The cistanche formula tea is a health food with anti-fatigue effect, which is made of cistanche, Herba epimedium, Acanthopanax, jujube and Zhengshan seed as raw materials. Studies have shown that the raw materials of cistanche formula tea contain more potential anti-fatigue components, such as polysaccharides, saponins, polyphenols and flavonoids in cistanche, Herba epimedium and black tea, which can delay peripheral and central fatigue and reduce excessive accumulation of free radicals by regulating metabolism, antioxidant effect and neuroprotection [1-4]. Therefore, identifying the contents of flavonoids, saponins, polysaccharides and polyphenols in Cistanche formula tea is of great significance for determining the main components that play an anti-fatigue role, so as to carry out precise quality control of the product and further study the possible anti-fatigue mechanism. In this study, UV spectrophotometry was used to determine and analyze the contents of total flavonoids, total polyphenols, total polysaccharides and total saponins in Cistanche formula tea, which provided experimental basis for product quality control and reference for related research on formula tea.
1.Materials and methods
1.1 Materials and reagents
Cistanche recipe tea.
Rutin standard, gallic acid standard, D-anhydrous glucose, syringin; Concentrated sulfuric acid (analytically pure),; Perchloric acid (analytically pure); Methanol, ethanol, ether, glacial acetic acid and phenol (analytically pure); Folinol; Sodium nitrite, vanillin, sodium hydroxide and sodium carbonate; Aluminum nitrate.
Rutin standard, gallic acid standard, D-anhydrous glucose, syringin; Concentrated sulfuric acid (analytically pure),; Perchloric acid (analytically pure); Methanol, ethanol, ether, glacial acetic acid and phenol (analytically pure); Folinol; Sodium nitrite, vanillin, sodium hydroxide and sodium carbonate; Aluminum nitrate.
1.2 Instruments and equipment
UV-1900 ultraviolet-visible spectrophotometer (Shanghai Meishe Instrument Co., LTD.), analytical balance, electric thermostatic water bath, CNC ultrasonic cleaner, low temperature and high speed centrifuge and rotary hair steamer.
1.3 Preparation of reference product solution
1.3.1 Preparation of rutin solution
Rutin reference 0.1000g was accurately weighed, placed in a 100mL volumetric bottle, filled with 70% ethanol to the scale, and ultrasonic dissolved to obtain 1mg·mL-1 rutin reserve solution. The standard rutin solution of 1mg·mL-1 was accurately removed and 10mL was placed in a 50mL volumetric bottle. The standard rutin solution of 200μg·mL-1 (total flavone control solution) was obtained with 70% ethanol and then prepared for color development.
1.3.2 Preparation of gallic acid solution
Accurately weigh 0.0093g gallic acid standard product into 100mL volumetric bottle, add distilled water to the scale, shake well, to obtain 93μg·mL-1 gallic acid standard product solution. Remove 5mL of gallic acid standard solution and put it into 25mL volumetric bottle. Then the distilled water was adjusted to scale to obtain 18.6μg·mL-1 gallic acid standard solution (total polyphenol reference solution) for color development.
1.3.3 D-glucose solution was prepared
Accurately weigh 0.01025g D-anhydrous glucose reference product, dissolve it with water and put it in a 100mL brown volumetric bottle to obtain 102.5μg·mL-1 D-glucose standard solution (total polysaccharide reference solution) for color preparation.
1.3.4 Preparation of syringin solution
Accurately weigh 0.01005g of syringin reference product, dissolve it with methanol and put it in a 10mL brown volumetric bottle to obtain 1005μg·mL-1 syringin standard solution (total saponin reference product solution) for color development.
1.4 Preparation of test product solution
1.4.1 Preparation of total flavonoids and total polyphenols sample solution
Precise weighing of C. deserticsa formula tea about 0.25g, 3 parts per batch, a total of 9 parts, placed in a 50mL volumetric bottle, adding 20mL70% ethanol for 20min, removing the supernatant and placing it in a 100mL volumetric bottle, repeated ultrasound for 5 times, combined with the supernatant liquid cooling, with 70% ethanol fixed volume to the scale, to obtain the test product solution for color preparation.
