Method for extracting and purifying Hubei polygonatum kingianum stem and leaf total phenols

Abstract

The invention belongs to the technical field of extraction processes, and particularly relates to a method for extracting and purifying Hubei polygonatum kingianum stem and leaf total phenols. The method comprises the following steps: extracting the Hubei polygonatum kingianum stem and leaf total phenols by adopting an ultrasonic combined double-enzyme extraction method, purifying the Hubei polygonatum kingianum stem and leaf total phenols by adopting a macroporous adsorption resin method, screening through a large number of experiments to obtain an optimal extraction and purification process, and the method has the advantages of high extraction rate, good purity and the like. And the Hubei polygonatum kingianum stem and leaf total phenol extract has antioxidant activity. By adopting the extraction and purification process disclosed by the invention, the economic value of the Hubei polygonatum kingianum stems and leaves can be improved, resource waste is reduced, and the extraction and purification process has a very good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of extraction technology, and specifically relates to a method for extracting and purifying total phenols from stems and leaves of Hubei polygonatum. Background Art

[0002] Hubei Polygonatum (Polygonatum zanlanscianense Pamp.) is a perennial herbaceous plant of the genus Polygonatum. The stem and leaves are distinct. The stem surface is yellow-green, square or flat with deep longitudinal grooves. The stem is about 65-150 cm long and 0.5-1.2 cm in diameter. The leaves are elliptical, rectangular, lanceolate, and strip-shaped. The leaf area is 7.5-10.3 cm long and 0.8-2.4 cm wide. Hubei Polygonatum is a commonly used Chinese medicine in the folks. The resources are large. In some areas, it can be used as a substitute or a confused product of Polygonatum. However, there are few studies on Hubei Polygonatum. The rhizome of Hubei Polygonatum is rich in bioactive substances, such as polysaccharides, flavonoids, saponins, polyphenols, amino acids and other chemical components. Among them, polysaccharides, flavonoids, and saponins account for the main content. Therefore, the main application part of Hubei Polygonatum is its underground dried rhizome. Its research also focuses on the processing, extraction of active ingredients, and pharmacological effects of roots and rhizomes. However, during the cultivation and production process, the above-ground non-medicinal parts of Hubei Polygonatum sibiricum, such as the above-ground parts, which account for more than 1 / 2 of the whole plant, are often discarded. The above-ground non-medicinal stems and leaves of Hubei Polygonatum sibiricum are rich in a variety of biologically active substances, mainly including total phenols, polysaccharides, flavonoids, saponins, amino acids and other chemical components. Phenolic components are the dominant components of the stems and leaves of Hubei Polygonatum sibiricum, which is of practical significance for the comprehensive utilization of Hubei Polygonatum sibiricum.

[0003] Due to the structural complexity and instability of phenolic substances, traditional extraction methods such as solvent extraction and metal ion precipitation, although they can achieve the extraction of phenolic components to a certain extent, have problems such as low extraction efficiency, long extraction time, and low purity. In addition, different phenolic substances may require different extraction and purification conditions, which seriously limits the efficient extraction and purification of phenolic components.

[0004] Therefore, it is necessary to optimize the extraction and purification method of phenolic components in the stems and leaves of Hubei Polygonatum, and formulate personalized and precise extraction plans, so as to improve the comprehensive utilization of Hubei Polygonatum resources and better expand its application areas. Summary of the invention

[0005] In view of the problems of the prior art, the present invention provides a method for extracting and purifying total phenols from the stems and leaves of Hubei Polygonatum cyrtonema.

[0006] A total phenolic extract of polygonatum stems and leaves is prepared according to the following method:

[0007] Step 1: Extraction of total phenols from stems and leaves of Polygonatum sibiricum:

[0008] (1) adding powder of stem and / or leaf of Polygonatum sibiricum to ethanol aqueous solution and ultrasonicating; the solid-liquid ratio of the powder to the ethanol aqueous solution is 1:(10-70); the concentration of the ethanol aqueous solution is 30-90%; and the ultrasonication time is 20-80 min;

[0009] (2) adding double enzymes, adjusting the pH value of the solution, performing enzymolysis, taking out and filtering, and obtaining a total phenol extract of stems and leaves of polygonatum; the dosage of the double enzymes is 5-65 U / mL, the double enzymes are cellulase and pectinase, and the ratio of the cellulase to the pectinase is (1-3):(1-3); the pH value is 2-5; and the enzymolysis time is 0.25-3 h;

[0010] Step 2: Purification.

