CN114694776A - Refining of pueraria flower isoflavone and research method of lipid-lowering effect thereof - Google Patents
Refining of pueraria flower isoflavone and research method of lipid-lowering effect thereof Download PDFInfo
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Abstract
The invention discloses a refining method of pueraria flower isoflavone and a research method of lipid-lowering effect of pueraria flower isoflavone, wherein an HPD-100 type macroporous resin is utilized to optimize a refining process of pueraria flower isoflavone and evaluate the in-vitro lipid-lowering activity of pueraria flower isoflavone. The result shows that the purification process of the pueraria flower isoflavone comprises the following steps of: the pueraria flower isoflavone concentration is 100mg/mL, the pH value is 6.0, the sample loading volume is 20mL, and the desorption parameters are as follows: the eluent is 70% ethanol (v/v) with pH of 5.0, the elution flow rate is 2.0mL/min, and the purity of the refined pueraria flower isoflavone can reach 81.47%; the inhibition rate of the refined pueraria flower isoflavone on pancreatic lipase, cholesterol esterase and cholesterol micelle solubility can reach 40.73%, 52.32% and 46.17%, and a certain dosage effect relationship exists. The flos Puerariae Lobatae isoflavone can reduce cholesterol micelle solubility by inhibiting activity of pancreatic lipase and cholesterol esterase to reduce blood lipid.
Description
Technical Field
The invention relates to the technical field of extraction of medicinal plant components, in particular to a refining method of pueraria flower isoflavone and a research method of lipid-lowering effect of pueraria flower isoflavone.
Background
Obesity has become a worldwide health problem, and lipid overdosing is one of the important factors leading to overweight. Inhibiting the absorption of dietary fat into the digestive system of a human body is one of the important ways of losing weight. Pancreatic lipase and cholesterol esterase are key enzymes for digesting and absorbing dietary fat and cholesterol, the activities of the pancreatic lipase and the cholesterol esterase are inhibited, and the cholesterol-reducing micelle solubility is a general index for evaluating the in-vitro lipid-reducing and weight-losing.
The flower of Pueraria lobata is the flower bud of Pueraria lobata, also called the flower of Pueraria lobata. The flos Puerariae Lobatae has various biological activities such as relieving hangover, reducing blood lipid, resisting tumor, and resisting oxidation, and flos Puerariae Lobatae isoflavone is one of main bioactive components of flos Puerariae Lobatae, and has various biological activities. In recent years, some research results are obtained in the aspects of extraction, purification and structure identification of pueraria flower isoflavone.
However, the application of pueraria flower isoflavone in the fields of food, cosmetics and medicine health is limited due to the laggard separation and purification technology of pueraria flower isoflavone and low product purity.
Disclosure of Invention
1. Technical problem to be solved
The invention aims to solve the problems that the prior art falls behind the separation and purification technology of pueraria flower isoflavone and the product purity is not high, which limits the application of pueraria flower isoflavone in the fields of food, cosmetics and medical health, and provides a method for researching the refining and lipid-lowering effect of pueraria flower isoflavone.
2. Technical scheme
In order to achieve the purpose, the invention adopts the following technical scheme:
the research on the refining process of the pueraria flower isoflavone comprises the following steps:
step 1: extracting isoflavone from flos Puerariae Lobatae;
step 2: research on the adsorption rate and desorption rate of pueraria flower isoflavone, wherein the adsorption rate is calculated according to formula (1), the desorption rate is calculated according to formula (2),
in the formula: a. the0The absorbance value (250nm) of the pueraria flower isoflavone of the stock solution is obtained;
A1the absorbance value of the solution after adsorption (250 nm);
A2absorbance values for the elution solution (250 nm);
and step 3: the resin optimization research specifically comprises the following steps:
step 3.1: respectively taking 5gAB-8, NKA-9, D101, HPD-100 and L step A-10 type macroporous adsorption resin, pretreating, placing in a triangular flask, respectively adding 50mg/mL of crude extract of pueraria flower isoflavone 20mL, shaking at room temperature for 2h, filtering, measuring the absorbance value of the filtrate at 250nm, and calculating the adsorption rate;
step 3.2: respectively adding 20mL of 70% ethanol aqueous solution into the resin subjected to adsorption saturation, oscillating at room temperature for 2h, filtering, measuring the absorbance value (250nm) of the filtrate, and calculating the desorption rate;
and 4, step 4: the method for researching the influence of the pueraria flower isoflavone concentration on the adsorption rate specifically comprises the following steps:
step 4.1: 50mL of pueraria flower isoflavone solutions with different mass concentrations are taken;
step 4.2: mixing with 20g of pretreated HPD-100 type macroporous resin, oscillating at room temperature for 2h, filtering, measuring absorbance value (250nm) of filtrate, and calculating adsorption rate;
and 5: the method for researching the influence of the ethanol concentration on the desorption rate of the pueraria flower isoflavone specifically comprises the following steps:
step 5.1: taking 50mL of ethanol solutions (v/v) with different concentrations respectively;
step 5.2: adding 20g of HPD-100 type macroporous resin with saturated adsorption, oscillating at room temperature for 2h, filtering, measuring the absorbance value (250nm) of the filtrate, and calculating the desorption rate;
step 6: the method for researching the influence of the pH value of the eluent on the desorption rate specifically comprises the following steps:
step 6.1: taking 20g of HPD-100 type macroporous resin with saturated adsorption into a triangular flask, and respectively adding 50mL of 70% (v/v) ethanol solution to obtain a mixed solution;
step 6.2: adjusting the mixed solution to different pH values, oscillating for 2h at room temperature, filtering, measuring the absorbance value (250nm) of the filtrate, and calculating the desorption rate;
and 7: the method for researching the influence of the elution flow rate on the desorption rate specifically comprises the following steps:
step 7.1: filling HPD-100 type macroporous resin (pretreated) into a chromatographic column (phi 35mm is multiplied by 500mm), and balancing with deionized water for 12 hours;
step 7.2: adding 20mL of 100mg/mL pueraria flower isoflavone solution into the upper layer of the column, and eluting with 70% ethanol solution with pH of 5.0 at different flow rates after the solution completely enters the resin layer;
step 7.3: the eluate was collected, and the absorbance (250nm) of the filtrate was measured to calculate the desorption rate.
