CN113662974A - Preparation and purification method of water chestnut shell polyphenol extract - Google Patents
Preparation and purification method of water chestnut shell polyphenol extract Download PDFInfo
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Abstract
The invention discloses a preparation method of a water chestnut shell polyphenol extract, which is characterized in that water chestnut shells are water chestnut shells in early mature stage (stage one); the preparation method comprises the following steps: (1) cleaning water caltrop shells, drying, and pulverizing to obtain water caltrop shell powder; (2) extracting water caltrop shell powder with 60% ethanol in 80 deg.C constant temperature water bath for 2 hr, performing ultrasonic treatment for 15min, centrifuging, collecting supernatant, concentrating under reduced pressure, and lyophilizing to obtain crude extract; (3) dissolving the crude extract in appropriate amount of water, adding 3 times of petroleum ether, repeatedly extracting for 3 times, mixing the obtained water phases, concentrating under reduced pressure, and freeze drying to obtain polyphenol extract. Further, the invention provides a purification method of the water chestnut shell polyphenol extract. The method can improve the yield of the water chestnut shell polyphenol extract, can obtain the water chestnut shell polyphenol extract with higher purity, is favorable for the activity research and further development and utilization of the water chestnut shells, and provides powerful technical support for the utilization of the water chestnut shells.
Description
Technical Field
The invention belongs to the technical field of natural medicines, and particularly relates to a preparation method and a purification method of a water chestnut shell polyphenol extract.
Background
Water caltrop (water chestnut) is an annual aquatic plant, and there are about 30 species and varieties worldwide. In China, water chestnuts have more than 3000 years of cultivation history, are distributed and cultivated most in Yangtze river basins, and are good products for both medicine and food since ancient times. The water chestnut is rich in starch, can be directly eaten, can also be used for preparing water chestnut powder, brewing wine or medicines, and the fresh stem leaves of the water chestnut are usually used as the feed of poultry and livestock, but the water chestnut shells as the by-products are generally not effectively developed and utilized. The water chestnut shells contain active ingredients such as polyphenol, polysaccharide, saponin and the like, wherein the polyphenol is a main active ingredient of water chestnut, and the gallic acid and the derivatives thereof are one of main members of the water chestnut polyphenol. The prior art shows that the water chestnut shell polyphenol extract has the biological activities of resisting breast cancer, reducing blood sugar, resisting oxidation and the like. Therefore, the further research and application of the water chestnut shell polyphenol extract have great prospect.
The water caltrops are generally matured in September, and the states of water caltrops are different in the maturation process: the freshly watered water chestnut shells are jade and soft, and then become hard and yellowish brown.
The macroporous adsorption resin is a macromolecular adsorption resin which does not contain exchange groups and has a macroporous structure, has a good macroporous network structure and a larger specific surface area, and can selectively and physically adsorb organic matters in a solution. The different components can be separated and purified by utilizing the selective adsorption and screening effects of the macroporous adsorption resin on the different components through the adsorption and desorption processes. The method for purifying the water chestnut shell polyphenol extract by using macroporous adsorption resin is available, but detailed research cannot be carried out, and a preferred purification method is provided.
Disclosure of Invention
In order to fully utilize natural resources, improve the yield of the water chestnut shell polyphenol extract and the content of polyphenol in the water chestnut shell polyphenol extract and solve the problems in the prior art, the invention provides a preparation and purification method of the water chestnut shell polyphenol extract.
The invention selects the water chestnut shells in early mature stage to prepare the water chestnut shell polyphenol extract.
Preferably, the water chestnut shell polyphenol extract is prepared according to the following operation steps:
(1) cleaning water caltrop shells, drying, and pulverizing to obtain water caltrop shell powder;
(2) extracting water caltrop shell powder with 60% ethanol in 80 deg.C constant temperature water bath for 2 hr, performing ultrasonic treatment for 15min, centrifuging, collecting supernatant, concentrating under reduced pressure, and lyophilizing to obtain crude extract;
(3) dissolving the crude extract in appropriate amount of water, adding 3 times of petroleum ether, repeatedly extracting for 3 times, mixing the obtained water phases, concentrating under reduced pressure, and freeze drying to obtain polyphenol extract.
Preferably, the water chestnut shells are water chestnut shells of water chestnut or water chestnut without water chestnut.