[0011] Preferably, the Huangjin is selected from Hubei Polygonatum.

[0012] Preferably, in step 2, the purification method is: adding the sample solution to a chromatography column filled with a macroporous resin, and after reaching adsorption equilibrium, washing impurities with distilled water, and then eluting with eluent ethanol to obtain a purified solution; the sample solution is the total phenol extract of polygonatum stems and leaves obtained in step 1 (2); the concentration of the sample solution is 0.1-1.5 mg / mL, the flow rate is 0.5-2.5 mL / min, and the sample volume is 15-25 mL; the concentration of the eluent is 15%-95%, the flow rate is 0.5-2.5 mL / min, and the elution volume is 200-300 mL.

[0013] Preferably, the macroporous resin is pretreated, and the pretreatment method is as follows: inject anhydrous ethanol into the macroporous resin and soak it for 24-36 hours, then wash it with pure water until there is no alcohol taste, then soak it in 5-10% NaOH solution for 6-8 hours, then wash it with pure water until the eluate is neutral, finally soak it in 5-10% hydrochloric acid solution for 6-8 hours, then wash it with pure water until the pH value of the macroporous resin reaches neutral, and filter it to obtain the product.

[0014] Preferably, in step 1, the solid-liquid ratio of the powder to the ethanol aqueous solution is 1:40; the concentration of the ethanol aqueous solution is selected from 60%; and the ultrasonic time is selected from 60 min.

[0015] Preferably, in step 1, the dosage of the dual enzyme is 55 U / mL, the ratio of the cellulase to the pectinase is 2:1; the pH value is 3.5; and the enzymolysis time is 2 h.

[0016] Preferably, in step 2, the macroporous resin is selected from AB-8 macroporous resin; the concentration of the loading solution is 0.5 mg / mL, the flow rate is 1.0 mL / min, and the loading volume is 20 mL; the concentration of the eluent is 75%, the flow rate is 1.0 mL / min, and the elution volume is 200 mL.

[0017] The present invention also provides a method for preparing the above-mentioned total phenolic extract of polygonatum stems and leaves, which is characterized by comprising the following steps:

[0018] Step 1: Extraction of total phenols from stems and leaves of Polygonatum sibiricum:

[0019] (1) adding powder of stem and / or leaf of Polygonatum sibiricum to ethanol aqueous solution and ultrasonicating; the solid-liquid ratio of the powder to the ethanol aqueous solution is 1:(10-70); the concentration of the ethanol aqueous solution is 30-90%; and the ultrasonication time is 20-80 min;

[0020] (2) adding double enzymes, adjusting the pH value of the solution, performing enzymolysis, taking out and filtering, and obtaining a total phenol extract of stems and leaves of polygonatum; the dosage of the double enzymes is 5-65 U / mL, the double enzymes are cellulase and pectinase, and the ratio of the cellulase to the pectinase is (1-3):(1-3); the pH value is 2-5; and the enzymolysis time is 0.25-3 h;

[0021] Step 2: Purification.

[0022] The present invention also provides the use of the total phenol extract of the stems and leaves of polygonatum in preparing antioxidant drugs or health products.

[0023] An antioxidant pharmaceutical composition or health product is prepared by taking the total phenol extract of polygonatum stems and leaves as active ingredients and adding pharmaceutically acceptable excipients or auxiliary ingredients.

[0024] The invention optimizes the selection of various conditions in the extraction and purification process of the phenolic components of the above-ground part (stems and leaves) of Hubei polygonatum, effectively improves the extraction rate of the total phenols of the stems and leaves of Hubei polygonatum, and determines the optimal purification conditions through dynamic adsorption and desorption tests. The purification process can significantly improve the purity of the total phenols of the stems and leaves of Hubei polygonatum, and proves that the total phenol extract of the stems and leaves of Hubei polygonatum has antioxidant activity. Therefore, the extraction and purification method of the invention can effectively improve the comprehensive utilization of Hubei polygonatum resources and reduce resource waste.

