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.

Hypoglycemic effects of Rhodiola crenulata (HK. f. et. Thoms) H. Ohba in vitro and in vivo and its ingredient identification by UPLC-triple-TOF/MS.

Rhodiola crenulata (HK. f. et. Thoms) H. Ohba (RC), mainly distributed in the highly cold region of China, has long been used as a medicine/healthy food for eliminating fatigue and increasing blood circulation. This study aimed to evaluate the inhibitory effects of the RCRS extract on α-amylase and α-glucosidase (sucrase and maltase) in vitro and in vivo, and tentatively analyze and identify its chemical ingredients using UPLC-Triple-TOF/MS. The Rhodiola crenulata RCRS extract had strong inhibitory activities against α-amylase, sucrase and maltase with an IC50 of 0.031 mg mL-1, 0.142 mg mL-1 and 0.214 mg mL-1, respectively. Furthermore, the RCRS extract could significantly decrease the postprandial blood glucose (PBG) level of normal mice in a starch tolerance test, and reduce the PBG levels of diabetic mice in a starch/maltose/sucrose tolerance test. UHPLC-Triple-TOF-MS/MS analysis indicated that hydroxybenzoic acids, hydroxycinnamic acids, alcohol glycosides, flavonols and their derivatives were the main active ingredients in the RCRS extract. The results demonstrate that the RCRS extract of Rhodiola crenulata could be employed as a healthy food or medicine for controlling postprandial blood glucose levels.

Bioactivity-guided isolation of cyclooxygenase-2 inhibitors from Saussurea obvallata (DC.) Edgew. Using affinity solid phase extraction assay.

ETHNOPHARMACOLOGICAL RELEVANCE: Saussurea obvallata (DC.) Edgew. is a traditional Tibetan medicine used for the treatment of inflammation-related diseases, but the scientific validation was very limited. AIM OF THE STUDY: This study aimed to rapid screen and targeted isolate cyclooxygenase-2 (COX-2) inhibitors from S. obvallata extract. MATERIALS AND METHODS: An efficient ligand-fishing method based on affinity solid phase extraction (A-SPE) combining with HPLC was developed. The identified COX-2 inhibitors were separated using preparative liquid chromatography. In vitro COX-2 inhibition assays were employed to confirm the inhibitory activities of the isolated compounds. In addition, the effect of the isolated compounds on the production of prostaglandin E2 (PGE2) and the expression of COX-2 in LPS-induced RAW 264.7 were evaluated. RESULTS: A total of four phenylpropanoids, isolariciresinol, syringaresinol, pinoresinol and balanophonin were targeted isolated as COX-2 inhibitors with IC50 values of 36.4 ± 2.6 µM, 23.1 ± 1.8 µM, 3.6 ± 0.3 µM and 12.1 ± 0.9 µM, respectively. The isolated compounds significantly inhibited LPS-induced NO production in a dose-dependent manner. And, the results of the inhibitory effect on the release of PGE2 and the expression of COX-2 in LPS-induced macrophages were consistent with A-SPE analysis. CONCLUSION: The present work demonstrated that the developed A-SPE-HPLC method could successfully targeted isolated COX-2 inhibitors from S. obvallata extract. And, the isolation results indicated that the therapeutic effect of S. obvallata on inflammation-related diseases was partly based on the COX-2 active ingredients.

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.

Comprehensive screening and separation of cyclooxygenase-2 inhibitors from Pterocephalus hookeri by affinity solid-phase extraction coupled with preparative high-performance liquid chromatography.

Pterocephalus hookeri, a classical Tibetan herb, is mainly used to treat rheumatoid arthritis (RA) and contains various constituents potentially with cyclooxygenase-2 (COX-2) selective inhibition. A novel strategy for screening and target separating COX-2 inhibitors from the extracts of P. hookeri based on affinity solid-phase extraction (ASPE) column combined with preparative high-performance liquid chromatography (pre-HPLC) was successfully developed. The potential COX-2 inhibitors of P. hookeri were screened and recognized by the ASPE-HPLC system, which strategy is to analyze the compounds isolated by the ASPE column. Then, the active compounds were targeted separated by pre-HPLC according to real-time chromatograms. The control drugs celecoxib and glipizide were analyzed to verify the specificity and accuracy of the developed method. As a result, two pure compounds with COX-2 binding affinities were successfully separated from P. hookeri. They were characterized as swertisin and scopoletin using 1H- and 13C NMR spectroscopy, and the in vitro COX-2 inhibitory activities were verified. Compounds with COX-2 inhibitory activities could be screened and targeted separated from crude extracts by this strategy, which indicated that the proposed method was feasible, robust and effective for rapid separation of COX-2 inhibitors from natural products.

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.

Alkaloids and phenolics identification in fruit of Nitraria tangutorum Bobr. by UPLC-Q-TOF-MS/MS and their a-glucosidase inhibitory effects in vivo and in vitro.

