Method for extracting and purifying insoluble bound phenols in peony root for oil

Abstract

The present application relates to the technical field of biological extraction, and provides a method for extracting and purifying insoluble bound phenols in oil use peony root, which comprises the following steps: firstly, obtaining the extraction residue of the oil use peony root through ethanol extraction; then, treating the residue by steam explosion pretreatment and ultrasonic-assisted hydrolysis to obtain a crude extract; and finally, purifying the crude extract by ultrasonic-assisted macroporous resin to obtain a purified product. In the method, the total phenol content of the crude extract reaches 185.59 mg GAE / g dw, and the total phenol content of the purified product increases to 254.74 mg GAE / g dw, which increases by 37.3%. In addition, compared with the crude extract, the antioxidant and anti-tyrosinase activities of the purified product are obviously increased, and the total content of 5 typical compounds increases by about 8 times. The present application provides a brand-new and excellent process for the extraction and purification of bound phenols in peony root, and the obtained purified product is expected to be used as a natural and green whitening additive in skin care products.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological extraction, and particularly relates to a method for extracting and purifying insoluble bound phenols in roots of Paeonia ostii. BACKGROUND

[0002] Moutan (tree peony), also known as rich and noble flower and white two gold, belongs to the Paeoniaceae and Paeonia. Moutan was introduced into the United Kingdom in the late 18th century and widely cultivated in Europe in the early 20th century. It has been introduced to more than 30 countries around the world for cultivation. Moutan contains 118 active ingredients, including tannins, polyphenols, flavonoids and various terpenes, which may be related to the detoxification, promotion of metabolism, anticancer, anti-inflammatory and prevention of cardiovascular diseases of moutan cortex.

[0003] With the development of moutan planting industry, Paeonia ostii for oil is also widely planted for the purpose of moutan seed oil. However, the content of paeonol in the root bark is less than 1.2%, which does not meet the medicinal material standard specified in the Chinese Pharmacopoeia, so it is often discarded as waste, which not only wastes valuable biological resources, but also causes environmental pollution and reduces the income of farmers.

[0004] In practice, researchers found that the "waste" could release more active substances through specific separation and extraction processes, especially insoluble bound phenols (IP), which are tightly bound to cellulose, hemicellulose and some proteins through covalent bonds. In previous studies, the IP in the roots and leaves of moutan was subdivided into bound phenols (IB), esterified bound phenols (IE) and glycosylated bound phenols (IG) by acid-base treatment. The total content of them in the roots is 14.06 mg GAE / g dw, and the total content in the leaves is 8.67 mg GAE / g dw, accounting for 15.98% and 12.69% of the total phenol content in the roots and leaves, respectively. This shows that the roots and leaves of moutan contain a certain proportion of bound phenols. However, the process of acid-base treatment is time-consuming and energy-consuming, and IB, IE and IG show low antioxidant activity and anti-tyrosinase activity.

[0005] At present, there is no method for rapidly and effectively extracting insoluble bound phenols in the roots of Paeonia ostii in the art. SUMMARY

[0006] Therefore, the present application provides a method for extracting and purifying insoluble bound phenols in the roots of Paeonia ostii. The extraction method provided by the present application can rapidly and effectively extract insoluble bound phenols in the roots of Paeonia ostii. The total phenol content in the obtained crude extract is high, and after further purification, the total phenol content in the obtained purified product is higher, and the antioxidant activity and anti-tyrosinase activity are high.

[0007] To achieve the above object, the present application provides the following technical solutions.

[0008] An extraction method of insoluble bound phenols in Paeonia suffruticosa root for oil, comprising the following steps:

[0009] Mixing the powder of Paeonia suffruticosa root and ethanol solution for ultrasonic-assisted extraction to obtain extraction residue;

[0010] Mixing the extraction residue and water for steam explosion treatment to obtain pretreated material;

[0011] Mixing the pretreated material, sodium hydroxide, EDTA and ascorbic acid for ultrasonic-assisted hydrolysis, and centrifuging the obtained material liquid to obtain supernatant;

[0012] Freeze-drying the supernatant to obtain crude extract of insoluble bound phenols.

[0013] Preferably, the temperature of the steam explosion treatment is 105-125℃, and the time is 5-15min; the ratio of the extraction residue to water is 1:10g / mL-1:50g / mL.

[0014] Preferably, after obtaining the pretreated material, further comprising adding water to the pretreated material.

[0015] Preferably, in the mixed solution obtained by mixing the pretreated material, sodium hydroxide, EDTA and ascorbic acid, the concentration of sodium hydroxide is 2-6M, the concentration of EDTA is 5-15mM, and the mass fraction of ascorbic acid is 0.5-1.5%.

[0016] Preferably, the ultrasonic-assisted hydrolysis has an ultrasonic power of 120-400W, an ultrasonic frequency of 20-60kHz, a hydrolysis time of 2-8h, and a hydrolysis temperature of room temperature.

[0017] The present application also provides a purification method of insoluble bound phenols in Paeonia suffruticosa root for oil, comprising the following steps:

[0018] Extracting Paeonia suffruticosa root by the extraction method described in the above scheme to obtain crude extract of insoluble bound phenols;

[0019] Dissolving the crude extract of insoluble bound phenols in water to obtain crude extract solution, and then performing ultrasonic-assisted adsorption on the crude extract solution by using macroporous resin to obtain adsorption resin;

[0020] Performing ultrasonic-assisted desorption on the adsorption resin by using ethanol solution to obtain purified product of insoluble bound phenols.

[0021] Preferably, the operation condition of the ultrasonic-assisted adsorption comprises: the total phenol content in the crude extract solution is 2-4 mg GAE / mL, the pH value is 3-5, the ultrasonic power is 140-250 W, the adsorption time is 40-120 min, and the adsorption temperature is room temperature.

[0022] Preferably, the macroporous resin is D101, D101C, LX12, LX83, XDA6 or AB8.

[0023] Preferably, the volume concentration of the ethanol solution is 40-80%; and the operation condition of the ultrasonic-assisted desorption comprises: the ultrasonic power is 140-250 W, the desorption time is 30-60 min, and the desorption temperature is room temperature.

