Miao medicine Rui cat stick, that is, Stalked Shiwei, Guizhou’s water quality is hard multi-ethnic areas, urinary calculi disease is more common, and Rui cat stick (stalked Shiwei) is often used to treat the disease, is commonly used in Guizhou folk ethnic medicine. In the book of Shennong Herbal Classics, Youxianshiwei was recorded to have a variety of medicinal effects, including diuresis and drenching, clearing lung and cough, cooling blood and stopping bleeding. Based on these traditional uses, there have been many Chinese patent medicines on the market, which are mainly used to achieve diuretic and drenching effects, including compound Shiwei tablets, compound Shiwei granules and compound Shiwei capsules.
Shiwei rhizome is rich in flavonoids, phenolic acids, polysaccharides and other chemical components, but there are few studies on the synergistic effect of polysaccharides in drug efficacy. The research group has carried out a large number of chemical compositions and pharmacological effects of the phyllostachy produced in Guizhou, and selected a group of active compounds with good diuretic and eluviatory effects, which have been verified in vivo and in vitro experiments. However, traditional Chinese medicine and ethnic medicine are mostly the result of multi-component regulation. In order to study the cooperative mechanism of polysaccharide in diuresis and diuresis, this research group intends to study its polysaccharide and total sugar.
Shiwei rhizome is rich in flavonoids, phenolic acids, polysaccharides and other chemical components, but there are few studies on the synergistic effect of polysaccharides in drug efficacy. The research group has carried out a large number of chemical compositions and pharmacological effects of the phyllostachy produced in Guizhou, and selected a group of active compounds with good diuretic and eluviatory effects, which have been verified in vivo and in vitro experiments. However, traditional Chinese medicine and ethnic medicine are mostly the result of multi-component regulation. In order to study the cooperative mechanism of polysaccharide in diuresis and diuresis, this research group intends to study its polysaccharide and total sugar.
Instruments and Materials
1.1 Main Instruments
Ultraviolet visible Spectrophotometer (Shanghai Meishe Instrument Co., LTD.); Centrifuge; Electric blast drying oven; Analytical balance; Water bath.
1.2 Reagents and materials
Anhydrous ethanol, petroleum ether, 95% ethanol, n-butanol (analytical pure AR, Tianjin Fuyu Fine Chemical Co., LTD.); 5% phenol, 98% concentrated sulfuric acid (Chongqing Chuandong Chemical Group Co., LTD.); Distilled water, glucose (analytical pure AR, Sinopharm Group Chemical Reagents Co., LTD.).
1.3 Sample
Sharp Cat Stick
Methods and Results
2.1 Preparation of reference solution
After drying the anhydrous glucose at 105℃ to constant weight, the sample of 545.6mg was accurately weighed and placed into a 1000mL measuring bottle, then dissolved with water and diluted to the scale, and a glucose solution with a concentration of 545.6mg/L could be obtained after full mixing. Next, 15mL was precisely absorbed from this solution, and the volume was fixed to 25mL, so as to prepare a control product solution with a concentration of 327.36mg/L. Then 0.0mL, 0.3mL, 0.6mL, 0.9mL, 1.2mL, 1.5mL and 1.8mL of glucose reference solution were absorbed into a 25mL colorimetric tube, supplemented to 2mL by distilled water, and then 3mL of concentrated sulfuric acid and 1mL of 5% phenol solution were precisely added. The mixture was thoroughly mixed and heated in boiling water for 15 minutes. Remove and cool in cold water for 5 minutes, remove again and measure absorption at a wavelength of 490nm. Draw A standard curve by using absorbance A as the ordinate and concentration C as the abscissa.
2.2 Preparation of test product solution
The test sample of stembulb was first air-dried, then crushed, and then dried to constant weight at 60℃. Then,the test sample of about 1.0g was accurately weighed, degreasing and pigment extraction were carried out according to the operation steps under “2.3.2”, and the monosaccharides, oligosaccharides and alcohol-soluble impurities were removed. Subsequently, the sample was extracted three times in a water bath at 100 ° C, and finally the filtrate was combined and concentrated, and the volume was fixed to 100mL. Finally, 10mL of the concentrated solution was precisely absorbed and diluted to 50mL with water to obtain the required experimental solution.
