1 and 2), together with a known analog terpendole L (3), were isolated from the solid fermentation culture of a mine soil-derived fungus Tolypocladium sp. XL115. Their structures and relative configurations were determined by comprehensive spectroscopic data analysis, as well as by comparison of their NMR data with those related known compounds. Compound 3 exhibited remarkable antibacterial activity against Micrococcus luteus with an MIC value of 6.25 µg/mL, and compounds 1 and 3 displayed moderate antifungal activity selectively against tested strains with MIC values of 25-50 µg/mL.

Phytochemical Analysis, Antioxidant and Analgesic Activities of Incarvillea compacta Maxim from the Tibetan Plateau.

Incarvillea compacta Maxim is a traditional Tibetan plant widely used to treat rheumatic pain and bruises. We conducted qualitative analyses by liquid chromatography-mass spectrometry and quantitative analyses of the total phenols, flavonoids, and alkaloids content of different extracts of I. compacta Maxim. Antioxidant and analgesic activity were analyzed. The results showed that the methanol extract had the highest content of the various ingredients. A total of 25 constituents were identified, of which compounds 1-23 were found for the first time in this plant. The water extract had the highest capacity to clear free radicals in the antioxidant test. The water extract had dose-dependent analgesic effects in the first and second phase in a formalin test. The latency of pain from a hot-plate test was augmented by the water extract when the dose was greater than or equal to 30 g/kg. The water extract significantly decreased the amount of writhing in a dose-dependent manner compared with the control group in the acetic acid-induced writhing test. These results showed that I. compacta Maxim is a new antioxidant and analgesic agent, and this study provides information on its ingredients for further study.

Target separation of flavonoids from Saxifraga tangutica using two-dimensional hydrophilic interaction chromatography/reversed-phase liquid chromatography.

An orthogonally (80.3%) preparative two-dimensional hydrophilic interaction chromatography/reversed-phase liquid chromatography method has been established for the isolation and purification of flavonoids from Saxifraga tangutica. Initially, flavonoids were enriched by means of a middle-pressure chromatographic tower (containing middle chromatogram isolated gel). In the first dimension, a XION preparative column was used to separate the flavonoid fractions under the guidance of characteristic ultraviolet absorption spectra of flavonoids and nine flavonoid fractions were obtained. Then, the coeluted flavonoid fractions were selected for further purification via reversed-phase liquid chromatography with the parent ion peak of quercetin (303), kaempferol (287), or isorhamnetin (317). Several flavonoids could be separated from each hydrophilic interaction chromatography fraction; furthermore, flavonoids with poor resolution in one-dimensional liquid chromatography were isolated in two-dimensional liquid chromatography due to the orthogonality. In addition, this technique was valuable for trace flavonoids, which were concentrated in the first stage and separated in the second stage. In total, 18 flavonoids with either quercetin, kaempferol, or isorhamnetin parent nuclei were targetedly obtained, and 15 flavonoids were obtained for the first time from S. tangutica. These results established that the off-line two-dimensional hydrophilic interaction chromatography/reversed-phase liquid chromatography technique was efficient for the isolation of flavonoids from Saxifraga tangutica.

Efficient Separation of Four Antibacterial Diterpenes from the Roots of Salvia Prattii Using Non-Aqueous Hydrophilic Solid-Phase Extraction Followed by Preparative High-Performance Liquid Chromatography.

An efficient preparative procedure for the separation of four antibacterial diterpenes from a Salvia prattii crude diterpenes-rich sample was developed. Firstly, the XION hydrophilic stationary phase was chosen to separate the antibacterial crude diterpenes-rich sample (18.0 g) into three fractions with a recovery of 46.1%. Then, the antibacterial fractions I (200 mg), II (200 mg), and III (150 g) were separated by the Megress C18 preparative column, and compounds tanshinone IIA (80.0 mg), salvinolone (62.0 mg), cryptotanshinone (70.0 mg), and ferruginol (68.0 mg) were produced with purities greater than 98%. The procedure achieved large-scale preparation of the four diterpenes with high purity, and it could act as a reference for the efficient preparation of active diterpenes from other plant extracts.

Rapid and sensitive determination of multiple endocrine-disrupting chemicals by ultrasound-assisted in situ derivatization dispersive liquid-liquid microextraction coupled with ultra-high-performance liquid chromatography/tandem mass spectrometry.