[0024] Preferably, the ultrasonic-assisted adsorption is carried out by using a static adsorption method; and the ultrasonic-assisted desorption is carried out by using a static desorption method.

[0025] The present application provides a method for extracting insoluble bound phenols from Paeonia suffruticosa Andr., which comprises the following steps: mixing the powder of Paeonia suffruticosa Andr. and an ethanol solution to carry out ultrasonic-assisted extraction, so as to obtain an extraction residue; mixing the extraction residue and water to carry out steam explosion treatment, so as to obtain pretreated materials; mixing the pretreated materials, sodium hydroxide, EDTA and ascorbic acid, and then carrying out ultrasonic-assisted hydrolysis, centrifuging the obtained hydrolysis liquid, and obtaining supernatant; and freeze-drying the supernatant, so as to obtain a crude extract of insoluble bound phenols. The present application firstly extracts the extraction residue of Paeonia suffruticosa Andr. by using an ethanol solution, and then treats the residue by using the method of steam explosion pretreatment and ultrasonic-assisted hydrolysis, so as to obtain a crude extract of insoluble bound phenols (IPs). The method provided by the present application has high extraction rate, and the total phenol content in the obtained crude extract is high. The principle of the steam explosion (SE) pretreatment is that the plant material is placed in a high-temperature and high-pressure environment, water vapor molecules will enter the plant interstice, the heat energy is rapidly converted into mechanical energy, and then acts on the inside of the material, so as to produce micropores, thereby decomposing the raw material with less energy. The material pretreated by steam explosion is more beneficial to the release of bioactive components in the subsequent extraction process. The principle of the ultrasonic-assisted hydrolysis is that the ultrasonic wave (US) causes the interaction between two media, and can produce a series of thermal, mechanical, optical and electrical effects. These effects can be attributed to the cavitation effect of the ultrasonic wave. The cavitation effect can cause the vibration, expansion, compression, collapse and closure of the cavitation nucleus in the liquid. The high shear force carried by the sound wave itself can promote the mass transfer, so that the US-assisted is beneficial to the separation and release of plant active substances from the plant material, thereby improving the extraction rate.

[0026] The application further provides a method for purifying insoluble bound phenols in peony root for oil, and the crude extract obtained by the above scheme is subjected to ultrasonic-assisted adsorption by using a macroporous resin, and then subjected to ultrasonic-assisted desorption by using an ethanol solution, so as to obtain purified insoluble bound phenols (PIPs). The method provided by the application has good purifying effect, the total phenol content of the obtained purified product is high, and the antioxidant activity and anti-tyrosinase activity of the purified product are high.

[0027] The results of the examples show that the total phenol content of the crude extract (IPs) obtained by the application reaches 185.59 mg GAE / g dw, and the total phenol content of the purified product (PIPs) increases to 254.74 mg GAE / g dw. The test results of antioxidant activity and anti-tyrosinase activity show that the sum of the five typical compounds (main components for antioxidant and anti-tyrosinase) in the crude extract and the purified product is 0.404 mg / g (IPs) and 3.233 mg / g (PIPs) respectively, and the content of the typical compounds in the purified product is about 8 times higher than that in the crude extract, wherein the content of benzoylpaeoniflorin increases by 10.3 times, and the content of paeoniflorin increases by 9.7 times.

[0028] In summary, the application provides a brand-new excellent process for the extraction and purification of insoluble bound phenols in peony root, and the obtained purified product is expected to be used as a natural green whitening additive in skin care products. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 Total phenol content of the crude extract obtained under different steam explosion treatment times;

[0030] Figure 2 Influence of different ultrasonic-assisted conditions on the total phenol content extracted by alkaline hydrolysis;

[0031] Figure 3 Influence of different pH values on the adsorption capacity of LX-83 resin;

[0032] Figure 4 Influence of the concentration of the crude extract solution on the adsorption capacity of the resin;

[0033] Figure 5 Adsorption isotherm of LX-83 resin under different ultrasonic-assisted conditions;

[0034] Figure 6 Influence of adsorption time on the adsorption capacity of the resin;

[0035] Figure 7 Pseudo-first-order kinetics fitting curve of the adsorption process;

[0036] Figure 8 Pseudo-second-order kinetics fitting curve of the adsorption process;

[0037] Figure 9 Scanning electron micrograph of LX-83 macroporous resin after adsorption with or without ultrasonic assistance (magnification 250 times);

[0038] Figure 10 Effect of different ethanol concentrations on the desorption amount of the adsorbed resin;

[0039] Figure 11 Effect of ultrasonic power on the desorption amount of the adsorbed resin;

[0040] Figure 12 Comparison of total phenol contents in IP (crude extract obtained without steam explosion), IPs and PIPs;

[0041] Figure 13 Test results of DPPH scavenging rates of Vc, IPs and PIPs;

[0042] Figure 14 Test results of reducing capacities of Vc, IPs and PIPs;

[0043] Figure 15 Test results of anti-tyrosinase activities of kojic acid (KA), IPs and PIPs;

[0044] Figure 16 Chromatogram of high performance liquid chromatography-mass spectrometry for determination of five typical compounds in IPs and PIPs. DETAILED DESCRIPTION

[0045] The present application provides a method for extracting insoluble bound phenols from Paeonia suffruticosa Andr. roots for oil use, comprising the following steps:

[0046] Mixing the powder of Paeonia suffruticosa Andr. roots with an ethanol solution for ultrasonic-assisted extraction to obtain an extraction residue;

[0047] Mixing the extraction residue with water for steam explosion treatment to obtain a pretreated material;

[0048] Mixing the pretreated material, sodium hydroxide, EDTA and ascorbic acid for ultrasonic-assisted hydrolysis, and centrifuging the obtained material liquid to obtain a supernatant;

[0049] Freeze-drying the supernatant to obtain a crude extract of insoluble bound phenols.

[0050] The present application mixes the powder of the root of the oil peony and the ethanol solution to perform ultrasonic-assisted extraction to obtain an extraction residue. In the present application, the powder of the root of the oil peony is preferably prepared by the following method: collecting the root system of the peony, washing with water to remove the soil on the surface, drying, crushing and sieving to obtain the powder. The peony is preferably 5-year-old peony, the drying temperature is preferably 60 DEG C, and the drying is performed until the constant weight. The mesh size of the sieve is preferably 50 mm, and the undersize is taken.

