| Gelsemium elegans Benth (GEB) also named the bowel grass, poisonous root, big tea medicine, Graceful Jesamine, Pueraria Mirifica, is the root, caulis, leaf of the plant Graceful Jesamine of Loganiacae. The drug is warm in nature and pungent in flavour. It was reported that the total alkaloids of Gelsemium have various pharmacology function, as if easing pain, dispersing pupil, promoting the phagocytosis of macrophages, and strong anti-inflammation, repressing the aggregation of blood platelets. Some research units have applied to treat the liver cancer, the stomach cancer and the esophagus cancer by taking orally Gelsemium or injecting total alkaloids. The foreign literature reported that the total alkaloids also have the function of anti-acetylcholine. Gelsemium's extracts have strong cell toxicity. In recent years, Gelsemium's extracts has been used to treat some dermatitis diseases, nervous dropsies and regulated the nerve system. Especially important, Gelsemium has already been made into injection and used for the treatment of the psoriasis and the erythema lupus, ideal clinical result was obtained.Psoriasis is a kind of chronic, inflammatory epidermal disease with theexcessive proliferative and abnormal polarization of the epidermal cells. Psoriasis is hard to be cured because of its higher incidence and recrudescence. This kind of disease also influences the sufferer's life quantity seriously. Psoriasis is the main research content in the dermatology realm. Now the medicine treating psoriasis at home and abroad are the anti-tumor medicine, the anti-metabolism medicine, the anti-inflammation medicine and the regulator of immunity. Because of the serious toxicity and side effect, their clinical application is limited. Up to present no ideal medicine curing psoriasis has been found.Aims at the above-mentioned problem, our experiment studies the anti-psoriasis function of Gelsemium which is enrich in our country using of the modern medical science technique. Firstly, we seek a simple and efficient process for extracting total alkaloids from Gelsemium through the method of orthogonal-design; then purify the compound of Koumine (Kou) and identify its structure by the methods of Foruier transform infrared (FITR), ultraviolet (UV) absorption spectrum, thin-layer chromatogram (TLC), Foruier nuclear magnetic resonance (FTNMR); further we observe its therapeutic effects on experimental animal psoriasis models, the levels of IL-2 in serum. Our research will provide the theories basis for the treatment of psoriasis of Kou.Objective:1. To seek a simple and efficient process for extracting total alkaloids from Gelsemium, investigate the yield under the different condition, extract total alkaloids using the best craft; and to establish a method for isolation and identification of Koumine (Kou), the biggest content, also the less toxicity compound in total alkaloid.2. To study the possible mechanism of Kou treating psoriasis and the effect of Kou on the epithelial mitosis and epidermal differentiation was detected using mouse vaginal mucous and mouse-tail as experimental models. The level of IL-2 in serumwas also detected. Methods:1. According to orthogonal-design, three factors in extraction total alkaloids were optimized, i.e. the solvent, solid/liquid ratio of extracting system and reflux time.2. Separation and purification of Kou were performed on silica gel column chromatography and its nature was identified by TLC and UV. The structure of Kou was identified by the methods of 'H-nuclear magnetic resonance (^H-NMR) and 13C-nuclear magnetic resonance (13C-NMR), distortionless enhancement by polarization transfer (DEPT), element analysis etc.3. Different groups of vaginal mucous and mouse-tail mouse were treated with methotrexate (MTX) or Kou (6 mg/ kg, 30 mg/ kg, 150 mg/ kg). Blank control group, MTX positive medicine group were set at the same time. Effects of Kou on the epithelial mitosis and epidermal differentiation were observed by microscope. The levels of IL-2 in serum of mice were detected with Enzyme-linked immunoabsorption assay (ELJSA).Result:1. According to orthogonal-design, the best craft of total alkaloids extraction were optimized. In the condition of chloroform as the solvent, solid/liquid ratio of 1:7.5 and reflux time of 3h with 3 repetitions, high content of alkaloids in the extract was obtained. The yield of total alkaloids is 1.42% through the experiment of verification.The extracts can be made sure as alkaloid by the reaction of precipitate and color. We know that the total alkaloids contain more than seven components by TLC method. TLC-plate is silica gel G (0.5%KOH), the developer is chloroform-carbinol(94:6) and the chromogenic agent is modified bismuth iodide.2. Prism form of Koumine was isolated from Gelseminum total alkaloids. Its nature was identified by UV. The peaks of absorption spectrum are 262.20nm and 222.00nm. It is close to the peaks of Kou standard (264.40nm, 221.20nm ) which was reported by literatures. The characteristic peaks of extracts were showed in infrared absorption spectra(4000~400 cm"1). There are characteristic peaks in 3066.1, 2946-2856, 2791^ 2768.8, 1628-1418, 1443.4, 1238, 1078, 991, 925.2, 751.6 cm"1. In addition, the sample's 2H NMR, 13C NMR were recognized and compared with alkaloids of indole which have the similar structure. We got the data in accordance with the report. The sample's *H NMR and 13C NMR have characteristics and reappearance. Element analytical results showed that the sample was the same as the theory data. Kou (molecular formula: C20H22N2O) : C78.43%, H7.19%, N9.15%; the sample: C78.6%, H7.36%, N9.09%o The sample is proved to be Kou according to the result of above-mentioned UV, FTIR, FTNMR and element analysis. The structure identification of Gelsemium total alkaloids extracts is the basis of developing safety and effective clinical preparation and establishing fingerprint diagram of Chinese herbal medicine.3. Influence of Kou on general behavior of experimental animal psoriasis models: all groups of mice in the process using medicine had fine hair, and the activity was normal, their body weight showed no obviously difference within the groups (P>0.05). It is proved that there is no side effect of Kou on the growth of mice.Influence of Kou on the epithelial mitosis of mice: 30mg/kg and 150mg/kg dosage groups of Koumine tested showed a remarkable inhibiting effect on mouse vaginal epithelial mitosis, and the suppression was in dose-dependent manner. The MTX groups also had inhibiting effect. The metotic index (MI) of blank control groups was 10.58±3.04, the MTX groups was 6.20±1.65 (P<0.05) and 150mg/kg Kouwas 4.86±2.36 (P<0.05).Influence of Kou on the formation of epidermal granular layer in the scales of mouse-tail: 30mg/kg and 150mg/kg dosage groups of Koumine tested showed a remarkable promoting effect on mouse-tail, and the promotion was in dose-dependent manner too. The scale with granular (SG) of mouse-tail of blank control groups was 6.83±1.83, the MTX groups was 14.83±2.48 (P<0.01) and 150mg/kg Kou was 15.00+3.03 (P<0.01).4. Influence of Kou on the levels of JL-2 in serum of experimental animal psoriasis models: compared with the blank control groups, different concentrations of Kou decreased the level of JL-2 obviously. The levels of JL-2 of blank control groups was 157.56±40.54, the MTX groups was 27.89±7.74 (P<0.01) and 150mg/kg Kou was 27.89±7.74 (P<0.01).Conclusion:1. In the condition of chloroform as the solvent, solid/liquid ratio of 1:7.5 andreflux time of 3h with 3 repetitions, high content of total alkaloids was obtained.2. Prism form of Kou was isolated from total alkaloids of Gelseminum.3. Higher and medium dosage groups of Kou tested showed a remarkable inhibiting effect on mouse vaginal epithelial mitosis and promoting effect on the formation of epidermal granular layer in the scales of mouse-tail. Kou has certain anti- psoriasis activity.4. Decreasing the level of IL-2 in serum, repressing the high cell immunity and improving the immunity state maybe are the anti-psoriasis mechanism of Kou.
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