Lin-Lin Jing

Anti-hypoxic activity at simulated high altitude was isolated in petroleum ether extract of Saussurea involucrata.

ETHNOPHARMACOLOGICAL RELEVANCE Rhodiola algida, Saussurea involucrata, and other herbs grown in Qinghai-Tibetan plateau have long been used to prevent and treat acute mountain sickness. AIM OF THE STUDY To screen and identify the anti-hypoxic constituents in the herbs grown in Qinghai-Tibetan plateau of Northwestern China. MATERIALS AND METHODS The anti-hypoxic activities of 20 selected plateau herbs were examined against two positive controls, Rhodiola algida and acetazolamide, using the normobaric hypoxia model of mice. The herb with the highest activity was successively extracted with 70% ethanol, petroleum ether, chloroform, ethyl acetate and n-butanol. The extract with the highest activity was identified by comparing the survival time of mice under normobaric hypoxia condition after being subjected to different extracts. The identified extract was further tested by simulating high altitudes through an acute decompression model and a chronic decompression model for mice. RESULTS The herb found to have the highest anti-hypoxic activity was Saussurea involucrate (Kar. et Kir.) Sch.-Bip, and the most effective fraction was in the petroleum ether extract. Administration of petroleum ether extract of Saussurea involucrata (PESI) to mice at 50mg/kg significantly decreased the mortality of animals under acute decompression conditions. Changes in biochemical indicators for glycometabolism and energy metabolism, including adenosine triphosphate (ATP) content and adenosine triphosphatase (ATPase) activity in brain and cardiac muscle, lactic acid (LAC) and lactate dehydrogenase (LDH) in blood and cardiac muscles, blood sugar, and glycogen content in liver and skeletal muscle were reversed under chronic decompression conditions. CONCLUSIONS Saussurea involucrata (Kar. et Kir.) Sch.-Bip exhibits high anti-hypoxic activity that may be effective in preventing acute mountain sickness, and the active constituents are mainly in the petroleum ether extract.

Antioxidant potential, total phenolic and total flavonoid contents of Rhododendron anthopogonoides and its protective effect on hypoxia-induced injury in PC12 cells

BackgroundRhododendron anthopogonoides Maxim, a kind of traditional Tibetan medicine, has been used to remove body heat, body detoxification, cough, asthma, stomachic and swelling, eliminate abundant phlegm and inflammatory for a long time. In the present study, the total phenols and total flavonoid contents as well as antioxidative properties of the crude extract and solvent fractions of R. anthopogonoides were determined using seven antioxidant assays. Additionally, the protective effect of the extracts on hypoxia-induced injury in PC12 cells was also investigated.MethodsThe content of total flavonoid and total phenolic was determined by the aluminum colorimetric method and Folin-Ciocalteu assay, respectively. In vitro antioxidant study, the effect of the crude extract and solvent fractions on total antioxidant activity, reducing power, DPPH radical scavenging, ABTS radical scavenging, superoxide radical scavenging, hydroxyl radical scavenging and nitric oxide radical scavenging were examined. The correlation between the phenolic and flavonoid content of the extracts and their antioxidant properties also analyzed. Furthermore, the protective effect of extracts on hypoxia-induced damage on PC12 cells was investigated by cell viability, lactate dehydrogenase (LDH) release, malondialdehyde (MDA) production and the activities of antioxidant enzymes.ResultsOur results showed that ethyl acetate and n-butanol fractions had higher content of phenolics and flavonoid compounds than other fractions. Except ABTS radical assay, n-butanol fraction exhibited the strongest antioxidant activity. While the hexane fraction showed the lowest antioxidant activity. Ethyl acetate also presented excellent antioxidant activity, which was just lower than n-butanol fraction. Significant correlation between the phenolic, flavonoid content of the extract and fractions with antioxidant assay excluding ABTS, OH scavenging assay was observed. Moreover, ethyl acetate and n-butanol fractions showed protective effect in PC12 cell under hypoxia condition, while crude extract and water fraction had no protective effect. In contrast, hexane fraction exhibited strong cytoprotective effect. Further study indicated that pretreatment of PC12 cells with ethyl acetate and n-butanol fractions, prior to hypoxia exposure, significantly increased the survival of cells and the activities of SOD, CAT, GSH-Px and T-AOC, as well as reduced the level of LDH and MDA. The gathered data demonstrated that ethyl acetate and n-butanol fractions were able to protect PC12 cells against hypoxia induced injury through direct free radical scavenging and modulation of endogenous antioxidant enzymes.ConclusionThese findings suggested that ethyl acetate and n-butanol fractions of R. anthopogonoides had significant antioxidant activity and could prevent PC12 cells against hypoxia-induced injury. So it might be regarded as an excellent source of antioxidants and had great potential to explore as therapeutic agent for preventing hypoxia related sickness in future.