Preferably, the extraction of the pueraria flower isoflavone in the step 1 specifically comprises the following steps:
step 1.1, taking pueraria flower as a raw material, crushing the pueraria flower to pass through a 80-mesh sieve, wherein the solid-to-liquid ratio (g/mL) is 1:16, the ethanol concentration (v/v) is 74%, the ultrasonic power is 260W, the ultrasonic time is 40min, the ultrasonic temperature is 76 ℃, and the extraction is carried out for 2 times;
step 1.2: then merging the filtrate, concentrating in vacuum, and freeze-drying to obtain the pueraria flower isoflavone crude product.
Preferably, the resin with the highest adsorption rate and desorption rate in the step 3.2 is the purified resin of the pueraria flower isoflavone.
Preferably, the mass concentrations of the different pueraria flower isoflavone solutions in the step 4.1 are respectively 12.5, 25, 50, 100, 200, 400 and 800 mg/mL.
Preferably, the concentration of the ethanol solution (v/v) in step 5.1 comprises 30%, 40%, 50%, 60%, 70%, and 80%.
Preferably, the pH of the mixed solution in step 6.2 includes 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0.
Preferably, the flow rates in step 7.2 include 1.0, 2.0, 3.0, 4.0 and 5.0 mL/min.
The invention also provides a research on the lipid-lowering effect of the pueraria flower isoflavone, which comprises the following steps:
s1: the method for researching the influence of the pueraria flower isoflavone on the pancreatic lipase activity specifically comprises the following steps: taking 350 mu L of buffer solution, 150 mu L of enzyme solution and 50 mu L of pueraria flower isoflavone samples with different mass fractions respectively, putting the buffer solution and the enzyme solution into a centrifuge tube, adding 450 mu L of substrate to start reaction, incubating at 37 ℃ for 2h, centrifuging (8000r/min,1min), measuring the absorbance value (400nm) of supernatant, replacing pueraria flower isoflavone with deionized water in a control group, and calculating the pancreatic lipase inhibition rate according to the formula (3).
In the formula: a. theCThe absorbance value of the control group is obtained; a. theSIs the absorbance value of the sample;
s2: the method for researching the influence of the pueraria flower isoflavone on the activity of the cholesterol esterase specifically comprises the following steps: taking 925 mu L substrate buffer solution, 50 mu L enzyme solution and 25 mu L pueraria flower isoflavone samples with different mass fractions respectively, placing the samples in a centrifuge tube, incubating for 5min at 25 ℃, measuring the absorbance value (405nm), adding 25 mu L deionized water into a control group, and calculating the cholesterol esterase inhibition rate formula (4).
In the formula: a. theCThe absorbance value of the control group is obtained; a. theSIs the absorbance value of the sample;
s3: the method for researching the influence of the pueraria flower isoflavone on the solubility of cholesterol micelles comprises the following steps: preparing with 15mmol/L phosphate buffer solution (pH 7.4), containing sodium taurocholate (10mmol/L), cholesterol (2mmol/L), oleic acid (5mmol/L) and NaCl (132mmol/L), treating with ultrasonic wave (400Hz) for 1h, incubating at 37 ℃ for 24h, mixing 1mL of pueraria flower isoflavone samples with different mass fractions with 5mL of cholesterol micelles, incubating at 37 ℃ for 2h, centrifuging (8000r/min,10min), measuring the cholesterol content of a supernatant by using a total cholesterol kit, adding 1mL of deionized water into a control group, and calculating the inhibition rate according to formula (5).
