Study On Of Analgesic Effective Part Of Paecilomyces Hepialid And Its Sustained-release Preparations

Paecilomyces hepialid chen&dai is a fungus belonging to Paecilomyces Bain of Moniliaceae, and isolated from the Cordyceps sinensis(Berk) Sacc. The mycelia of the fungus is cultivated by artificial deep fermentation, and has been used widely as a substitute of Cordyceps sinensis in medicine and health foods. In this paper, the acetic acid induced writhing method was used to screen the populations and active sites with high analgesic effect of paecilomyces hepialid, then developed analgesic release tablets and established its quality inspection method. The main contents are as follows:1. Screening of populations with high analgesic activity in Paecilomyces hepialidMycelias isolated and cultured from 8 wild populations were used to feed mice for three days according to two dosages, then acetic acid induced writhing in mice method was adopted and the analgesic percentage was calculated to screen the active populations of analgesia. The results showed that, there were seven populations can significantly reduce the writhing times caused by acetic acid in mice, significantly,with statistical significance(P<0.05, P<0.01). Among them, the mycelia named XGL00 and collected from the town of Tiger-Leaping Gorge in Shangrila, has a analgesic percentage of 42.41% with a high dosage, and 57.07% with low dosage,both of which were higher than any other mycelium of the 13 populations. Therefore XGL001 was selected as the mycelia that has the most analgesic activity.among them2. Comparison of analgesic activity of extracting parts of Paecilomyces hepialid mycelia2.1 Separation and comparison of analgesic activity among different polar extracting parts from cultures and myceliaThe culture( cultivated in solid media, containing mycelia and media) and mycelia( cultivated in liquid media, free of the culture medium) of strain XGL001 was used as raw material, which were heated respectively to reflux with 90% ethanol and water to obtain alcohol extracts and water extracst, the alcohol extracts were then extracted with petroleum ether, ethyl acetate, n-butyl alcohol respectively, to obtain petroleum ether portion, ethyl acetate portion, n-butyl alcohol portion and water portion.The six parts of extracts were tested respectively by acetic acid writhing method.The results showed that the alcohol extracts of culture, alcohol extracts of material,petroleum ether portion of material and ethyl acetate fraction of material had significant analgesic activity(P<0.05 or P<0.01). The tendency of a nalgesic activity was ethyl acetate fraction> alcohol extract> petroleum ether portion.2.2 Determination of nucleosides, polysaccharides, cordyceps acid and total flavonoids of each portionThe content of nucleosides components was determined by phenol-sulfuric acid method, polysaccharides and cordyceps acid were determined by using ultraviolet spectrophotometric and nucleosides were determined by using HPLC. And HPLC methodological study was approached for determinatioin simultaneously of 5nucleosides. The HPLC method was performed on an Waters Sun Fire C18(5μm,4.6×250mm) column with methanol-water gradient elution as the mobile phase. The detection wavelength was 260 nm and the colunmn temperature was controlled at30℃. The results showed that the parts that had remarkable analgesic activity were relatively those had higher content of nucleosides, Cordyceps acid and total flavonoids, but the water extracts containing high levels of polysaccharides did not show analgesic activity, so it is speculated that the analgesic active ingredients in Paecilomyces hepialid are not polysaccharides. But further tests are necessary to separate and confirm the analgesic activity parts or ingredients.3. Chemical constituents study of the petroleum ether fraction of Paecilomyces hepialidTo investigate the components of extracts fraction of petroleum ether with both higher analgesic activity and higher extraction rate by silica gel column chromatography, two compounds were separated and the chemical structure were dentified by physical and chemical constants and NMR spectral data analysis, namely ergosterol and β-sitosterol.4. Preparation of Paecilomyces hepialid culture sustained-release tabletThe gel matrix sustained-release coated tablets prescription of the alcohol extracts from the culture was screened by single factor test and orthogonal test.Taking releasing rate in vitro as an index, types and dosages of HPMC, granulation or not, contents of fillers and absorbents, membranes thickness and the type of porogen were explored respectively on release effect. The optimized formulation was as the follows: Micro silica gel dosage is 25%, HPMC(K100M) dosage is 25%, after granulating pressed into tablets;The prescription of coating liquid: 5% of EC, 4% of PEG 6000 as porogen, diethyl phthalate as plasticizer for the amount of 20% EC.Coated membranes weight increased by 1%. The release profile of the tablets was fitted with the Higuchi model, 12 h release 94.11%. The the screened costed tablets were prepared with 12 h stained-release, good reproducibility and stability.5. Quality examination of coating sustained-release tablets of Paecilomyces hepialid myceliaThe relevant requirements of the sustained-release tablets under the reference to the appendix of Chinese Pharmacopoeia edition 2010 volume 2. The appearance,tablet weight variation, content determination and in vitro release rate was investigated. The results showed that weight variation of the same batch of sustained release tablets was in the range of 92.5% to 107.5% range, which met the requirements of Chinese Pharmacopoeia; The average content of Uridine in the sustained-release tablets was 4.85 g, the RSD value is 2.37%; The tablets sustainable released for 12 h in water with the 12 h accumulated release rate up to 96.99%. This standard can control the quality of sustained release tablets well.