Plant active component in camphor tree seed kernel and separation and purification method thereof

Abstract

The invention relates to the field of extraction and purification of active substances of natural products, and discloses plant active ingredients in camphor tree seed kernels and a separation and purification method thereof.The method comprises the steps that fresh camphor tree seed kernels and water are mixed, wet-process superfine grinding is conducted through a colloid mill at the room temperature, centrifugal separation is conducted to obtain a middle-layer water phase, and the middle-layer water phase is statically or dynamically adsorbed through HJ-18 macroporous resin; and desorbing with 80% ethanol, collecting a desorption solution, carrying out rotary evaporation, and freeze-drying to obtain the water-soluble active component of the camphor tree seed kernel. The invention provides a new process for enriching and purifying the active components of the camphor tree seed kernels, the active components of the camphor tree seed kernels are enriched and purified by taking water as an extracting agent and combining with macroporous resin, the operation is simple and convenient, the enrichment efficiency is high, the use amount of organic solvents such as methanol and ethanol is reduced, and the process is safe and environment-friendly. In addition, compared with an ethanol extraction method, the method has the advantages that the camphor tree seed kernel protein is not denatured, and the quality of the camphor tree seed kernel protein is improved; according to the technical scheme, a new solution is provided for comprehensive utilization of camphor tree seed resources.

Description

Technical Field

[0001] The invention relates to the field of extraction and purification of active substances in natural products, in particular to a plant active component in camphor tree seed kernel and a separation and purification method thereof. Background Art

[0002] Plant active ingredients such as polyphenols, flavonoids, alkaloids, etc. are a class of natural organic compounds produced by secondary metabolism in plants and are widely present in various tissues of plants. Plant active ingredients have excellent physiological activities, such as: scavenging free radicals, anti-oxidation, anti-aging, anti-bacterial and anti-inflammatory, improving intestinal flora, regulating immunity, anti-tumor and anti-cancer effects.

[0003] In recent years, recycling natural active substances (plant protein, polysaccharide polyphenol, flavonoids, alkaloids, saponin, etc.) from agricultural and sideline products is an effective method to improve the resource utilization of agricultural and sideline products. It is a more mature method to combine solvent extraction separation and macroporous resin purification to obtain plant active ingredients in plant raw materials. For example, application publication number CN103012544A discloses a method for extracting saponin and polysaccharide from tea oil seed cake, including petroleum ether degreasing, water extraction of saponin and polysaccharide, macroporous resin purification of saponin and other steps, extracting saponin and polysaccharide from tea oil seed cake. Compared with the water emulsification extraction method, this method needs to use toxic, flammable and explosive organic solvents, there are safety hazards such as flammable and explosive in the production process, and there are problems such as organic solvent residues in plant by-products.

[0004] In (Research on the process of removing phenol and glucosinolate from rapeseed by glycolic acid and its influence on protein quality, Food Industry Science and Technology, Zhang Yimeng, 2020), rapeseed was dephenolized and desulfurized with ethanol, and the aqueous phase protein obtained after oil extraction was extracted by an energy-saving and environmentally friendly water-based method, and high-quality protein with a purity of 63.63% was obtained. In order to reduce the influence of ethanol treatment on protein dissolution, this method pre-treats whole rapeseed with ethanol to remove polyphenols and glucosinolates in rapeseed, thereby obtaining high-quality aqueous phase protein. However, (Dissolution behavior and mechanism of alcohol-denatured soy protein under physical modification conditions, Food and Fermentation Industry, Hua Yufei, 2004) pointed out that ethanol solution will cause alcohol denaturation of protein, change the structure of protein, and thus affect the functional properties of protein. The α-helix degree of the denatured conformation of alcohol-denatured protein is higher than that of the natural conformation, and after treatment with other denaturation methods such as heat denaturation, the protein conformation generally changes from regular α-helix and β-folding to irregular random coil conformation. Therefore, although ethanol pretreatment can obtain high-purity proteins, there may be a risk of denaturing the protein structure.

