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.

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.

Actividad antiproliferativa de Aldama arenaria (Baker) E. E. Schill. & Panero / Antiproliferative activity from Aldama arenaria (Baker) E. E. Schill. & Panero

El extracto de cloroformo (CE) y las fracciones obtenidas de las raíces de Aldama arenaria se evaluaron para determinar su actividad antiproliferativa in vitro contra 10 líneas celulares tumorales humanas [leucemia (K-562), mama (MCF-7), ovario que expresa un fenotipo resistente a múltiples fármacos (NCI/ADR-RES), melanoma (UACC-62), pulmón (NCI-H460), próstata (PC-3), colon (HT29), ovario (OVCAR-3), glioma (U251) y riñón (786-0)]. CE presentó actividad antiproliferativa débil a moderada (log GI50 medio 1.07), mientras que las fracciones 3 y 4, enriquecidas con diterpenos de tipo pimarane [ent-pimara-8 (14), ácido 15-dien-19-oico y ent-8(14),15-pimaradien-3ß-ol], presentaron actividad moderada a potente para la mayoría de las líneas celulares, con un log GI50 medio de 0.62 y 0.59, respectivamente. Los resultados mostraron una acción antiproliferativa in vitro prometedora de las muestras obtenidas de A. arenaria, con los mejores resultados para NCI/ADR-RES, HT29 y OVCAR-3, y valores de TGI que van desde 5.95 a 28.71 µg.mL-1, demostrando que los compuestos de esta clase pueden ser prototipos potenciales para el descubrimiento de nuevos agentes terapéuticos.

Chloroform extract (CE) and fractions obtained from Aldama arenaria roots were evaluated for their in vitro antiproliferative activity against 10 human tumor cell lines [leukemia (K-562), breast (MCF-7), ovary expressing a multidrug-resistant phenotype (NCI/ADR-RES), melanoma (UACC-62), lung (NCI-H460), prostate (PC-3), colon (HT29), ovary (OVCAR-3), glioma (U251), and kidney (786-0)]. CE presented weak to moderate antiproliferative activity (mean log GI50 1.07), whereas fractions 3 and 4, enriched with pimarane-type diterpenes [ent-pimara-8(14),15-dien-19-oic acid and ent-8(14),15-pimaradien-3ß-ol], presented moderate to potent activity for most cell lines, with mean log GI50 of 0.62 and 0.59, respectively. The results showed promising in vitro antiproliferative action of the samples obtained from A. arenaria, with the best results for NCI/ADR-RES, HT29, and OVCAR-3, and TGI values ranging from 5.95 to 28.71 µg.mL-1, demonstrating that compounds of this class may be potential prototypes for the discovery of new therapeutic agents.

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.

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.

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.

Trace anti-inflammatory ß-carboline alkaloid identified in Arenaria kansuensis by two-dimensional chromatography coupled with UniElut C18AEX based solid-phase extraction re-enrichment technology.

Traditional Chinese medicine is important for discovery of drug precursors. However, information about trace chemical composition of them is very limited due to the lack of appropriate enrichment and chromatographic purification methods In our work, A. kansuensis was taken as an example, a novel two-dimensional reversed-phase/hydrophilic interaction liquid chromatography coupled with UniElut C18AEX solid-phase extraction re-enrichment method based on anti-inflammatory bioactivity-guided assay was developed for gathering and purifying trace ß-carboline alkaloids with high purity from the ethyl acetate extract of A. kansuensis. Extraction with ethyl acetate as the first enrichment method, then, a UniElut C18AEX column was employed to re-enrich trace fraction which was hardly detected by diode array detector during high performance liquid chromatography analysis, eighteen grams of UniElut C18AEX was used as sorbent material to pack a 60mL pipette tip for the extraction of ß-carboline alkaloids from 100mL of ethyl acetate sample. The whole extraction process was finished in 10min, and the volume of eluent used was only 120mL. The enriching fraction (100mg) was used for the following two-dimensional purification. First-dimensional preparation was carried on a RP-Megress-C18 prep column, and four anti-inflammatory fractions were obtained from the 100mg re-enriching sample with a recovery of 66.9%. A HILIC-XAmide prep column was selected for the second dimensional preparation. Finally, two pair of analogue ß-carboline alkaloids and one other ß-carboline alkaloid were purified from A. kansuensis. The purity of the isolated compounds was ≫>98%, which indicated that the method was efficient to re-enrich and manufacture single trace ß-carboline alkaloids with high purity from A. kansuensis. Additionally, this method showed great potential to serve as a good example for the purification and enrichment of analogue structure anti-inflammation carboline alkaloids from other plant materials.

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.

Two-dimensional chromatography based on on-line HPLC-DPPH bioactivity-guided assay for the preparative isolation of analogue antioxidant compound from Arenaria kansuensis.

