Preparation and application of a hesperidin immunoaffinity column
A hesperidin and immunophilic technology, which is applied in the preparation of test samples, chemical instruments and methods, material separation, etc., can solve the problems that the same extraction and purification method and chromatographic conditions cannot be applied to different preparations and medicinal materials, etc. Significant purification effect, improved efficiency and strong specificity
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Embodiment 1
[0030] Example 1: Preparation of Hesperidin Immunoaffinity Purification Column
[0031] A preferred embodiment of the present invention to prepare the hesperidin immunoaffinity purification column is as follows:
[0032] 1. Agarose Gel Activation
[0033] Take 2% agarose gel sepharose 4B and wash it with 20 times the volume of distilled water to remove the remaining ethanol. Use a funnel to filter out the water. Weigh 5 grams of the wet gel after filtering out the water, add 7.5 ml of 0.8M NaOH, 2 ml of 30% epichlorohydrin, 2 mg / ml of sodium borohydride NaBH4, 5 ml, shake the table at 25 ° C React for 8 hours.
[0034] After the reaction, wash with 50 ml of distilled water to remove the mixed epichlorohydrin in the gel.
[0035] 2. Anti-hesperidin IgG monoclonal antibody combined with sepharose
[0036] The anti-hesperidin antibody was dissolved in 20mM PBS pH7.4, the final concentration was 2mg / ml, and the total volume was 10ml.
[0037] The sepharose was washed three t...
Embodiment 2
[0046] Example 2: Purification and detection of hesperidin components in Shuganzhitong pills by using hesperidin immunoaffinity column
[0047] 1. Sample processing of Shugan Zhitong Pills:
[0048] Take 10g of this product, grind it finely, take about 2g, accurately weigh, accurately add 50ml of methanol and ultrasonically extract for 30 minutes, make up the weight, centrifuge for 3 minutes (centrifugal speed 2500 rpm), and accurately measure 1ml to 100ml of the supernatant In the bottle, add water to the mark.
[0049] 2. Equilibrate the hesperidin immunoaffinity purification column at room temperature for 30 minutes.
[0050] 3. Take out the immunoaffinity column, connect the injection port with the syringe barrel, and connect the syringe to the air-controlled operating frame.
[0051] 4. Wash the affinity column with deionized water for 3 times, 10 ml each time, adjust the air pump pressure of the stomata operating frame, so that the liquid flows out at a flow rate of 3 ...
Embodiment 3
[0055] Example 3: Purification and detection of hesperidin components in Yiqi Yangxue Oral Liquid by using hesperidin immunoaffinity column
[0056] 1. Sample processing of Yiqi Yangxue Oral Liquid:
[0057]Accurately measure 3ml of this product, put it in a 10ml measuring bottle, add an appropriate amount of methanol, ultrasonicate (power 250W, frequency 33kHz) for 30 minutes, let it cool, add methanol to the scale, shake well, and accurately measure the supernatant from 1ml to 25ml In the bottle, add water to the mark. Precisely measure 1ml and put it in a 50ml measuring bottle, add water to the mark.
[0058] 2. Equilibrate the hesperidin immunoaffinity purification column at room temperature for 30 minutes.
[0059] 3. Take out the immunoaffinity column, connect the injection port with the syringe barrel, and connect the syringe to the air-controlled operating frame.
[0060] 4. Wash the affinity column with deionized water for 3 times, 10 ml each time, adjust the air p...
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