Targeted isolation of cyclooxygenase-2 inhibitors from Saussurea obvallata using affinity ultrafiltration combined with preparative liquid chromatography.

Saussurea obvallata (S. obvallata) is widely used in Qinghai-Tibet Plateau with high medicinal and edible values of reducing inflammation. But, the individual components and mechanisms of action still ill-defined. In this work, an integrated method using affinity ultrafiltration combined with preparative liquid chromatography was developed to identify and separate cyclooxygenase-2 (COX-2) inhibitors from S. obvallata. The sample was pretreated using on-line medium pressure liquid chromatography to yield the active fraction. Then, the potential COX-2 ligands were screened out using affinity ultrafiltration from the targeted fraction and the identified compounds were isolated via preparative liquid chromatography. As a result, four main compounds, coniferin (1), syringin (2), roseoside (3) and grasshopper ketone (4) were targeted isolated with IC50 values of 12.34 ± 1.81, 4.04 ± 0.43, 13.91 ± 2.46 and 7.97 ± 1.21 µM, respectively. Results of this work demonstrated that the developed strategy was effective for the targeted separation of COX-2 inhibitors from natural product extracts.

ABSCISIC ACID-INSENSITIVE 5-ω3 FATTY ACID DESATURASE3 module regulates unsaturated fatty acids biosynthesis in Paeonia ostii.

Paeonia ostii is an authorized novel vegetable oil crop due to its seeds rich in unsaturated fatty acids (UFAs) especially α-linolenic acid (ALA), which overweight the current available edible oil. However, little is known on the regulation mechanism of UFAs biosynthesis during its seed development. Here, we used transcriptome and proteome data combining phytochemistry means to uncover the relationship between abscisic acid (ABA) signaling and UFAs biosynthesis during P. ostii seed development. Based on transcriptome and proteome analysis, two desaturases of omega-6 and omega-3 fatty acid, named as PoFAD2 and PoFAD3 responsible for ALA biosynthesis were identified. Then, an ABSCISIC ACID-INSENSITIVE 5 (ABI5) proteins was identified as an upstream transcriptional factor, which activated the expression of PoFAD3 instead of PoFAD2. Moreover, silencing of PoABI5 repressed the response of PoFAD3 to ABA. This study provides the first view on the connection between the function of ABA signaling factors and ALA biosynthesis in the P. ostii seed, which lays the foundation for studies on the regulatory mechanism of ABA signaling involved in the UFAs synthesis during seeds development, meanwhile, it will shed light on manipulation of ALA content for satisfying human demands on high quality of edible oil or healthy supplement.

Quantitative analysis of resveratrol derivatives in the seed coats of tree peonies and their hypoglycemic activities in vitro/vivo.

Tree peonies are well-known horticultural and medicinal plants. The tree peony seeds, as emerging woody oil crops, recently have attracted great attention for their metabolites and bioactivities. In this study, the phytochemicals isolated from tree peony seed coats were systematically investigated. Seven polyphenolics were separated and prepared, mainly belonging to resveratrol derivatives. There was a great variation in the seed coat polyphenolic content among eight Paeonia species, and the contents of the resveratrol trimers and dimers were significantly higher in the seed coats of Paeonia ostii than other species. Based on the HPLC fingerprint characteristics and chemometric analysis, a clear discrimination among Paeonia plants was found, including the composition patterns and contents of the constituents. Moreover, the characteristic phytochemicals (vateriferol and trans-ε-viniferin) could significantly reduce the starch-mediated levels of postprandial blood glucose in diabetic/normal mice. In addition, in vitro enzyme tests showed that the two compounds could effectively and competitively inhibit α-glucosidase, with the IC50 values of 3.01 and 7.75 µM, respectively, indicating that vateriferol and trans-ε-viniferin could be therapeutic potential agents for hyperglycemia and diabetes mellitus.

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.

Phytochemical profiles and the hypoglycemic effects of tree peony seed coats.