[0051] In the present application, the volume concentration of the ethanol solution is preferably 40-60%, more preferably 60%, and the solid-liquid ratio of the powder of the root of the oil peony and the ethanol solution is preferably 1:10 g / mL-1:20 g / mL, more preferably 1:15 g / mL. The ultrasonic power of the ultrasonic-assisted extraction is preferably 140-250 W, more preferably 180 W, the extraction time is preferably 20-60 min, more preferably 30 min, and the extraction temperature is preferably room temperature. After the extraction is completed, solid-liquid separation is performed, and the obtained solid is the extraction residue. In the present application, the solid obtained by the solid-liquid separation is preferably freeze-dried, and the obtained dried powder is subjected to subsequent treatment.

[0052] After obtaining the extraction residue, the present application mixes the extraction residue and water to perform steam explosion treatment to obtain pretreated material. In the present application, the temperature of the steam explosion treatment is preferably 105-125 DEG C, more preferably 121 DEG C (when the steam explosion temperature is 121 DEG C, the corresponding pressure is 0.2 MPa), and the time is preferably 5-15 min, more preferably 10-15 min, most preferably 15 min. The steam explosion treatment is preferably performed in an autoclave. The solid-liquid ratio of the extraction residue and water is preferably 1:10 g / mL-1:50 g / mL, more preferably 1:30 g / mL. In the present application, the water is preferably deionized water, which will not be described in detail. After obtaining the pretreated material, the present application preferably further comprises supplementing water to the pretreated material, and the amount of the supplemented water is the amount of the water before the steam explosion.

[0053] After obtaining the pretreated material, the present application mixes the pretreated material, sodium hydroxide, EDTA and ascorbic acid to perform ultrasonic-assisted hydrolysis, and centrifuges the obtained material liquid to obtain supernatant. In the present application, in the mixed solution obtained by mixing the pretreated material, sodium hydroxide, EDTA and ascorbic acid, the concentration of sodium hydroxide is preferably 2-6 M, more preferably 4 M, the concentration of EDTA is preferably 5-15 mM, more preferably 10 mM, and the mass fraction of ascorbic acid is preferably 0.5-1.5%, more preferably 1%.

[0054] In the present application, the ultrasonic power for the ultrasonic-assisted hydrolysis is preferably 120-400 W, more preferably 150-380 W, and most preferably 180 W, the ultrasonic frequency is 20-60 kHz, preferably 40 kHz, the hydrolysis time is preferably 2-8 h, more preferably 4 h, and the hydrolysis temperature is preferably room temperature.

[0055] After the ultrasonic-assisted hydrolysis is completed, the pH value of the obtained hydrolysis solution is preferably adjusted to 2 by using hydrochloric acid, and then centrifugation is performed; the rotation speed for the centrifugation is preferably 10,000 rpm, and the centrifugation time is preferably 10 min.

[0056] After the supernatant is obtained, the supernatant is freeze-dried to obtain a crude extract of insoluble bound phenols.

[0057] The present application also provides a method for purifying insoluble bound phenols in the roots of Paeonia suffruticosa Andr. for oil, comprising the following steps:

[0058] The roots of Paeonia suffruticosa Andr. for oil are extracted by using the extraction method described in the above scheme to obtain a crude extract of insoluble bound phenols.

[0059] The crude extract of insoluble bound phenols is dissolved in water to obtain a crude extract solution, and then the crude extract solution is subjected to ultrasonic-assisted adsorption by using a macroporous resin to obtain an adsorption resin.

[0060] The adsorption resin is subjected to ultrasonic-assisted desorption by using an ethanol solution to obtain a purified product of insoluble bound phenols.

[0061] Firstly, the roots of Paeonia suffruticosa Andr. for oil are extracted by using the extraction method described in the above scheme to obtain a crude extract of insoluble bound phenols, and the specific extraction conditions are not described herein.

[0062] After obtaining the insoluble bound phenol crude extract, the present application dissolves the insoluble bound phenol crude extract in water to obtain a crude extract solution, and then performs ultrasonic-assisted adsorption on the crude extract solution by using a macroporous resin to obtain an adsorbed resin. In the present application, the macroporous resin is preferably D101, D101C, LX12, LX83, XDA6 or AB8, and more preferably LX83. Before use, the macroporous resin is preferably pretreated, and the pretreatment is preferably as follows: the macroporous resin is soaked in an ethanol solution with a volume fraction of 95% for 12 h, then the ethanol solution is removed, the macroporous resin is washed with deionized water until neutral, then the macroporous resin is sequentially soaked in a 5% hydrochloric acid solution (v / v) and a 5% NaOH solution (w / v), and finally the macroporous resin is washed with deionized water until neutral and stored in a 95% ethanol solution. The soaking time in the hydrochloric acid solution is preferably 4-5 h, and the soaking time in the NaOH solution is preferably 4-5 h.

[0063] In the present application, the operating conditions of the ultrasonic-assisted adsorption are preferably as follows: the total phenol content in the crude extract solution is 2-4 mg GAE / mL, and more preferably 3 mg GAE / mL, the pH value is 3-5, and preferably 4, the ultrasonic power is 140-250 W, and more preferably 180 W, and the adsorption time is 40-120 min, and more preferably 120 min. In a specific embodiment of the present application, a 5 wt% hydrochloric acid solution is preferably used to adjust the pH value of the crude extract solution. The ultrasonic-assisted adsorption is preferably performed by using a static adsorption method. In a specific embodiment of the present application, the container containing the crude extract solution and the macroporous resin is placed in a shaking table, and the static adsorption is performed under shaking of the shaking table, and the ultrasonic is applied at the same time.

