| Objective: The artificial cultivated mycelia of Paecilomyces militaris can be used as substitute for Cordyceps sinensis(Berk.) G.H. Sung, J.M. Sung. But due to the limitation of it’s development and application, the scope for practical application of mycelia of P. militaris is narrow. This paper was aimed to screen strains and extracted fractions that have high analgesic activity from Paecilomyces militaris, and optimize the extraction process, grope and improve the preparation technology and quality evaluation of the sustained release capsule of the effective extracts of Paecilomyces militaris, in order to provide the experimental basis for enlarging the application of Paecilomyces militaris.Methods: 1. Screening of Analgesic activity of strains: 10 strains of mycelia or culture(mixture of mycelia and medium) of Paecilomyces militaris that were isolated from 5 different populations of Cordyceps militaris(Link. Fries) Link in Mid-Yunnan region were selected to investigated the analgesic effect. By using the pharmacological model of writhing body test in mice, with NS as snegative control and Rutondine as positive control, mice were orally given 10 kinds of mixed suspension of culture or mycelia of Paecilomyces militaris according to three dosages of low, medium and high(0.05 g/ml, 0.1 g/ml, 0.2 g/ml) or two dosages of low and high(0.05 g/ml, 0.1 g/ml) in three days continuously. Then the incubation period and the times of writhe body in mice induced by glacial acetic acid was observed and analgesia percentage was calculated to compare and screen out the strains of Paecilomyces militaris with significant analgesic action.2. The optimization of extraction process of Paecilomyces militaris: A High Performance Liquid Chromatographic method was established to determine the content of cordycepin which was taken as indexes to optimize the extraction process of Paecilomyces militaris with alcohol. Orthogonal test method was used to investigate three factors of alcohol concentration, alcohol volume ratio, and the combination of extraction times and time to influence on the extraction efficiency of cordycepin. The chromatographic condition of HPLC was: a reversed-phase Therom C18 column(250 × 4.6 mm, 5 μm). The mobile phase consisted of methanol-water(14:86 v/v) at a flow rate of 1.0 ml/min. Detection was performed at wavelength of 260 nm.The column temperature was 30℃. After extracted by alcohol, the residue of mycelia was dealed with water to extracted Cordycepic polysaccharides, and colorimetric method by using phenol—concentrated sulfuric acid as chromogenic agent was used to determine the content of Cordycepic polysaccharides in the extract under 490 nm.3. Separation and evaluation of analgesic activity of the effective parts: The alcohol extraction part from culture and mycelia of Paecilomyces militaris was extracted by systematic solvent extraction with petroleum ether, ethyl acetate, n-butanol respectively. HPLC method was established to determine the content of cordycepin in each extract part. The chromatographic condition of HPLC was: a reversed-phase Waters C18 column(250 × 4.6 mm, 5 μm). The mobile phase consisted of methanol-water(12:88 v/v) at a flow rate of 1.0 ml/min. Detection was performed at wavelength of 260 nm.The column temperature was 30℃; With Glucan as a reference substance, colorimetric method by using phenol—concentrated sulfuric acid as chromogenic agent was used to determine the content of Cordycepic polysaccharides of the extraction parts under 490 nm; With D- mannitol as a reference substance; UV method was used to determine the content of Cordyceps acid of the extraction parts under 412 nm; With Rutin as a reference substance, UV method was used to determine the content of flavonoids of the extraction parts under 508 nm; Qualitative identification of cordycepin and flavonoids ingredients by TLC wasadopted.; By using the writhing body test in mice to investigate the analgetic action of the extract parts, with NS as the negative control, Rutondine as the positive control. Culture, mycelia of Paecilomyces militaris and the extract parts were prepared with a certain concentration of mixed suspension, which were given orally in mive according to two dosages of low and high(0.05 g/ml, 0.1 g/ml) in three days continuously. Then the incubation period and the times of writhe body in mice induced by glacial acetic acid was observed and analgesia percentage was calculated. Combination with the results of determination of the content and analgesic action, the effective analgesic parts of culture and mycelia of Paecilomyces militaris were compare and screened.4. The preparation technology and quality evaluation of sustained-release capsule: The effective analgesic parts of Paecilomyces militaris was screened out to prepare sustained-release capsule. With the cumulative release as investigate indicators, Single factor method and L9(34) orthogonal design experiment were used to select the dosages of stearyl alcohol, PEG-4000 and PEG-6000. The content and content uniformity of cordycepin in sustained-release capsules were evaluated by using HPLC, and the