In the formula: n is a radical of hydrogenCCholesterol content/mmol/L of control group; n is a radical ofSIs the cholesterol content/mmol/L of the sample;
s4: data processing, wherein experimental data are expressed in x +/-s, SPSS 19.0 software is adopted for data difference significance analysis, and Origin 8.5 software is adopted for plotting.
Preferably, in S1:
the preparation method of the enzyme solution (10mg/mL) comprises the following steps: dissolving 1g of pancreatic lipase in 10mL of deionized water, vortexing for 2min, centrifuging (8000r/min,10min), and taking supernatant for later use;
buffer solution: 100mM Tris solution (Tris) pH 8.2;
the preparation method of the substrate (p-nitrophenyl laurate 0.08%) comprises the following steps: prepared with 5mM phosphate buffer pH5.0 (containing 1% Triton X-100).
Preferably, in S2:
the preparation method of the enzyme solution (25 mug/mL) comprises the following steps: dissolving 0.25mg of porcine pancreatic cholesterol esterase in 10mL of deionized water, centrifuging (8000r/min,10min), and taking supernatant for later use;
the preparation method of the substrate buffer solution comprises the following steps: 0.1mol/L phosphate buffer solution (pH 7.04) (containing 0.1mol/L NaCl, 0.02mol/L p-nitrophenyl butyrate and 6mmol/L sodium taurocholate) is prepared.
3. Advantageous effects
Compared with the prior art, the invention has the advantages that:
(1) in the invention, the pueraria flower isoflavone is refined by using macroporous resin and the lipid-lowering and weight-losing activity of the pueraria flower isoflavone is discussed. The HPD-100 type macroporous adsorption resin is the best resin for refining the pueraria flower isoflavone, and the adsorption parameters are as follows: the concentration of pueraria flower isoflavone is 100mg/mL, the pH value is 6.0, the volume is 20mL, and the desorption parameters are as follows: 70% ethanol water solution (pH5.0), flow rate is 2.0mL/min, and the refined flos Puerariae Lobatae isoflavone has purity of 81.47%.
(2) In the invention, the refined pueraria flower isoflavone shows a certain inhibition effect on pancreatic lipase, cholesterol esterase and cholesterol micelle solubility, and has a certain dose effect relationship, the inhibition rate is slightly lower than orlistat, and the pueraria flower isoflavone reduces the cholesterol micelle solubility by inhibiting the activities of the pancreatic lipase and the cholesterol esterase to play a role in reducing fat and losing weight.
Drawings
FIG. 1 is a graph comparing the adsorption and desorption rates of isoflavone from different macroporous resins;
FIG. 2 is a graph comparing the effect of different concentrations of isoflavones on adsorption rate;
FIG. 3 is a graph comparing the effect of different pH values of the adsorption solution on the adsorption rate;
FIG. 4 is a graph comparing the effect of different ethanol concentrations on desorption rate;
FIG. 5 is a graph comparing the effect of eluent pH on desorption rate;
FIG. 6 is a graph showing the effect of elution flow rate on desorption rate;
FIG. 7 is a graph showing the elution profile of pueraria flower isoflavones;
FIG. 8 is a graph comparing the effect of different pueraria flower isoflavone concentrations on pancreatic lipase activity;
FIG. 9 is a graph comparing the effect of different concentrations of isoflavone on cholesterol esterase activity;
FIG. 10 is a graph comparing the effect of different concentrations of pueraria flower isoflavones on the solubility of cholesterol micelles.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
Example 1:
the research on the refining process of the pueraria flower isoflavone comprises the following steps:
step 1: extracting pueraria flower isoflavone;
step 2: research on the adsorption rate and desorption rate of pueraria flower isoflavone, wherein the adsorption rate is calculated according to formula (1), the desorption rate is calculated according to formula (2),
in the formula: a. the0The absorbance value (250nm) of the pueraria flower isoflavone of the stock solution is obtained;
A1the absorbance value of the solution after adsorption (250 nm);
A2absorbance values for the elution solution (250 nm);
and step 3: the resin optimization research specifically comprises the following steps:
step 3.1: respectively taking 5gAB-8, NKA-9, D101, HPD-100 and L step A-10 type macroporous adsorption resin, pretreating, placing in a triangular flask, respectively adding 50mg/mL of crude extract of pueraria flower isoflavone 20mL, shaking at room temperature for 2h, filtering, measuring the absorbance value of the filtrate at 250nm, and calculating the adsorption rate;
step 3.2: respectively adding 20mL of 70% ethanol aqueous solution into the resin with saturated adsorption, oscillating at room temperature for 2h, filtering, measuring the absorbance value (250nm) of the filtrate, calculating the desorption rate, and taking the resin with the highest adsorption rate and desorption rate as the refined resin of the pueraria flower isoflavone;
and 4, step 4: the method for researching the influence of the pueraria flower isoflavone concentration on the adsorption rate specifically comprises the following steps:
