Study On Dermal Antiallergic Activity Of Natural Herb Extract

19 kinds of natural herbs including Dictamnus dasycarpus,Chamomile,Purslane,Licorice,Sop Hora flavescens,Smilax,Cranberry,Saponaria saponaria,Kochia scoparia,Earthen peel,Cactus,cork,Mugwort,Scutellaria,Magnolia officinalis,Centella asiatica,Astragalus,Chenpi,Lithospermum,were extracted,separated and investigated for anti-allergic activity in the thesis,and 5 kinds of plant extracts with good anti-allergy ability were found.Among them,the Dictamnus dasycarpus water extract with better anti-allergy ability was separated and purified further.Then,the molecular structure and anti-allergy ability of the purified Dictamnus dasycarpus polysaccharides were characterized and investigated.Finally,based on Dictamnus dasycarpus polysaccharides and the perferred herb extracts,a combination of natural herb extracts with antiallergic and synergistic effects was obtained,which provides data support for the research on the anti-allergic ability of natural herbs.The main research contents are as follows:19 kinds of natural herbs were extracted with 60% ethanol to obtain crude extracts,and the hyaluronidase inhibition rate of these crude extracts were determined.Among these crude extracts,Chamomile,Dictamnus dasycarpus,Licorice,Scutellaria,Tangerine peel,Purslane,Centella asiatica and Saponaria had high hyaluronidase inhibition rate,which was higher than 70%.it was shown these nine kinds of herbs has anti-allergy activity.After screening,the 9 kinds of crude extracts were extracted further with petroleum ether,ethyl acetate and n-butanol respectively,and the hyaluronidase inhibition rate experiment was performed.The results showed five extracts had hyaluronidase inhibition rate higher than 80%.The herb extracts included Portulaca oleracea ethyl acetate extract(92.31%),Chamomile water extract(94.87%),Cactus water extract(89.13%),Licorice water extract(88.46%)and Dictamnus dasycarpus water extract(87.18%).The seeding density and culture time of RBL-2H3 cells were optimized for growth and detection of the cells.The best conditions were obtained: the seeding density is 2.0×105 cells/mL,the seeding time is between 12 h and 24 h.Within the range,the cell survival rate is the highest.By evaluating the effect of different concentrations of C48/80 on cell viability,the optimal stimulation concentration range was 10 μg /mL to 100 μg/mL under stimulation time at 40 min,where where was no toxic effect on RBL-2H3 cells.By evaluating the effect of C48/80 stimulation concentration on the ability of RBL-2H3 cells to degranulate and release β-Hex,the optimal stimulation concentration of C48/80 was 00 μg/mL,The release rate of β-Hex of RBL-2H3 cells reached a peak value of 39.93%.So,the conditions for the β-Hex release inhibition of RBL-2H3 cells were summarized below.The seeding density of RBL-2H3 cells was 2.0×105cells/mL,the seeding time was within the range of 12 h to 24 h,and the stimulation time of C48/80 was 40 min,the stimulation concentration is 100 μg/mL.Dictamnus dasycarpus polysaccharide(DDPS)was separated from Dictamnus dasycarpus and purified,and two polysaccharide components,DDPS-1 and DDPS-2,were separated by DEAE column.Structural characterization,as well as investigation of the anti-allergic activity of DDPS were carried out.High-performance gel filtration chromatograp Hy(GFC),ion chromatograp Hy(IC),and infrared spectroscopy(IR)were used to analyze the molecular weight,monosaccharide composition and glycosidic bond types of DPPS-1.The CCK-8 method was used to detect the toxicity of DDPS to RBL-2H3 cells.C48/80 was used as an indicator to induce RBL-2H3 cells to degranulate and release β-hexosaminidase(β-HEX),and DDPS inhibited C48/80-induced RBL-2H3 degranulation,to evaluate the anti-allergic activity of DDPS.The protective performance of DPPS on cell membranes was tested through the modified red blood cell hemolysis test.The antioxidant capacity of DDPS was obtained through the DPPH free radical scavenging test.The results showed that DDPS-1 is composed of fucose,glucose,xylose,rhamnose,and galactose,with a molar ratio of 0.41:1.38:3.15:15.4:79.61.α-glycosidic bonds is dominate between these monosaccharides,DPPS-1 has a molecular weight of 2577 Da.When DDPS-1 at 1 mg/mL,the inhibitory rate of β-HEX released by activation and degranulation of RBL-2H3 cells reached28.54%.;As DDPS-1 at 5 mg/mL,the inhibitory rate of RBC hemolysis under the protection of DDPS reached 62.1%.When DDPS-1 at 10mg/mL,the scavenging free radicals DPPH rate was40.06%.These xperimental results indicated that DPPS has anti-allergy ability.Through the RBL-2H3 cells viability experiment,it was verified that Licorice water extract is toxic to RBL-2H3 cells as the concentration more than 5.0 mg/mL.Chamomile water extract and Cactus water extract in the experimental concentration range(0.1 to 10.0mg/mL)has no effect on RBL-2H3 cells viability.Through the β-Hex release inhibition rate experiment of RBL-2H3 cells,it was shown that all of Chamomile,Licorice and Cactus water extracts have β-Hex release inhibition ability.As Chamomile water extract at 1.0 mg/mL,Cactus water extract at 0.5 mg/mL and Licorice water extract at 0.5 mg/mL,β-Hex release inhibition rate was 21.42%,30.29%and32.43%,respectively.Through the inhibition rate of β-Hex release from RBL-2H3 cells,the polysaccharides from Dictamnus dasycarpus and Chamomile water extract,Licorice water extract and Cactus water extract had good anti-allergenic activity.As the compound of Dictamnus dasycarpus and Chamomile water extracts as 1:1 at the concentration of 0.1 mg/mL,0.5 mg/mL and 1.0 mg/mL,β-Hex release inhibition reaches 37.18%,36.21% and 43.36%,respectively.The following natural product compound combinations have better β-Hex release inhibition ability than single extract under the following concentration conditions: as the compound of Dictamnus dasycarpus and Licorice water extracts as 1:1 at the concentration of 0.1 mg/mL,β-Hex release inhibition reached40.00%.As the four herb water extracts mixed together in the equal proportion at a concentration of 0.5 mg/mL and 1.0 mg/mL,β-Hex release inhibition rate reached 35.42% and 39.97%.It was proved that these herb extracts compound has the better anti-allergic ability than the single herb,and there was the synergistic effect among them.