chromatographic condition of HPLC was: a reversed-phase Waters C18 column(250 × 4.6 mm, 5 μm). The mobile phase consisted of methanol-water(12:88 v/v) at a flow rate of 1.0 ml/min. Detection was performed at wavelength of 260 nm.The column temperature was 30 ℃ and the injection volume was 10 μL.Results:1.The analgesic activity of strains: Compared with NS, the times of writhe body of the three dosages of low, middle and high of mycelia ZX4(Zixi Mountain, Chuxiong)and mycelia SM5 declined obviously(P<0.01), and all of the culture and mycelia of ZX4(Zixi Mountain, Chuxiong), the mycelia SM5(Dashao, Songming), the low dosage of mycelia XS13(Zhaotong), the low dosage of mycelia XS2(Xishan Mountain) and the low dosage of mycelia of ZT12(Zhaotong) have declined significantly(P<0.05); the analgesia percentage of the three dosage of mycelia ZX4 were 27%, 46% and 78%; and the three dosa of mycelia SM5 were 54%, 63% and 76%. The incubation period of the low dosage of mycelia XS2 and the low dosage of mycelia XS13 had extended significantly(P < 0.05).2. The optimum process conditions of extraction: Under the condition of HPLC, the precision of RSD was 0.30%, the stability of RSD was 1.27% and the RSD of reproducibility was 2.93%. The results showed the extract ratio of cordycepin was the highest with the process parameters of 90% alcohol, 10 times of alcohol volume ratio, 3 times with 1h for every time. And the water extract ratio of cordyceps polysaccharide was the highest in the mycelia residue from which cordycepin was extracted in advance with 95% alcohol, 6 times of alcohol volume ratio, 3 times, with 1h each time. Combination with the extract ratio of cordycepin and cordyceps pthe olysaccharide, the extract process parameters were determined as 90% alcohol, 10 times of alcohol volume ratio, 3 times with 1h for every time to gain alcohol extract; Then after alcohol were volatilized, the residue was extracted 2 times with 10 times and eight times of water, 1.5 h for every time, to gain the water extract.3. Evaluation of the effective analgesic parts: Under the condition of HPLC, cordycepin has a good linear relationship in 10 ~ 200 μg/ml(R2= 0.9999), the stability of RSD was 0.34 % and RSD of reproducibility was 1.71%, the recovery was 98.89%, RSD was 2.42%. The linear relationship and accuracy of UV method was good to determine the content of cordycepic polysaccharides, cordyceps acid and flavonoids in cultivates or mycelia of Paecilomyces militaris and each extract components. The TLC spots of cordycepin and flavonoids were clear. The result of twisting body test in mice showed that compared with NS, the times of writhe body in mice of the low-dose ethyl acetate extract of mycelia declined significantly(P<0.01), the analgesia percentage was 54.51%; and all of the low-dose and high-dose of petroleum ether extract and the high-dose of water-course of the culture, the low-dose of the mycelia, the low-dose of alcohol of the mycelia and the low-dose of petroleum ether extract of the mycelia declined(P<0.05), the analgesia percentage were 44.66%, 42.07%, 50.16%, 43.30%,44.31% and 46.53% respectively; The incubation period of the the low-dose and the high-dose of water-course of culture had significant difference(P<0.05).4. The preparation technology and quality evaluation of sustained-release capsule:The linear equation of HPLC method for determined the content of cordycepin in sustained-release capsules was Y=48.057X+1.1276, R2=0.9996.The recovery were 103.94%, 98% and 99.37% respectively, RSD were 1.94%, 1.8% and 0.93%. Single factor method for optimized the best dosage of stearyl alcohol and the best dosage of PEG-4000 for 10 capsules were 0.8 g and 1.8g respectively. The orthogonal test design optimized the prescription of sustained release capsules was: stearyl alcohol: PEG-4000: PEG-6000 =2.5:2:4.5:1. The accumulative release rate of three batchs of capsules on 6 h and 12 h were more than 60% and 90% respectively, the average content of cordycepin was 986.82 μg/g. The result of content uniformity test(A+1.80S≤15.0) is corresponding to the relevant regulation of Pharmacopoeia of Chinese(2010 edition).Conclusion: By using the pharmacological model of writhing body test in mice, combine the results of the times of writhe body, the incubation period and the analgesia percentage, to screen the strain of Paecilomyces militaris with high analgesic activity. And the extraction process conditions of Paecilomyces militaris were confirmed according to the content of cordycepin which was taken as indexes, and the verification experiment proved that the conditions was convenient and feasible. The established methods of HPLC to determine the content of cordycepin were stable and reliable, and could be adapted to evaluate the content of effective analgesic parts and the quality control of the preparation. The preparation of sustained-release capsules was simple. The finished product’s release rate was stable, the content was uniform, and easy to carried and used, and lay a theoretical basis for the popularization and application in the future. |
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