Study Of Components And The Assaying Of Polysaccharide Leaves Of Urtica Cannabina L. And Its Anti-inflammation Action

Urtica cannabina L. (Urticaceae) is widely distributed in northwest, northeast and Northren China, as well as Sichuan province. The parts of the plant as medical use are whole herb and roots. It has the effects of expeling wind-evil and removes wetness, promoting the circulation of blood, relaxing the muscular spasm and relieving toxin of snakes. Recently U. dioica L. has been greatly studied oversea. The components and pharmacological activity of U. dioica L. had been reported. Further more the extract of U. dioica L. has already been made into many drugs for disease such as joint muscular rheumatism and benign prostate hyperplasia. Studies on U. fissa Pritz., U. angustifolia Fisch.ex Homem. and U. cannabina L. had also been carried out by domestic scholars. Pharmaco-empirical study shows good bacteriostatic activity of U. cannabina L. seeds. The water extract of its roots could dose-dependent degrade blood pressure of dog and strengthen heart function of rabbit or toad. The leaves water extract of this plant also had distinguished bacteriostatic action. We are promoted to investigate on its leaves'component, activity of polysaccharide and polysaccharide assaying. Objective: To study polarity components in U. cannabina L. using silica gel column chromatogram, ploymide column chromatogram, and sephadex column chromatogram. To establish the structures of compounds isolated from Urtica cannabina L. with the aids of spectrosopic methods including UV, IR, 1H-NMR, 13C-NMR, MS. To study the anti-inflammation action of polysaccharide. To determine the polysaccharide by colourimetry with sulfuric acid-phenol as colorimetric reagent.Methods: 1. Extraction, isolation, and structure identification of U.cannabina L. leaves (UCLL): Dried UCLL (5.4 Kg) was extracted with 70% ethanol three times and the solvent was evaporated under reduced pressure, yielded the extract. The extract was diluted with water and extracted with petroleum ether, ethyl acetate and n-butanol respectively, and after evaporated the solvent, we got each fraction.Two methods were used to isolate EtOAc-fraction. One part (11.1 g) was fractionated by polymide column chromatogram. From 30% and 70% ethanol elutions we got the white powders which were further purified with silica gel column chromatography and yielded compound 1 (31 mg) and compound 2 (22 mg). The other part (9.4 g) was isolated by silica gel column chromatography directly and got some white powders. With further isolation and purification we got compound 3 to 7 (each 2 mg, 28 mg, 24 mg, 40 mg, 20 mg).We used two methods in isolation of n-butanol fraction. One part was isolated by silica gel and ODS column chromatography. And finally we got compound 5(24 mg) and 8(5 mg). The other part was isolated by D101 macroporous adsorbent and yielded compound 9(60 mg) with further purification. Identification of compounds got from leaves of U. cannabina L. was done with the aids of UV, IR, NMR and MS.2. Study of polysaccharides anti-inflammation activity: Study on the inhibition of ear swelling induced by dimethylbenzene in mice: The preparation of test extracts. Purified urtica polysaccharides were abtained from dried leaves of U. cannabina L. by defatting with petroleum ether and Sevage method. Then we dissolved it with water. The concentration of the solution was 0.2 mg/ml. To study the inhibition of different concentration extracts on swelling of ear induced by dimethylbenzene in mice, aspirin was the comparition sample.3. Determination of UCLL polysaccharides: The purified polysaccharides were determined by colourimetry with sulfuric acid-phenol as colourimetric reagent: (1) Extraction and purification of polysaccharides: UCLL was refluxed by hot water to extract polysaccharides purified by degrease and deproteinization. (2) Preparation of phenol: After distillation, the phenol was diluted with water, reserved in refrigerator. (3) Preparation of sample: The sample was extracted with water by supersound, concentrated and metered volumn by water. (4) Preparation of standard curve: Prepared appropriate concentration reference and choosed suitable coloration reagent, wave and determine absorbility. (5) Experiment of reproducibility: the sample was injected 5 times sequencely and determined the absorbability. (6) Experiment of recovery: weighed out sample, added the polysaccharide respectively, determined the absorbability and calculated recovery. (7) Experiment of precision: Weighed out 5 shares of TDBF to make 5 samples, determined the absorbability and calculated recovery. (8) Experiment of stability: In one hour, determined the absorbability of the sample solution, polysaccharides solution, and glucose solution.Results: 1. From U. cannabina L. leaves 9 compounds were isolated. Among them 8 compounds were identified on the basis of spectroscopic methods including UV, IR, NMR, and MS. They areβ-sitosterol(1), daucosterol(2), hexadecanoic acid(4), uracil(5), (2S,3S,4R,8E)-2-[(2′R)-2′-hydroxydocosanosylamino] -8(E)-octadecane-1,3,4-triol(6), 1-O-β-D-glucopyranose -(2S,3S,4R,8E)-2-[(2′R)-2′-hydroxydocosanosylamino]-8(E)-octadecane-1,3,4-triol(momor-cerebroside)(7), thymine(8), kalium nitricum(9). Amone them, compound 5, 6, 7, 8 were separated for the first time from this genus.2. The result of anti-inflammation action of UCLL polysaccharides: Anti-inflammation experiment results showed that polysaccharides of U. cannabina L. leaves could inhibit swelling of ear induced by dimethylbenzene in mice (P<0.01). The low dose did the same effect as aspirin (P>0.05) and the high dose was higher (P<0.01). It indicated that UCLL polysaccharides did have anti-inflammation action and dose dependent.3. The results of polysaccharides determination: Sulfuric acid-phenol was choosen as color reagent; the detection wave length was 490 nm. The regression equation was C = 0.0633A + 0.0002, r = 0.9997, (n = 5). The content of polysaccharide in UCLL was 5.96%, reproducibility: RSD = 4.13%; recovery: RSD = 2.17%, stability of sample solution: RSD = 1.43%.Conclusions: The results showed that the solvents and methods of extraction used in these experiments are practicable. With silica gel, polymide and Sephadex LH-20 column chromatogram, the components in UCLL were successfully separated and purified. 8 compounds were isolated and identified, among them 4 were separated for the first time from this genus. It provided the foundation for investigation bioactivity of UCLL. From the results of anti-inflammation experiment we learned that polysaccharide of U. cannabina L. leaves has obvious anti-inflammation activity. The determination of UCLL polysaccharides were done by colourimetry with sulfuric acid-phenol as colourimetric reagent, glucose as the control article. The method was simple, specific and repoductive.