Keywords: pine pollen; Flavonoids; Macroporous adsorption resin; purify
Pine pollen is the dried pollen of Pinus massoniana. Pinus tabulaeformis Or several plants belonging to the same family. It has astringent and hemostatic effects, and can be used for treating traumatic hemorrhage, eczema, impetigo, skin erosion and purulent fluid dripping. Pine pollen contains flavonoids, polysaccharides, choline, fatty acids, protein and trace elements. Clinically proved at home and abroad, pine pollen has many pharmacological effects, such as anti-fatigue, lowering blood sugar, anti-aging, inhibiting prostatic hyperplasia, protecting the liver, regulating immune function, losing weight and beautifying, and is known as the "king of pollen". In this paper, the purification technology of flavonoids in pine pollen was studied systematically in order to provide reference for the further development and utilization of pine pollen.
1 experimental materials
1. 1 instrument
FA 1004N Analytical Balance (Shanghai Second Analytical Instrument Factory); UV765 ultraviolet-visible spectrophotometer (Shanghai Precision Scientific Instrument Co., Ltd.); RE-52 rotary evaporator (Shanghai Anting Experimental Instrument Co., Ltd.); Chromatographic column 2cm×50cm (Jinzhou Yousheng Huabo Distribution Office); D 10 1 macroporous resin (made in pesticide plant); AB- 8 and X-5 (produced by Tianjin Nankai University Chemical Plant)
1.2 drug testing
Pine pollen (purchased from Shaanxi medicinal materials market and identified as the pollen of Pinus massoniana, a Pinaceae plant, by Professor Lei Guolian of Pharmacognosy Department); Rutin reference substance (purchased from China Institute for the Control of Pharmaceutical and Biological Products, batch number:100080-200707); Other reagents such as ethanol, sodium nitrite, aluminum nitrate and sodium hydroxide are analytically pure, and distilled water is used in the experiment.
2 determination method of total flavonoids
2. 1 determination of total flavonoids: NaNO2-Al(NO3)3-NaOH colorimetric method. The relationship between concentration Y(mg/mL) of rutin standard solution and absorbance x is y = 4.1164x-0.0368 (R2 = 0.0998).
2.2 Preparation of Flavonoids Sample Solution: Take 700g of pine pollen, add 65438 00 times of 60% ethanol, extract twice, each time 65438±0.5h, filter the extract, recover ethanol under reduced pressure, concentrate until there is no alcohol smell, and dilute it to 5000 ml with water as the test solution. (The content of total flavonoids is 0.497 mg/ml)
Study on purification process of 3
3. Pretreatment of1Macroporous Resin
Clean the chromatographic column to prevent other substances from polluting the resin, drain the water in the column, add 95% ethanol to the chromatographic column, put the new resin into the column, soak it for 24 hours, use 95% ethanol to pass through the resin layer at a flow rate of 2BV/h until the effluent of 1ml and 4ml distilled water is not white and turbid, and then rinse it with distilled water at the same flow rate until it has no alcohol smell; Use 2BV5%HCl to pass through the resin layer at a flow rate of 6BV/h and soak for 4h, then use distilled water at the same flow rate to make the effluent PH neutral, use 2BV2%NaOH to pass through the resin layer at a flow rate of 6BV/h and soak for 4h, and then use distilled water at the same flow rate to make the effluent PH neutral.
3.2 resin screening
3.2. 1 Static adsorption of total flavonoids from pine pollen by different macroporous resins
Accurately weigh 65,438 0 g of each of the three resins after treatment, put them in a 50mL triangular bottle with a stopper and a grinding mouth, add 20mL of the precisely measured test solution, oscillate on an oscillator for 65438±02h to make them fully absorbed, and take 65438±0ml of the upper liquid to determine the content of total flavonoids. See table 1 for the results.
3.3 Investigation on Static Elution Performance of Different Adsorption Resins for Total Flavonoids from Pine Pollen Experimental resin static elution
Using the resin saturated with total flavonoids in the previous step, the surface moisture was filtered, 80% ethanol was added accurately, and the total flavonoids content was determined by oscillating 12h on an oscillator. The results are shown in Table 2.