step 4.1: taking 50mL of pueraria flower isoflavone solutions with different mass concentrations respectively, wherein the mass concentrations of the pueraria flower isoflavone solutions are respectively 12.5, 25, 50, 100, 200, 400 and 800 mg/mL;
step 4.2: mixing with 20g of pretreated HPD-100 type macroporous resin, oscillating at room temperature for 2h, filtering, measuring absorbance value (250nm) of filtrate, and calculating adsorption rate;
and 5: the method for researching the influence of the ethanol concentration on the desorption rate of the pueraria flower isoflavone specifically comprises the following steps:
step 5.1: taking 50mL of ethanol solutions (v/v) with different concentrations, wherein the concentration of the ethanol solution (v/v) comprises 30%, 40%, 50%, 60%, 70% and 80%;
step 5.2: adding 20g of HPD-100 type macroporous resin with saturated adsorption, oscillating at room temperature for 2h, filtering, measuring the absorbance value (250nm) of the filtrate, and calculating the desorption rate;
and 6: the method for researching the influence of the pH value of the eluent on the desorption rate specifically comprises the following steps:
step 6.1: taking 20g of HPD-100 type macroporous resin with saturated adsorption into a triangular flask, and respectively adding 50mL of 70% (v/v) ethanol solution to obtain a mixed solution;
step 6.2: adjusting the pH value of the mixed solution to different pH values, oscillating for 2h at room temperature, filtering, measuring the absorbance value (250nm) of the filtrate, and calculating the desorption rate, wherein the pH value of the mixed solution comprises 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0;
and 7: the method for researching the influence of the elution flow rate on the desorption rate specifically comprises the following steps:
step 7.1: filling HPD-100 type macroporous resin (pretreated) into a chromatographic column (phi 35mm is multiplied by 500mm), and balancing with deionized water for 12 hours;
step 7.2: adding 20mL of 100mg/mL pueraria flower isoflavone solution into the upper layer of the column, and eluting with 70% ethanol solution with pH of 5.0 at different flow rates including 1.0, 2.0, 3.0, 4.0 and 5.0mL/min after the solution completely enters the resin layer;
step 7.3: the eluate was collected, and the absorbance (250nm) of the filtrate was measured to calculate the desorption rate.
In the invention, the enzyme standard instrument F50: tecan group, Switzerland; UV-1700 type ultraviolet-visible spectrophotometer: shimadzu, Japan; analytical balance model AL 104: Mettler-Tollido instruments (Shanghai) Inc.; ALPHA1-4LDPLUS model vacuum freeze dryer: marin Christ, Germany; RT-25 air-flowing type ultrafine grinder: beijing kujieyi mechanical devices, Inc.; KS-600N type ultrasonic extractor: kogaku Kogyo instruments and devices, Inc.; RE-52C rotary evaporator: consolidate City Yaohua instruments, Inc.
In the invention, the puerarin standard substance is as follows: the Chinese food and drug testing institute; AB-8, NKA-9, HPD-100, D101, LSA-10 type macroporous resins: tianjin Kaishu resin science and technology Co., Ltd; porcine pancreatic lipase (100U/mg), porcine pancreatic cholesterol esterase (100U/mg), p-nitrophenyl laurate, p-nitrophenylbutyrate: sigma, USA; orlistat capsule (orlistat): chongqing Huasen pharmaceutical Co., Ltd; total cholesterol assay kit: nanjing is built into a bioengineering institute; other reagents are domestic analytical purifiers.
Example 2
The implementation contents of the above embodiments can be referred to the above description, and the embodiments herein are not repeated in detail; in the embodiment of the present application, the difference from the above embodiment is:
the pueraria flower isoflavone extraction method specifically comprises the following steps:
step 1.1, taking pueraria flower as a raw material, crushing and sieving the pueraria flower by a sieve of 80 meshes, wherein the solid-to-liquid ratio (g/mL) is 1:16, the concentration (v/v) of ethanol is 74%, the ultrasonic power is 260W, the ultrasonic time is 40min, the ultrasonic temperature is 76 ℃, and the extraction is carried out for 2 times;
step 1.2: then merging the filtrate, concentrating in vacuum, and freeze-drying to obtain the pueraria flower isoflavone crude product.
Example 3
The research on the lipid-lowering effect of the pueraria flower isoflavone comprises the following steps:
s1: the method for researching the influence of the pueraria flower isoflavone on the pancreatic lipase activity specifically comprises the following steps: taking 350 mu L of buffer solution, 150 mu L of enzyme solution and 50 mu L of pueraria flower isoflavone samples with different mass fractions respectively, putting the buffer solution and the enzyme solution into a centrifuge tube, adding 450 mu L of substrate to start reaction, incubating at 37 ℃ for 2h, centrifuging (8000r/min,1min), measuring the absorbance value (400nm) of supernatant, replacing pueraria flower isoflavone with deionized water in a control group, and calculating the pancreatic lipase inhibition rate according to the formula (3).