Protective Effect of Tempol Against Hypoxia-Induced Oxidative Stress and Apoptosis in H9c2 Cells

Background Hypoxia-induced oxidant stress and cardiomyocyte apoptosis are considered essential processes in the progression of heart failure. Tempol is a nitroxide compound that scavenges many reactive oxygen species (ROS) and has antioxidant and cardioprotective effects. This study aimed to investigate the protective effect of Tempol against hypoxia-induced oxidative stress and apoptosis in the H9c2 rat cardiomyoblast cell line, in addition to related mechanisms. Material/Methods H9c2 cells were pre-treated with Tempol, followed by hypoxia (37°C, 5% CO2, and 95% N2) for 24 h. Cell viability was detected using MTT assay. ROS level was evaluated using DCFH-DA. Lactate dehydrogenase (LDH), creatinine kinase (CK), malondialdehyde (MDA), catalase (CAT), and superoxide dismutase (SOD) were evaluated using the relevant kits. Cell apoptosis was determined by Annexin V/7-AAD double labelling. The expression of apoptosis-related molecules was assessed with RT-PCR analysis and Western blotting. Results Tempol protected H9c2 cells against hypoxia-induced injury, with characteristics of increased the cell viability and reduced LDH and CK release. Tempol also reduced oxidant stress by inhibiting ROS generation and lipid peroxidation, as well as enhancing antioxidant enzyme activity. Moreover, Tempol pretreatment upregulated the expression of Bcl-2 and downregulated the expression of Bax and caspase-3, thereby reducing hypoxia-induced apoptosis in H9c2 cells. Conclusions These results indicate that Tempol reduces the hypoxia-induced oxidant stress and apoptosis in H9c2 cells by scavenging free radicals and modulating the expression of apoptosis-related proteins.

A new anti-fibrinolytic hemostatic compound 8-O-acetyl shanzhiside methylester extracted from Lamiophlomis rotata.

BACKGROUND Fibrinolysis prevents blood clots from growing and becoming problematic. Antifibrinolytics are used as inhibitors of fibrinolysis. Aprotinin was doubted after identification of major side effects, especially on kidney. Lysine analogues has their own defects and whether they are adequate substitutes for aprotinin is still under doubt. Lamiophlomis rotata (Benth.) Kudo. was previous found to have hemostatic activity. But the active compound in L. rotata and its hemostatic mechanism were unknown. OBJECTIVES To find the major hemostatic compound in L. rotata and identify its haemostasis mechanism. METHODS Traumatic hemorrhage model and coagulant activity assays were monitored in mice and platelets in drug treatment group and control group. Hyperfibrinolysis model was established by intravenous administration of urokinase in mice. Capillary blood clotting time (CBCT), activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen and euglobulin clot lysis time (ECLT) were measured. RESULTS The anti-fibrinolytic activity come from 8-O-Acetyl shanzhiside methylester (ASM) one of the highest iridoid glycosides contents in TIG extracted from L. rotata. ASM significantly (P<0.05) shorten CBCT and reduced blood loss volume in vivo, but did not influence mice APTT, PT or TT. In particular, it significantly prolonged ECLT in hyperfibrinolysis mice. It indicated that ASM could inhibit fibrinolysis. ASM was also effective in CBCT, traumatic bleeding volume and ECLT in hyperfibrinolysis mice model. CONCLUSIONS ASM was the major hemostatic compound in L. rotata. The haemostasis mechanism of ASM was achieved by anti-fibrinolytic activity. ASM was a new fibrinolysis inhibitor as iridoid glycoside compound.