[0064] After obtaining the adsorbed resin, the present application performs ultrasonic-assisted desorption on the adsorbed resin by using an ethanol solution to obtain a purified insoluble bound phenol product. In the present application, the volume concentration of the ethanol solution is preferably 40-80%, and more preferably 60%. The operating conditions of the ultrasonic-assisted desorption are preferably as follows: the ultrasonic power is 140-250 W, and preferably 180 W, the desorption time is 30-60 min, and preferably 30 min, and the desorption temperature is room temperature. The ultrasonic-assisted desorption is preferably performed by using a static desorption method. In a specific embodiment of the present application, the container containing the saturated resin and the ethanol solution is placed in a shaking table, and the static desorption is performed under shaking of the shaking table, and the ultrasonic is applied at the same time.

[0065] After the desorption is completed, the present application preferably performs freeze-drying on the obtained desorption solution, and the obtained powder is the final purified product.

[0066] In the present application, the total phenol content of the purified product obtained under the optimal conditions is 254.74 mg GAE / g dw.

[0067] The technical solutions in the present application will be clearly and completely described below in combination with the embodiments in the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without creative effort belong to the scope of protection of the present application.

[0068] The materials and reagents used in the embodiments are as follows:

[0069] The root system of 5-year-old peony was harvested from the peony plantation of Na'an Biotechnology Co., Ltd. in Changzhi, Shanxi Province. The collected roots were washed under running water for 2-3 times to remove the surface soil, then dried at 60°C to a constant weight, ground in a grinder, passed through a 50 mm sieve to obtain powder, and stored at room temperature for standby use.

[0070] Six kinds of macroporous resins (LX-83, LX-12, XDA-6, D101, D101C and AB-8) were purchased from Shaanxi Lanxiao Biotechnology Co., Ltd., and their physical properties are shown in Table 1. Mass spectrometry grade acetonitrile and methanol were purchased from Thermo Fisher Scientific-China. All the standards and other reagents were purchased from Source Leaf Biotech Co., Ltd. (Shanghai, China).

[0071] Table 1 Properties of six kinds of macroporous resins

[0072]

[0073] In the embodiments, the test method of total phenol content (TPC) of the crude extract and the purified product is as follows:

[0074] 300 μL of Folin-Ciocalteu phenol reagent, 1.5 mL of 7 wt% Na2CO3 aqueous solution, 100 μL of sample, 3 mL of deionized water, mixed and incubated for 2 h, then the absorbance was tested at a wavelength of 760 nm, and the total phenol content (expressed in mg GAE / g) was calculated by formula (1):

[0075] A = 1.808C + 0.056 Formula (1);

[0076] In formula (1), A is the absorbance, C is the concentration, mg GAE / mL;

[0077] After obtaining the concentration data, the TPC content was calculated according to TPC = CV / m, wherein V is the volume of the solution (mL); and m is the weight of the sample (g).

[0078] In the following embodiments, each experiment was repeated at least 3 times, the experimental results were expressed using the mean value ± standard deviation, the variance data was analyzed using SPSS software, and the data was statistically calculated. P < 0.05 indicates significance.

[0079] Example 1: Effect of Steam Explosion Time on Extraction Rate

[0080] The powder of peony root for oil extraction was mixed with a 60% ethanol solution at a ratio of 1g:15mL, and extracted for 30min under ultrasonic assistance at 180W to obtain the residue.

[0081] Deionized water was added to the residue at a ratio of 1g:30mL, and the mixture was placed in an autoclave for steam explosion treatment at a temperature of 121℃ for 5min, 10min, 15min, and 30min. Water was then added to the pressure before pressurization, followed by the addition of NaOH, EDTA, and ascorbic acid. The resulting mixture contained 4M NaOH, 10mM EDTA, and 1wt% ascorbic acid. The mixture was then subjected to ultrasonic-assisted hydrolysis at 360W and 40kHz for 4h at room temperature. The pH was then adjusted to 2 with HCl, and the mixture was centrifuged at 10000rpm for 10min. The supernatant was then freeze-dried under vacuum to obtain the crude extract, which was stored at 4℃.

[0082] In addition, an experimental group without steam explosion was set up as a control. All other conditions were the same as in Example 1, and the crude extract obtained was denoted as IP.

[0083] The total phenol content of the crude extract obtained under different steam explosion treatment times was tested, and the results are as follows: Figure 1 As shown, Figure 1 The lowercase letter 'ae' in the formula was statistically significant (p<0.05). According to... Figure 1 It can be seen that the total phenolic content of the crude extract obtained after steam explosion pretreatment showed a trend of first increasing and then decreasing. In the experimental group without steam explosion, the total phenolic content of the crude extract was 112.26 mg GAE / g dw (IP). During steam explosion for 5–15 min, the total phenolic content of the crude extract gradually increased to 148.75 mg GAE / g dw, while it decreased to 81.87 mg GAE / g dw at 30 min. This indicates that excessively high steam explosion levels are not more conducive to the release of phenolic substances. High temperature and high pressure not only destroy the covalent bonds between insoluble bound phenols and hemicellulose lignin and some proteins, but also further damage the internal structure of polysaccharides, proteins, and phenolic compounds, ultimately impairing biological activity and total phenolic yield. Therefore, steam explosion treatment at 121℃ for 15 min is the optimal condition and was used as the treatment condition for subsequent experiments.

[0084] Example 2: Effect of Ultrasonic Power on Extraction Rate

[0085] The release of phenolic substances also had significant differences under different ultrasonic powers. This embodiment verifies the effect of ultrasonic power on the extraction rate of TPC in the ultrasonic-assisted alkaline hydrolysis process. The experimental process is the same as that in Example 1. The temperature of the steam explosion treatment is 121℃, and the time is 15 min. The ultrasonic power in the ultrasonic-assisted hydrolysis process is 144W, 180W, 216W or 360W, respectively. Other conditions are the same as those in Example 1. The specific test results are shown in Table 2. Figure 2 Figure 2 The results in Table 2 show that in the ultrasonic-assisted alkaline hydrolysis at 144-216W, the TPC of the extract is improved. With the further increase of ultrasonic intensity, their TPCs show a downward trend. The mechanical effect produced by this ultrasonic effect can destroy the structure of the cell wall, thereby helping the alkaline hydrolysis and promoting the release of active substances from the cell wall and intracellular, and improving the extraction rate of target compounds. However, too strong mechanical action can lead to the degradation and destruction of polyphenol structure and reduce the active substances. Finally, the maximum TPC of 185.59mg GAE / g dw under the alkaline hydrolysis at 180W for 4h superimposed with steam explosion pretreatment, and the extract is named as IPs, which is applied to the subsequent experiments.

