Mmune-Enhancing And Antioxidant Activities Of Cyrtomium Macrophyllum In Mice

Cyrtomium macrophyllum (Makino) Tagawa f. muticum (Christ) Ching et Shing (Dryopteridaceae), recorded as Daye Guanzhong in Traditional Chinese Medicines. According to the "Flora of China" records, the height of the plant is 70 cm, the petiole length is 58 cm, the base diameter is 5 mm; the height and width of the round lanceolate leaves are 42 cm and 18 cm, respectively; leathery leaves with sparsely hairy abaxially scales; multi-line sori irregular, no indusia.Cyrtomium macrophyllum is a common medicinal plant found in the southwest of China. Decoctions of C. macrophyllum are used in folk medicine for the treatment of a variety of diseases, including flu, epidemic encephalitis B, hemostasis and dysfunctional uterine bleeding. Previous studies suggested that the rhizome of Cyrtomium may contain various pharmacological actions such as anti-oxidative, anti-bacterial, anti-parasite, anti-viral and anti-cancer. These activities are mediated by active components such as flavonoids, phloroglucinol derivatives, triterpenes, tannis, essential oil and polysaccharides. However, there are too little information on the components and bioactivities of most fern species including Cyrtomium macrophyllum. Therefore, this study selected the Cyrtomium macrophyllum and cyclophosphamide-induced immunosuppressed mice for research. The immuno-enhancement and anti-oxidation activity were conducted after the safety evalution. The immuno-enhancement effects and mechanism of Cyrtomium macrophyllum aqueous extract was conducted in the study. The active ingredients polyphenols were isolated and identified, the anti-oxidation activity was researched to the further study of Cyrtomium macrophyllum pesticide effect and exploration. The details are divides into six parts as follows:Experiment 1 Preparation of Cyrtomium macrophyllum aqueous extract and determination of safety Cyrtomium macrophyllum aqueous extract (CMAE) was extracted by water decoction. The polyphenols, flavonoids and polysaccharides contents of CMAE were measured by Folin-Ciocalteau, Sodium nitrite-aluminium nitrite and Phenol-Sulfuric acid meathod, respectively. In order to evaluate the safety of Cyrtomium macrophyllum aqueous extract and provide theory evidence for guiding clinical medication, acute toxicity and sub-chronic toxicity test were conducted. The two tests were carried out according to the methods stipulated by the experiment technical literature of veterinary drugs. The result showed that its polyphenols, flavonoids and polysaccharides contents were 272.47±12.89 mg·g-1,182.44±2.71 mg·g-1 and 43.73±1.21 mg·g-1, respectively. At acute toxicity test, the result showed that the mice had some untoward reactions an hour after administration, such as poor spirit, anorexia and so on, but they restored to normal quickly, and then did not appear any paradoxical reaction again. There was no death in all the mice within 14 days, and the maximum dosage was 5000 mg·kg-1. At sub-chronic long-term toxicity test during the administration period, the body weight of mice in high dose group was decreased, but there were no significant differences between them. The total white cell count of high dose group was significantly larger than control group. There were no significant differences in ALT, AST, TG and UN among all groups. There were no significant differences in viscera index and histopathology among all groups. These results indicated that Cyrtomium macrophyllum aqueous extract had no toxicity, long-term toxicity, and special toxicity. Therefore, the clinical application of Cyrtomium macrophyllum aqueous extract was safe.Experiment 2 Effects of CMAE on proliferation and induction of cytokines of splenic lymphocytes in mouse In order to study the action mechanism of CMAE on immune enhancement, the effects of CMAE on lymphocyte proliferation and expression of IL-2 and IL-6 mRNA were tested. In lymphocyte proliferation test, eight kinds of concentrations of CMAE alone or with ConA, LPS was added to the culture system of mouse spleen lymphocytes, cultured for 48 h with MTT method for the determination of lymphocyte proliferation (A570) changes. Results indicated that the CMAE could significantly promote the secretion of T and B lymphocyte proliferation. At the concentration of 250 μg·mL-1 or 15.63 μg·mL-1 CMAE, the T or B lymphocyte proliferation was significantly higher than that in control group. In cytokine expression test, CMAE with three concentrations (7.82、15.63and 31.25 μg·mL-1) were added into the cultivating system, taking ConA as control. After cultivation of 24 h, lymphocyte cells were collected, total RNA was extracted, and then