| Avascular necrosis of the femoral head is a kind of common and stubborn disease in department of orthopaedics.It can be further developed to arthritis,with a high rate of mutilation,which has serious impacts on the patients' life and work. Against the disease ,there are no effective and reliable drugs at present. The prescription of Fuhuo capsule was a empirical formula summarized by traditional Chinese physician in our hospital in years of clinical practice,which had been prooved effective by a great quantity of clinical observations.The prescription was composed of Radix Dipsaci,Rhizoma Drynariae,Colla cornus cervi,Radix Rehmanniaae preparata,Rhizoma Zingiberis Preparata,Radix Angelicae Pubescentis,Cortex magnoliae officinalis,White mustard seed,Fructus Chaenomelis Cortex Cinnamomi,Herba Ephedrae and Radix Glycytthizae,which has the function of anti-necrosis of the femoral head.It had being made into pellet by manufacturing laboratory of our hospital .Because the different active substance had the different physico-chemical property, the previous preparation was less reasonable. As to keep as many effective components as possible, and reduce the dosage and enhance the effectiveness and convenience of administration at the same time, the experiment was carried out to make it into capsule by optimizing the extraction and forming condition,and then make the quality standard of the capsule.The principal elements of experiment were as follows:Objective: 1. To optimize the extraction , impurity removal technique and the inclusion process of volatile oil,and then add appropriate adjuvant to the intermediate product as the powder to granulation. 2. To establish methods for identifying Radix Rehmanniae preparata, Rhizoma Drynariae and Radix Dipsaci by TLC. 3. To develop methods for determining the contents of Naringin,AsperosaponinⅥin Fuhuo capsule by HPLC.To develop a method to determine the contents of Polysaccharide in Fuhuo capsule by UV spectrophotometry.Methods: Orthogonal design was carried out for choosing the preparation process. 1. With the volume of water ,extracting time and times as evaluation factors,and the content of polysaccharide and the yield of powdered extract as indexes,the optimal extracting condition for Radix Rehmanniae preparata,White mustard seed,Radix Glycytthizae,Cortex Cinnamomi and Herba Ephedrae was investigated. With the concentration of water extract, the amount of the flocculant, the temperature to flocculate as factors ,and polysaccharide retention and the yield of powdered extract as indexes, the optimal condition of flocculation for water extract was investigatied. 2. With the concentration of alcohol,volume of alcohol, extracting time and times as factors,and the yield of Naringin,AsperosaponinⅥand powdered extract as indexes, the optimal extracting condition with alcohol for Rhizoma Drynariae,Radix Dipsaci,Fructus Chaenomelis,Pubescentis and Cortex magnoliae officinalis was investigatied. 3. With the comminution degree,volume of water, soaking time ,extracting time as factors, and the yield of volatile oil from Rhizoma Zingiberis Preparata as index , the optimal extracting condition for volatile oil was investigatied.With the ratio of volatile oil toβ-CD, inclusion time and temperature as factors,and the comprehensive score of transfer rate and inclusion rate of oil as indexes,the optimal inclusion process of volatile oil was studied. 4. The physico-chemical property of intermediate product was investigatied,and then to inspect if the granulation process can go successfully or not and the yield of granula so as to choose the appropriate adjuvant for granulation. 5. Identification of Radix Rehmanniae preparata:silica gel G(254) was used as the coating material and ethyl acetate-ligarine(60～90℃)(1:1)as the mobile phase, examining under ultraviolet light(254nm). Identification of Radix Dipsaci:silica gel G was used as the coating material and the upper solution of n-butanol- acetic acid-water(4:1:5) as the mobile phase , spray with 10% sulphuric acid–alcohol solution, heat at the temperature of 105℃for several minutes and examine in daylight. Identification of Rhizoma Drynariae:silica gel G was used as the coating material and the upper solution of dimethyl benzene-acetic ether- methanoic acid-water (1:10:2.5:3) as the mobile phase,spray with aluminium trichloride- ethanol solution and examine under ultraviolet light(365nm). 