[0086] Example 3 Effect of pH on adsorption and effect of ethanol concentration on desorption

[0087] LX-83 resin 1g and 50mL crude extract solution (obtained by dissolving the crude extract solution in deionized water, and the total phenol content in the extract solution is 3.0mg GAE / g dw) were placed in a 300mL conical flask, and the pH value was adjusted to 1, 2, 3, 4, 5, 6, 7 with 5% hydrochloric acid and 5% NaOH respectively, and oscillated at 25℃ and 150rpm for 12h. The adsorption amount was calculated according to formula (2).

[0088] Figure 3 The effect of different pH values on the adsorption amount of LX-83 resin, wherein the different lowercase letters a-d have statistical significance (p<0.05). The pH value directly affects the ionization degree of polyphenols, which further affects the adsorption of polyphenols by the resin. As shown in Table 3, when the pH value is 7, the adsorption amount decreases rapidly, indicating that this condition is not conducive to the adsorption of phenols in the sample. When the pH value is 4-5, the adsorption amount of polyphenols reaches a very high level, which is also the most suitable pH for the preservation of polyphenols. Under acidic conditions, polyphenols are more likely to exist in molecular form rather than ionic form, which can promote the adsorption of macroporous resin to polyphenols. Therefore, pH=4 is selected as the best condition for the subsequent experiments. Figure 3

[0089] Example 4 Selection of macroporous resin

[0090] ​​The macroporous resin was soaked in 95% ethanol solution overnight, then the ethanol solution was removed, washed with deionized water to neutral, and finally soaked in 5% hydrochloric acid (v / v) and 5% NaOH (w / v) for 4h respectively, removed the solution, washed with deionized water to neutral, and then stored in 95% ethanol solution.

[0091] Take 1g macroporous resin and add to a 300mL conical flask, add 50mL crude extract solution (obtained by dissolving the crude extract under the optimal conditions in Example 1 in deionized water, the total phenol content in the extract was 3mg GAE / mL), shake in a constant temperature shaking incubator at 25℃, 150rpm for 10h, measure the total phenol content in the extract, and calculate the adsorption capacity according to formula (2).

[0092] Then remove the extract, wash the macroporous resin with deionized water, add 50mL of 60% ethanol, and shake at 25℃, 150rpm for 10h, test the total phenol content in the desorption solution, calculate the desorption capacity according to formula (3), and calculate the desorption ratio according to formula (4), and calculate the recovery rate according to formula (5).

[0093]

[0094]

[0095]

[0096]

[0097] In formula (2)~formula (5), q a and q b are the equilibrium adsorption capacity and desorption capacity respectively; C0is the initial concentration of total phenols in the extract; C a is the total phenol concentration in the solution after adsorption; C b is the total phenol concentration in the solution after desorption, V a is the volume of the extract, V b is the volume of ethanol, m is the mass of the resin, D is the desorption ratio, and R is the recovery rate.

[0098] The adsorption, desorption and recovery rate of six kinds of macroporous resins on IPs are shown in Table 2.

[0099] Table 2 Adsorption, desorption and recovery rate of six kinds of macroporous resins on IPs

[0100]

[0101] From the data in Table 2, it can be seen that LX-83 and XDA-6 showed the highest adsorption capacity, but LX-83 was better than XDA-6 in desorption, obtaining the highest recovery rate of 26.21%. The main bioactive phenols of IPs are gallic acid, catechin and ellagic acid. LX-83 is a non-polar resin, with the largest specific surface area among the six resins, showing obvious advantages in adsorption and desorption, while XDA-6 is moderately polar / weakly polar, which may be too tight to bind with acidic phenolic acids, showing the disadvantage of low desorption efficiency. Therefore, LX-83 was selected as the resin used in subsequent purification.

[0102] Example 5 Adsorption isotherm experiment

[0103] The total phenol content of the crude extract was diluted with deionized water to 0.298, 0.596, 1.192, 1.788, 2.384, 2.98, 3.576 and 5.96 mg GAE / mL, respectively, to obtain crude extract solutions of different concentrations. These solutions (50 mL) were added to 300 mL conical flasks, 1 g of LX-83 macroporous resin was added, and the mixed solutions were shaken (150 rpm), adsorbed under ultrasonic assistance at 0 (shaker 150 rpm), 144, 180, 216 W, and adsorbed at 25°C for 10 h, and the adsorption capacity was calculated according to formula (2).

[0104] To further study the adsorption characteristics of the resin, Langmuir and Freundlich models were selected to analyze the data, and the two models are shown in formula (6) and formula (7).

[0105] The Langmuir model is:

[0106] The Freundlich model is:

[0107] In formula (6) and formula (7): q m is the maximum theoretical adsorption capacity (mg), q e is the adsorption capacity, Ce is the concentration of polyphenols in the solution, K L is the Langmuir constant, n and K F are Freundlich constants, K F represents the theoretical saturation adsorption capacity, and n represents the adsorption driving force, n>1 indicates that the adsorption is beneficial.

[0108] Figure 4 The effect of crude extract solution concentration on the adsorption capacity of the resin. According to Figure 4It can be seen that when the concentration of the crude extract solution is lower than 2.98 mg GAE / mL, the adsorption capacity of LX-83 increases rapidly with the increase of the concentration, and after this turning point, the adsorption capacity of LX-83 resin for polyphenols remains basically unchanged or slightly increases. Therefore, the concentration of the crude extract solution of 2.98 mg GAE / mL (about 3.0 mg GAE / mL) is selected as the optimal sample concentration.

[0109] Figure 5 For the adsorption isotherms of LX-83 resin under different ultrasonic assistance conditions, the adsorption isotherms are fitted to Langmuir and Freundlich models, and the results are shown in Table 3.