RATIONALE: Endocrine-disrupting chemicals (EDCs) in environment samples and food stuffs are an increasing serious public health issue due to their potency to interfere and deregulate several aspects of the endocrine system. Because of their extremely low abundance, it remains a challenging task to develop a sensitive detection method. METHODS: 4'-Carbonyl chloride rosamine (CCR) was used as a derivatization reagent for EDCs for the first time. A new ultrasound-assisted in situ derivatization/dispersive liquid-liquid microextraction (UA-DLLME with in situ derivatization) method for multiple EDCs including five estrogens, two alkylphenols, eight bisphenols, seven parabens and triclosan coupled with ultra-high-performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) has been developed and validated. RESULTS: The ionization efficiency of EDCs was greatly enhanced through the introduction of a permanent charged moiety of CCR into the derivatives during electrospray ionization (ESI)-MS analysis. The main variables potentially affecting the UA-DLLME with in situ derivatization process are optimized. The recoveries and matrix effects of 23 EDCs for the spiking samples were in the range of 83.0-116.0% and 85.8-114.6%, respectively. Good method reproducibility was achieved. CONCLUSIONS: The limits of detection (LODs) for 23 EDCs were 0.05-0.40 ng/L and 0.03-0.25 ng/g (dry weight, d.w.) for environment samples and food stuffs, respectively. The proposed method has been demonstrated to be suitable for simultaneous determination of multiple EDCs in real samples with high sensitivity, speediness, and good sample clean-up ability. Copyright © 2017 John Wiley & Sons, Ltd.

Anti-inflammatory bioactive equivalence of combinatorial components ß-carboline alkaloids identified in Arenaria kansuensis by two-dimensional chromatography and solid-phase extraction coupled with liquid-liquid extraction enrichment technology.

Bioactive equivalent combinatorial components play a critical role in herbal medicines. However, how to discover and enrich them efficiently is a question for herbal pharmaceuticals researchers. In our work, a novel two-dimensional reversed-phase/hydrophilic interaction high-performance liquid chromatography method was established to perform real-time components trapping and combining for preparation and isolation of coeluting components. Arenaria kansuensis was taken as an example, and solid-phase extraction coupled with liquid-liquid extraction as a simple and efficient method for enriching trace components, reversed phase column coupled with hydrophilic interaction liquid chromatography XAmide column as two-dimensional chromatography technology for isolation and preparation of coeluting constituents, enzyme-linked immune-sorbent assay as bio-guided assay, and anti-inflammatory bioactivity evaluation for bioactive constituents. A combination of 12 ß-carboline alkaloids was identified as anti-inflammatory bioactive equivalent combinatorial components from A. kansuensis, which accounts for 1.9% w/w of original A. kansuensis. This work answers the key question of which are real anti-inflammatory components from A. kansuensis and provides a fast and efficient approach for discovering and enriching trace ß-carboline alkaloids from herbal medicines for the first time. More importantly, the discovery of bioactive equivalent combinatorial components could improve the quality control of herbal products and inspire a herbal medicine based on combinatorial therapeutics.

Preparative isolation of flavonoid glycosides from Sphaerophysa salsula using hydrophilic interaction solid-phase extraction coupled with two-dimensional preparative liquid chromatography.

An offline preparative two-dimensional reversed-phase liquid chromatography/hydrophilic interaction liquid chromatography coupled with hydrophilic interaction solid-phase extraction method was developed for the preparative isolation of flavonoid glycosides from a crude sample of Sphaerophysa salsula. First, the non-flavonoids were removed using an XAmide solid-phase extraction cartridge. Based on the separation results of three different chromatographic stationary phases, the first-dimensional preparation was performed on an XAqua C18 prep column, and 15 fractions were obtained from the 5.2 g target sample. Then, three representative fractions were selected for additional purification on an XAmide preparative column to further isolate the flavonoid glycosides. In all, eight flavonoid glycosides were isolated in purities over 97%. The results demonstrated that the two-dimensional liquid chromatography method used in this study was effective for the preparative separation of flavonoid glycosides from Sphaerophysa salsula. Additionally, this method showed great potential for the separation of flavonoid glycosides from other plant materials.

Efficient separation of high-purity compounds from Oxytropis falcata using two-dimensional preparative chromatography.

The separation of high-purity compounds from traditional Tibetan medicines plays an important role in investigating their bioactivity. Nevertheless, it is often quite difficult to isolate compounds with high purity because of the complexity of traditional Tibetan medicines. In this work, an offline two-dimensional reversed-phase preparative method was successfully developed for the separation of high-purity compounds from Oxytropis falcata. Based on the analysis results, an ODS C18 prep column was used for first-dimensional preparation, and 14.8 g of the crude sample was separated into five fractions with a recovery of 74.6%. Then, an XAqua C18 prep column was used to isolate high-purity compounds in the second-dimensional preparation because its separation selectivity is different with the ODS C18 stationary phase. As a result, eight compounds in the crude sample were isolated in more than 98% purity. This is the first report of trans-cinnamic acid (1) and trifolirhizin (2) from Oxytropis falcata. This method has the potential to be an efficient separation method of high-purity compounds from Oxytropis falcata and it shows great promise for the separation of high-purity compounds from complex samples.