6. Determination of the contents of active substance. (1) Chromatogram condition: choose appropriate stationary phase and adjust the composition,proportion and flow rate of mobile phase to separate the peak of Naringin, or Asperosaponin well and meet the requirements of number of theoretical plates. UV spectrophotometry : choose a suitable coloration method and the wavelengths of maximal absorption. (2)The investigation of linear range: a series of the reference solutions were prepared and the regression equation was obtained with the contents of reference as abscissa and the peak area(absorbability in UV absorption spectrophotometr)as ordinate. (3) The investigation of technology : investigate the precision, reproducibility, stability and recovery (4) Determination of the sample:take three batch of samples for six portion and prepare for the test solution, determine the content of active substance in samples.Results: 1. The optimal extracting condition with water was to add 10 times volume of water and reflux 3 times ,1.5 hour once.The optimal flocculation condition was that the ratio of the volume of water extract to weight of dried medicinal herb was 10:1,at the temperature of 80℃with adding 2% flocculant. 2. The optimal extracting condition with alcohol was to add 8 times amount of 60% alcohol,and reflux 3 times with 1 hour once. 3. The optimal extraction process of volatile oil was to add 10 times volume of water into the fine powder ,to soak for 1 hour and to extract for 10 hour.The optimal inclusion condition was volatile oil:β-CD=1:10(ml:g),inclusion temperature at 60℃,including for 3 hour. 4. Mixture of powdered extract ,Colla cornus cervi and adjuvant was used to granulate with 95% of alcohol by sieving 20 mesh.After being dried at the temperature of 60℃,it would be mixed with the inclusion compound which has being pulverised and sieved by 40 mesh. 5. Radix Rehmanniae preparata, Rhizoma Drynariae and Radix Dipsaci all had clear map of TLC. Sample solutions showed the same color of dots in the corresponding position with reference solutions ,and negative control solutions showed no interference. 6. Naringin chromatographic conditions: Diamond (250mm x 4.6mm,5um) column; mobile phase : methanol-water-acetic acid (42:55:3);flow rate 1.0ml/min; column temperature: 30°C; The detection wavelength was set at 283nm. Regression equation : y=539.84x+10.634 r =0.9997. Naringin showed good linear relationship in 0.21ug~1.51ug. The RSD of the precision, reproducibility and stability in an eight-hour period were 0.56%,0.71% and 1.23% respectively. The average recovery and RSD were 99.14% and 0.95%. The content of Naringin in each capsule was not less than 1.10mg. 7. AsperosaponinⅥchromatographic conditions : Diamond (250mm x 4.6mm. 5um) was used with water-acetonitrile (70:30) as the mobile phase ,flow rate was 1.0ml/min,column temperature: 30℃, detection wavelength was set at 212 nm. Regression equation y=4.1379x+13.706,r=0.9996. AsperosaponinⅥshowed good linear relationship in 50.04~417ug/ml. The RSD of the precision, reproducibility and stability in an eight-hour period were 0.99%,1.06% and 1.16% respectively. The average recovery rate and RSD were 99.3% and 0.84%. The content of AsperosaponinⅥin each capsule was not less than 4.5mg. 8. Phenol - sulfuric acid method was used for the determination of polysaccharide. The maximal absorption wavelength was 490nm. Regression equation y=0.0017x-0.0053, r = 0.9997. Glucose showed good linear relationship in 192~480ug. The RSD of the precision, reproducibility and stability in an eight-hour period were 0.62%,1.78% and 1.51%, the average recovery and RSD were 99.44% and 1.43%. The content of polysaccharide in each capsule was not less than 9.4mg.Conclusion: Through the orthognoal design ,the optimal extracting and forming process of Fuhuo capsule was determined. The process was stabile with a high content of active substance. The TLC methods had good specificity and clear spots ,which can be use for the accurate identification of the traditional Chinese medicine in preparation ; HPLC and UV spectrophotometry methods were stable, accurate, specific. They can effectively control the quality of the preparation.
|
PDF Full Text Request