[0110] Table 3 Isotherm parameters of LX-83 resin adsorbing IPs at 25℃

[0111]

[0112] The results show that IPs have different fitting results and parameters for two different isothermal adsorption models. Under four ultrasonic powers, the adsorption process can be better described by using the Langmuir adsorption model, and the R L 2 (0.9504, 0.9278, 0.9576 and 0.9809) are slightly larger than R F 2 (0.8951, 0.8040, 0.7652 and 0.8195). The Langmuir adsorption model describes monolayer adsorption, that is, each adsorption site can only adsorb one molecule, the adsorption energy is uniformly distributed, and the small molecules adsorbed do not migrate on the surface of the macroporous resin, which indicates that the adsorption process of IPs is more reasonably described as monolayer adsorption.

[0113] Example 6 Adsorption kinetics experiment

[0114] LX-83 resin 1 g and 50 mL of IP solution containing 3.0 mg GAE / mL were placed in a 300 mL conical flask (×4 groups), and the adsorption was carried out at 25℃ under 0 (shaker 150 rpm), 144, 180 and 216 W ultrasonic assistance, and then the total phenol content of the solution was collected and determined at 0, 5, 10, 20, 30, 60, 90, 120, 180 and 240 min, respectively.

[0115] Two mathematical models, i.e. pseudo-first-order and pseudo-second-order, are selected in this example, as shown in formula (8) and formula (9):

[0116] Pseudo-first-order: q t = k i t 1 / 2 +C formula (8);

[0117] Pseudo-second order: ln(q t -q e ) = -k i t + lnq e Equation (9);

[0118] where: q t is the adsorption capacity (mg) at interval time t, t represents the adsorption time (min), k i is the pseudo-first order and pseudo-second order kinetic constant.

[0119] Figure 6 is the effect of adsorption time on the adsorption capacity of the resin. According to Figure 6 It can be seen that without US, the adsorption of phenol on LX-83 resin increased rapidly before 40 minutes, and tended to equilibrium after 40 minutes. The adsorption capacity increased rapidly within the first 20 minutes, and gradually tended to equilibrium after 20 minutes. This indicates that there are many adsorption sites in the resin before the adsorption begins, and there is a concentration difference between the resin and the solution, which makes it easier to adsorb phenol. Subsequently, the concentration of phenol in the solution is close to that in the resin, and the adsorption sites are reduced, which together lead to the slowing down of the adsorption efficiency. In addition, LX-83 macroporous resin has the highest adsorption capacity at 180W, reaching 69.23mg GAE / g dw, which is increased by 324.57% compared with 21.33mg GAE / g dw without US assistance. However, the higher the power of ultrasound is not better, because the maximum adsorption capacity of macroporous resin decreases to 56.3mg GAE / g dw at 216W. This may be due to the destruction of the physical structure of macroporous resin caused by high ultrasonic power.

[0120] Figure 7 is the pseudo-first order kinetic fitting curve of the adsorption process, Figure 8 is the pseudo-second order kinetic fitting curve of the adsorption process; Table 4 is the pseudo-first order and pseudo-second order dynamic model parameters of the adsorption process of IP.

[0121] Table 4 Pseudo-first order and pseudo-second order dynamic model parameters of the adsorption process of IP

[0122]

[0123] According to Figures 7-8It can be seen from the data in Table 4 that the adsorption process of IPs at four levels of 0 W (R2= 0.9962), 144 W (R2= 0.9960), 180 W (R2= 0.9906) and 216 W (R2= 0.9923) is also consistent with the pseudo-second-order kinetics equation. It is found that the ultrasonic-assisted treatment in the adsorption process not only shortens the adsorption time, but also improves the adsorption capacity. The reason may be that the ultrasonic destroys the structure of the resin surface, increases the specific surface area of the resin, and the relatively mild low-power ultrasonic in the adsorption process promotes the mass transfer of polyphenols, thereby accelerating the achievement of adsorption equilibrium and increasing the adsorption capacity. However, after a high degree of ultrasonic enhancement, the adsorption capacity of the resin decreases, which may be due to the heat effect of ultrasonic, which leads to temperature rise, which is not conducive to the combination of phenolic substances and resin, and the temperature rise also leads to more water vapor entering the cavitation bubble, thereby reducing the cavitation effect of US.

[0124] Figure 9 Scanning electron micrographs of LX-83 macroporous resins after adsorption with or without ultrasonic assistance (magnification 250 times), wherein (a) is the control group (without ultrasonic, adsorption in a fluidized bed), (b) is column chromatography adsorption, (c) is shaking bed adsorption, (d) is ultrasonic-assisted adsorption at 144 W ultrasonic power, (e) is ultrasonic-assisted adsorption at 180 W ultrasonic power, and (f) is ultrasonic-assisted adsorption at 216 W ultrasonic power. According to Figure 9 It can be seen that the surfaces of the LX-83 macroporous resins in the control group, after column chromatography and after shaking bed show smooth surfaces, while the resins treated by ultrasonic assistance show rough and cracked surfaces. Compared with the smooth surface, the rough surface of the latter may be the main reason for improving the adsorption efficiency and increasing the adsorption capacity. In addition, as the ultrasonic power increases, the damage to the resin surface becomes more serious. In particular, at 216 W, large cracks and damage appear, which is the reason for the decrease in adsorption capacity compared with 180 W. These results further prove the previous guess that 180 W is a suitable ultrasonic-assisted adsorption power.

[0125] Example 7 Effect of ethanol concentration on desorption

[0126] The resins after adsorption at a pH of 4 were desorbed with 50 mL of ethanol with a volume fraction of 0% (i.e. deionized water), 20%, 40%, 60%, 80% and 100%, respectively, and the desorption conditions were the same as in Example 3. After desorption, the desorption amount was calculated according to formula (3).

[0127] Figure 10 The effect of different ethanol concentrations on the desorption amount of the adsorbed resin, wherein the lowercase letters a-d are statistically significant (p < 0.05). According to Figure 10It can be seen that the change of ethanol concentration has a significant effect on the desorption amount. When the ethanol concentration is from 0% to 60%, the desorption amount gradually increases, and when the ethanol concentration is from 60% to 100%, the desorption amount gradually decreases. Therefore, 60% ethanol is selected as a suitable desorption solvent.