[0005] In (The Effect of Alcohol Washing Pretreatment on Camphor Kernel Protein Isolate Products and Their Protein Structure, China Oils and Fats, Sun Dan, 2022), after pre-treating defatted camphor kernel powder with ethanol of different volume fractions, high-purity camphor kernel protein isolate was prepared by alkali dissolution and acid precipitation. Although this method can effectively remove phenolic substances with a volume fraction of 55% to 85% ethanol, and the alcohol treatment has no obvious effect on the secondary structure of camphor kernel protein. However, the alkali dissolution and acid precipitation process will lead to the destruction of hydrogen bonds between camphor kernel protein molecules, a reduction in β-folding and β-turns, and an increase in random coils and α-helices. Therefore, finding a suitable high-quality protein extraction method is crucial for the resource utilization of agricultural and sideline products.

[0006] Camphor tree is widely cultivated in the south of the Yangtze River in my country as a landscape tree, with an annual output of more than 5 million tons. However, camphor seed, except for being used to cultivate camphor seedlings, has not been rationally developed and utilized so far, and falls off every time the fruit matures, seriously polluting the urban and rural environment. Camphor seed kernel is rich in medium-chain oil (56-60%) and protein (18-25%), and is the only natural medium-chain oil and protein resource in the world and unique to China. At present, the literature reports on the development and utilization of camphor seed mainly focus on the extraction technology, physiological effects (biological activity) and transformation and modification of camphor seed kernel oil, but there are few research reports on the separation, identification and utilization of protein, polysaccharide, polyphenol, flavonoid and alkaloid substances with a content of more than 40% in camphor seed kernel.

[0007] Using water as separation medium and adopting emulsification extraction-demulsification separation (water emulsification extraction method) to extract camphor tree seed kernel oil, not only can high-quality medium-chain oil (medium-chain fatty acid content is greater than 95%) be obtained, but also the structure and biological activity of substances such as protein, polysaccharide, polyphenol, flavonoid, alkaloid in by-products will not be destroyed. However, in industry, the middle layer water phase in the process of water extraction of vegetable oil is not effectively utilized and directly discharged, which causes energy waste and environmental pollution. Therefore, in order to realize the high-value utilization of camphor tree seeds, it is of great significance to enrich and recycle the biologically active substances in the middle layer water phase of the process of water extraction of camphor tree seed kernel oil. Summary of the invention

[0008] In view of the above-mentioned problems, the present invention proposes a plant active ingredient in camphor tree seed kernel and a method for separating and purifying the same. An emulsified phase (camphor tree seed kernel oil), an aqueous phase (intermediate aqueous phase) and a residue phase are separated from the camphor tree seed kernel by a water emulsification extraction method, and the aqueous phase (intermediate aqueous phase) is enriched and purified by macroporous resin adsorption to obtain the plant active ingredient of the camphor tree seed kernel. The method has the advantages of being simple, green and pollution-free, and low in cost. In addition, the prepared plant active ingredient of the camphor tree seed kernel has high yield, high extraction rate and high quality, thereby improving the comprehensive utilization efficiency of camphor tree seed resources and meeting the requirements of more fields.

[0009] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0010] The first aspect of the present invention provides a method for separating and purifying plant active ingredients in camphor tree seed kernels, comprising the following steps:

[0011] Step S1: fresh camphor tree seed kernels are mixed with water, and the mixture is ultrafinely ground by a colloid mill to obtain slurry I, and the mixture is centrifuged to obtain an upper emulsified phase I, an intermediate aqueous phase I, and a lower slag phase I; the intermediate aqueous phase I and the lower slag phase I are ultrafinely ground by a colloid mill to obtain slurry II, and the mixture is centrifuged to obtain an upper emulsified phase II, an intermediate aqueous phase II, and a lower slag phase II; the intermediate aqueous phase II is collected and refrigerated for later use;

[0012] Step S2: using HJ-18 macroporous resin to perform static or dynamic adsorption on the active ingredients of camphor tree seed kernel in the intermediate layer water phase II, wherein during static adsorption, whether the adsorption equilibrium is reached is determined by the adsorption amount of polyphenols by the resin; during dynamic adsorption, the intermediate layer water phase II flows through the resin filler at a flow rate of 1.5 BV / h according to 5 to 10 column volumes, so that the resin is fully adsorbed, and the water phase after flowing through the macroporous resin is collected;