Furthermore, the polyphenol extract contains gallic acid and pentagalloyl glucose, and can be applied to preparation of gallic acid and pentagalloyl glucose.
Furthermore, the water chestnut polyphenol extract has MDA-MB-231 triple negative breast cancer cell line activity, and can be used for preparing triple negative breast cancer resistant medicines.
The invention provides a method for purifying a water chestnut shell polyphenol extract, which comprises the following specific operations:
(1) carrying out pretreatment on AB-8 macroporous adsorption resin, and then filling the column by a wet method;
(2) sampling the water chestnut shell polyphenol extract at the flow rate of 0.5ml/min, wherein the sampling concentration is 0.5mg/ml, and the pH of the sampling solution is 2.5;
(3) eluting with 50% ethanol solution with pH of 4.5 at flow rate of 0.5 ml/min;
(4) collecting eluate, concentrating under reduced pressure, and lyophilizing to obtain refined polyphenol extract.
The invention has the advantages that:
the inventor finds that the water chestnut shells can be extracted at different stages to obtain different polyphenol extracts and polyphenol contents. In addition, the water caltrops are various in variety, and the amount of polyphenol extracts obtained by extracting different water caltrops and the polyphenol content of the polyphenol extracts are different. The prior art extracts polyphenol in water chestnut shells, but water chestnut shells in different mature stages and water chestnut varieties are not distinguished and cannot be fully utilized.
The invention selects the water chestnut shells of the water chestnut at the early stage of maturation (stage one) to prepare the polyphenol extract, obviously improves the yield of the polyphenol extract, the polyphenol content in the polyphenol extract and the contents of gallic acid and pentagalloyl glucose in the polyphenol extract, and can be further used for preparing the gallic acid and the pentagalloyl glucose. In addition, the water chestnut shells of the water chestnut or the water chestnut without water chestnut are selected for preparing the polyphenol extract, and the polyphenol content of the obtained polyphenol extract is higher. The invention also provides a purification method of the water chestnut shell polyphenol extract, which can remove other impurities and obviously improve the polyphenol content in the water chestnut shell polyphenol extract. The water chestnut shell polyphenol extract with high yield and high purity can be obtained by combining the preparation method and the purification method of the invention, and a technical basis is provided for further research on the water chestnut shell polyphenol extract and effective utilization of the water chestnut shells.
Drawings
In the attached figure 1, the water chestnut in the different maturation stages are stage four, stage three, stage two and stage one from left to right
FIG. 2 shows the polyphenol content in the water chestnut shells of the south lake at different maturation stages, wherein P is less than 0.01
FIG. 3 is water caltrop and water caltrop shell of different varieties of water caltrops
FIG. 4 shows polyphenol content in water chestnut shells of different varieties of water chestnut, wherein P is less than 0.01
FIG. 5 influence of macroporous adsorbent resin on adsorption of water chestnut shell polyphenol extract
FIG. 6 shows the effect of macroporous adsorbent resin on the desorption of polyphenol extract from water chestnut shells
FIG. 7 influence of the concentration of the loading solution on the adsorption of the polyphenol extract of water chestnut shells
FIG. 8 shows the influence of pH value of the sample solution on the adsorption of water chestnut shell polyphenol extract
FIG. 9 Effect of sample flow Rate on adsorption of Trapa natans L polyphenol extract
FIG. 10 shows the effect of volume fraction (ethanol ratio) of resolving liquid on the desorption of polyphenol extract from pericarpium Trapae
FIG. 11 influence of pH of resolving solution on the desorption of polyphenol extract from pericarpium Trapae
FIG. 12 influence of flow rate of eluent on the desorption of polyphenol extract from Equisetum Chinense Maxim
Detailed Description
The invention is further described with reference to the following figures and examples.
Example 1 content of polyphenols, Gallic acid and Pentagoyl Glucose in Water chestnut shells of Trapa natans L at different maturation stages and determination thereof
According to the maturity degree, the water chestnut is divided into four maturity stages: stage one, stage two, stage three and stage four (fig. 1). The appearance of the water chestnut in the first stage is emerald green, the peel is soft, and the length of the fruit is about 2 cm; the appearance of the water chestnut in the second stage is emerald green and slightly whitened, the peel is initially hardened, and the length of the fruit is about 3 cm; the appearance of the water chestnut in the third stage is emerald green and whitish, the peel is hardened, and the length of the fruit is about 4 cm; the water chestnut in the fourth stage is white and brown, the peel is hard, and the length of the fruit is about 5 cm.