From Table 1 and Table 2, it can be seen that D-1kloc-0/performs best in both saturated adsorption capacity and elution rate, so D-10/macroporous adsorption resin is selected to purify total flavonoids from pine pollen.
3.5 D 10 1 macroporous resin purification process.
3.5. 1 sample flow rate
3g of treated D 10 1 resin was filled into the column by wet method, and 20 ml of four test solutions (total flavone concentration was 0.497 mg mL- 1) were mixed with 0.5 mL min- 1, 1.0 mL min-65438 respectively.
It can be seen from Table 3 that it is better to control the flow rate of the sample at1.0 ~1.5min-1.
Sample concentration
Add 20 ml of three test solutions to the resin column, control the flow rate at 1 ml min- 1, collect the effluent, measure the absorbance at 500nm, and calculate the adsorption rate. The results are shown in Table 4.
As can be seen from Table 4, the resin adsorption rate of the three samples is related to the concentration, and the higher the concentration, the higher the adsorption rate.
pH value
Take 20mL of test solution, adjust the pH value to 3, 4, 5, 6 and 7 with1mol L-1HCl, and control the flow rate at1ml min-1adsorption, and at1ml min-.
It can be seen from Table 5 that it is better to adjust the PH value of the sample solution to 5.0-6.0.
Sample loading
Add the test solution into the resin column, control the flow rate 1ml min- 1 for adsorption, measure and calculate the content of total flavonoids at 500nm, and draw the leakage curve of d1kloc-0/macroporous resin. See figure 1.
It can be seen from Figure 1 that the macroporous resin is nearly saturated when the effluent volume of the sample is 6BV. At this time, the ratio of resin to raw materials is about 1:3.
Eluent concentration
Take 20mL of the test solution (the concentration of total flavonoids is 0.497 mg mL-1), adsorb dynamically at the flow rate of1ml min-1,and then elute with 6BV water at the flow rate of1ml min-1. Then elute with 10%, 30%, 50%, 70% and 90% ethanol in turn at the flow velocity gradient of1ml min-1,collect the eluate, determine the content of flavonoids in the eluate, and calculate the desorption rate of flavonoids. The results are shown in Table 6.
As can be seen from Table 6, 50% ethanol has the best desorption effect.
2.3.6 elution speed
At the flow rate of 1 ml min- 1, take 20mL of each of the four test solutions for adsorption, and elute with 50% ethanol at the flow rates of 0.5 ml min- 1, 1.0 ml min- 1.5 ml respectively.
As can be seen from Table 7, the elution rate is 0.5 ~ 1.0 ml min- 1.
3.5.7 Purity Detection of Total Flavonoids from Purified Pine Pollen
As can be seen from Table 8, the purity of total flavonoids from pine pollen can reach above 80% after purification by macroporous resin, and the reproducibility is good.
4 conclusion
The results showed that D 10 1 macroporous adsorption resin had a good purification effect on flavonoids from pine pollen, and the adsorption rate and resolution rate were high. The effects of several factors on the loading effect and elution effect during the purification of flavonoids from pine pollen by D 10 1 macroporous adsorption resin were studied. The final process is as follows: the sample concentration is 0.358 mg/mL, the sample flow rate is 1 mL/min, and 3BV is eluted with 50% ethanol solution at the speed of 1 mL/min. The purity of total flavonoids in pine pollen can reach above 80%.
refer to
[1] Propaganda Department of china health care association Health Professional Committee. Pharmacological research and clinical application of Guozhen pine pollen [J]. Health Consultant, 2005, 17: 48-5 1.
[2] Wang min Research progress on components and pharmacological effects of pine pollen [J]. Anhui Medical College, 2008, 12 (4): 357-359.
[3] National Pharmacopoeia Committee. People's Republic of China (PRC) * * * and National Pharmacopoeia [M]. Part I: China Medical Science and Technology Press, 20 10:333.
About the author:
Peng Xiujuan (198 1-), female, from Linyi, Shandong Province, graduate student, lecturer, research direction: basic research on pharmacodynamics of traditional Chinese medicine.