In the formula: a. theCThe absorbance value of the control group is obtained; a. theSIs the absorbance value of the sample;
s2: the method for researching the influence of the pueraria flower isoflavone on the activity of the cholesterol esterase specifically comprises the following steps: taking 925 mu L substrate buffer solution, 50 mu L enzyme solution and 25 mu L pueraria flower isoflavone samples with different mass fractions respectively, placing the samples in a centrifuge tube, incubating for 5min at 25 ℃, measuring the absorbance value (405nm), adding 25 mu L deionized water into a control group, and calculating the cholesterol esterase inhibition rate formula (4).
In the formula: a. theCThe absorbance value of the control group is obtained; a. theSIs the absorbance value of the sample;
s3: the method for researching the influence of the pueraria flower isoflavone on the solubility of cholesterol micelles specifically comprises the following steps: preparing with 15mmol/L phosphate buffer solution (pH 7.4), containing sodium taurocholate (10mmol/L), cholesterol (2mmol/L), oleic acid (5mmol/L) and NaCl (132mmol/L), treating with ultrasonic wave (400Hz) for 1h, incubating at 37 ℃ for 24h, mixing 1mL of pueraria flower isoflavone samples with different mass fractions with 5mL of cholesterol micelles, incubating at 37 ℃ for 2h, centrifuging (8000r/min,10min), measuring the cholesterol content of a supernatant by using a total cholesterol kit, adding 1mL of deionized water into a control group, and calculating the inhibition rate according to formula (5).
In the formula: n is a radical ofCCholesterol content/mmol/L of control group; n is a radical ofSIs the cholesterol content/mmol/L of the sample;
s4: data processing, test data to
Data were analyzed for significance using SPSS 19.0 software and plotted using Origin 8.5 software.
Example 4
The implementation contents of the above embodiments can be referred to the above description, and the embodiments herein are not repeated in detail; in the embodiment of the present application, the difference from the above embodiment is:
in S1: the preparation method of the enzyme solution (10mg/mL) comprises the following steps: dissolving 1g of pancreatic lipase in 10mL of deionized water, vortexing for 2min, centrifuging (8000r/min,10min), and taking supernatant for later use;
buffer solution: 100mM Tris solution (Tris) pH 8.2;
the preparation method of the substrate (p-nitrophenyl laurate 0.08%) comprises the following steps: prepared with 5mM phosphate buffer pH5.0 (containing 1% Triton X-100).
Example 5
The implementation contents of the above embodiments can be referred to the above description, and the embodiments herein are not repeated in detail; in the embodiment of the present application, the difference from the above embodiment is:
in S2: the preparation method of the enzyme solution (25 mug/mL) comprises the following steps: dissolving 0.25mg of porcine pancreatic cholesterol esterase in 10mL of deionized water, centrifuging (8000r/min,10min), and taking supernatant for later use;
the preparation method of the substrate buffer solution comprises the following steps: 0.1mol/L phosphate buffer solution (pH 7.04) (containing 0.1mol/L NaCl, 0.02mol/L p-nitrophenyl butyrate and 6mmol/L sodium taurocholate) is prepared.
Example 6
The implementation contents of the above embodiments can be referred to the above description, and the embodiments herein are not repeated in detail; in the embodiment of the present application, the difference from the above embodiment is:
analysis of experimental results:
in this embodiment, the macroporous resin is a column filler of a refined isoflavone compound which is widely selected, and due to differences in polarity and particle size, there are differences in the degree of difficulty in adsorption and dissociation of different isoflavone compounds.
In the embodiment, as can be seen from fig. 1, the HPD-100 type macroporous resin has better adsorption and dissociation properties on pueraria flower isoflavone, the adsorption rate is 89.61% at most, the desorption rate is 83.86% at most, and the polarity and particle size of the HPD-100 type macroporous resin are better than those of other types of macroporous resin, so that the HPD-100 type macroporous resin is suitable for separating pueraria flower isoflavone and can be used for the subsequent refining process.
In this example, as can be seen from fig. 2, the adsorption rate tends to increase and then decrease with the increase of the isoflavone concentration of pueraria lobata, and when the isoflavone concentration of pueraria lobata reaches 100mg/mL, the adsorption rate reaches a maximum of 88.41%, and thereafter the adsorption rate gradually decreases. It may be that the high concentration of pueraria flower isoflavone blocks the holes of the resin, which results in the decrease of the adsorption rate, so that the concentration of the subsequent pueraria flower isoflavone adsorption solution is set as 100 mg/mL.
In this example, as can be seen from fig. 3, the adsorption rate tends to increase and decrease with the increase of the pH of the adsorption solution, and when the pH of the adsorption solution is 6.0, the adsorption rate is 87.49% at the highest. The weak acidic nature of the pueraria flower isoflavones causes the highest adsorption rate to occur at pH6.0, and thus the pH of the subsequent adsorption solution is adjusted to 6.0.