2.3 Determination of polysaccharide
2.3.1 Preparation of semi-permeable film Take 10mL collodion solution and slowly pour it into 150mL conical bottle. During this process, carefully rotate the conical bottle so that the collodion forms a film of uniform thickness on the wall of the bottle. Subsequently, pour the excess collodion liquid back into the original bottle. Next, place the conical bottle upside down on the iron ring of the rack and wait for the remaining collodion to drain completely. When the solution is fully evaporated, gently touch the surface of the collodion with your fingers until it no longer feels sticky. At this time, fill the conical bottle with water, soak for 10min, pour the water in the bottle, and then open a part of the film in the bottle, add an appropriate amount of water between the film and the bottle wall, so that the film is separated from the bottle wall, gently remove the film, the semi-permeable film is filled with water and suspended, and the water in the bag can gradually ooze out.
2.3.2 Extraction and refining of polysaccharides from the air-dried samples of Qianproduced stembulb Shiwei, the samples were first crushed and dried to constant weight at 60℃. Then, 30g of the stalked sphygma powder was accurately weighed, wrapped in paper and placed in Soxhlet extractor. After reflux extraction of petroleum ether, the fat and pigment were removed. The extraction was repeated twice, each time lasting 3 hours. The liquid after extraction was discarded, and after the solvent was removed from the residue, the reflux extraction of 80% ethanol was carried out twice, each lasting 4 hours. After the residue was treated again, distilled water was added and extracted three times in a water bath at 100℃, using 250mL, 200mL and 200mL of water, each extraction time was 2 hours. The extracted medicinal liquid was then combined, concentrated by extraction filtration and then centrifuged using the Sevage method to remove the protein at a rotational speed of 3000r/min, thus obtaining a polysaccharide solution. The next step is to place this solution in a semi-permeable membrane for drip dialysis for 24 hours, followed by the addition of anhydrous ethanol to achieve a mass fraction of 80% ethanol. This mixture was left to rest for 24 hours in a refrigerator at 4° C. Finally, the polysaccharide precipitate was obtained by centrifugation, washed twice with acetone, chloroform and anhydrous ethanol, and finally dried to constant weight at 60 。 C to obtain the purified polysaccharide. It was stored in a dryer for later use.
2.3.3 Determination of conversion factors 4.0mg (6 parts in total) of P.japonicum polysaccharide dried to constant weight at 60℃ was accurately weighed, dissolved and contained to 50mL. Then, 2mL solution was precisely measured and placed into 25mL colorimetric tube, followed by 3mL concentrated sulfuric acid and 1mL 5% phenol solution. After fully mixing, the colorimetric tube was heated in boiling water for 15 minutes. Then take out the colorimetric tube and put it in cold water to cool for 5 minutes. Finally, its absorption was measured at 490nm wavelength. Results: The mean conversion factor f = 1.164, RSD was 0.3%.
2.3.4 Methodological investigation In the precision test, we accurately absorbed 2.0mL of the sample solution of Qianproduced Stalksphaeria, and carried out six times of rapid continuous absorbance determination. According to the conditions of the standard curve, the results showed that the average absorbance was 0.4420, and the relative standard deviation (RSD) was 0.95%, which indicated that the instrument performed well in terms of precision.
In the stability test, we also accurately absorbed 2.0mL of Shiwei test product solution, and carried out the operation according to the requirements of the standard curve. The absorbance was measured every 0.5 hours, and a total of six measurements were made. The results showed that the mean absorbance was 0.4417 and the RSD was 0.64%, which indicated that the instrument had good stability.
During the repeatability test, we precisely weighed 6 samples of 1.0g of Qianproduced stalkplant powder, and prepared the test liquid according to the preparation method of the test product, and measured it according to the conditions of the standard curve. The results showed that the average content was 4.950%, and the RSD was 0.8%, indicating that it had good repeatability.
In the recovery test, we took 9 samples of 0.5g of the powder from Guizhou, extracted by petroleum ether and ethanol, and added glucose control solution (3 samples each) according to 80%, 100% and 120% of the sample volume, respectively. Then, the test solution was prepared and determined according to the preparation method of the test product of the Stalkwort. The results showed that the average recovery was 99.40% and RSD was 0.94%, which indicated that the method had a good recovery.
2.3.5 Determination of Polysaccharide in samples First, 1.0g of the test product was accurately weighed and a total of 6 samples were prepared. Then, the test solution was prepared according to the established preparation method of the test product solution, and then the corresponding determination was carried out.