1.4.2 Preparation of total polysaccharide sample solution
Precise weighing of about 1.0g, 3 parts per batch, 9 parts in total, placed in a 100mL round-bottom flask, added 15mL distilled water, extracted by reflux at 100℃ for 1h, placed at room temperature, filtered with a degrease cotton into a centrifuge tube, and added anhydrous ethanol until the volume fraction of ethanol in solution was 65%, shaken, and left for overnight in a refrigerator at 4℃ (12h). On the second day, the extraction solution was centrifuged for 10min at 3000r· Min-1, the supernatant was discarded, and 5mL 65% ethanol solution was added to wash and precipitate. The above operation was repeated three times. The precipitation left after centrifugation was dissolved by adding water and then fixed in a 100mL volumeter bottle to obtain the experimental sample solution for color development.
1.4.3 Preparation of total saponin sample solution
Precise weighing of about 1.0g of cistanche formula tea, 3 parts per batch, a total of 9 parts, placed in 100mL round-bottom flask, adding 50mL75% ethanol to reflux extraction at 80℃ for 2 times, 1h each time. After cooling, it was centrifuged at 5000r·min-1 for 10min, combined with the extraction liquid, collected the centrifugal supernatant, concentrated to dry on a rotary evaporator, and the concentrate was added with distilled water to a 100mL volumeter bottle. 1mL of the superant was transferred to the chromatographic column containing D101 macroporous adsorption resin (10mL syringe was used as the chromatographic column, and the packing was filled to about 6cm), then the column was washed with 25mL water, and the eluent was discarded and eluted with 25mL70% ethanol. The eluent was collected and transferred to the evaporating dish, and evaporated to dry in a water bath at 60℃. The evaporating substance in the evaporating dish was dissolved with 5mL methanol and filtered by 0.45μm filter membrane to obtain the sample solution. Take 1mL and transfer it to a test tube with plug, and dry it in a water bath at 70℃ for color development.
1.5 Method of color development
1.5.1 Color development of total flavone sample and rutin control product solution
Add 0.4mL 5% sodium nitrite into the total flavone sample and rutin control product solution respectively, and let it stand for 5min, add 0.4mL 10% aluminum nitrate, and let it stand for 5min, then add 4mL 4% sodium hydroxide, add distilled water and let it stand for 10mL, shake well, and let it stand for 15min, that is, color development is complete.
1.5.2 Color development of total polyphenol sample and gallic acid control product solution
Add 0.5mL folin phenol solution to the total polyphenol sample and gallic acid control solution respectively, add distilled water to 6mL, shake well, add 1.5mL10% sodium carbonate, and then add distilled water to 10mL. Heat the water bath at 75℃ for 10min, cool away from light, that is, the color development is complete.
1.5.3 Color development of total polysaccharide sample and D-glucose control product solution
Add 1mL 5% phenol solution to the total polysaccharide sample and D-glucose control solution respectively, shake well, quickly add 5mL concentrated sulfuric acid, shake well, heat in boiling water bath for 15min, then cool in ice water bath for 5min, add water and set volume to 10mL, that is, color development is complete.
1.5.4 Color development of total saponin sample and syringin control solution
The total saponin sample and the control product of syringin were added with 0.2ml 5% vanillaldehyde and glacial acetic acid solution to dissolve the residue, then 0.8mL perchloric acid was added, mixed well and transferred to the test tube. The lid was tightly closed and heated in a water bath at 60℃ for 20min, and then removed. After cooling in the ice bath for 5min, 5mL of glacial acetic acid was added accurately, and the color development was completed.
1.6 Drawing of standard curve
1.6.1 Rutin standard curve drawing. Precise removal of 200μg·mL-1 rutin reference solution 0.5mL, 1.0mL, 1.5mL, 2.0mL, 2.5mL and 3.0mL into a 10mL volumetric bottle and color rendering according to 1.5.1. The accompanying reagent was used as a blank control, and the absorbance was measured at 510nm wavelength, with the concentration (μg·mL-1) as the horizontal coordinate X and the absorbance as the vertical coordinate Y, for linear fitting.
1.6.2 gallic acid standard curve was drawn. Precise removal of 18.6μg·mL-1 gallic acid control solution 0.5mL, 1.0mL, 1.5mL, 2.0mL and 2.5mL into a 10mL volumetric bottle and color rendering according to 1.5.2. The accompanying reagent was used as a blank control, and the absorbance was measured at 760nm, with the concentration (μg·mL-1) as the abscess X and the absorbance as the ordinate Y for linear fitting.