[0025] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0026] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the gallic acid standard curve.

[0028] Figure 2 This is a graph showing the influence of various factors on the total phenol extraction rate of the stems and leaves of Hubei Polygonatum.

[0029] Figure 3 This is the effect of sample solution concentration on adsorption rate.

[0030] Figure 4 This is the effect of eluent concentration on desorption rate.

[0031] Figure 5 This is the effect of sample flow rate on adsorption effect.

[0032] Figure 6 This is the effect of eluent flow rate on desorption effect.

[0033] Figure 7 (A, B) Mean fluorescence intensity results measured by flow cytometry; (C) ROS level results. DETAILED DESCRIPTION

[0034] In the following examples and experimental examples, reagents and raw materials not specifically described are all commercially available products.

[0035] Embodiment 1 Extraction method of total phenols from stems and leaves of Hubei Polygonatum

[0036] Hubei Polygonatum was collected from Group 15, Hongqi Village, Daoming Town, Chongzhou City, Sichuan Province. The stems and leaves of the above-ground parts were separated and washed, dried in an oven at 60°C to constant weight, and crushed through a No. 5 sieve to obtain Hubei Polygonatum stem powder and leaf powder. 0.5g Hubei Polygonatum stem powder and 0.5g leaf powder were accurately weighed and placed in a clean and dry beaker. A 60% ethanol aqueous solution was added at a liquid-to-solid ratio of 1:40. After shaking, it was placed in a 300W, 50°C constant temperature ultrasonic machine for 60 minutes, and then taken out, the solution pH was adjusted to 3.5, 55U / mL of double enzymes were added according to the ratio of cellulase: pectinase (2:1), and the mixture was fully shaken. It was placed in a constant temperature water bath at 50°C for enzymolysis for 2h, taken out and shaken, and filtered to obtain the Hubei Polygonatum stem and leaf total phenol extract.

[0037] Example 2: Purification method of total phenols from stems and leaves of Hubei Polygonatum

[0038] 1. Macroporous resin pretreatment

[0039] Anhydrous ethanol is injected into the AB-8 type macroporous resin for soaking. After soaking for 24 hours, it is rinsed with pure water until there is no alcohol smell. Then, the macroporous resin is soaked in 5% NaOH solution for 6 hours, and then washed with pure water again until the eluate is neutral. The macroporous resin is then soaked in 5% hydrochloric acid solution for 6 hours, and then washed with pure water again until the pH value of the macroporous resin reaches neutral. Finally, it is filtered to obtain the pretreated macroporous resin.

[0040] 2. Purification method of total phenols from stems and leaves of Hubei Polygonatum

[0041] 20.0 g of pretreated macroporous resin was wet-processed, and the total phenol extract of the stems and leaves of Hubei polygonatum obtained in Example 1 was used as the loading solution. It was loaded into a chromatography column (Ф2.5 cm×30 cm) at a loading concentration of 0.5 mg / mL and a loading flow rate of 1.0 mL / min. The loading amount was 20 mL. After reaching adsorption equilibrium, impurities were washed with distilled water, and then eluted with 75% ethanol at a flow rate of 1.0 mL / min. The elution volume was 200 mL. Every 10 mL of the effluent was collected to obtain purified total phenols of the stems and leaves of Hubei polygonatum.

[0042] The technical solution of the present invention is further illustrated by experiments below.

[0043] Experimental Example 1 Optimization of Extraction Process of Total Phenols from the Stems and Leaves of Hubei Polygonatum