In this example, as can be seen from fig. 4, the desorption rate increases first and then decreases as the ethanol concentration increases, and at the ethanol concentration of 70%, the maximum desorption rate appears to be 82.35%, and the 70% ethanol polarity is suitable for the separation of the pueraria flower isoflavone from the resin, so the elution solution is defined as 70% ethanol aqueous solution.
In this example, as can be seen from fig. 5, the desorption rate increases and then decreases as the pH of the eluent increases, and the maximum desorption rate of 81.52% occurs at a pH of 5.0, and therefore, the pH of the eluent is adjusted to 5.0.
In this embodiment, as can be seen from fig. 6, the desorption rate tends to increase first and then decrease as the elution flow rate increases, and when elution is performed at a speed of 2.0mL/min, the maximum desorption rate is 82.35%, the elution flow rate is too high, and the elution of pueraria flower isoflavone is not complete enough, which leads to a decrease in desorption rate, so the elution flow rate is adjusted to 2.0 mL/min.
In this example, the refining process parameters of pueraria flower isoflavone are as follows: the chromatographic column (phi 35mm multiplied by 500mm) is filled with pretreated HPD-100 type macroporous resin, the balance is carried out for 12 hours by deionized water, and the adsorption parameters are as follows: the concentration of the pueraria flower isoflavone is 100mg/mL, the pH6.0 is realized, and the volume is 20 mL; desorption parameters: 70% ethanol aqueous solution (pH5.0), flow rate 2.0 mL/min.
In the present example, as can be seen from fig. 7, after purification by HPD-100 type macroporous resin, a single component peak appears in the elution curve, the number of the collection tubes is (22-30), vacuum concentration and freeze drying are performed to obtain a purified component a of pueraria flower isoflavone1(purity 81.47%)。
In this example, as can be seen from fig. 8, the lipase inhibition rate shows a linear increasing trend with the increase of the pueraria flower isoflavone concentration, and after the concentration exceeds 0.2mg/mL, the inhibition rate becomes gentle, and at the concentration of 1.0mg/mL, the maximum inhibition rate is 40.73%, which is slightly lower than the inhibition rate of orlistat (44.36%). Pancreatic lipase is a key enzyme for dietary lipolysis, and can reduce fat absorption and reduce fat utilization rate by inhibiting the activity of pancreatic lipase. The flos Puerariae Lobatae isoflavone can inhibit pancreatic lipase activity, and reduce fat and weight by reducing fat digestion and decomposition.
In the present example, as can be seen from fig. 9, as the concentration of isoflavone of pueraria lobata increases, the activity of cholesterol esterase shows a certain inhibition effect, and the inhibition rate shows a linear increasing trend, wherein the increasing trend is gentle when the concentration is higher than 0.6mg/mL, and the inhibition rate is 52.32% at the highest when the concentration is 1.0mg/mL, which is slightly lower than the inhibition rate of orlistat (54.28%). Inhibiting cholesterol esterase activity, reducing cholesterol ester in diet, decomposing into cholesterol, inhibiting cholesterol absorption, and reducing blood fat and body weight]. The pueraria flower isoflavone can inhibit the decomposition of dietary cholesterol ester into free cholesterol to enter a digestive system, and has certain lipid-lowering and weight-losing activity.
In this example, as can be seen from fig. 10, the inhibition rate of cholesterol micelle solubility linearly increases with the increase of the concentration of pueraria flower isoflavone, and at a concentration of 1.0mg/mL, the inhibition rate has a maximum value of 46.17%, which is slightly lower than that of orlistat (48.41%). The cholesterol is transported to the digestive system by virtue of cholesterol micelles, and the higher the solubility is, the higher the absorption and utilization rate is[25]. The pueraria flower isoflavone can reduce the solubility of cholesterol micelle, reduce the bioavailability of cholesterol and play roles in reducing fat and losing weight.