As emerging woody oil crops, the tree peony seeds recently have been attracting great attention for their metabolites and bioactivities. In this research, the phytochemical profiles of the seed coats of tree peonies from different production regions were investigated systematically. Twelve phytochemicals were separated and prepared, mainly belonging to stilbenes. A great variation in stilbene content was detected in the three Paeonia plants, and Paeonia ostii seed coats (POSC) had significantly higher contents of the stilbene compounds than other species. There were nineteen significant correlations between ecogeographical factors and the predominant compounds. A clear discrimination among the species was observed in their HPLC fingerprint and chemometric analysis. Furthermore, POSC extracts could significantly reduce the starch mediated PBG (postprandial blood glucose) levels in normal/diabetic mice. Meanwhile, in vitro enzyme tests revealed that the predominant compounds, suffruticosol B and ampelopsin D, could effectively and competitively inhibit α-glucosidase, indicating that POSC could be a natural source of hypoglycemics in the food and drug fields.

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.

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.

Flavonoid compounds isolated from Tibetan herbs, binding to GABAA receptor with anxiolytic property.

ETHNOPHARMACOLOGICAL RELEVANCE: Previously, the phytochemical constituents of Biebersteinia heterostemon Maxim (BHM) and Arenaria kansuensis Maxim (AKM) were studied and the evaluation of anxiolytic effect based on their extracts was also investigated. The two traditional Tibetan herbs, BHM and AKM, have been widely used in Qinghai-Tibet Plateau for cardiopulmonary disorders and neuropsychiatric diseases. The anxiolytic activities of a number of agents mediated by α2/3-containing GABAA receptors (GABAARs) have been demonstrated through the genetic and pharmacological studies. Flavonoids, such as flavones and flavanols, are a class of ligands that act at GABAARs and exhibit anxiolytic effects in vivo. Here, the flavonoids are the predominant constituents isolated from BHM and AKM. And our purpose is to investigate structure-activity relationships of the flavonoid compounds with binding to BZ-S of GABAAR complexes, and to search for anxiolytic constituents that lack undesirable-effects such as sedation and myorelaxation. MATERIALS AND METHODS: The flavonoid constituents were separated and purified through the repeatedly silica gel or/and C18 column chromatography. The affinities of the compounds for BZ-S of GABAARs were detected by the radioreceptor binding assay with bovine cerebellum membranes, in which the different recombinant subunits-containing GABAARs were expressed in HEK 293T cells. The behavior tests, including elevated plus maze, locomotor activity, holeboard, rotarod and horizontal wire, were used to determine and evaluate the anxiolytic, sedative, and myorelaxant effects of these flavonoids. RESULTS: Eleven total flavonoid compounds were obtained from the Tibetan herbs (BHM and AKM). The flavones with 6-and/or 8-OMe possessed the most potent binding affinity to GABAARs, which were based on the result of structure-activity relationships analysis. Demethoxysudachitin (DMS, Ki = 0.59 µM), a flavone that binds to recombinant α1-3/5 subunit-containing GABAARs, was isolated from BHM, and exhibited high anxiolytic activity, without inducing sedation and myorelaxation. Moreover, the anxiolytic effect of DMS was antagonized by flumazenil, suggesting that a mode of action was mediated via the BZ-S of GABAARs. CONCLUSIONS: This present study indicated that the flavones, especially DMS, are novel GABAAR ligands and therapeutic potential candidates for anxiety.

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.

Ultrasonic extraction of anthocyanins from Lycium ruthenicum Murr. and its antioxidant activity.

The ultrasonic extraction (UE) technology, possessed the advantages of effective, energy-saving, and environmental-friendly, was applied to extract the anthocyanin from Lycium ruthenicum (LR). The extraction parameters of UE were optimized by response surface methodology (RSM) with Box-Behnken design (BBD). Anthocyanin composition in LR fruits grown in China was systematically identified and quantified by HPLC-ESI-MS. The result showed that PRG was the major anthocyanin, and delphinidin, petunidin, and malvidin were the major anthocyanidins in LR fruits. There was the same anthocyanin composition of LR and great variation in anthocyanins content of LR from different areas in China. However, there was no significant difference between wild and cultivated LR in the same region. A clear separation of LR according to geographical origins was revealed by hierarchical cluster analysis (HCA) and principal component analysis (PCA), and the discrimination model for the anthocyanin concentrations were developed using these two analysis methods. Furthermore, on-line HPLC-DPPH assay and scavenging activity of three kinds of radicals (DPPH·, ·OH, and O 2 - · ) in vitro were well applied to evaluate the antioxidant activity of the LR anthocyanin extract (LRAE). And its results indicated the LRAE could be a credible antioxidant agent for applications in cosmetics, food, and medicine.