[0044] 1. Experimental Methods

[0045] Accurately weigh 0.5g of Hubei Polygonatum stem powder and 0.5g of leaf powder, place them in a clean and dry beaker, add different concentrations of ethanol solution (30%, 40%, 50%, 60%, 70%, 80%, 90%) at a liquid-to-solid ratio of 1: (10, 20, 30, 40, 50, 60, 70), shake well, place in a 300W, 50℃ constant temperature ultrasonic machine for a certain time (20min, 30min, 40min, 50min, 60min, 70min, 80min), then take it out and adjust the solution pH (2.0, 2.5, 3.0, 3.5, 4.0, 4. 5, 5.0), add a certain amount of double enzyme (5U / mL, 15U / mL, 25U / mL, 35U / mL, 45U / mL, 55U / mL, 65U / mL) according to the ratio of cellulase: pectinase (1:1, 2:1, 3:1, 1:2, 1:3, 2:3, 3:2), shake well, place in a constant temperature water bath at 50°C for enzymolysis for a certain time (0.25h, 0.3h, 1h, 1.5h, 2h, 2.5h, 3h), take out, shake well and filter, and obtain the total phenol extract of Hubei Polygonatum cyrtonema stems and leaves. The total phenol extraction rate was determined by Folin-Ciocalteu method. The specific conditions for optimization are shown in Table 1

[0046] Determination of total phenol extraction rate: prepare a gallic acid standard solution with a mass concentration of 0.25 mg / mL, respectively aspirate gradient concentrations (0, 0.08, 0.12, 0.16, 0.20, 0.24 and 0.28 mL) of standard solutions, place them in a 10 mL volumetric flask, add 6.4 mL of distilled water and 0.4 mL of Folin-phenol reagent to each volumetric flask in sequence, shake evenly, then add 1 mL of 20% Na2CO3 solution respectively, shake well to mix, let stand at room temperature in the dark for 2 h, measure the absorbance at 764 nm, draw a standard curve, and calculate the total phenol extraction rate based on the standard curve.

[0047] Table 1

[0048]

[0049] 2. Experimental Results

[0050] Gallic acid standard curve Figure 1 As shown, the regression equation is Y=19.691X+0.0853, R2=0.9993, and the regression coefficient indicates that gallic acid maintains good linear absorbance in the mass concentration range of 10-70 μg / mL.

[0051] Depend on Figure 2(a) It can be seen that in the initial stage, the total phenol extraction rate will increase with the increase of the solid-liquid ratio, and when the latter increases to 1:40, the extraction rate reaches a peak value, and then the extraction rate decreases when the solvent ratio is increased. Increasing the solid-liquid ratio in the early stage is conducive to increasing the molecular diffusion rate, so it is positively correlated with the extraction rate in this process. After the total phenol dissolution reaches the peak value, due to the continuous increase of the solvent, other substances will be further dissolved, and the two are negatively correlated at this time. Therefore, the optimal liquid-to-solid ratio is determined to be 1:40.

[0052] Depend on Figure 2 (b) It can be seen that when the ethanol concentration is 60%, the corresponding extraction rate reaches the peak value, so the optimal ethanol concentration is determined to be 60%.

[0053] Depend on Figure 2 (c) It can be seen that the total phenol in the aerial part reaches the maximum dissolution amount at 60 minutes of ultrasound. After 60 minutes, the extraction rate decreases and stabilizes, indicating that within a certain range, the ultrasound time is positively correlated with the total phenol extraction rate. If it exceeds the range, it will not only increase energy consumption, but also destroy the material structure of the extract or cause more impurities to dissolve. Therefore, the optimal ultrasound time is determined to be 60 minutes.

[0054] Depend on Figure 2 (d) It can be seen that the total phenol extraction rate reached the maximum value when the enzymatic hydrolysis time was 2 hours. After 2 hours, most of the cellulose and pectin in the cell wall were decomposed as the enzymatic hydrolysis time was extended, resulting in a large amount of substances other than the active ingredients in the cells being dissolved, and the extraction rate was reduced. Finally, the optimal enzymatic hydrolysis time was determined to be 2 hours.

[0055] Cellulase and pectinase are stable in acidic environments and unstable in alkaline environments, so the scope of investigation is set in acidic environments. Figure 2 (e) It can be seen that as the pH value of enzymatic hydrolysis increases, the total phenol extraction rate increases first and then decreases. The extraction rate reaches the maximum when the pH value of enzymatic hydrolysis is 3.5, at which time the dual enzyme activity is the highest. Therefore, the optimal pH value of enzymatic hydrolysis is 3.5.