[0013] Step S3: if static adsorption is adopted in step S2, the resin is recovered by filtration or centrifugation after adsorption equilibrium, and the aqueous phase enriched with the macroporous resin is collected at the same time; the resin is statically desorbed by using 80% ethanol solution, and whether the desorption equilibrium is reached is determined by the desorption amount of polyphenols in the supernatant; if dynamic adsorption is adopted in step S2, the adsorption saturated HJ-18 resin is dynamically desorbed by using 80% ethanol solution, and the elution flow rate is controlled to be 3.0 BV / h;

[0014] Step S4: After static desorption equilibrium, the eluate is filtered or centrifuged to recover the eluate, and the eluate is subjected to reduced pressure rotary distillation to recover the ethanol, and after removing the eluent, the eluent is vacuum freeze-dried to obtain the plant active ingredients of the camphor tree seed kernel; after the dynamic desorption is completed, the eluate is recovered, and the eluate is subjected to reduced pressure distillation to remove the eluent, and after vacuum freeze-drying to obtain the enriched plant active ingredients of the camphor tree seed kernel.

[0015] The present invention adopts a water emulsification extraction method to separate an emulsified phase (camphor tree seed kernel oil), an aqueous phase (middle layer aqueous phase) and a slag phase from camphor tree seed kernels, and then enriches and purifies the aqueous phase (middle layer aqueous phase) through macroporous resin adsorption to obtain camphor tree seed kernel plant active ingredients. Macroporous resin belongs to functional polymer materials and is a type of organic polymer adsorbent. Its pore size and specific surface area are relatively large, and it has a three-dimensional spatial pore structure inside the resin. It has many advantages such as high physical and chemical stability, large specific surface area, large adsorption capacity, good selectivity, fast adsorption speed, mild desorption conditions, convenient regeneration treatment, long service life, suitable for forming a closed-loop cycle, saving costs, and is insoluble in acid, alkali and various solvents. The invention provides a process for enriching the active components of camphor tree kernel plants in the middle layer water phase of the process of emulsifying and extracting camphor tree kernel oil with a macroporous resin. The method for enriching and purifying with a macroporous resin has the advantages of being simple, green and pollution-free, and low cost. Moreover, the prepared active components of camphor tree kernel plants have high yield, high extraction rate, and high quality, and can meet the requirements of more fields. In addition, the active substances and proteins in the water phase can be separated by macroporous resin treatment, so that not only the active substances of camphor tree kernel can be enriched, but also the quality of the protein in the water phase of camphor tree kernel can be improved, thereby providing a theoretical basis for the industrial application of water emulsification extraction of camphor tree kernel oil.

[0016] In a preferred technical solution, the solid-liquid ratio of fresh camphor tree seed kernels to water in step S1 is 1:(3-5); the ultrafine grinding time is 4-6 minutes, the centrifugal separation speed is 4000-6000rpm, and the time is 15-30 minutes.

[0017] In a preferred technical solution, the HJ-18 macroporous resin in step S2 is pretreated before static or dynamic adsorption, and the pretreatment method is: the macroporous resin is immersed in 95% ethanol with a volume of 3 to 4 times the volume of the resin, and after the resin is fully swollen for 12-24 hours, the resin is rinsed with distilled water until there is no alcohol taste, then soaked in 1M HCl solution for 6-12 hours, rinsed with purified water until the resin is neutral, and then soaked in 1M NaOH solution for 6-12 hours, and rinsed with purified water until the resin is neutral, so as to obtain the treated macroporous resin, and the resin is soaked in purified water for standby use.

[0018] In a preferred technical solution, the specific process of static adsorption in step S2 is: adding the macroporous resin to the intermediate layer aqueous phase II, sealing and placing it in a constant temperature shaker at 150 r / min, and oscillating and adsorbing at room temperature until adsorption equilibrium.

[0019] In a preferred technical solution, the adsorption amount in step S2 is calculated according to the following formula:

[0020]

[0021] Where: Qe is the adsorption amount, mg / g; C0 is the initial polyphenol concentration in the intermediate aqueous phase II, mg / mL; C e is the polyphenol concentration at adsorption equilibrium, mg / mL; V i is the volume of the intermediate layer aqueous phase II, mL; W is the wet weight of the macroporous adsorption resin, g.