Drying and pulverizing water chestnut shells of the four stages of the water chestnut in the south lake respectively to prepare powder. Weighing about 0.5g of powder of the four stages of the Nanhu lake water chestnut shells by an electronic balance respectively, pouring the powder into a 25ml volumetric flask, adding 15ml of 60% ethanol, extracting for 2h in a constant-temperature water bath kettle at 80 ℃, performing ultrasonic treatment for 15min, pouring into a 50ml centrifugal tube, and centrifuging the extract in a centrifuge at 3000r/min for 2 min. After centrifugation, the supernatant was taken with a disposable dropper into a 20ml centrifuge tube for use. The polyphenol content was determined according to the Folin phenol method. The contents of gallic acid and pentagalloylglucose were measured by HPLC, and the experimental results are shown in Table 1 below.
TABLE 1 content of gallic acid and pentagalloylglucose in the water chestnut hull of the water chestnut in different maturation stages of the south lake
The result shows that along with the continuous maturation of water caltrops, the polyphenol content in water caltrops shells is reduced, wherein the polyphenol content in the first stage is the highest and can reach 25.50 percent (figure 2); with the continuous maturation of water caltrops, the contents of gallic acid and pentagalloylglucose are reduced, wherein the contents of gallic acid and pentagalloylglucose in stage one are also highest, respectively 0.60% and 1.70%.
Therefore, the water chestnut shells of the first stage of the south lake water chestnut are selected for extracting polyphenol, the obtained polyphenol amount is higher than that of the water chestnut shells of other mature stages, the water chestnut shells are further used for preparing gallic acid and pentagalloyl glucose, and the yield of the obtained gallic acid and pentagalloyl glucose is higher.
Example 2 content of polyphenols, Gallic acid and Pentagoyl Glucose in Water chestnut shells of different species of Water chestnut and measurement thereof
And (3) the water caltrops of different varieties of water caltrops in the same harvest time (stage three): peeling water chestnut shells of Jinhuaqing water chestnut, Shaobai water chestnut, Shaoxing red water chestnut, Wujiao red water chestnut and Trapa acornis (figure 3), cleaning, freeze-drying for later use, pulverizing all dried water chestnut shells, and subpackaging in bags for marking for later use.
Weighing about 0.5g of powder of the shells of the Trapa acornis nakai, the Trapa japonica Thunb, the Shaobanchang Trapa japonica Thunb, the Trapa acornis nakai by using an electronic balance respectively, pouring into a 25ml volumetric flask, adding 15ml of 60% ethanol, extracting in a 80 ℃ constant temperature water bath kettle for 2h, performing ultrasonic treatment for 15min, pouring into a 50ml centrifugal tube, centrifuging the extract in the centrifugal machine at 3000r/min for 2min, and repeating for 3 times for each group. After centrifugation, the supernatant was taken with a disposable dropper into a 20ml centrifuge tube for use. The polyphenol content was determined according to the Folin phenol method. The contents of gallic acid and pentagalloylglucose were measured by HPLC, and the results are shown in Table 2.
TABLE 2 Gallic acid and Pentagoyl glucose content in pedicellus et pericarpium Trapae of different varieties of Trapae
The results show that the polyphenol content of the water chestnut shells of the Jinhuaqing water chestnut and the water chestnut shells of the water chestnut without water chestnut are respectively 17.71 percent and 17.11 percent, which are obviously higher than that of the water chestnut shells of other three types of water chestnut (figure 4). The pentagalloyl glucose content of the water chestnut shells of the Jinhuaqing water chestnut and the water chestnut shells of the water chestnut without the water chestnut is respectively 1.04 percent and 1.28 percent, and is also obviously higher than that of the water chestnut shells of other three types of water chestnut. In addition, the diamond shell of the water chestnut has the highest gallic acid content.