2-[2-(2-Hy­droxy­eth­oxy)phen­yl]-4,4,5,5-tetra­methyl-2-imidazoline-1-oxyl 3-oxide

In the title compound, C15H21N2O4, the nitronyl nitroxide unit displays a twisted conformation. The crystal structure is stabilized by non-classical C—H⋯O and C—H⋯π hydrogen bonds, which build up a three-dimensional network.

Synthesis of Glycosylated Chrysin Derivatives Via Ester Linkers

A series of glycosylated chrysin derivatives have been synthesized in good yields with simple procedures and mild reaction conditions. Six different kinds of sugar moieties were introduced through each ester linker.

5,6-Dihy-droxy-7,8-di-meth-oxy-flavone.

The title compound (systematic name: 5,6-dihydroxy-7,8-dimethoxy-2-phenylchromen-4-one), C17H14O6, is a flavone that was isolated from the petroleum ether-soluble fraction of the rare traditional Chinese medicinal herb Saussurea involucrata. The flavone molecule is almost planar, with a dihedral angle between the planes of the benzopyran-4-one group and the attached phenyl group of 1.89 (6)°. The 5-hydroxy group forms a strong intramolecular hydrogen bond with the carbonyl group, resulting in a six-membered hydrogen-bonded ring. The 6-hydroxy group also forms an intramolecular O—H⋯O contact. In the crystal, the molecules are linked by O—H⋯O and C—H⋯O hydrogen bonds and π–π interactions [3.37 (2)–3.39 (2) Å], which build up a three–dimensional network.

2-[4-(2-Hy-droxy-eth-oxy)phenyl]-4,4,5,5-tetra-methyl-2-imidazoline-1-oxyl 3-oxide.

In the title compound, C15H21N2O4, the imidazoline ring displays a twisted conformation. The dihedral angle between the mean plane of the imidazoline ring and the benzene ring is 33.50 (12)°. In the crystal, molecules are connected by O—H⋯O hydrogen bonds, forming a zigzag chain along the c axis. The chains are linked by C—H⋯O and C—H⋯π interactions.

