Extraction And Separation Of Antioxidant And Anti-inflammatory Active Constituents Of Herbaceous Peony Flower

Herbaceous peony (Paeonia lactiflora Pall. Ranunculaceae), a perennial root herbaceous plant, is grown on dry open stony slopes, riverbanks and sparse woodland edges. The dried root without bark of herbaceous peony is one of the most well-known herbs in East Asia with a long history. Herbaceous peony not only has lots of pharmacological activities including anti-inflammatory, analgesic, antibacterial, antioxidant, anticancer, antidepressant and anti-hepatic fibrosis effects, but also has abilities to improve and treat autoimmune diseases, cardiovascular and cerebrovascular diseases as well as neurodegenerative diseases. Herbaceous peony flower including abundant nutrients is non-toxic, which makes it have a certain therapeutic effect. The purpose of this study is to assess the antioxidant and anti-inflammatory activities of herbaceous peony flower, isolate and indentify the bioactive constituents in order to provide a basis for the further integration used in medicine and the development in new functional foods of herbaceous peony flower.1. The antioxidant activities of extracts from herbaceous peony flowerThe total phenolic contents of different extracts from herbaceous peony flower were investigated by ferrous tartrate colorimetry. The results showed that all of extracts contained polyphenols, of which methanol extract from herbaceous peony flower was the highest of all (44.35mg/g). The antioxidant potency of the extracts from herbaceous peony flower were investigated by three systems in vitro including DPPH free radical scavenging activity, hydroxyl radical scavenging activity, superoxide radical scavenging activity. When the original extract was diluted100times, DPPH free radical scavenging activities of the water extract and methanol extract from herbaceous peony flower were92.33%and90.31%, respectively, which were stronger than that of100μg/mL quercetin (88.74%); when the original extract was diluted200 times, the hydroxyl radical scavenging activities of five extracts were all stronger than that of100μg/mL quercetin; when the original extract was diluted50times, the superoxide radical scavenging activity of the methanol extract from herbaceous peony flower was58.73%. which was stronger than that of100μg/mL quercetin (49.23%). The methanol extract and water extract of herbaceous peony flower exhibited strong free radical scavenging activity which might be associated with the presence of polyphenols.2. The antioxidant and anti-inflammatory activities of different fractions from herbaceous peony flowerTo determine whether the petroleum ether, ethyl ether, ethyl acetate, n-butanol and water fractions of herbaceous peony flower have antioxidant activity, we evaluated DPPH free radical scavenging activity, hydroxyl radical scavenging activity, superoxide radical scavenging activity, total antioxidant activity and total reducing power of them. Our results showed that the antioxidant activity of ethyl acetate fraction was the strongest, followed by ethyl ether fraction. For ethyl acetate fraction and ethyl ether fraction, IC50values of DPPH free radical scavenging activity were4.71μg/mL and7.31μg/mL, which were lower than that of positive control BHA (7.79μg/mL); inhibition ratios of protein damage were81.36%and88.45%at the concentration of1mg/mL and superoxide radical scavenging rates were77.73%and60.51%at the concentration of400μg/mL, respectively. Moreover, the total antioxidant activity and total reducing power of them were enhanced with the increase of concentration; ethyl acetate fraction exhibited the stronger total antioxidant activity and total reducing power than positive control BHA. We determined the total phenolic content and total flavonoid content of different fractions from herbaceous peony flower with Folin-Ciocalteu colorimetry and sodium nitrite-aluminum nitrate-sodium hydroxide colorimetry, respectively. Our datas showed that total phenolic contents of ethyl acetate fraction and ethyl ether fraction were158.73mg/g and190.48mg/g; total flavonoid contents of them were58.62mg/g and43.66mg/g, respectively. We determined the cytotoxicity on RAW264.7cells and inhibitory effect on LPS-induced apoptosis of the fractions from herbaceous peony flower by MTT assay; the results showed that all of fractions from herbaceous peony flower had no cytotoxicity on RAW264.7cells and could inhibited LPS-induced the decrease of cell vitality, of which the ethyl acetate fraction exhibited the strongest inhibitory effect with83.96%cell survival rate at the concentration of of400μg/mL. We evaluated the anti-inflammatory effect of different fractions from herbaceous peony flower on LPS-exposed RAW264.7cells by measuring the production of nitric oxide (NO). Compared with the LPS group, ethyl acetate fraction and ethyl ether fraction reduced NO levels to13.61%and13.46%in LPS-treated RAW264.7cells, respectively, exhibited strong NO inhibitory activity.3. Extraction and separation of antioxidant and anti-inflammatory active constituents of herbaceous peony flowerThrough isolating the ethyl acetate and ethyl ether extracts by silica gel column chromatography (chloroform:methanol=40:1,20:1,10:1,5:1,2.5:1,0:1) and Sephadex LH-20gel column chromatography we obtained seven elementary substances, named them A1-A7. Compared with the standard through HPLC-MS, A1, A2and A3were identified as1,2,3,4,6-O-pentagalloylglucose, methyl gallate and astragalin; the contents of these three compounds in the dry powder of herbaceous peony flower were5.09%,3.30%and3.39%, respectively. In addition, it is speculated that A4, A5, A6, A7were quercetin-3-O-β-D-glucoside, kaempferol-7-O-β-D-glucoside, kaempferol-3-O-glucoside-2"-gallate and quercetin-3-O-glucosi-de-2"-gallate, respectively, through HPLC-MS analysis combined with relevant literatures.In summary, our results suggested that the herbaceous peony flower had a variety of protective effects for organism including antioxidant and anti-inflammatory activities and might be a potential drug for treating oxidative stress and inflammation-related diseases. The antioxidant and anti-inflammatory activities of herbaceous peony flower might be associated with the presence of active constituents, such as1,2,3,4,6-O-pentagalloylglucose, methyl gallate, astragalin, quercetin-3-O-β-D-glucoside, kaempferol-7-O-β-D-glucoside, etc. The chemical components isolated from the herbaceous peony flower provided material base for the research of different pharmacological effects.