[0022] In a preferred technical solution, the polyphenol concentration is determined by the Folin phenol method, and the specific operation process is as follows:

[0023] (1) Sample treatment: Take 0.5 mL of the sample to be tested, add 2 mL of anhydrous ethanol, and centrifuge at 3500 rpm for 5 min to obtain the supernatant;

[0024] (2) Determination of polyphenol content in samples: 1 mL of supernatant was taken, 0.5 mL of Folin-phenol reagent was added, mixed evenly, 2 mL of 7.5% Na2CO3 solution was added, and the total volume was made up to 10 mL with ultrapure water. The mixture was vortexed for 1 min, reacted in a 70°C water bath for 30 min, and cooled naturally to room temperature. A standard curve was established using distilled water as a blank and gallic acid as a reference to evaluate the polyphenol content. The absorbance value was measured at a wavelength of 765 nm, and the polyphenol concentration (mg GAE / mL) was calculated.

[0025] In a preferred technical solution, the specific process of dynamic adsorption in step S2 is: the pre-treated macroporous resin is wet loaded into the chromatography column, after being balanced with distilled water, the intermediate layer aqueous phase II is passed through the chromatography column at a flow rate of 1.5 BV / h and the effluent is collected; the specific process of dynamic desorption in step S3 is: first, 3 to 5 BV of distilled water is used to flow through the resin filler at a flow rate of 3.0 BV / h to remove protein and pigment, and the effluent is cooled to 0.1% by volume. 280nm When the pH is 0, 5 to 10 BV of 80% ethanol solution eluent is passed through the resin packing at a flow rate of 3.0 BV / h.

[0026] In a preferred technical solution, the specific process of static desorption in step S3 is as follows: the macroporous resin material obtained by filtration or centrifugation is rinsed with distilled water to remove surface proteins and pigments, and then the water is absorbed with filter paper, and 80% ethanol solution is added as an eluent according to a solid-liquid ratio of 1: (5-10) (w / v), and the mixture is sealed and placed in a constant temperature shaker at 150 r / min, and desorbed by shaking at room temperature for 1 to 2 hours, and then allowed to stand and settle.

[0027] In a preferred technical solution, the desorption amount in step S3 is calculated according to the following formula:

[0028]

[0029] Where: Qd is the desorption amount, mg / g; C d is the concentration of polyphenols in the desorption solution, mg / mL; V d is the volume of desorption liquid, mL; W is the wet weight of macroporous adsorption resin, g.

[0030] A second aspect of the present invention provides an extract of plant active ingredients in camphor tree seed kernels, which is obtained according to any of the above-mentioned methods for separating and purifying plant active ingredients in camphor tree seed kernels.

[0031] The third aspect of the present invention provides a protein in camphor tree seed kernel, which is prepared by obtaining an aqueous phase after static adsorption of a macroporous resin or an aqueous phase after dynamic flow through a macroporous resin in any of the above-mentioned methods for separating and purifying plant active ingredients in camphor tree seed kernels, and then drying it.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The method of the present invention makes full use of the intermediate layer water phase produced during the water emulsification extraction of camphor tree seed kernel oil, and while effectively enriching the plant active ingredients in the intermediate layer water phase, improves the quality of the water phase protein, which is conducive to the full utilization of camphor tree seed resources. The extraction method is mild in condition, simple and fast in operation, and has high enrichment efficiency. While obtaining high-quality camphor tree seed kernel oil, it effectively enriches the active substances in the intermediate layer water phase and improves the quality of the protein in the intermediate layer water phase. In addition, the macroporous resin material in the present invention can be reused after regeneration, reducing production costs. Only low-concentration NaOH and HCl solutions are used in the purification process of the method, and other organic solvents are not used. Only ethanol is used in the resin desorption process at the same time, and the extraction reagent is non-toxic, harmless, and pollution-free. It is a safe and environmentally friendly method for enriching and purifying plant active ingredients in the intermediate layer water phase of the water emulsification extraction of camphor tree seed kernel oil. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 This is a graph showing the adsorption and desorption performance of different types of macroporous resins on the active ingredients of camphor tree seed kernels;