In the invention, the pueraria flower isoflavone is refined by using macroporous resin and the lipid-lowering and weight-losing activity of the pueraria flower isoflavone is discussed. The HPD-100 type macroporous adsorption resin is the best resin for refining the pueraria flower isoflavone, and the adsorption parameters are as follows: the concentration of the pueraria flower isoflavone is 100mg/mL, the pH value is 6.0, the volume is 20mL, and the desorption parameters are as follows: 70% ethanol water solution (pH5.0), flow rate is 2.0mL/min, and the refined flos Puerariae Lobatae isoflavone has purity of 81.47%; the refined pueraria flower isoflavone shows a certain inhibition effect on pancreatic lipase, cholesterol esterase and cholesterol micelle solubility, and has a certain dose effect relationship, the inhibition rate is slightly lower than orlistat, the pueraria flower isoflavone reduces the cholesterol micelle solubility to play a role in reducing fat and losing weight by inhibiting the activities of the pancreatic lipase and the cholesterol esterase, and the activity and the action mechanism of the pueraria flower isoflavone for reducing fat and losing weight in vivo are discussed in subsequent subject researches.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (10)
1. The research on the refining process of the pueraria flower isoflavone is characterized by comprising the following steps of:
step 1: extracting isoflavone from flos Puerariae Lobatae;
step 2: research on the adsorption rate and desorption rate of isoflavone in flos Puerariae Lobatae, wherein the adsorption rate is calculated according to formula (1), the desorption rate is calculated according to formula (2),
in the formula: a. the0The absorbance value (250nm) of the pueraria flower isoflavone of the stock solution is obtained;
A1the absorbance value of the solution after adsorption (250 nm);
A2absorbance values for the elution solution (250 nm);
and step 3: the resin optimization research specifically comprises the following steps:
step 3.1: respectively taking 5gAB-8, NKA-9, D101, HPD-100 and L step A-10 type macroporous adsorption resin, pretreating, placing in a triangular flask, respectively adding 50mg/mL of crude extract of pueraria flower isoflavone 20mL, shaking at room temperature for 2h, filtering, measuring the absorbance value of the filtrate at 250nm, and calculating the adsorption rate;
step 3.2: respectively adding 20mL of 70% ethanol aqueous solution into the resin subjected to adsorption saturation, oscillating at room temperature for 2h, filtering, measuring the absorbance value (250nm) of the filtrate, and calculating the desorption rate;
and 4, step 4: the method for researching the influence of the pueraria flower isoflavone concentration on the adsorption rate specifically comprises the following steps:
step 4.1: 50mL of pueraria flower isoflavone solutions with different mass concentrations are taken;
step 4.2: mixing with 20g of pretreated HPD-100 type macroporous resin, oscillating at room temperature for 2h, filtering, measuring absorbance value (250nm) of filtrate, and calculating adsorption rate;
and 5: the method for researching the influence of the ethanol concentration on the desorption rate of the pueraria flower isoflavone specifically comprises the following steps:
step 5.1: taking 50mL of ethanol solutions (v/v) with different concentrations respectively;
step 5.2: adding 20g of HPD-100 type macroporous resin with saturated adsorption, oscillating at room temperature for 2h, filtering, measuring the absorbance value (250nm) of the filtrate, and calculating the desorption rate;
step 6: the method for researching the influence of the pH value of the eluent on the desorption rate specifically comprises the following steps:
step 6.1: taking 20g of HPD-100 type macroporous resin with saturated adsorption into a triangular flask, and respectively adding 50mL of 70% (v/v) ethanol solution to obtain a mixed solution;
step 6.2: adjusting the mixed solution to different pH values, oscillating for 2h at room temperature, filtering, measuring the absorbance value (250nm) of the filtrate, and calculating the desorption rate;
and 7: the method for researching the influence of the elution flow rate on the desorption rate specifically comprises the following steps:
step 7.1: filling HPD-100 type macroporous resin (pretreated) into a chromatographic column (phi 35mm is multiplied by 500mm), and balancing with deionized water for 12 hours;
step 7.2: adding 20mL of 100mg/mL pueraria flower isoflavone solution into the upper layer of the column, and eluting with 70% ethanol solution with pH of 5.0 at different flow rates after the solution completely enters the resin layer;
step 7.3: the eluate was collected, and the absorbance (250nm) of the filtrate was measured to calculate the desorption rate.
2. The research on the refining process of pueraria flower isoflavone according to claim 1, wherein the extraction of pueraria flower isoflavone in the step 1 specifically comprises the following steps:
step 1.1, taking pueraria flower as a raw material, crushing and sieving the pueraria flower by a sieve of 80 meshes, wherein the solid-to-liquid ratio (g/mL) is 1:16, the concentration (v/v) of ethanol is 74%, the ultrasonic power is 260W, the ultrasonic time is 40min, the ultrasonic temperature is 76 ℃, and the extraction is carried out for 2 times;
step 1.2: then merging the filtrate, concentrating in vacuum, and freeze-drying to obtain the pueraria flower isoflavone crude product.
3. The research on the refining process of pueraria flower isoflavone according to claim 1, wherein the resin having the highest adsorption and desorption rates in step 3.2 is a refined resin of pueraria flower isoflavone.
4. The research on the refining process of pueraria flower isoflavone according to claim 1, wherein the mass concentrations of the different solutions of pueraria flower isoflavone in the step 4.1 are 12.5, 25, 50, 100, 200, 400 and 800mg/mL, respectively.
5. The research on the purification process of pueraria flower isoflavone according to claim 1, wherein the concentration of the ethanol solution (v/v) in the step 5.1 includes 30%, 40%, 50%, 60%, 70%, and 80%.
6. A study on a refining process of pueraria flower isoflavone as claimed in claim 1, wherein the pH of the mixture solution in step 6.1 is 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0.