Therefore, the shells of the Jinhuaqing water chestnut and the water chestnut without water chestnut are selected for extracting the polyphenol, the obtained polyphenol amount is higher than that of the shells of the water chestnut of Shao Bo, Shao xing hong water chestnut and the water chestnut of south lake, the further preparation of the pentagalloyl glucose is carried out, and the yield of the obtained pentagalloyl glucose is higher.
Example 3 preparation of Polyphenol extract from Equisetum Tinctoria
Taking water caltrop, peeling water caltrop shells, cleaning, drying and crushing to obtain water caltrop shell powder. Taking water chestnut shell powder, and mixing the water chestnut shell powder and the water chestnut shell powder according to a material-liquid ratio of 1: adding 60% ethanol solution into 25 g/ml, extracting in 80 deg.C constant temperature water bath for 2 hr, ultrasonic treating for 15min, and centrifuging at 3000r/min for 10 min. Repeating the extraction for 2 times, and concentrating under reduced pressure to obtain crude extract. Dissolving the crude extract in appropriate amount of water, adding 3 times of petroleum ether, repeatedly extracting for 3 times, mixing the obtained water phases, concentrating under reduced pressure, and freeze drying to obtain polyphenol extract.
Example 4 Trapa natans polyphenol extract and polyphenol content and triple negative breast cancer cytotoxic activity thereof
According to example 3, 0.5g of water chestnut shells of the Trapa acornis at different maturity stages (stage one, stage two, stage three and stage four) and water chestnut shells of different varieties (Jinhuaqing Trapa, Shaobai, Shaoxing Hongling, Wujiao Hongling and Trapa acornis) at the same maturity stage (stage three) are respectively taken to prepare polyphenol extracts, and the weight of each obtained polyphenol extract is recorded. The polyphenol content of each polyphenol extract is measured according to a Folin phenol method, the activity of each polyphenol extract on MDA-MB-231 triple negative breast cancer cell strains is measured respectively, the activity measuring method is an MTT method, and the experimental results are shown in the following tables 3 and 4.
TABLE 3 Water caltrop polyphenol extracts at different maturation stages, their polyphenol contents and MDA-MB-231 triple negative breast cancer cytotoxic activity assay results
TABLE 4 determination results of water chestnut polyphenol extracts of different varieties, their polyphenol contents and MDA-MB-231 triple negative breast cancer cytotoxic activity
The experimental results prove that: (1) the water chestnut shells of the Trapa acornis nakai at the first mature stage are selected for preparing the water chestnut shell polyphenol extract, the preparation yield is the highest, and the yields at the second stage are far higher than those at the third stage and the fourth stage. The ripening processes of water caltrops of different varieties of water caltrops are similar, namely the processes that the color is changed from light to dark and the texture is changed from soft to hard, and the change of the polyphenol content in water caltrop shells is also similar. Therefore, the water chestnut shells of the water chestnut at the first maturation stage are selected for preparing the polyphenol extract, so that the preparation yield can be obviously improved.
(2) The water chestnut shell polyphenol extract in the first stage has the highest polyphenol content compared with other ripe stages. The water chestnut shells of the first-stage water chestnut are used for preparing the polyphenol extract to obtain the water chestnut shell polyphenol extract with higher polyphenol content, which is beneficial to the subsequent preparation of the high-purity water chestnut shell polyphenol extract and the research on the activity of the water chestnut shell polyphenol.
(3) The water chestnut shell polyphenol extract of water chestnut has the highest polyphenol content, and the polyphenol content of water chestnut shells of water chestnut with no horns is much higher than that of water chestnut of Shaobai, Shaoxing water chestnut and Trapa acornis. Therefore, the water chestnut shell polyphenol extract prepared from the water chestnut and the water chestnut without horns can obtain the extract with higher polyphenol content, and can be further used for preparing the high-purity polyphenol extract.
(4) Each water chestnut shell polyphenol extract has MDA-MB-231 triple negative type breast cancer cytotoxic activity, and the extract with higher polyphenol content has higher cytotoxic activity. The water chestnut shell polyphenol extract can be applied to further research, development and preparation of health-care food and medicines related to prevention and treatment of triple negative breast cancer, and in addition, the higher the polyphenol content is, the higher the research, development and application value of the water chestnut shell polyphenol extract is.