[0128] The resin adsorbed under the condition of pH 4 is desorbed with 50 mL of 60% ethanol by volume, and the desorption is carried out under the assistance of ultrasound at 0 W, 144 W, 180 W and 216 W, respectively, for 120 min, and the desorption amount at different times is tested.

[0129] Figure 11 The effect of ultrasonic power on the desorption amount of the adsorbed resin is shown in Table 3. Figure 11 It can be seen that the desorption capacity of the macroporous resin is the highest at 180 W, and in addition, the ultrasonic-assisted desorption is significantly faster (about 20 min to complete desorption) compared with the oscillation treatment (about 60 min to complete desorption). Therefore, the present application selects 180 W as the optimal power for ultrasonic-assisted desorption.

[0130] Based on the above results, the optimal conditions for purifying the crude extract of insoluble bound phenols are as follows: the macroporous resin is LX-83, the adsorption conditions are pH 4, the total phenol content in the crude extract solution is 3.0 mg GAE / mL, and the adsorption is assisted by ultrasound at 180 W for 120 min; and the desorption conditions are that 60% ethanol is used as the desorption liquid, and the desorption is assisted by ultrasound at 180 W for 30 min. In Example 2, the total phenol content of the crude extract (IPs) obtained under the optimal conditions is 185.59 mg GAE / g dw, and the total phenol content of the purified product (PIPs) obtained by purification under the above optimal conditions is 254.74 mg GAE / g dw, which is increased by 37.3%; Figure 12 The total phenol contents of IP (crude extract obtained without steam explosion), IPs and PIPs are compared.

[0131] The crude extract (IPs) obtained under the optimal conditions and the purified product (PIPs) obtained under the optimal conditions are used for subsequent bioactivity tests.

[0132] Example 8 Bioactivity test

[0133] 1. Test method

[0134] (1) Antioxidant activity

[0135] The test method is as follows: 2 mL of sample solution of different concentrations (solvent is water, and the specific concentration is shown in Table 4) is taken, and 0.5 mL of 1% DPPH solution is added, and the mixture is mixed and reacted in the dark for 30 min. The absorbance at 517 nm is measured, and the antioxidant activity is calculated according to the formula: antioxidant activity (%) = (A0-A1) / A0×100%, where A0 is the absorbance of the control sample, and A1 is the absorbance of the sample. Figure 13Add 2 mL of DPPH reaction solution, shake well, react in the dark for 40 min, and measure its absorbance at 517 nm. The blank is 95% ethanol to replace the sample solution, and 2 mL of 95% ethanol is added to 2 mL of DPPH reaction solution as a control; the clearance rate is measured according to formula (10).

[0136]

[0137] In formula (10), A1 is the absorbance of the reaction solution; A0 is the absorbance of the blank; A c The absorbance of the control group; the higher the scavenging rate, the higher the antioxidant activity.

[0138] (2) Iron ion reducing power

[0139] 1.5 mL of sample solutions of different concentrations (solvent is water, see specific concentrations) Figure 14 Mix with 3 mL of FRAP working solution, and incubate the reaction solution in a dark incubator at 37°C for 30 min. Measure the absorbance at 593 nm to indicate the reducing power; the higher the absorbance, the better the reducing power.

[0140] (3) Antityrosinase activity

[0141] 60 μL of sample solutions of different concentrations (solvent is water, see specific concentrations) Figure 15 The sample solution was mixed with 60 μL (46 U / mL) of tyrosinase solution and 20 μL of phosphate buffer (pH = 6.8) in a 96-well plate and incubated at 25 °C for 15 min. Then, 60 μL (5 mM) of L-DOPA was added to initiate the reaction. Similarly, a blank was prepared by adding the sample solution to all reaction reagents without adding tyrosinase. After incubation at 25 °C for 15 min, the absorbance was measured at 475 nm. The inhibition rate was determined according to Equation (11).

[0142]

[0143] In formula (11): A represents the absorbance of L-DOPA+PBS+tyrosinase; B represents the absorbance of the L-DOPA+PBS mixture; C represents the absorbance of L-DOPA+PBS+tyrosinase+sample; D represents the absorbance of L-DOPA+PBS+sample.

[0144] (4) HPLC-MS

[0145] Qualitative and quantitative analysis of each phenolic component was performed using HPLC-MS method, using a high resolution mass spectrometer, a C18 reverse phase column (100 mm x 2.1 mm, 3 μm particle size), mobile phase: water + 0.1% formic acid (A) and acetonitrile (B), flow rate: 0.3 mL / min; elution gradient: 0.0-4.0 min, 13% B; 4.0-5.0 min, 13%-35% B; 5.0-9.0 min, 35%-85% B; 9.0-15.0 min, 85%-90% B; 15.0-16.0 min, 90%-100% B; 16.0-20.0 min, 100% B; 20.0-21.0 min, 100%-5% B; 21.0-25.0 min, 5% B. Mass spectrometry conditions: electrospray ionization source (ESI), negative ion scan mode. Capillary temperature was 320 °C, sheath gas flow rate 40 arb, auxiliary gas flow rate 10 arb, spray voltage 2500 V, full scan scan range 100-1000 m / z. Qualitative and quantitative analysis was performed using external standard method and standards, expressed in mg / g.

[0146] 2. Test results

[0147] (1) Vc was generally used as a positive control for free radical scavenging and metal cation reduction. In this example, Vc, IPs and PIPs were used at five different concentrations (20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL, solvent deionized water) to evaluate their antioxidant performance.

[0148] Figure 13 DPPH scavenging rate test results for Vc, IPs and PIPs; Figure 14 Reducing power test results for Vc, IPs and PIPs; Figures 13-14 In Table 2, different capital letters (A-C) indicate the significance (p < 0.05) of different samples at the same concentration, and different lowercase letters (a-e) indicate the significance (p < 0.05) of the same sample at different concentrations.

[0149] According to Figures 13-14 It can be seen that at the same concentration, the activity of PIPs obtained after ultrasonic-assisted purification is significantly enhanced in terms of free radical scavenging and iron ion reducing power. In particular, PIPs exhibit similar reducing power to Vc in terms of iron ion reducing power.