[0056] Depend on Figure 2 (f) It can be seen that the total phenol extraction rate when the dual enzyme ratio is 2:1 is higher than the extraction rate under other ratio conditions. The reason is presumably that the binding ability between cellulose molecules is stronger than that of pectin, and the ability to hinder the dissolution of active ingredients is stronger. Therefore, the dual enzyme ratio of this study was determined to be cellulase: pectinase = 2:1.

[0057] Depend on Figure 2 (g) It can be seen that the total phenol extraction rate tends to be stable after 55 U / mL. Cellulose and pectin are widely distributed in plant cell walls and are obstacles to the dissolution of active ingredients during the extraction process. Therefore, the optimal dual enzyme dosage was finally determined to be 55 U / mL.

[0058] The above results show that the optimal extraction conditions are a solid-liquid ratio of 1:40, an enzymatic hydrolysis pH of 3.5, an enzymatic hydrolysis time of 2 h, an extract ethanol concentration of 60%, an ultrasonic time of 60 min, a dual enzyme ratio (cellulase: pectinase) of 2:1, and a dual enzyme dosage of 64 U / mL. Under these conditions, the total phenol extraction rate is 7.03%.

[0059] Experimental Example 2 Optimization of the purification process of total phenols from the stems and leaves of Hubei Polygonatum

[0060] 1. Experimental Methods

[0061] (1) Macroporous resin pretreatment

[0062] According to the macroporous resin pretreatment method in Example 2, 8 different types of macroporous resins (D3520, AB-8, HPD750, HPD-600, NKA-9, XAD-2, HPD-100, S-8) were prepared.

[0063] (2) Selection of macroporous resin model

[0064] The best macroporous resin for separation and purification of total phenol was screened by static adsorption-desorption test; 5.0g of each pretreated macroporous resin was accurately weighed, and 50mL of total phenol extract with a mass concentration of 0.5mg / mL was added respectively, and then placed in an oscillator with a speed of 120r / min and a constant temperature of 25℃, and the oscillation was continued for 24h. When the vibration degree reached the adsorption equilibrium, the concentration of total phenol in the supernatant was measured. After the adsorption equilibrium, the resin was washed with distilled water, filtered and dried, and 50mL of 70% ethanol was added for desorption. It was continuously shaken in a water bath constant temperature oscillator for 24h under the same conditions. After adsorption equilibrium, the total phenol concentration of the supernatant was measured again, and the adsorption rate and desorption rate of 8 different macroporous resins were calculated according to formulas (2-1) and (2-2):

[0065] Adsorption rate (%) = (C0V0-C1V1) / C0V0×100% (2-1)

[0066] Desorption rate (%) = C2V2 / (C0V2-C1V1)×100% (2-2)

[0067] In formulas (2-1) and (2-2), C0, C1, and C2 represent the concentration of total phenol extract, the total phenol concentration in the supernatant after adsorption equilibrium, and the total phenol concentration in the eluate, respectively, and the units are all mg / mL; V0, V1, and V2 represent the volume of total phenol extract, the volume of filtrate after adsorption equilibrium, and the volume of eluate, respectively, and the units are all mL.

[0068] (3) Static adsorption-desorption test of macroporous resin

[0069] Accurately weigh 4.0 g of the pretreated macroporous resin, add 50 mL of 0.5 mg / mL total phenol crude extract, and follow the rest of the experimental steps (2). With a cycle of 1 hour, the effects of different loading solution concentrations (0.1, 0.25, 0.5, 0.75, 1.0, 1.5 mg / mL) on the adsorption rate and the effects of different eluent concentrations (15%, 25%, 35%, 45%, 55%, 65%, 75%, 85%, 95%) on the desorption rate were investigated.

[0070] (4) Dynamic adsorption-desorption test of macroporous resin

[0071] 20.0 g of pretreated macroporous resin was loaded into a chromatography column (Ф2.5 cm×30 cm) using a wet method at a loading solution concentration of 0.5 mg / mL. The sample was loaded at different loading solution flow rates (0.5, 1, 1.5, 2, 2.5 mL / min), and the effluent was collected every 10 mL. The total phenol concentration of the effluent was measured to determine the optimal loading solution flow rate and loading amount.