[0036] Figure 2 This is the static adsorption curve of HJ-18 macroporous resin on the active ingredients of camphor tree seed kernel;

[0037] Figure 3This is a graph showing the effect of ethanol concentration on the desorption of active ingredients from camphor tree seed kernels;

[0038] Figure 4 This is the static desorption curve of HJ-18 macroporous resin for active ingredients in camphor tree seed kernels;

[0039] Figure 5 It is the dynamic breakthrough curve of HJ-18 macroporous resin at different flow rates;

[0040] Figure 6 This is the dynamic desorption curve of HJ-18 macroporous resin at different eluent flow rates;

[0041] Figure 7 This is the HPLC chromatogram of the active ingredients of camphor tree seed kernel before enrichment and purification, with a concentration of 4 mg / mL;

[0042] Figure 8 This is the HPLC chromatogram of the enriched and purified active ingredients from camphor tree seed kernels, with a concentration of 2 mg / mL;

[0043] Fig. 9 is the adsorption and desorption performance graph of HJ-18 resin in 10 adsorption-desorption cycles;

[0044] Fig.10 This is a diagram of the aqueous solution before and after macroporous resin enrichment. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0046] Example 1

[0047] The present invention provides a method for separating and purifying plant active ingredients in camphor tree seed kernels, comprising the following steps:

[0048] a) picking fresh and mature camphor seeds from the Qingshan Lake Campus of Nanchang University, removing the peel and pulp of the camphor seeds, drying at (40±1)° C. to constant weight, shelling and color sorting to obtain camphor seed kernel entities;

[0049] b) mixing fresh camphor tree seed kernels with water at a solid-liquid ratio of 1:4, and subjecting the mixture to ultrafine grinding by colloid milling for 5 minutes to obtain slurry I, and subjecting the mixture to centrifugal separation at a centrifugal speed of 4800 rpm for 20 minutes to obtain an upper emulsified phase I, an intermediate aqueous phase I, and a lower slag phase I; subjecting the intermediate aqueous phase I and the lower slag phase I to ultrafine grinding by colloid milling for 5 minutes to obtain slurry II, and subjecting the mixture to centrifugal separation at a centrifugal speed of 4800 rpm for 20 minutes to obtain an upper emulsified phase II, an intermediate aqueous phase II, and a lower slag phase II; collecting the intermediate aqueous phase II and refrigerating it for later use;

[0050] c) Pre-treat HJ-18 macroporous resin: the specific surface area of ​​macroporous resin is ≥ 650m 2 / g, particle size is 0.30-1.25mm; soak the macroporous resin in 95% ethanol of 3 times the volume of the resin, wait for the resin to fully swell, swell for 24 hours, rinse the resin with distilled water until there is no alcohol smell, then soak it in 1M HCl solution for 12 hours, rinse the resin with purified water until it is neutral, soak it in 1M NaOH solution for 12 hours, rinse the resin with purified water until it is neutral, and then get the treated macroporous resin, soak the resin in purified water for standby use;

[0051] d) Add the macroporous resin to the intermediate aqueous phase II, seal it and place it in a constant temperature shaker at 150 r / min, and oscillate and adsorb at room temperature until adsorption equilibrium; calculate the adsorption amount and adsorption rate according to the following formula:

[0052]

[0053] Where: Q e is the adsorption amount, mg / g; A is the adsorption rate, %; C0 is the initial polyphenol concentration in the intermediate aqueous phase II, mg / mL; C e is the polyphenol concentration at adsorption equilibrium, mg / mL; V i is the volume of the intermediate layer aqueous phase II, mL; W is the wet weight of the macroporous adsorption resin, g.