[0150] (2) Kojic acid (KA) is the most common tyrosinase inhibitor and used as a positive control for inhibitory effect. Five different concentrations (20, 40, 60, 80, 100 μg / mL, solvent: deionized water) of KA, IPs and PIPs were used to evaluate their antioxidant properties.

[0151] Figure 15 Results of the anti-tyrosinase activity test for KA, IPs and PIPs, Figure 15 Different capital letters (A-C) represent the significance (p<0.05) of different samples at the same concentration, and different small letters (a-e) represent the significance (p<0.05) of the same sample at different concentrations. According to Figure 15 It can be seen that the inhibition rate of kojic acid on tyrosinase is 75.65% at 100 μg / mL, while the inhibition rate of IPs is 12.19% and that of PIPs is 29.00% at the corresponding concentration. The inhibition activity of PIPs is 2.37 times that of IPs. The above results show that the double optimization of the extraction and purification process of the insoluble bound phenols in the oil tree peony root can effectively increase the content of phenolic acids in the extract, and enhance the antioxidant and anti-tyrosinase activities.

[0152] 3. Quantitative analysis of typical compounds

[0153] By comparing m / z, fragments and retention time with standards and calibration curves, a total of 5 typical compounds were identified and quantified, and the results are shown in Table 5 and Figure 16 Table 6.

[0154] Table 5. Determination of the content of typical compounds in IPs and PIPs by high performance liquid chromatography-mass spectrometry

[0155]

[0156] The sum of the 5 typical compounds in Table 5 is 0.404 mg / g (IPs) and 3.233 mg / g (PIPs), respectively, which is about 8 times higher in PIPs than in IPs.

[0157] Paeoniflorin is a monoterpene compound and also a signature compound in peony. It has various anti-inflammatory and DNA-protecting effects. The content of paeoniflorin in IPs and PIPs is 0.277 mg / g and 2.673 mg / g, respectively. Although paeoniflorin mainly exists in free form in peony, the extracted peony root residues still contain a considerable amount of paeoniflorin. After ultrasonic-assisted purification, the content of paeoniflorin in PIPs increases by 9.7 times. In addition, benzoylpaeoniflorin also increases by 10.3 times. This shows that the purification method of the present application has an ideal effect on paeoniflorin and its derivatives.

[0158] Based on the above results, the total phenol content in the crude extract was increased from 112.26 mg GAE / g dw (IP) to 148.75 mg GAE / g dw (IPs) under the optimal conditions (121℃, 15min) by steam explosion treatment, about 32.50% increase. In the flow bed (1:50 = LX-83:IPs solution) of pH = 4 and 3.0 mg GAE / mL, after 120min of ultrasonic assisted adsorption at 180W and 30min of ultrasonic assisted desorption at 180W, PIPs were obtained. Compared with IPs, the total phenol content of PIPs increased to 254.74 mg GAE / g dw, about 71.2% increase. The adsorption process of IPs was better described by Langmuir adsorption model, which belonged to monolayer adsorption, and the fitting kinetic curve of IPs was pseudo-second-order kinetics equation. Compared with IPs, PIPs had higher antioxidant and anti-tyrosinase activities, which might be related to the increase of paeonol and its derivatives, gallic acid and p-hydroxybenzoic acid in PIPs. In summary, the new extraction and purification method of the present application released more active substances from the oil tree peony root, which provided a basis for the utilization of the oil tree peony root, especially in the whitening and skin care products.

[0159] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for extracting insoluble bound phenols from the root of oil peony, characterized in that, Includes the following steps: The oil was extracted with ultrasonic-assisted extraction by mixing peony root powder and ethanol solution to obtain the extraction residue; The extracted residue and water are mixed and subjected to steam explosion treatment to obtain pretreated material; the temperature of the steam explosion treatment is 105~125℃ and the time is 5~15min; the material-to-liquid ratio of the extracted residue and water is 1:10g / mL~1:50g / mL. The pretreated material, sodium hydroxide, EDTA, and ascorbic acid are mixed and then subjected to ultrasonic-assisted hydrolysis. The resulting solution is centrifuged to obtain a supernatant. The ultrasonic power for the ultrasonic-assisted hydrolysis is 120-400W, the ultrasonic frequency is 20-60kHz, the hydrolysis time is 2-8h, and the hydrolysis temperature is room temperature. In the mixture obtained by mixing the pretreated material, sodium hydroxide, EDTA, and ascorbic acid, the concentration of sodium hydroxide is 2-6M, the concentration of EDTA is 5-15mM, and the mass fraction of ascorbic acid is 0.5-1.5%. The supernatant was freeze-dried to obtain a crude extract of insoluble bound phenols.

2. The extraction method according to claim 1, characterized in that, After obtaining the pretreated material, the process also includes adding water to the pretreated material.

3. A method for purifying insoluble bound phenols from the root of oil peony, characterized in that, Includes the following steps: The root of peony for oil extraction was extracted according to the extraction method described in any one of claims 1 to 2 to obtain a crude extract of insoluble bound phenols; The crude extract of insoluble bound phenols was dissolved in water to obtain a crude extract solution. Then, the crude extract solution was subjected to ultrasonic-assisted adsorption using a macroporous resin to obtain an adsorption resin. The operating conditions for ultrasonic-assisted adsorption included: the total phenol content in the crude extract solution was 2-4 mg GAE / mL, the pH value was 3-5, the ultrasonic power was 140-250 W, the adsorption time was 40-120 min, and the adsorption temperature was room temperature. The adsorption resin was subjected to ultrasonic-assisted desorption using an ethanol solution to obtain a purified product of insoluble bound phenol; the volume concentration of the ethanol solution was 40-80%; the operating conditions for ultrasonic-assisted desorption included: ultrasonic power of 140-250W, desorption time of 30-60min, and desorption temperature of room temperature.

4. The purification method according to claim 3, characterized in that, The macroporous resin is D101, D101C, LX12, LX83, XDA6, or AB8.

5. The purification method according to claim 3, characterized in that, The ultrasound-assisted adsorption is performed using a static adsorption method; the ultrasound-assisted desorption is performed using a static desorption method.

Images