reverse transcription. The effect of CAME on expression of IL-2 and IL-6 mRNA was determined by fluorescence quantitative PCR. The results showed that CMAE could significantly promote the expression of IL-2 and IL-6 mRNA. At the concentration of 15.63 μg·mL-1 or 31.25 μg-mL"1 CMAE, the IL-2 or IL-6 mRNA was significantly higher than that in control group.Experiment 3 CMAE can enhance the immunity in immunosuppressed mice 90 male BALB/c mice were randomly divided into six groups. The mice in the three experimental groups and model control group were given cyclophosphamide at 80 mg·kg-1·d-1 via intraperitoneal injection for 3 days. Three CMAE groups were administrated 25,50, or 100 mg·kg-body weight CMAE via gavage. The healthy control group and the model control group were given physiological saline, and the positive control group was orally administered 100 mg-kg-1 CMAE, lasting 18 days. In the last administration after 24 h,3 mice of each group was detected macrophage phagocytic index by carbon clearance test; 3 mice was measured lymphocyte proliferation by MTT method. The remaining mice were sacrificed by cervical dislocation after weighing, Acquisition of visceral and weighing. The results showed that 3 groups of CMAE in a dose-dependent manner increased spleen index, thymus index, spleen lymphocyte proliferation and macrophage phagocytic index, and the cytokines activity of high dose group returned to normal level. The results indicated that CMAE can reverse the immune organ atrophy induced by cyclophosphamide immunosuppressed mice, enhance the cellular immunity and macrophage function.Experiment 4 The study of antioxidant activity of CMAE in vitro The antioxidant activity of Cyrtomium macrophyllum aqueous extract was determined with experiment of the scavenging power of DPPH, hydroxyl radical, superoxide anion and the total reducing power. Vc was used as the positive control. In the hydroxyl radical scavenging assay, the IC50 values of CMAE and Vc were 157.59±6.82 μg-mL-1 and 201.37±5.71 μg-mL’1, respectively. At the same dose, the hydroxyl radical scavenging ability of CMAE is better than that of Vc, respectively. In the DPPH radical scavenging, superoxide anion radical scavenging and total reducing power assay, the IC50 values of CMAE were 14.85±0.21 μg·mL-1 59.12±0.77 μg·mL-1 and 196.36 ±5.68 μg-mL-1, respectively, while the IC50 values of Vc 11.01±0.09μg-mL’1、43.57±0.26 μg·mL-1 and 106.71±3.61 μg-mL-1, respectively. At the same dose, the DPPH radical scavenging, superoxide anion radical scavenging and reducing power of phenolic extract was lower than that of Vc.Experiment 5 CMAE can enhance antioxidation activity in immunosuppressed mice 60 male BALB/c mice were randomly divided into six groups. The mice in the three experimental groups and model control group were given cyclophosphamide at 80 mg·kg-1·d-1 via intraperitoneal injection for 3 days. Three CMAE groups were administrated 25,50, or 100 mg-kg-1 body weight CMAE via gavage. The healthy control group and the model control group were given physiological saline, and the positive control group was orally administered 100 mg-kg-1 CMAE, lasting 18 days. In the last administration after 24 h, the mice were sacrificed by cervical dislocation. The biochemical parameters were measured. The results showed that CMAE100 groups of SOD, CAT, GSH-Px and TAOC activity increased significantly, the level of MDA decreased significantly compared with the model group. The results indicated that CMAE can effectively remove free radicals.Experiment 6 CMP can enhance antioxidation activity in immunosuppressed mice The CMP was extracted and purified by HPD600, and the components were characterized by UHPLC-LTQ-Orbitrap. At the same time, the antioxidant activity of CMP was test in immunosuppressed.60 male BALB/c mice were randomly divided into six groups. The mice in the three experimental groups and model control group were given cyclophosphamide at 80 mg·kg-1·d-1 via intraperitoneal injection for 3 days. Three CMP groups were administrated 50,100, or 200 mg·kg-1 body weight CMP via gavage. The healthy control group and the model control group were given physiological saline, and the positive control group was orally administered 200 mg-kg-1 CMP, lasting 18 days.24h later of the last administration, the mice were sacrificed by cervical dislocation. The biochemical parameters were measured. The results showed that CMP200 groups of SOD, CAT, GSH-Px and TAOC activity increased significantly, the level of MDA decreased significantly compared with the model group. By the HPD600 and UHPLC-MS/MS, there were characteristic 11 polyphenols compound. The results indicated that CMP can effectively remove free radicals.