[0072] 20.0 g of pretreated macroporous resin was loaded into a chromatography column (Ф2.5 cm×30 cm) by a wet method at a loading solution concentration of 0.5 mg / mL and a loading solution flow rate of 1.0 mL / min. 20 mL of the sample was loaded, and after reaching adsorption equilibrium, impurities were washed with distilled water, followed by elution with 75% ethanol at flow rates of 0.5, 1.0, 1.5, 2.0, and 2.5 mL / min, respectively; 10 mL of the effluent was collected, the total phenol concentration of the effluent was measured, and the optimal elution solution flow rate and elution volume were determined.

[0073] (5) Calculation of total phenol purity

[0074] The total phenol extract before purification and the eluate after purification with macroporous resin were measured respectively, and vacuum distilled at a constant temperature of 50°C until there was no alcohol taste, and then freeze-dried and weighed. Among them, before purification: the solution volume was 650mL, the total phenol concentration was 0.36mg / mL, and the sample mass after freeze-drying was 1g; after purification: the solution volume was 100mL, the total phenol concentration was 0.21mg / mL, and the sample mass after freeze-drying was 30mg. The total phenol purity before purification and after purification with macroporous resin was calculated according to formula (2-3).

[0075] Total phenol purity P / % = [(C×V) / M]×100% (2-3)

[0076] In formula (2-3): C, total phenol concentration before / after purification, mg / mL; V, measured solution volume, mL; M, mass of sample solution after freeze-drying, mg.

[0077] 2. Experimental Results

[0078] 1. Macroporous resin model selection

[0079] As shown in Table 2, from the perspective of dependent variable adsorption rate, AB-8 macroporous resin has the highest adsorption rate for total phenol extract, which is 90.57±1.04%. Under the experimental conditions, the adsorption effect is significantly better than other types of resins (P<0.05). From the perspective of dependent variable desorption rate, the desorption rate of AB-8 macroporous resin for total phenol crude extract is 80.62±0.69%, which is not significantly different from HPD-750 resin, but significantly higher than other types of resins (P<0.05). Considering the adsorption rate and desorption rate, AB-8 macroporous resin is selected as the best resin for the separation and purification of total phenol. Different letters in each column in Table 2 represent significant differences (P<0.05), and the same letters represent insignificant differences (P>0.05).

[0080] Table 2 Comparison of adsorption and desorption performance of 8 macroporous resins for total phenol extracts

[0081]

[0082] 2. Results of macroporous resin static adsorption-desorption test

[0083] Depend on Figure 3 It can be seen that with the increase of sample concentration, the adsorption rate gradually increased, and the maximum adsorption rate was achieved when the sample concentration was 0.5 mg / mL; when the sample concentration exceeded 0.5 mg / mL, the adsorption rate slowly decreased. Therefore, the sample concentration with the best adsorption rate in this experiment was 0.5 mg / mL.

[0084] Depend on Figure 4 It can be seen that before the eluent concentration is 75%, the desorption rate shows an overall upward trend, and after exceeding 75%, the desorption rate decreases. Therefore, the optimal eluent concentration should be 75%.

[0085] 3. Results of dynamic adsorption-desorption test of macroporous resin

[0086] Depend on Figure 5 It can be seen that at the same flow rate, as the volume of the effluent gradually increases, the total phenol content therein also shows an increasing trend, which indicates that the adsorption rate decreases with the increase of the effluent volume. Under the condition of the same effluent volume, there are obvious differences in the total phenol adsorption rates corresponding to different flow rates. Specifically, when the loading flow rate is controlled at 1.0mL / min, the adsorption rate reaches the highest value. Therefore, the optimal loading flow rate is 1.0mL / min, and the optimal loading volume under this condition is 20mL.

[0087] Depend on Figure 6It can be seen that under the conditions of elution flow rates of 1.5, 2.0, and 2.5 mL / min, the peak value was observed to gradually decrease with the increase of flow rate, and the peak shape gradually became broad, and a significant tailing phenomenon appeared, all of which led to a decrease in the desorption rate. In contrast, when the flow rate was reduced to 0.5 and 1.0 mL / min, the peak shape of the elution curve showed more concentrated and symmetrical characteristics. In particular, when the flow rate was 1.0 mL / min, the peak value reached the highest, and the desorption rate at this time also reached the maximum. Therefore, the optimal elution flow rate should be 1.0 mL / min, and under this elution flow rate condition, the optimal elution volume is 200 mL.