Chemical Constituents of Saussurea involucrata with Anti-Hypoxia Activity

Saussurea involucrata (Kar. et Kir.) Sch.-Bip., belonging to the Asteraceae family, or more commonly known as the snow lotus, is a rare traditional Chinese medicinal herb on the verge of extinction and has been listed as a second-grade national protected wild plant in China [1]. According to the theories of traditional Chinese medicine, S. involucrata shows efficacy in warming kidney, activating “yang,” expelling wind, eliminating dampness, inducing menstruation, and promoting blood circulation [2]. Modern pharmacological studies have demonstrated that S. involucrata has lots of activities, such as free radical scavenging, anti-fatigue [3], anti-inflammation [4], and anticancer [5] activities. Our previous studies have shown that the petroleum ether extract of S. involucrata has significant anti-hypoxia activity [6], but the chemical constituents responsible for this activity are not clear. In the present study, we isolated, elucidated, and identified four flavones with excellent anti-hypoxia activity from the petroleum ether extract of S. involucrata. Four flavones are known, but they were isolated from that plant for the first time. Air-dried S. involucrata (10 kg) was milled and extracted at reflux temperature with EtOH–H2O (150 L, 70:30, v/v) three times. The extract were filtered and concentrated in a rotary evaporator to generate a crude extract (2.4 kg). The crude extract (500 g) was suspended in H2O (2.0 L) and re-extracted successively with petroleum ether, chloroform, EtOAc, and n-butanol. The extract from each agent was then filtered and concentrated in vacuo to generate the petroleum ether extract (36.86 g), the chloroform extract (57.88 g), the ethyl acetate extract (60.34 g), and the n-butanol extract (144.87 g). The petroleum ether extract (35 g) was applied to a silica gel column eluted with a petroleum ether–EtOAc gradient (10:0 to 1:2, v/v) to give 10 fractions (Fr.1–Fr.10). Fraction 2 was chromatographed several times on a silica gel column to afford compound 1 (33.2 mg). Compound 2 (38.5 mg) was recrystallized in EtOAc from Fr. 3. Compounds 3 (19.7 mg) and 4 (13.5 mg) were recrystallized in MeOH from Fr. 5 and Fr. 6. The structure was characterized by ESI-MS and 1H and 13C NMR. Furthermore, their identity was verified through comparison with literature spectral data. The compounds were identified as mosloflavone (1), moslosooflavone (2), negletein (3), and 5,6-dihydroxy-7,8dimethoxyflavone (4). Mosloflavone (5-hydroxy-6,7-dimethoxyflavone) (1) [7], yellow needles, mp 163–164 C. ESI-MS m/z 299 [M + H]+. 1H NMR (600 MHz, CDCl3, , ppm, J/Hz): 12.68 (1H, s, 5-OH), 7.88–7.87 (2H, d, J = 7.8, H-2 , 6 ), 7.50–7.54 (3H, m, H-3 , 4 , 5 ), 6.66 (1H, s, H-3), 6.55 (1H, s, H-8), 3.92 (3H, s, 6-OCH3), 3.96 (3H, s, 7-OCH3). 13C NMR (150 MHz, CDCl3, , ppm): 182.7 (C=O, C-4), 163.9 (C-2), 158.9 (C-9), 153.3 (C-7, 5), 153.0 (C-6), 131.8 (C-4 ), 131.3 (C-1 ), 129.1 (C-3 , 5 ), 126.2 (C-2 , 6 ), 106.3 (C-10), 105.6 (C-3), 90.6 (C-8), 60.8 (OCH3), 56.3 (OCH3). Moslosooflavone (5-hydroxy-7,8-dimethoxyflavone) (2) [8], yellow needles, mp 166–167 C. ESI-MS m/z 299 [M + H]+. 1H NMR (600 MHz, CDCl3, , ppm, J/Hz): 12.57 (1H, s, 5-OH), 7.96–7.95 (2H, d, J = 6.6, H-2 , 6 ), 7.58–7.56 (3H, m, H-3 , 4 , 5 ), 6.68 (1H, s, H-3), 6.44 (1H, s, H-6), 3.96 (3H, s, OCH3), 3.95 (3H, s, OCH3). 13C NMR (150 MHz, CDCl3, , ppm): 182.7 (C=O, C-4), 163.9 (C-2), 158.7 (C-9), 157.6 (C-7, 5), 149.5 (C-8), 131.9 (C-4 ), 131.4 (C-1 ), 129.2 (C-3 , 5 ), 126.3 (C-2 , 6 ), 105.4 (C-10), 105.0 (C-3), 95.8 (C-6), 61.7 (OCH3), 56.3 (OCH3).

5,6-Dihydroxy-7,8-dimethoxyflavone

The title compound (systematic name: 5,6-dihydroxy-7,8-dimethoxy-2-phenylchromen-4-one), C17H14O6, is a flavone that was isolated from the petroleum ether-soluble fraction of the rare traditional Chinese medicinal herb Saussurea involucrata. The flavone molecule is almost planar, with a dihedral angle between the planes of the benzopyran-4-one group and the attached phenyl group of 1.89 (6)°. The 5-hydroxy group forms a strong intramolecular hydrogen bond with the carbonyl group, resulting in a six-membered hydrogen-bonded ring. The 6-hydroxy group also forms an intramolecular O—H⋯O contact. In the crystal, the molecules are linked by O—H⋯O and C—H⋯O hydrogen bonds and π–π interactions [3.37 (2)–3.39 (2) Å], which build up a three–dimensional network.