[0054] The polyphenol concentration was determined by the Folin phenol method, and the specific operation process was as follows:

[0055] (1) Sample treatment: Take 0.5 mL of the sample to be tested, add 2 mL of anhydrous ethanol, and centrifuge at 3500 rpm for 5 min to obtain the supernatant;

[0056] (2) Determination of polyphenol content in samples: 1 mL of supernatant was taken, 0.5 mL of Folin-phenol reagent was added, mixed evenly, 2 mL of 7.5% Na2CO3 solution was added, and the total volume was made up to 10 mL with ultrapure water. The mixture was vortexed for 1 min, reacted in a 70°C water bath for 30 min, and cooled naturally to room temperature. A standard curve was established using distilled water as a blank and gallic acid as a reference to evaluate the polyphenol content. The absorbance value was measured at a wavelength of 765 nm, and the polyphenol concentration (mg GAE / mL) was calculated.

[0057] In the static adsorption process, the static adsorption curve is established with the adsorption time as the horizontal axis and the adsorption amount as the vertical axis, such as Figure 2 As shown in the figure, it can be seen that the adsorption of active ingredients of camphor tree seed kernel by HJ-18 macroporous resin increases rapidly within the first hour, and the adsorption equilibrium can be reached after 2 hours of adsorption. e It is 100.65 mg / g, and the adsorption rate A is 86.67%.

[0058] e) After adsorption equilibrium, the resin is recovered by filtration or centrifugation, and the aqueous phase after static adsorption of the macroporous resin is collected; the macroporous resin material obtained by filtration or centrifugation is rinsed with distilled water to remove surface proteins and pigments, and then the water is absorbed with filter paper, and 80% ethanol solution is added as eluent according to a solid-liquid ratio of 1:5 (w / v), and the resin is sealed and placed in a constant temperature shaker at 150r / min, and desorbed by shaking at room temperature to reach desorption equilibrium, and then allowed to settle; the desorption amount and desorption rate are calculated according to the following formula:

[0059]

[0060] Where: Q d is the desorption amount, mg / g; D is the desorption rate, %; C d is the concentration of polyphenols in the desorption solution, mg / mL; C0 is the initial polyphenol concentration in the intermediate aqueous phase II, mg / mL; C e is the polyphenol concentration at adsorption equilibrium, mg / mL; V d is the volume of desorption liquid, mL; V i is the volume of the intermediate layer aqueous phase II, mL; W is the wet weight of the macroporous adsorption resin, g.

[0061] In the static desorption process, the static desorption curve is established with the desorption time as the horizontal axis and the desorption amount as the vertical axis, such as Figure 4 As shown in the figure, it can be seen that the desorption amount of the active ingredients of camphor tree seed kernel by HJ-18 macroporous resin increases rapidly within the first hour, and the desorption equilibrium can be reached after 2 hours of desorption. d It is 96.42 mg / g, and the desorption rate D is 95.80%.

[0062] f) After static desorption equilibrium, the eluent is filtered or centrifuged to recover the eluent, and the eluent is subjected to reduced pressure rotary distillation to recover the ethanol at a rotary distillation temperature of 40° C. After removing the eluent, the eluent is vacuum freeze-dried to obtain the active ingredients of the camphor tree seed kernel.

[0063] Example 2

[0064] The present invention provides a method for separating and purifying plant active ingredients in camphor tree seed kernels, comprising the following steps:

[0065] a) picking fresh and mature camphor seeds from the Qingshan Lake Campus of Nanchang University, removing the peel and pulp of the camphor seeds, drying at (40±1)° C. to constant weight, shelling and color sorting to obtain camphor seed kernel entities;

[0066] b) mixing fresh camphor tree seed kernels with water at a solid-liquid ratio of 1:5, and subjecting the mixture to ultrafine grinding by wet method of a colloid mill for 4 min to obtain slurry I, and subjecting the mixture to centrifugal separation at a centrifugal speed of 6000 rpm for 15 min to obtain an upper emulsified phase I, an intermediate aqueous phase I, and a lower slag phase I; subjecting the intermediate aqueous phase I and the lower slag phase I to ultrafine grinding by wet method of a colloid mill for 4 min to obtain slurry II, and subjecting the mixture to centrifugal separation at a centrifugal speed of 6000 rpm for 15 min to obtain an upper emulsified phase II, an intermediate aqueous phase II, and a lower slag phase II; collecting the intermediate aqueous phase II and refrigerating it for later use;