Functional MYB transcription factor encoding gene AN2 is associated with anthocyanin biosynthesis in Lycium ruthenicum Murray.

BACKGROUND: Lycium ruthenicum Murray is an important economic plant in China and contains higher levels of anthocyanins in its fruits than other Lyciums. However, the genetic mechanism of anthocyanin production in this plant is unknown. RESULTS: Based on previous transcriptome analysis, LrAN2 and LbAN2, encoding MYB transcription factors, were isolated from L. ruthenicum and L. barbarum, respectively. Both genes contained two introns, encoded 257 amino acids with two-Aa difference, and carried the unabridged HTH-MYB, MYB-like DNA-binding, and SANT domains. In the phylogenetic trees, LrAN2 and LbAN2 were found to be closely related to NtAN2, which regulates anthocyanin biosynthesis in tobacco. Overexpression of LrAN2 and LbAN2 induced anthocyanin biosynthesis in all tissues of tobacco. The anthocyanin content in the leaves of transgenic lines with LbAN2 was lower than LrAN2. It indicated that the function of LbAN2 was weaker than LrAN2. The AN2 transcript could be detected only in the fruits of L. ruthenicum and increased during fruit development, accompanied by anthocyanin accumulation. In natural population, the alleles LrAN2 and LrAN2 were associated strictly with L. ruthenicum and L. barbarum, respectively. Moreover, an AN2 genetic diversity study suggested that Lyciums with yellow, white, purple, and jujube red fruits were derived from L. ruthenicum. CONCLUSIONS: Two AN2 alleles, from L. ruthenicum and L. barbarum, were functional MYB transcriptor regulating anthocyanin biosynthesis. The functional diversity and high expression level of LrAN2 could be the reason for high anthocyanin content in the fruit of L. ruthenicum. Lyciums with yellow, white, purple, and jujube red fruits were derived from L. ruthenicum based on AN2 sequence diversity. The results may be advantageous in identifying new varieties and breeding new cultivars.

Flavonoids isolated from Tibetan medicines, binding to GABAA receptor and the anticonvulsant activity.

BACKGROUND: Our previous studies on Asterothamnus centrali-asiaticus Novopokr. (ACN) and Arenaria kansuensis Maxim. (AKM) had led to the isolation of some phytochemical constituents and evaluation of anticonvulsant effect based on their extracts. ACN and AKM have been widely used in traditional Tibetan herbs for neuropsychiatric diseases and cardiopulmonary disorders. PURPOSE: The purpose is to investigate structure-activity relationships of flavonoids isolated from ACN and AKM, for binding to the benzodiazepine site (BZ-S) of γ-aminobutyric acid type A (GABAA) receptor complex, and to search for anticonvulsant compounds without undesirable effects such as myorelaxation and sedation. STUDY DESIGN AND METHODS: The affinities of these flavonoids for the BZ-S of GABAA receptors were determined by [3H]flunitrazepam binding to mouse cerebellum membranes in vitro. And the anticonvulsant, myorelaxant and sedative effects were determined by pentylenetetrazol (PTZ)-induced seizure and electrogenic seizure protection, rotarod test and locomotor activity test, respectively. RESULTS: Fifteen and thirteen flavonoids were isolated from ACN and AKM, respectively. Structure-activity relationships analysis indicated that 6-and/or 8-OMe flavones exhibited the most potent binding affinity to GABAA receptors. Furthermore, 2',4',5,7-tetrahydroxy-5',6-dimethoxyflavone (DMF, IC50 value of 0.10 µM), a flavone isolated from ACN, presented high anticonvulsant activity against chemical-induced seizures and electrogenic seizures, without myorelaxation and sedation. CONCLUSION: This study suggested that these flavones, especially DMF, are new BZ receptor ligands and prospective therapeutic candidates for seizures.

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.