Example 5 purification of an extract of Trapa natans L by macroporous adsorbent resin
1 macroporous adsorbent resin pretreatment
Respectively taking 10g of AB-8, D101, HP100, HPD400 and NKA-9 macroporous adsorption resins, filling the macroporous adsorption resins into a column by a wet method with 20ml of absolute ethyl alcohol, and soaking for 24 hours. The mixture is rinsed with 30ml of absolute ethyl alcohol and then rinsed three times with 100ml of water. The resin was rinsed sequentially with 3 resin volumes of 5% hydrochloric acid, water, and 4% sodium hydroxide solution. Rinsing with water until the water is neutral. And draining water in the resin for later use.
2 calculation of adsorption Rate and desorption
The polyphenol content is determined according to the Folin phenol method, and the adsorption rate and desorption rate are calculated according to the following formula.
Adsorption rate ═ C0-C1)/C0
Desorption rate ═ C2V2/[(C0-C1)V1
In the formula C0、C1、C2Before adsorption, after adsorption and after desorption polyphenol mass concentration, V1For adsorption of volume of polyphenol solution, V2As volume of solution after desorption
3 selection of macroporous adsorbent resin
Respectively weighing 1.0g of pretreated AB-8, D101, HP100, HPD400 and NKA-9 macroporous adsorption resins, placing in a 50ml conical flask, adding 25ml of 6mg/ml water chestnut shell polyphenol extract, and sealing with tinfoil paper. Placing in a constant temperature oscillator at 30 deg.C and 100r/min for sufficiently oscillating and adsorbing for 8h, and arranging 3 parts in parallel for each group. Taking supernatant, and determining the concentration of the compound before adsorption according to Folin phenol method0) And after adsorption (C)1) And calculating the mass concentration of polyphenol and calculating the adsorption rate. From the experimental results (FIG. 5), it is found that the adsorption rate of AB-8 to polyphenol extract is the highest, and the adsorption rate is 79.31%, and the adsorption rates of other 4 kinds of macroporous adsorption are HPD100, HP20, D101 and NKA-9 in order from the highest to the lowest.
And (3) respectively carrying out suction filtration on the five adsorbed macroporous adsorption resins by using a suction filtration device, washing out surface residual substances by using 3ml of distilled water, and sucking out surface water. Placing in a 50ml conical flask, adding 25ml volume fraction 60% ethanol, sealing with tinfoil paper, and desorbing in a constant temperature oscillator at 30 deg.C and 100r/min for 8 hr to obtain 3 parts of the total amount. Taking supernatant, and respectively measuring mass concentration (C) of desorbed polyphenols by Folin phenol method2) The desorption rate was calculated. From the experimental results (fig. 6), the desorption rate of the AB-8 macroporous adsorbent resin was the highest, and was 95.28%.
In conclusion, among the five macroporous adsorption resins AB-8, D101, HP100, HPD400 and NKA-9, the adsorption rate and desorption rate of the AB-8 macroporous adsorption resin to the water chestnut shell polyphenol extract are the highest, and the method is more suitable for purifying the water chestnut shell polyphenol extract.
3 sample loading condition optimization
Putting 1.0g of the pretreated AB-8 macroporous adsorption resin into conical flasks respectively, adding 25ml of water chestnut shell polyphenol extract solution (loading solution) with mass concentration of 0.125, 0.25, 0.5, 1 and 2mg/ml, placing in a constant temperature oscillator at 30 ℃ and 100r/min for adsorption for 4h, and arranging 3 parts in parallel for each group. Taking the supernatant, and determining the concentration of the pre-adsorbed compounds (C) by the Folin phenol method0) And after adsorption (C)1) And calculating the mass concentration of polyphenol and calculating the adsorption rate. From the experimental results (fig. 7), it can be seen that the adsorption rate of the macroporous resin to the water chestnut shell polyphenol is 85.14% when the loading solution mass concentration is 0.5mg/ml, and the adsorption rate tends to increase when the loading solution concentration is increased from 0.25mg/ml to 0.5mg/ml, and the loading solution concentration continues to increase and the adsorption rate tends to decrease, so the optimal loading solution concentration is 0.5 mg/ml.