2.3.2 Extraction and refining of polysaccharides from the air-dried samples of Qianproduced stembulb Shiwei, the samples were first crushed and dried to constant weight at 60℃. Then, 30g of the stalked sphygma powder was accurately weighed, wrapped in paper and placed in Soxhlet extractor. After reflux extraction of petroleum ether, the fat and pigment were removed. The extraction was repeated twice, each time lasting 3 hours. The liquid after extraction was discarded, and after the solvent was removed from the residue, the reflux extraction of 80% ethanol was carried out twice, each lasting 4 hours. After the residue was treated again, distilled water was added and extracted three times in a water bath at 100℃, using 250mL, 200mL and 200mL of water, each extraction time was 2 hours. The extracted medicinal liquid was then combined, concentrated by extraction filtration and then centrifuged using the Sevage method to remove the protein at a rotational speed of 3000r/min, thus obtaining a polysaccharide solution. The next step is to place this solution in a semi-permeable membrane for drip dialysis for 24 hours, followed by the addition of anhydrous ethanol to achieve a mass fraction of 80% ethanol. This mixture was left to rest for 24 hours in a refrigerator at 4° C. Finally, the polysaccharide precipitate was obtained by centrifugation, washed twice with acetone, chloroform and anhydrous ethanol, and finally dried to constant weight at 60 。 C to obtain the purified polysaccharide. It was stored in a dryer for later use.
2.3.3 Determination of conversion factors 4.0mg (6 parts in total) of P.japonicum polysaccharide dried to constant weight at 60℃ was accurately weighed, dissolved and contained to 50mL. Then, 2mL solution was precisely measured and placed into 25mL colorimetric tube, followed by 3mL concentrated sulfuric acid and 1mL 5% phenol solution. After fully mixing, the colorimetric tube was heated in boiling water for 15 minutes. Then take out the colorimetric tube and put it in cold water to cool for 5 minutes. Finally, its absorption was measured at 490nm wavelength. Results: The mean conversion factor f = 1.164, RSD was 0.3%.
2.3.4 Methodological investigation In the precision test, we accurately absorbed 2.0mL of the sample solution of Qianproduced Stalksphaeria, and carried out six times of rapid continuous absorbance determination. According to the conditions of the standard curve, the results showed that the average absorbance was 0.4420, and the relative standard deviation (RSD) was 0.95%, which indicated that the instrument performed well in terms of precision.
In the stability test, we also accurately absorbed 2.0mL of Shiwei test product solution, and carried out the operation according to the requirements of the standard curve. The absorbance was measured every 0.5 hours, and a total of six measurements were made. The results showed that the mean absorbance was 0.4417 and the RSD was 0.64%, which indicated that the instrument had good stability.
During the repeatability test, we precisely weighed 6 samples of 1.0g of Qianproduced stalkplant powder, and prepared the test liquid according to the preparation method of the test product, and measured it according to the conditions of the standard curve. The results showed that the average content was 4.950%, and the RSD was 0.8%, indicating that it had good repeatability.
In the recovery test, we took 9 samples of 0.5g of the powder from Guizhou, extracted by petroleum ether and ethanol, and added glucose control solution (3 samples each) according to 80%, 100% and 120% of the sample volume, respectively. Then, the test solution was prepared and determined according to the preparation method of the test product of the Stalkwort. The results showed that the average recovery was 99.40% and RSD was 0.94%, which indicated that the method had a good recovery.
2.3.5 Determination of Polysaccharide in samples First, 1.0g of the test product was accurately weighed and a total of 6 samples were prepared. Then, the test solution was prepared according to the established preparation method of the test product solution, and then the corresponding determination was carried out.
2.4 Determination of total sugar
2.4.1 Extraction of total sugar and preparation of the test product solution After selecting the air-dried samples of the dried Qian, the samples were crushed and then dried at 60℃ until the samples reached the constant weight state. Then, 1.0g of Shiwei powder was accurately weighed and put into Soxhlet extractor. Petroleum ether was used for reflux extraction to remove lipids and pigments from the sample. The process was repeated twice, each time lasting 3 hours. Upon completion of the extraction, the extraction solution was discarded. Then, the solvent in the residue is volatilized, and 50mL of water is added to the residue in a water bath at 100° C for three times of extraction, and the extraction time is 3 hours, 2 hours and 2 hours. Finally, the filtrate is combined, concentrated, and the volume is adjusted to 100mL. Take out 2mL of mother liquor from it, and then dilute the constant volume to 25mL to obtain the required extraction liquid.
2.4.2 Determination of the total sugar of the sample Accurately weigh the 1.0g Stalkwort sample (atotal of 6 parts), and then prepare the test solution according to the predetermined preparation method of the test product solution, and carry out the relevant determination.
2.4.2 Determination of the total sugar of the sample Accurately weigh the 1.0g Stalkwort sample (atotal of 6 parts), and then prepare the test solution according to the predetermined preparation method of the test product solution, and carry out the relevant determination.
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