7. Research on a pueraria flower isoflavone refining process according to claim 1, wherein the flow rate in the step 7.2 includes 1.0, 2.0, 3.0, 4.0, and 5.0 mL/min.
8. The research on the lipid-lowering effect of the pueraria flower isoflavone is characterized by comprising the following steps of:
s1: the method for researching the influence of the pueraria flower isoflavone on the pancreatic lipase activity specifically comprises the following steps: taking 350 mu L of buffer solution, 150 mu L of enzyme solution and 50 mu L of pueraria flower isoflavone samples with different mass fractions respectively, putting the buffer solution and the enzyme solution into a centrifuge tube, adding 450 mu L of substrate to start reaction, incubating at 37 ℃ for 2h, centrifuging (8000r/min,1min), measuring the absorbance value (400nm) of supernatant, replacing pueraria flower isoflavone with deionized water in a control group, and calculating the pancreatic lipase inhibition rate according to the formula (3).
In the formula: a. theCThe absorbance value of the control group is obtained; a. theSIs the absorbance value of the sample;
s2: the method for researching the influence of the pueraria flower isoflavone on the activity of the cholesterol esterase specifically comprises the following steps: taking 925 mu L substrate buffer solution, 50 mu L enzyme solution and 25 mu L pueraria flower isoflavone samples with different mass fractions respectively, placing the samples in a centrifuge tube, incubating for 5min at 25 ℃, measuring the absorbance value (405nm), adding 25 mu L deionized water into a control group, and calculating the cholesterol esterase inhibition rate formula (4).
In the formula: a. theCThe absorbance value of the control group is obtained; a. theSIs the absorbance value of the sample;
s3: the method for researching the influence of the pueraria flower isoflavone on the solubility of cholesterol micelles specifically comprises the following steps: preparing by 15mmol/L phosphate buffer solution (pH 7.4), containing sodium taurocholate (10mmol/L), cholesterol (2mmol/L), oleic acid (5mmol/L) and NaCl (132mmol/L), treating for 1h by ultrasonic wave (400Hz), incubating for 24h at 37 ℃, mixing 1mL of isoflavone samples with different mass fractions with 5mL of cholesterol micelles, incubating for 2h at 37 ℃, centrifuging (8000r/min,10min), measuring the cholesterol content of a supernatant by using a total cholesterol kit, adding 1mL of deionized water into a control group, and calculating the inhibition rate according to the formula (5).
In the formula: n is a radical ofCCholesterol content/mmol/L of control group; n is a radical ofSIs the cholesterol content/mmol/L of the sample;
s4: data processing, test data to
Data were analyzed for significance using SPSS 19.0 software and plotted using Origin 8.5 software.
9. The method for studying lipid-lowering effect of pueraria flower isoflavone of claim 8, wherein in S1:
the preparation method of the enzyme solution (10mg/mL) comprises the following steps: dissolving 1g of pancreatic lipase in 10mL of deionized water, swirling for 2min, centrifuging (8000r/min,10min), and taking supernatant for later use;
buffer solution: 100mM Tris solution (Tris) pH 8.2;
the preparation method of the substrate (p-nitrophenyl laurate 0.08%) comprises the following steps: prepared with 5mM phosphate buffer pH5.0 (containing 1% Triton X-100).
10. The method for studying lipid-lowering effect of pueraria flower isoflavone of claim 8, wherein in S2:
the preparation method of the enzyme solution (25 mug/mL) comprises the following steps: dissolving 0.25mg of porcine pancreatic cholesterol esterase in 10mL of deionized water, centrifuging (8000r/min,10min), and taking supernatant for later use;
the preparation method of the substrate buffer solution comprises the following steps: 0.1mol/L phosphate buffer solution (pH 7.04) (containing 0.1mol/L NaCl, 0.02mol/L p-nitrophenyl butyrate and 6mmol/L sodium taurocholate) is prepared.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20070003642A1 (en) * | 2002-12-24 | 2007-01-04 | Satoshi Wanezaki | Composition containing high concentration of isoflavone and having high solubility and process for producing the same |
| CN101269135A (en) * | 2008-04-02 | 2008-09-24 | 湖北中医学院 | Extraction and Purification Process of Pueraria Radix, Hawthorn and Safflower Flavonoids |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070003642A1 (en) * | 2002-12-24 | 2007-01-04 | Satoshi Wanezaki | Composition containing high concentration of isoflavone and having high solubility and process for producing the same |
| CN101269135A (en) * | 2008-04-02 | 2008-09-24 | 湖北中医学院 | Extraction and Purification Process of Pueraria Radix, Hawthorn and Safflower Flavonoids |
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| Title |
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| 吴威等: "葛花异黄酮精制及体外降脂活性研究", 《食品与机械》, no. 2022, 28 February 2022 (2022-02-28), pages 180 - 185 * |
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