| Three Chinese traditional medicines, opuntia milpa Alta, Sophora flavescens Var., dwarf lilyturf turber were chosen from a group of anti-allergic and anti-itching plants and by employing a variety of efficacy evaluation methods on the basis of anti-allergic, relieving itching and repair. The technologies borrowed from analytical chemistry, pharmacology and cell biology were applied to elucidate the foundation of anti-allergic materials, the effect of anti-allergy, the mechanism of efficacy, and the safety of the.extracts from opuntia milpa Alta, Sophora flavescens Var., and dwarf lilyturf turber. The aim of this research is to develop the anti-allergic ingredients that can be applied to protect skin, and to explore its mechanism, thus providing theoretical basis for market application and data support.Based on our previous work, ten kinds of plants were selected, and prepared respectively in the form of freeze-dried powder. Their Anti-allergic effect was evaluated by by inhibition capacity of hyaluronidase in vitro; Their anti-pruritic properties were determined by the skin itching induced by histamine phosphate on guinea pigs.The extracts of opuntia milpa Alta showed the best inhibition on hyaluronidase. The extracts of Sophora flavescens Var. had the strongest inhibitory effect of itchy skin of guinea pigs caused by histamine phosphate. And the extracts of dwarf lilyturf turber have the best repair effect of the stimulation to the skin.Mainly from the anti-allergic, relieving itching and repair three ways to solved the problem of cosmetics allergy,through uniform design experiment, optimizing the best ratio for 7:1:3. In order to the preparation method is simple operation and low cost, also realize industrialization easily, active carbon decoloring method and ion absorption of resin for process optimization were investigated.Preparation technics are as followes: sophora and dwarf lilyturf were each grinded in 20 meshes, 5 g of sophora and 15 g dwarf lilyturf were mixed and dissolved in 1100 m L of deionized water, heated to 90 ℃ for 1 h, then cooled to 50 ℃, 35 g cactus powder were added and the extraction was continued for another 1 h in 50 ℃. The mixture was then cooled to room temperature. After filtration and centrifugation on 5000 r?min-1 for 10 min, the supernatant was filtered through 0.8 μm apertures cardboard to obtain a clear filtrate. The filtrate was decolorized by activated carbon(1.0%) at 80 ℃ for 0.5 h, and was desalted through ion exchange resin(001×7 cation and anion resin D309). The resulting extract was concentrated and frozen dried to a powder, in which the contents of some active ingredients were determined to be: polysaccharide 34.6 mg?g-1, protein 38.4 mg?g-1, polyphenol 56.8 mg?g-1, oxymatrine 4.98 mg?g-1, and matrine 3.12 mg?g-1.Several techniques were applied to evaluate the effectiveness on anti-allergy of the extract, such as the inhibition activity of hyaluronidase, itching extent induced by histamine phosphate on guinea pig, the influence of low molecular dextran induced mice skin itching and the influence of blood capillary permeability of the abdominal in mice. The difference of the extract in high dose was not statistically significant difference statistically compared with to the fluocinolone group(p>0.05), suggesting the extract has the ability to relieve symptoms of skin itch caused by histamine phosphate, and has anti-pruritic effect. All three groups with different doses of the extract can reduce low molecular dextran to reduce the frequency of mice itching, and extend the latent period. The difference between high dose group and model group was statistically significant(**p<0.01); In the experiment of influence of blood capillary permeability of the abdominal cavity on mice, it showed that the extract in high dose, as well as fluocinolone ointment had a significant effect on the reduction of blood capillary permeability(*P<0.05) compared with the model group. In addition, the extract could reduce the performance of the skin irritation symptoms, and had anti-allergic effect to some extent.Safety evaluation of the extract was made by using the tests of erythrocyte hemolysis, BCOP and patch on human. Erythrocyte hemolysis experiment shows that the hemolysis rates are less than 5% of combination extract under the concentration range of the general cosmetics formula, the combination extracts are moderat,without stimulation, high safety, low concentration and high concentrations which can be added in the skin care security; BCOP experiments and human patch test show that the combination extracts is no irritating to the skin, to daub emulsion containedthe combination extract can greatly ease the stimulus to the skin damage, can protect skin cell membrane structural integrity and barrier function, improve skin resistance to a certain extent, has the effect of repair the damaged skin and relieve allergy symptoms. In addition, the applications of the combination of extracts in cosmetic formulations were studied, and achieved great success by obtaining different cosmetic recipes.The pathway of anti-allergy of the extract were investigated under the premise of safety. The DPPH radical scavenging test showed that the combination extract could remove excess free radicals, thereby reducing free radicals of mast cells and basophilic cell membrane damage, indirectly reduces the release of histamine, thus resulting an anti-allergic antipruritic effect. TEWL test indicated that the combination extracts can improve the skin barrier function and reduce the amount of the loss of moisture in the skin, thus to some extent, can improve the skin’s self protection ability and reduce the sensitivity. Composition analysis shows that the polysaccharide exacted from the combination extracts can mainly reduces moisture loss, and enhance the effect of barrier function.KU812 cell histamine release model induced by both PMA and A23187 was established to evaluate extract effect on histamine release, The results showed that after the induction by PMA and A23187, the content of amine in contrast model increased significantly, compared with the blank control group with significant difference(**p<0.01).The histamine content significantly decreased by using combination extract, high, medium and low inhibition rate respectively were 45.23%, 39.91% and 33.45%, suggesting that combination extract can inhibit the release of histamine, thus reducing allergy symptoms and have an anti-allergic effect.PMA combined calcium ionophore induction method is adopted to establish the KU812 mast cell degranulation in vitro model, the results show that KU812 cells will release TNF-α, IL-1β, IL-6, IL-8proinflammatory cytokines after induced by PMA and A23187, the combination of high, medium and low dose extract can inhibit the expression of proinflammatory cytokines separately and reflect the dose dependent. That combination extract could reduce allergic reaction in the skin pathological damage and other symptoms by releasing the inhibitive histamine and a series of proinflammatory factor. |
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