[0088] 4 Comparison of total phenol purity before and after purification

[0089] According to calculation, the total phenol purity was 23.4% before purification. After purification by AB-8 macroporous resin, the total phenol purity of the total phenol extract increased to 73.3%, a full 3.13 times increase, indicating that the purification process has excellent enrichment effect and strong feasibility.

[0090] The above results show that the optimal purification conditions are 0.5 mg / mL sample concentration, 1.0 mL / min sample flow rate, 20 mL sample volume, 75% eluent concentration, 1.0 mL / min elution flow rate, and 200 mL elution volume. The purity of total phenol is significantly improved after the purification process is optimized using AB-8 macroporous resin.

[0091] Experimental Example 3 Antioxidant activity of total phenolic extracts from stems and leaves of Polygonatum cyrtonema

[0092] 1. Experimental Methods

[0093] 1. Culture of HepG2 cells

[0094] HepG2 cells were placed in culture medium (10% fetal bovine serum, 1% double antibody, MEM) and transferred to a 37°C, 5% CO2 cell culture incubator for growth. The growth of HepG2 cells was observed. When they grew to about 80% abundance, they were digested and subcultured using trypsin, with a subculture interval of 3 to 4 days. HepG2 cells that were in the growth phase and growing well were selected and inoculated in a 96-well plate at a cell density of 100 μL / 5×10 4 The cells were cultured at 37°C for 24 hours.

[0095] 2. Determination of intracellular active ROS

[0096] Divided into 6 groups, each group configuration is as follows: the control group (Control group) only added the same amount of basal culture medium and cultured for 24h. The model group (Model group) added 500μmol / Lt-BHP induction solution on the basis of the control group and induced for 2h. The VC group was pretreated with an equal amount of VC solution with a density of 5μg / mL for 24h, and then added 500μmol / Lt-BHP induction solution for 2h. The Hubei Polygonatum stem and leaf total phenol extract group (PPP group) was divided into three groups according to the concentration of the extract, high, medium and low. After pretreatment for 24h, 500μmol / Lt-BHP induction solution was added for 2h. After the cell culture of each group was completed, the cell supernatant was removed, and finally each group was added with a concentration of 10μmol / L dichlorodihydrofluorescein-acetoacetate (DCFH-DA), and then transferred to a cell culture incubator at 37℃ for 20 minutes. After the culture was completed, the cells were washed with serum-free cell culture medium for 3 cycles, and the average fluorescence level in each group of cells was measured using flow cytometry.

[0097] 2. Experimental Results

[0098] from Figure 7 (AC) The results showed that compared with the Control group, the ROS level in the Model group increased to 62.72%. After using the positive control drug VC, the ROS level dropped to 16.58%. After using the total phenolic extract, ROS was lower than that in the Model group, and the low, medium and high dose groups dropped to 33.49%, 20.2% and 17.47%, respectively. The results showed that the total phenolic extract of the stems and leaves of Hubei Polygonatum can reduce the release of ROS in oxidatively damaged cells and alleviate oxidative damage to a certain extent.

[0099] The invention obtains the best extraction and purification process through a large number of experimental screenings, has the advantages of high extraction rate, good purity, etc., and the total phenol extract of the stem and leaves of Hubei polygonatum has antioxidant activity. The extraction and purification process of the invention can improve the economic value of the stem and leaves of Hubei polygonatum, reduce resource waste, and has good application prospects.

Claims

1. A total phenolic extract from stems and leaves of Polygonatum cyrtonema, characterized in that It is prepared as follows: Step 1: Extraction of total phenols from stems and leaves of Polygonatum sibiricum: (1) adding powder of stem and / or leaf of Polygonatum sibiricum to ethanol aqueous solution and ultrasonicating; the solid-liquid ratio of the powder to the ethanol aqueous solution is 1:(10-70); the concentration of the ethanol aqueous solution is 30-90%; and the ultrasonication time is 20-80 min; (2) adding double enzymes, adjusting the pH value of the solution, performing enzymolysis, taking out and filtering, and obtaining a total phenol extract of stems and leaves of polygonatum; the dosage of the double enzymes is 5-65 U / mL, the double enzymes are cellulase and pectinase, and the ratio of the cellulase to the pectinase is (1-3):(1-3); the pH value is 2-5; and the enzymolysis time is 0.25-3 h; Step 2: Purification.