Traditional Tibetan medicine is important for discovery of drug precursors. However, information about the chemical composition of traditional Tibetan medicine is very limited due to the lack of appropriate chromatographic purification methods. In the present work, A. kansuensis was taken as an example and a novel two-dimensional reversed-phase/hydrophilic interaction liquid chromatography(HILIC) method based on on-line HPLC-DPPH bioactivity-guided assay was developed for the purification of analogue antioxidant compounds with high purity from the extract of A. kansuensis. Based on the separation results of many different chromatographic stationary phases, the first-dimensional (1D) preparation was carried on a RP-C18HCE prep column, and 2 antioxidant fractions were obtained from the 800mg crude sample with a recovery of 56.7%. A HILIC-XAmide prep column was selected for the second-dimensional (2D) preparation. Finally, a novel antioxidant ß-carboline Alkaloids (Glusodichotomine AK) and 4 known compounds (Tricin, Homoeriodictyol, Luteolin, Glucodichotomine B) were purified from A. kansuensis. The purity of the compounds isolated from the crude extract was >98%, which indicated that the method built in this work was efficient to manufacture single analogue antioxidant compounds of high purity from the extract of A. kansuensis. Additionally, this method showed great potential in the preparation of analogue structure antioxidant compounds and can serve as a good example for the purification of analogue structure antioxidant carboline alkaloids and flavonoids from other plant materials.

Bioactivity and phytochemical characterization of Arenaria montana L.

The bioactivity (antioxidant and cytotoxic activities) of the aqueous and methanolic extracts of Arenaria montana L., a plant commonly used in Portuguese folk medicine, was evaluated and compared. Furthermore, the phytochemical composition was determined based on hydrophilic (sugars, organic acids and phenolic compounds) and lipophilic (fatty acids and tocopherols) compounds, in order to valorize this plant material as a functional food/nutraceutical. Fructose, oxalic acid, methyl-luteolin 2''-O-feruloylhexosyl-C-hexoside, α-tocopherol, and linoleic acid were the main individual compounds found in A. montana. In general, the aqueous extract showed higher antioxidant and cytotoxic activities than the methanolic extract; the latter showed activity only against HeLa and HepG2 cell lines. Both aqueous and methanolic extracts showed some hepatotoxicity but at higher doses than the ones active for tumor cell lines. Moreover, the aqueous extract of A. montana may be used as a functional food or nutraceutical due to the high antioxidant and cytotoxic activities, and due to the presence of bioactive compounds. As far as we know, this is the first report on the phytochemical composition and bioactivity of A. montana.

Inhibitory activity of chemical constituents from Arenaria serpyllifolia on nitric oxide production.

Five new compounds, including one new xanthone, 1-hydroxy-5-methoxyxanthone 6-O-ß-D-glucopyranoside (1), one new lignan, 3-(ß-D-glucopyranosyloxymethyl)-2-(4-hydroxy-3-methoxyphenyl)-5-(3-acetoxypropyl)-7-methoxy-(2R,3S)-dihydrobenzofuran (2), and three new γ-pyrones, japonicumone A 4'-O-ß-D-glucopyranoside (3), japonicumone B 3'-O-ß-D-glucopyranoside (4), and japonicumone B 4'-O-ß-D-glucopyranoside (5), together with eight known compounds (6-13) were isolated from the whole plants of Arenaria serpyllifolia. Their structures were elucidated on the basis of extensive spectroscopic analysis (UV, IR, HRESIMS, 1D- and 2D-NMR, and CD) as well as chemical methods. The isolated compounds were evaluated for their inhibitory effects on nitric oxide production in lipopolysaccharide-activated RAW 264.7 macrophages. Compounds 1-5, sacranoside A (9), and pedunculoside (13) showed potential nitric oxide inhibitory activities with IC50 values ranging from 14.92 µM to 52.23 µM.

Unusual oleanane-type saponins from Arenaria montana.

Three oleanane-type saponins, 3-O-ß-d-glucopyranosylechinocystic acid 28-O-ß-d-xylopyranosyl-(1→4)-[α-l-rhamnopyranosyl-(1→2)]-α-l-rhamnopyranosyl ester (1), 3-O-ß-d-glucopyranosylechinocystic acid 28-O-α-l-arabinopyranosyl-(1→3)-ß-d-xylopyranosyl-(1→4)-[α-l-rhamnopyranosyl-(1→2)]-α-l-rhamnopyranosyl ester (2), 3-O-ß-d-glucopyranosylcaulophyllogenin 28-O-ß-d-apiofuranosyl-(1→3)-ß-d-xylopyranosyl-(1→4)-[ß-d-apiofuranosyl-(1→3)]-α-l-rhamnopyranosyl-(1→2)-α-l-rhamnopyranosyl ester (3) were isolated from the whole plant of Arenaria montana. Their unusual structures for the Caryophyllaceae family were established mainly by 2D NMR techniques and mass spectrometry.

New cyclopeptides in Arenaria oreophila (Caryophyllaceae).

Two new cyclopeptides, arenariphilin G (1) and arenariphilin H (2), were isolated from the whole plants of Arenaria oreophila Hook. Their structures were determined as cyclo(Pro-Ile-Ser(2)-Gly-Ala(1)-Ala(2)-Val-Ser(1)) (1) and cyclo(Pro-Gly- Phe-Ser-Leu) (2), respectively, by detailed spectroscopic analysis.

[Chemical constituents from a Tibetan medicine Arenaria kansuensis I].

OBJECTIVE: To study the chemical constituents of Arenaria kansuensis. METHOD: Column chromatographic techniques were applied to isolated constituents. A combination of physico-chemical properties and spectroscopic analysis were used to identify structures of constituents. RESULT: Five compounds were obtained and elucidated as tricin (1), tricin-7-O-beta-D-glucopyranoside (2), tricin 4'-O-beta-guaiacylglyceryl ether (3), isoscoparin (4) and isovitexin (5). CONCLUSION: Compound 2, 3 and 5 were isolated from A. kansuensis for the first time, compound 3 was obtained from the family Caryophyllaceae for the first time.