The pH of the sample solution was adjusted with hydrochloric acid to obtain 0.5mg/ml sample solutions with pH values of 4, 3.5, 3, 2.5, and 2, respectively. Taking 1.0g of the pretreated AB-8 macroporous adsorption resin respectively, putting the pretreated AB-8 macroporous adsorption resin into a conical flask, and adding 25ml of sample solutions with pH values of 4, 3.5, 3, 2.5 and 2 respectively. Placing in a constant temperature oscillator at 30 ℃ and 100r/min for adsorption for 4h, and arranging 3 parts in parallel for each group. Taking the supernatant, and determining the concentration of the pre-adsorbed compounds (C) by the Folin phenol method0) And after adsorption (C)1) And calculating the mass concentration of polyphenol and calculating the adsorption rate. From the experimental results (fig. 8), it was found that the adsorption rate gradually changed as the pH of the sample solution decreased and decreased to 3.0, and the adsorption rate reached the maximum (91.15%) at pH 2.5, and further decreased to further pH, and the adsorption rate started to decrease, so that the pH of the sample solution was preferably in the range of 2.0 to 3.0, and the optimum pH was 2.5.
Taking 4.0g of each pretreated AB-8 macroporous adsorption resin, loading the resin on a column by a wet method, taking 0.5mg/ml sample loading liquid with the pH of 2.5, loading the sample at the flow rates of 0.5ml/min, 2.0ml/min and 5.0ml/min respectively, and collecting effluent liquid at the lower end, wherein 3 parts are parallelly arranged in each group. Taking the effluent, and determining adsorbed polyphenol mass concentration (C) according to Folin phenol method1) And calculating the adsorption rate. As shown in the experimental results (FIG. 9), the adsorption rate decreased with the increase of the flow rate, and the adsorption rate was the highest at 99% at a flow rate of 0.5ml/min, so that the concentration of the sample solution was selected to be 0.5 ml/min.
In summary, the optimal sampling conditions after optimization are as follows: the concentration of the sample loading solution is 0.5mg/ml, the pH of the sample loading solution is 2.5, and the sample loading flow rate is 0.5 ml/min.
4 Desorption Condition optimization
Taking 15g of the pretreated AB-8 macroporous adsorption resin into a 500ml conical flask, measuring 375ml of 0.5mg/ml water chestnut shell polyphenol extract solution into the 500ml conical flask, and placing the mixture in a constant temperature oscillator at 30 ℃ and 100r/min for full oscillation and adsorption for 4 hours. And (3) carrying out suction filtration on the adsorbed macroporous adsorption resin by using a suction filtration device, washing away residual substances on the surface by using 45ml of distilled water, and sucking dry the surface water for later use.
Taking 1.0g of the AB-8 macroporous adsorption resin subjected to adsorption drying treatment, respectively placing in a conical flask, respectively adding 25ml of ethanol solution with volume fractions of 10%, 30%, 50%, 70% and 90%, placing in a constant temperature oscillator at 30 ℃ and 100r/min for desorption for 7h, and arranging 3 parts in parallel for each group. Taking the supernatant, and determining the mass concentration of the polyphenol according to a Folin phenol method. From the experimental results (fig. 10), it is understood that the mass concentration of the desorbed polyphenol increases with the increase of the volume fraction of ethanol, the mass concentration of the polyphenol reaches 232.91mg/ml at the maximum when the volume fraction of ethanol reaches 50%, and the mass concentration of the desorbed polyphenol tends to decrease with the increase of the volume fraction of ethanol again, so that the optimal ethanol concentration of the desorption solution is 50%.
Respectively taking 1.0g of the AB-8 macroporous adsorption resin subjected to adsorption drying treatment, putting into a conical flask, respectively adding desorption solutions with pH values of 6, 5, 4, 3 and 2 (namely 50% ethanol solution, adjusting the pH value by hydrochloric acid), putting into a constant temperature oscillator at 30 ℃ and 100r/min for desorption for 7h, and parallelly arranging 3 parts in each group. Taking the supernatant, and determining the mass concentration of the polyphenol according to a Folin phenol method. From the experimental results (fig. 11), it was found that the desorbed polyphenol concentration tended to increase with decreasing pH, and reached the highest concentration at pH 4.0, but further decreased pH, and declined at pH 4.0, and reached the highest concentration (14.16 mg/ml), so the optimum pH of the desorption solution was 4.0.
