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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-β-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-β-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.
<br>Display Omitted<br>• Optimal dosages of phosphate and potassium fertilizer on <b>R. tanguticum</b> were firstly explored. • The U-shaped fluctuation curve of total anthraquinone content is firstly proposed. • Optimization of chromatographic columns was firstly proposed when detecting index constituents. • Total anthraquinone content of two-year-old plants had reached <b>Chinese Pharmacopoeia</b> standard.<br>The dried root of <b>Rheum tanguticum</b> plays an important role in formulations and prescriptions in traditional Chinese medicine and Kampo medicine. Due to over-exploitation, <b>R. tanguticum</b> resources have decreased sharply in recent years. The main objective of our investigation (a 3-year field experiment) was to explore the effect of different levels of phosphorus (superphosphate) and potassium (potassium sulfate) fertilizer on the biomass (root fresh weight, root increment, and root dry weight), yield, dry matter content, and anthraquinone content of this plant at different harvesting stages (green stage, growth stage, and wilting stage) under alpine conditions. The root fresh weight and root dry weight increased significantly at the wilting stage following treatment with 90 kg P2O5/ha (100% and 59%, respectively) in 2016 and 75 kg K2O/ha (43% and 41%, respectively) in 2015 compared to the control. The yield of root dry weight obtained from three-year-old <b>R. tanguticum</b> plants was 9200 kg/ha when 90 kg P2O5/ha of phosphorus fertilizer was applied, and 10,400 kg/ha when 75 kg K2O/ha of potassium fertilizer was applied. This yield reached a maximum at the wilting stage. The anthraquinone content of two-year-old <b>R. tanguticum</b> plants had already reached the standard level of the <b>Chinese Pharmacopoeia</b>; however, three-year-old plants had double the anthraquinone content of two-year-old plants. Phosphorus and potassium fertilizers had no obvious influence on the anthraquinone content of <b>R. tanguticum</b> at the same harvesting stage.
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 β-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.