1.6.3 D-glucose standard curve was drawn. Precise removal of 102.5μg·mL-1 D-glucose reference solution 0.5mL, 0.8mL, 1.1mL, 1.4mL, 1.7mL and 2.0mL into 10mL volumetric bottle, color rendering according to 1.5.3. The accompanying reagent was used as a blank control, and the absorbance was measured at the wavelength of 490nm, with the concentration (μg·mL-1) as the horizontal coordinate X and the absorbance as the vertical coordinate Y, for linear fitting.
1.6.4 The standard curve of syringin was drawn. Precise removal of 1005μg·mL-1 syringin reference solution 70μl, 100μL, 130μL, 160μL, 190μL, 220μL and 250μL into 10mL test tubes was carried out in a water bath at 70℃, and the dried product was developed according to 1.5.4. The accompanying reagent was used as a blank control, and the absorbance was measured at wavelength 553nm, with the concentration (μg·mL-1) as the horizontal coordinate X and the absorbance as the vertical coordinate Y, and the linear fitting was carried out.
1.6.2 gallic acid standard curve was drawn. Precise removal of 18.6μg·mL-1 gallic acid control solution 0.5mL, 1.0mL, 1.5mL, 2.0mL and 2.5mL into a 10mL volumetric bottle and color rendering according to 1.5.2. The accompanying reagent was used as a blank control, and the absorbance was measured at 760nm, with the concentration (μg·mL-1) as the abscess X and the absorbance as the ordinate Y for linear fitting.
1.6.3 D-glucose standard curve was drawn. Precise removal of 102.5μg·mL-1 D-glucose reference solution 0.5mL, 0.8mL, 1.1mL, 1.4mL, 1.7mL and 2.0mL into 10mL volumetric bottle, color rendering according to 1.5.3. The accompanying reagent was used as a blank control, and the absorbance was measured at the wavelength of 490nm, with the concentration (μg·mL-1) as the horizontal coordinate X and the absorbance as the vertical coordinate Y, for linear fitting.
1.6.4 The standard curve of syringin was drawn. Precise removal of 1005μg·mL-1 syringin reference solution 70μl, 100μL, 130μL, 160μL, 190μL, 220μL and 250μL into 10mL test tubes was carried out in a water bath at 70℃, and the dried product was developed according to 1.5.4. The accompanying reagent was used as a blank control, and the absorbance was measured at wavelength 553nm, with the concentration (μg·mL-1) as the horizontal coordinate X and the absorbance as the vertical coordinate Y, and the linear fitting was carried out.
1.7 Content determination
1.7.1 Total flavonoids
Accurately weigh the cistanche formula tea (0.25g, 3 batches, 3 parts per batch, a total of 9 parts), prepare the sample solution according to 1.4.1, accurately remove 2mL of each test product solution and place it in a 10mL volumetric bottle; According to 1.5.1 color development, the absorbance was measured at 510nm, and the total flavone content was calculated according to the linear regression equation.
1.7.2 Total polyphenols
Precision weigh the cistanche formula tea (0.25g, 3 batches, 3 parts per batch, a total of 9 parts), prepare the sample solution according to 1.4.1, accurately remove 1mL of each test product solution and place it in a 50mL volumetric bottle; According to 1.5.2 color development, absorbance was measured at 760nm, and the total polyphenol content was calculated according to linear regression equation.
1.7.3 Total polysaccharides
Accurately weigh the cistanche formula tea (about 1.0g, 3 batches, 3 parts per batch, a total of 9 parts), prepare the sample solution according to 1.4.2, accurately transfer 1mL of each test product solution into 15mL tube with plugs, add water and volume to 15mL according to 1.5.3 color development; Random solution was used as blank control solution, absorbance was measured at 490nm, and total polysaccharide content was calculated according to linear regression equation.
1.7.4 Total saponins
Accurately weigh the formula tea of cistanche deserticola about 1.0g, 3 batches, 3 parts per batch, a total of 9 parts, prepare sample solution according to 1.4.3, accurately remove 1mL of each test product solution, place it in 15mL test tube, steam it in a water bath, and develop color according to 1.5.4; The accompanying reagent was used as a blank control, the absorbance was measured at 553nm, and the total saponin content was calculated according to the linear equation.
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