Collecting the above AB-8 macroporous adsorbent resin after adsorption drying 3.9g, loading onto column by wet method, eluting with 50% ethanol solution with pH of 4.5, controlling volume flow rate at 0.5ml/min, 2.0ml/min and 5.0ml/min, each group being arranged in parallelPlacing 3 parts. Collecting the lower elution solution, and determining the mass concentration (C) of polyphenol by Folin's phenol method2) The desorption rate was calculated. The results show (FIG. 12) that the desorption efficiency decreases with increasing flow rate, and when the flow rate is 0.5ml/min, the desorption efficiency is the highest (the concentration of adsorbed water chestnut shell polyphenol is 198.30 μ g/ml), so the optimal volume concentration of desorption solution is 0.5 ml/min.
Purification of water chestnut shell polyphenol extract
The AB-8 macroporous adsorbent resin is pretreated according to the method. Adding 40g AB-8 macroporous adsorbent resin into 35cm × 2.5cm glass chromatographic column, and loading with water chestnut shell polyphenol extract solution with mass concentration of 0.5mg/ml and pH of 2.5 as loading solution at flow rate of 0.5ml/min to obtain 300ml loading amount. Eluting with 50% ethanol solution of pH 4.5 at flow rate of 0.5ml/min to complete elution. Collecting eluate, concentrating under reduced pressure, and lyophilizing to obtain refined water chestnut shell polyphenol extract. The following table 5 (three parallel groups) shows the results of the determination of polyphenol content in the water chestnut shell polyphenol extract before and after purification by means of the forskolin phenol method.
TABLE 5 Polyphenol content in Water chestnut shell polyphenol extracts before and after purification
The results show that: the polyphenol content in the refined polyphenol extract obtained after purification is obviously improved by about 1.3 times. Therefore, the purification method can effectively remove impurities in the water chestnut shell polyphenol extract and obtain the water chestnut shell polyphenol extract with higher polyphenol content. From the above example 4, it can be seen that the cytotoxic activity of the water chestnut shell polyphenol extract is in direct proportion to the polyphenol content, the purified polyphenol extract obtained by the purification method has higher cytotoxic activity, can be applied to the research and development of triple negative breast cancer health food and drugs, and has low impurity, low probability of generating interference or side reaction, and good safety. The purification method provides powerful technical support for further application of the water chestnut shell polyphenol extract.
The above embodiments are only used for illustrating the technical ideas and features of the present invention, and the scope of the present invention should not be limited thereby, and any modifications made based on the technical ideas and features of the present invention are within the scope of the present invention. The technology not related to the invention can be realized by the prior art.
Claims (8)
1. The preparation method of the water chestnut shell polyphenol extract is characterized in that the water chestnut shells are water chestnut shells in early mature stage I.
2. The method for preparing the water chestnut shell polyphenol extract as claimed in claim 1, wherein the method comprises the following steps:
(1) cleaning water caltrop shells, drying, and pulverizing to obtain water caltrop shell powder;
(2) extracting water caltrop shell powder with 60% ethanol in 80 deg.C constant temperature water bath for 2 hr, performing ultrasonic treatment for 15min, centrifuging, collecting supernatant, concentrating under reduced pressure, and lyophilizing to obtain crude extract;
(3) dissolving the crude extract in appropriate amount of water, adding 3 times of petroleum ether, repeatedly extracting for 3 times, mixing the obtained water phases, concentrating under reduced pressure, and freeze drying to obtain polyphenol extract.
3. The method for preparing water chestnut shell polyphenol extract according to claim 1, wherein the water chestnut shells comprise water chestnut shells of water chestnut, water chestnut without water chestnut and water chestnut in lake south.
4. The method for preparing the water chestnut shell polyphenol extract as claimed in claim 1, wherein the polyphenol extract contains gallic acid and pentagalloyl glucose, and can be used for preparing the gallic acid and the pentagalloyl glucose.
5. The method for preparing the water chestnut shell polyphenol extract as claimed in claim 1, wherein the water chestnut shell polyphenol extract can be used for preparing a medicine for resisting triple negative breast cancer.