2. The total phenolic extract from stems and leaves of Polygonatum cyrtonema according to claim 1, characterized in that The Huang Jing is selected from Hubei Polygonatum.

3. The total phenolic extract from stems and leaves of polygonatum according to claim 1, characterized in that: In step 2, the purification method is: adding the sample solution to a chromatography column filled with a macroporous resin, and after reaching adsorption equilibrium, washing impurities with distilled water, and then eluting with eluent ethanol to obtain a purified solution; the sample solution is the total phenol extract of polygonatum stems and leaves obtained in step 1 (2); the concentration of the sample solution is 0.1-1.5 mg / mL, the flow rate is 0.5-2.5 mL / min, and the sample volume is 15-25 mL; the concentration of the eluent is 15%-95%, the flow rate is 0.5-2.5 mL / min, and the elution volume is 200-300 mL.

4. The total phenolic extract from stems and leaves of Polygonatum cyrtonema according to claim 3, characterized in that: The macroporous resin is pretreated, and the pretreatment method is as follows: inject anhydrous ethanol into the macroporous resin and soak it for 24-36 hours, then wash it with pure water until there is no alcohol taste, then soak it in 5-10% NaOH solution for 6-8 hours, then wash it with pure water until the eluate is neutral, finally soak it in 5-10% hydrochloric acid solution for 6-8 hours, then wash it with pure water until the pH value of the macroporous resin reaches neutral, and filter it to obtain the resin.

5. The total phenolic extract from stems and leaves of polygonatum according to claim 1, characterized in that In step 1, the solid-liquid ratio of the powder to the ethanol aqueous solution is 1:40; the concentration of the ethanol aqueous solution is selected from 60%; and the ultrasonic time is selected from 60 minutes.

6. The total phenolic extract from stems and leaves of polygonatum according to claim 1, characterized in that In step 1, the dosage of the dual enzyme is 55 U / mL, the ratio of the cellulase to the pectinase is 2:1; the pH value is 3.5; and the enzymolysis time is 2 h.

7. The total phenolic extract from stems and leaves of Polygonatum cyrtonema according to claim 3, characterized in that In step 2, the macroporous resin is selected from AB-8 macroporous resin; the concentration of the loading solution is 0.5 mg / mL, the flow rate is 1.0 mL / min, and the loading volume is 20 mL; the concentration of the eluent is 75%, the flow rate is 1.0 mL / min, and the elution volume is 200 mL.

8. The method for preparing the total phenolic extract of stems and leaves of Polygonatum cyrtonema according to any one of claims 1 to 7, characterized in that: The steps include: Step 1: Extraction of total phenols from stems and leaves of Polygonatum sibiricum: (1) adding powder of stem and / or leaf of Polygonatum sibiricum to ethanol aqueous solution and ultrasonicating; the solid-liquid ratio of the powder to the ethanol aqueous solution is 1:(10-70); the concentration of the ethanol aqueous solution is 30-90%; and the ultrasonication time is 20-80 min; (2) adding double enzymes, adjusting the pH value of the solution, performing enzymolysis, taking out and filtering, and obtaining a total phenol extract of stems and leaves of polygonatum; the dosage of the double enzymes is 5-65 U / mL, the double enzymes are cellulase and pectinase, and the ratio of the cellulase to the pectinase is (1-3):(1-3); the pH value is 2-5; and the enzymolysis time is 0.25-3 h; Step 2: Purification.

9. Use of the total phenolic extract from stems and leaves of Polygonatum cyrtonema according to any one of claims 1 to 7 in the preparation of antioxidant drugs or health products.

10. An antioxidant pharmaceutical composition or health product, characterized in that: The invention is prepared by taking the total phenol extract of stems and leaves of polygonatum as claimed in any one of claims 1 to 7 as active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.

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