Nitraria tangutorum Bobr. (NTB), mainly distributed in the Qaidam Basin of Tibetan Plateau, have high economic, ecological and medicinal value. The chemical compositions in the NTB fruits were tentatively analyzed and characterized by applying UPLC-Q-TOF-MS/MS. Total 45 constituents, including 9 hydroxycinnamic acids derivatives, 12 flavonols, 4 flavonoids, 1 trolox derivative, 8 ß-carboline alkaloids, 4 tryptophan derivatives, and 7 other amino acid derivatives were identified by comparing with standard products, and analyzing their retention times, characteristic fragment ions and deprotonated molecule ions. The activity studies in vitro indicated that NTB-Z and NTB-C extracts had marked inhibitory effects against sucrase and maltase. Further sucrose/maltose/starch tolerance experiment demonstrated that both NTB-Z and NTB-C extracts at 400 mg/kg could markedly lower the postprandial blood glucose (PBG) level in diabetic animals. All these results indicated that the NTB fruits could be used as the functional health food or medicine for controlling postprandial blood glucose level.

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.

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.

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.

A new flavonol acylglycoside from the fruits of Nitraria tangutorum Bobr.

Phytochemical investigation on the ethanol extract of the fruits of Nitraria tangutorum Bobr. led to the isolation of a new flavonol acylglycoside tangutoside (7) and six known ones including p-formylphenol (1), 3-formylindole (2), rutin (3), prionitisid B (4), quercitrin (5) and flazin (6). Among them, compounds 2, 4, and 5 were isolated from Nitraria tangutorum Bobr. for the first time. Their structures were identified by spectroscopic methods and by comparison with data reported in the references. The compounds 3, 4, 5, and 7 were demonstrated to possess strong DPPH radical scavenging ability with IC50 values of 31.45 µg/mL, 19.21 µg/mL, 59.30 µg/mL, and 44.54 µg/mL, respectively.

Anti-rheumatoid arthritis effects of iridoid glucosides from Lamiophlomis rotata (Benth.) kudo on adjuvant-induced arthritis in rats by OPG/RANKL/NF-κB signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Lamiophlomisrotata (Benth.) Kudo. has been used to treat trauma bleeding, rheumatism, yellow water disease in traditional Chinese medicine. AIM: The aim of this work was to evaluate the anti-rheumatoid arthritis (RA) activities and underlying mechanisms of the total iridoid glucosides (TIG) from Lamiophlomisrotata (Benth.) Kudo. METHODS: The chemical constituents of TIG was analyzed by high-performance liquid chromatography (HPLC) with seven reference compounds (penstemonoside, chlorotuberside, shanzhiside methyl ester, phloyoside, 7-epliamalbide, phlorigidoside C and lamalbide). The anti-rheumatoid arthritis effects of TIG were investigated by arthritis indexes and paw swelling degrees, as well as histopathological and Micro-CT analysis in adjuvant-induced arthritis (AIA) rats. The impacts of TIG on the level of inflammatory cytokines (IL-1ß, TNF-α, IL-6, IFN-γ, IL-17 and IL-10), and the regulation of OPG/RANKL/NF-κB pathways were determined by the ELISA and western blot, respectively. RESULTS: TIG significantly reduced the arthritis indexes and paws swelling in AIA rats, attenuated the inflammation and bone destruction in joint tissues, reduced the generation of pro-inflammatory cytokines IL-1ß, TNF-α, IL-6, IFN-γ and IL-17, as well as increased the generation of anti-inflammatory cytokine IL-10 in serum. Moreover, TIG markedly inhibited the expression of p-IKK-α, p-IκB and p-p65, and decreased the ratio of OPG/RANKL in the synovial tissues. CONCLUSION: TIG possessed significant anti-RA activities on adjuvant-induced arthritis, which might be ascribed to the regulation of inflammatory cytokines IL-1ß, TNF-α, IL-6, IFN-γ IL-17 and IL-10, as well as inhibition of OPG/RANKL/NF-κB signaling pathways.

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.

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 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.

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.

ß-carboline alkaloids attenuate bleomycin induced pulmonary fibrosis in mice through inhibiting NF-kb/p65 phosphorylation and epithelial-mesenchymal transition.

