The Study Of HDAC Inhibition Activity Components In Aikeqing

Background and ObjectiveAcquired Immure Deficiency Syndrome(AIDS) is one of the most serious public health problems in the world. In the past research, treatment of HIV by HAART, RNA levels in plasma rapid decline in short-term, that cannot be detected. However, HIV-1 could still survive in resting CD4+ T cells and other long-lived cells. These cells are infected by latent HIV which constitutes HIV latent reservoir. If there is no attack or suppression of antiretroviral drugs, HIV-1 latent reservoir will continue to produce new HIV-1. Therefore, the development of agent that could active latent HIV-1 virus has become the emphasis and difficulty of anti HIV/AIDS. HDAC inhibitors can activate latent virus in the HIV patient who receives HAART treatment. Some natural HDAC inhibitors have been discovered, such as:Trichostatin A (TSA), resveratrol, quercetin, etc.There is a wide range of the type of the chemical structure.Therefore, research and development of natural HDAC inhibitors are important for activation of latent HIV virus reservoir.MethodsUnder the guidance of HDAC activity inhibition screening test results, active chinese herb in Aikeqing 2 has been screened; we extracted epimedium which found to be the effective HDAC inhibitor, compounds was isolated from epimedium extraction by using D101 macroporous resin, ODS, silica gel, Sephadex LH-20 gel column chromatography method. We identified compounds by spectroscopic and ESI-MS analysis. Then we employed HDAC inhibitor screening kit to examine the activity of compounds. Epimedium in the Aikeqing 2 inhibiting HDAC active ingredient is identified.Results1. Screening test by HDAC inhibitors, Aikeqing 2 methanol extract at a concentration of 0.25 mg/mL～1 mg/mL, HDAC inhibition rate is 23.39%-48.17%.2.Epimedium methanol extract at a concentration of 0.25 mg/mL～1mg/mL. HDAC inhibition rate is 36.02%～73.88%.3.15 different source, batch epimedium methanol extract has been screened. NO.15 epimedium holds the highest activity inhibition rate 1.87%～29.91% at 50 μg/mL～200 μg/mL.4. We found ethyl acetate fraction of epimedium #15 at a concentration of 100 μg/mL～400 μg/mL has the highest HDAC inhibition rate 24.3%～58.9%.12 compounds were obtained from active extracts by using column chromatography method.12 compounds were identified by spectroscopic analysis and ESI-MS as schizandriside (1), apigenin-8-C-glucoside (2), quercetin (3), quercetin-3-O-β-D-arabinopyranoside (4), kaempferol-3-O-α-L-arabinopyranoside (5), kaempferol-3-O-β-D-xylopyranoside (6), kaempferol-3-O-β-D-glucopyranoside (7), kaempferol-3-O-β-D-acetylglucopyranoside (8), kaempferol-3-O-[6"-O-(E)-p-coumaroyl]-β-D-glucopyranoside (9), kaempferol-3-O-[2", 4"-di-O-(E)-p-coumaroyl]-β-D-glucopyranoside (10), isorhamnetin-3-O-βD)-glucopyran-oside (11), isorhamnetin-3-O-β-D-xylopyranoside (12).5.Under the working concentration of 200 μM, the inhibition rate of icariin is only 15.40%. However, in the same concentration, the HDAC inhibition rate of other compounds such as kaempferol-3-O-α-L-arabinopyranoside (54.46%), kaempferol-3-O-β-D-xylopyranoside (45.86%), kaempferol-3-O-β-D-glucopyranoside (43.01%), isorhamnetin-3-O-β-D-xylopyranoside (39.67%), kaempferol-3-O-β-D-acetylglucopy-ranoside (39.61%), isorhamnetin-3-O-β-D-glucopyranoside (36.04%), quercetin (30.61%), quercetin-3-O-β-D-arabinopyranoside (25.29%), apigenin-8-C-glucoside (24.15%), kaempferol-3-O-[6"-O-(E)-p-coumaroyl]-β-D-glucopyranoside (23.18%), kaempferol-3-O-[2",4"-di-O-(E)-p-coumaroyl]-β-D-glucopyranoside (22.54%)are higher than icariin.3 compounds have high HDAC activity inhibition rate at a working concentration of 200μM. They are kaempferol-3-O-a-L-arabinopyranoside (54.46%), kaempferol-3-O-β-D-xylopy-ranoside (45.86%), kaempferol-3-O-β-D-glucopyranoside (43.01%).6.Under the working concentration of 100 μM, the inhibition rate of kaempferol is 21.11%. In the same concentration, the HDAC inhibition rate of its monoglycoside: kaempferol-3-O-α-L-arabinopyranoside (38.67%), kaempferol-3-O-β-D)-xylopyranoside (31.31%), kaempferol-3-O-β-D-glucopyranoside (26.50%), the activity inhibition rate of kaempferol lower than three of its monoflycoside. At working concentration of 50 μM-200 μM the activity inhibition rate of schizandriside are-0.88%～1.12%. In this concentration range, schizandriside basically does not affect the activity of HDAC. ConclusionWe found that epimedium(identified as Epimedium sagittatum (Sieb. et Zucc.) Maxim.) was an effective HDAC inhibitor in Aikeqing 2. We isolated 12 compounds in the ethyl acetate fraction which was effective.11 Compounds were obtained from epimedium sagittatum maxim for the first time.11 flavonoids had HDAC inhibitory activity. The HDAC inhibitory activity of 9 flavonoids (4-12) was first reported.