Preparative isolation of antioxidative compounds from Dracocephalum heterophyllum using off-line two-dimensional reversed-phase liquid chromatography/hydrophilic interaction chromatography guided by on-line HPLC-DPPH assay.

Traditional Tibetan medicine (TTM) has been valuable for the identification of new therapeutic leads. Nevertheless, reports about the chemical constituents of TTM are meager owing to the lack of suitable purification techniques. In this study, an off-line two-dimensional reversed-phase/hydrophilic interaction liquid chromatography (2D RP/HILIC) technique guided by on-line HPLC-DPPH has been established for the isolation of pure antioxidants from the extract of Dracocephalum heterophyllum. According to the chromatographic recognition outcome of the HPLC-DPPH system, the first-dimensional (1D) separation on the Megress C18 preparative column yielded 6 antioxidative fractions (61.4% recovery) from the ethyl acetate fraction (6.1 g). In the second-dimensional (2D) separation, a HILIC XAmide preparative column was employed. In total, 8 antioxidants were isolated from D. heterophyllum with a purity of >95%, which indicated the efficiency of the developed method to prepare antioxidative compounds with high purity from plant extracts. In addition, this method was highly efficient for the preparation of structural analogues of the antioxidative polyphenols and could be applied for the purification of structural analogues from other resources.

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.

Antihypoxic activities of constituents from Arenaria kansuensis.

BACKGROUND: In previous investigation, we have identified antioxidative effects of water-soluble ethanolic extracts (named as AKE) from Arenaria kansuensis and inferred that these extracts or their constituents may also have antihypoxic activity. A. kansuensis has been widely used in traditional Tibetan medicine for altitude sickness (AS) and has been known as the herb of anti-inflammatory and hypoxia resistance for a long time. PURPOSE: The purpose of this study is to evaluate protective effects of AKE and its major constituents against hypoxia-induced lethality in mice and RSC96 cells. STUDY DESIGN AND METHODS: Hypoxia-induced lethality in mice was investigated by 3 experimental animal models of hypoxia. Meanwhile, we established a RSC96 cell model of hypoxia which applied to screen and assess the anti-hypoxic activity of compounds isolated from A. kansuensis. RESULTS: Results indicated that AKE dose-dependently prolonged survival time of hypoxia induced lethality in mice compared to vehicle group and exhibited significantly anti-hypoxic effect. AKE also enhanced the number of red blood cells (RBC) and the concentration of hemoglobin (HB). 8 compounds were bio-guided separated and purified from AKE based on the animal model and cell model of hypoxia. Among which pyrocatechol (C16) and tricin 7-O-ß-d-glucopyranoside (C13) were confirmed to express better protective effects on cell damage induced by hypoxia, suggesting that these two compounds are major active constituents of AKE for anti-hypoxia. CONCLUSION: This study demonstrated that pyrocatechol and tricin 7-O-ß-d-glucopyranoside could be therapeutic candidates for treatment of AS. It is the first time to find the major active constituents of AKE for anti-hypoxia. Meanwhile, a RSC96 cell model of hypoxia was established to screen anti-hypoxic activity of compounds for the first time.

Erythritol Attenuates Postprandial Blood Glucose by Inhibiting α-Glucosidase.

Diabetes mellitus (DM) is a serious metabolic disorder, where impaired postprandial blood glucose regulation often leads to severe health complications. The natural chemical erythritol is a C4 polyol approved by the U.S. Food and Drug Administration for use as a sweetener. Here, we examined a potential role for erythritol in the control of postprandial blood glucose levels in DM. An anti-postprandial hyperglycemia effect upon erythritol administration (500 mg kg-1) was demonstrated in alloxan-induced DM model mice by monitoring changes in blood glucose after intragastric administration of drugs and starch. We also found that erythritol most likely exerts its anti-postprandial hyperglycemic activities by inhibiting α-glucosidase in a competitive manner. This was supported by enzyme activity assays and molecular modeling experiments. In the latter experiments, it was possible to successfully dock erythritol into the catalytic pocket of α-glucosidase, with the resultant interaction likely driven by electrostatic interactions involving Asp215, Asp69, and Arg446 residues. This study suggests that erythritol may not only serve as a glucose substitute but also be a useful agent in the treatment of DM to help manage postprandial blood glucose levels.