ETHNOPHARMACOLOGICAL RELEVANCE: The plant Arenaria kansuensis is used in traditional medicine to treat lung inflammation for a long time. However, the anti-pulmonary fibrosis effect and its corresponding bioactive constituents of this plant have not been studied extensively. AIM OF THE STUDY: The purpose of this study was to investigate the anti-pulmonary fibrosis effect and its corresponding bioactive constituents of A. kansuensis and its possible mechanism. MATERIALS AND METHODS: In vivo experiment, the anti-pulmonary fibrosis effects of the fraction (Part1) enriched from ethyl acetate extracts of the whole plant A. kansuensis were evaluated through bleomycin (BLM)-induced pulmonary fibrosis mice (five groups, n = 10) daily at doses of 50, 100 and 150 mg/kg for 15 days. In vitro experiment, the anti-inflammation and reversed epithelial-mesenchymal transition (EMT) effect of 12 ß-carboline alkaloids isolated from Part1 were evaluated through lipopolysaccharide (LPS)-induced RAW264.7 inflammatory cell model and TGF-ß1 induced A549 cell model. RESULTS: In this study, a fraction named Part1 extracted from Arenaria kansuensis presented strong anti-pulmonary fibrosis effect at the dose of 150 mg/kg. Vivo experiments showed that the survival rate and body weight of mice significantly increased after Part1 treatment. Part1 could significantly inhibit the initial of inflammation, deposition of collagen and expression of TGF-ß1 and α-SMA, moreover, the expression of E-cadherin was significantly elevated after administration of Part1. All the cure effects of Part1 were in dose dependent manner. A total of 12 ß-carboline alkaloids were identified in Part1 and they all showed suppressive effect on inflammatory cytokines including MCP-1, TNF-α, IL-6 and IL-1ß through inhibition of NF-kb/p65 phosphorylation, and that epithelial-mesenchymal transition (EMT) process was reversed by different compounds in different levels. The expression of indicators of EMT including α-SMA, vimentin and E-cadherin was significantly improved after given different ß-carboline alkaloids. CONCLUSIONS: This study showed that antifibrogenic effect of ß-carboline alkaloids was due to inhibiting the initial of inflammation through NF-kb/p65 pathway and reversing the process of EMT.

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.

Purification of Flavonolignan Diastereoisomers from Arenaria kansuensis by Two-Dimensional Liquid Chromatography Combined with Solid-Phase Extraction.

Herbal plants are significant for the reason that they have a great potential in discovering drug precursors. However, how to purify compounds with higher purity from them is a question which needs to be discussed. In present study, an offline 2D reversed-phase (RP) preparative liquid chromatography coupled with solid-phase extraction (SPE) method was successfully developed for the separation of flavonolignan diastereoisomers from Arenaria kansuensis. Based on the analysis of results, the major conclusion that we have drawn from it is a RP-SPE was selected for enriching target flavonolignan sample from A. kansuensis. After that, an ODS preparative column was used for 1D preparation, and the target sample (4.6 g) was divided into five fractions with a recovery of 83.9%. Then, a C18HCE preparative column, a polar-modified RP (polar-copolymerized) type, was used for isolating flavonolignan diastereoisomers in the 2D preparation. By establishing optimal 2D chromatography, hydrophilic interaction chromatography (HILIC) columns and normal-phase (NP) columns were tested simultaneously, and the result showed that diastereoisomers are not suitable for HILIC and NP chromatography mode. Our study resulted in a tricin and five analogous derivative flavonolignans with purity >98% were successfully purified from A. kansuensis. This is the initial report of Salcolin C, Salcolin B, Tricin 4'-O-(C-veratroylglycol) ether and 5'-methoxyhydnocarpin D from A. kansuensis. In addition, it tended to be the first time that Tricin 4'-O-(C-veratroylglycol) ether is isolated from natural resource. This method has great potential for efficiently isolating flavonolignan diastereoisomers from A. kansuensis, and it shows a great prospect for the separation of flavonolignans from complex samples.

On-line HPLC-DPPH bioactivity-guided assay for isolated of antioxidative phenylpropanoids from Qinghai-Tibet Plateau medicinal plant Lancea tibetica.

Lancea tibetica is an important traditional Tibetan medicinal plant that grows on the Qinghai-Tibet Plateau with great development potential in pharmaceutical industry. In this study, a combinative method using HPLC-DPPH and two-dimensional liquid chromatography has been developed to identify and separate antioxidants from Lancea tibetica. Under the target-guidance of HPLC-DPPH experiment, three antioxidant fractions from Lancea tibetica were recognized. Then, separation of the three fractions using two-dimensional semi-preparation liquid chromatography led to seven phenylpropanoids: (+)-pinoresinol-ß-D-glucoside (1), isoacteoside (2), acteoside (3), tibeticoside (4),epipinoresinol (5), anthelminthicol (6) and phillygenol (7). As a result, seven major antioxidants in Lancea tibetica were isolated with more than 96% purity. Furthermore, in vitro bioassay against DPPH revealed compounds 1-7 with IC50 values ranging from 6.16 ±â€¯0.08 to 25.09 ±â€¯0.11 (µM) and compounds 1, 2 and 3 showed activities stronger than the two reference antioxidants (vitamin C, rutin), with IC50 values of 6.16 ±â€¯0.08, 8.93 ±â€¯0.06 and 7.98 ±â€¯0.05 (µM), respectively. Results of the present study indicated that the method was an efficient technique to systematically screen and isolate antioxidants from medicine crops.