6. A method for purifying a water chestnut shell polyphenol extract comprises the following specific operation steps:
(1) carrying out pretreatment on AB-8 macroporous adsorption resin, and then filling the column by a wet method;
(2) sampling the water chestnut shell polyphenol extract at the flow rate of 0.5ml/min, wherein the sampling concentration is 0.5mg/ml, and the pH of the sampling solution is 2.5;
(3) eluting with 50% ethanol solution with pH of 4.5 at flow rate of 0.5 ml/min;
(4) collecting eluate, concentrating under reduced pressure, and lyophilizing to obtain refined polyphenol extract.
7. The method for purifying the water chestnut shell polyphenol extract as claimed in claim 6, wherein the water chestnut shell polyphenol extract is prepared from water chestnut shells in early maturation stage (stage one), and the specific operation steps comprise:
(1) cleaning water caltrop shells, drying, and pulverizing to obtain water caltrop shell powder;
(2) extracting water caltrop shell powder with 60% ethanol in 80 deg.C constant temperature water bath for 2 hr, performing ultrasonic treatment for 15min, centrifuging, collecting supernatant, concentrating under reduced pressure, and lyophilizing to obtain crude extract;
(3) dissolving the crude extract in appropriate amount of water, adding 3 times of petroleum ether, repeatedly extracting for 3 times, mixing the obtained water phases, concentrating under reduced pressure, and freeze drying to obtain polyphenol extract.
8. The method for purifying water chestnut shell polyphenol extract as claimed in claim 6, wherein the water chestnut shells comprise water chestnut shells of water chestnut, water chestnut without water chestnut and water chestnut in lake south.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114766671A (en) * | 2022-05-18 | 2022-07-22 | 黑龙江省科学院大庆分院 | Preparation method of black jerusalem artichoke polyphenol with activity of inhibiting colon cancer cell proliferation |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050281894A1 (en) * | 2002-11-08 | 2005-12-22 | Kin-Ping Wong | Extract of Trapa natans and methods of using the same |
| CN104546957A (en) * | 2014-12-24 | 2015-04-29 | 江苏省中国科学院植物研究所 | Water chestnut shell polyphenol extract as well as preparation method and application thereof |
| CN110003290A (en) * | 2019-04-22 | 2019-07-12 | 浙江工业大学 | A kind of mechanochemistry extracting method preparing hydrolyzable tannins crude extract from Shell of Water Chestnut |
| CN110652529A (en) * | 2018-06-28 | 2020-01-07 | 嘉兴学院 | Application of Trapa acornis nakai shell polyphenol extract in preparation of drug for resisting triple negative breast cancer |
| CN110652505A (en) * | 2018-06-28 | 2020-01-07 | 嘉兴学院 | Application of Trapa acornis nakai shell polyphenol extract in preparation of medicine for resisting HER-2 over-expression type breast cancer |
-
2021
- 2021-07-20 CN CN202110820546.1A patent/CN113662974A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050281894A1 (en) * | 2002-11-08 | 2005-12-22 | Kin-Ping Wong | Extract of Trapa natans and methods of using the same |
| CN104546957A (en) * | 2014-12-24 | 2015-04-29 | 江苏省中国科学院植物研究所 | Water chestnut shell polyphenol extract as well as preparation method and application thereof |
| CN110652529A (en) * | 2018-06-28 | 2020-01-07 | 嘉兴学院 | Application of Trapa acornis nakai shell polyphenol extract in preparation of drug for resisting triple negative breast cancer |
| CN110652505A (en) * | 2018-06-28 | 2020-01-07 | 嘉兴学院 | Application of Trapa acornis nakai shell polyphenol extract in preparation of medicine for resisting HER-2 over-expression type breast cancer |
| CN110003290A (en) * | 2019-04-22 | 2019-07-12 | 浙江工业大学 | A kind of mechanochemistry extracting method preparing hydrolyzable tannins crude extract from Shell of Water Chestnut |
Non-Patent Citations (2)
| Title |
|---|
| 严志萱: "《蔬菜设施栽培实用技术》", 31 August 2016, 浙江科学技术出版社 * |
| 赵小芳 等: "菱属植物化学成分与生物活性的研究进展", 《世界科学技术-中医药现代化》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114766671A (en) * | 2022-05-18 | 2022-07-22 | 黑龙江省科学院大庆分院 | Preparation method of black jerusalem artichoke polyphenol with activity of inhibiting colon cancer cell proliferation |
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