[0067] c) Pre-treat HJ-18 macroporous resin: the specific surface area of ​​macroporous resin is ≥ 650m 2 / g, particle size is 0.30-1.25mm; soak the macroporous resin in 95% ethanol of 4 times the volume of the resin, wait for the resin to swell fully, swell for 12 hours, rinse the resin with distilled water until there is no alcohol smell, then soak it in 1M HCl solution for 6 hours, rinse the resin with purified water until it is neutral, soak it in 1M NaOH solution for 6 hours, rinse the resin with purified water until it is neutral, and then get the treated macroporous resin, soak the resin in purified water for standby use;

[0068] d) The pretreated macroporous resin is wet loaded into a chromatography column with a size of Φ35 mm×600 mm, a column volume (BV) of 500 mL, and a height-to-diameter ratio of 15:1. After balancing with distilled water, 5 times the column volume of the intermediate layer aqueous phase II is passed through the chromatography column at a flow rate of 1.5 BV / h and the effluent is collected;

[0069] e) Dynamic desorption: First, use 3-5 BV of distilled water to flow through the resin packing at a flow rate of 3.0 BV / h to remove proteins and pigments. 280nm When the concentration is 0, 5 to 10 BV of 80% ethanol solution eluent is passed through the resin packing at a flow rate of 3.0 BV / h;

[0070] f) After the dynamic desorption is completed, the eluent is recovered and distilled under reduced pressure at a temperature of 45° C. After removing the eluent, vacuum freeze drying is performed to obtain the enriched camphor tree seed kernel plant active ingredients.

[0071] Example 3

[0072] A plant active ingredient extract from camphor tree seed kernels, wherein the plant active ingredients from camphor tree seed kernels are enriched to obtain the plant active ingredients from camphor tree seed kernels according to the separation and purification method for the plant active ingredients from camphor tree seed kernels of Example 2.

[0073] Example 4

[0074] A protein in camphor tree seed kernels, the aqueous phase after macroporous resin static adsorption collected in step e) of the method for separating and purifying plant active ingredients in camphor tree seed kernels in embodiment 1 or the effluent collected in step d) of the method for separating and purifying plant active ingredients in camphor tree seed kernels in embodiment 2, after drying, obtains a protein product.

[0075] Analysis of separation and purification effects

[0076] (I) HPLC analysis of active ingredients in camphor tree seed kernels before and after macroporous resin enrichment and purification

[0077] Sample processing: The plant active ingredients of camphor tree seed kernel before enrichment and purification by macroporous resin (4 mg / mL) and the plant active ingredients of camphor tree seed kernel after enrichment and purification by macroporous resin (2 mg / mL) were filtered through a 0.22 μm filter membrane, and an appropriate amount of the filtrate was taken for HPLC analysis.

[0078] Liquid chromatography conditions: Agilent 1260 high performance liquid chromatograph equipped with a UV detector and a Sepax C-18 reverse column (250 mm × 4.6 mm, 5.0 μm) was used for detection. The column temperature was 25 ° C and the flow rate was 1.0 mL / min. The mobile phase used a combination of two solvents: phase A (water containing 0.1% (v / v) formic acid) and phase B (acetonitrile). The gradient conditions of the mobile phase were: 0-8 min, 5-10% B; 8-11 min, 10-20% B; 11-28 min, 20-35% B; 28-38 min, 35-50% B; 38-58 min, 50-70% B; 58-60 min, 70-5% B. The injection volume was 5 μL, and the UV detection wavelength was 280 nm.

[0079] like Figure 7 and 8 As shown in the figure, it can be seen that compared with the HPLC chromatogram before macroporous resin adsorption ( Figure 7 ), HPLC chromatogram after macroporous resin adsorption ( Figure 8) was significantly increased, and the number of impurity peaks was significantly reduced, indicating that the macroporous resin has a good enrichment and purification effect on the active ingredients of camphor tree seed kernels.

[0080] (II) Total polyphenol content before and after macroporous resin enrichment and purification of active ingredients in camphor tree seed kernels

[0081] The content of total polyphenols in the intermediate layer aqueous phase II and the camphor tree seed kernel plant active ingredients obtained in Example 2 was determined by the Folin phenol method. The results were as follows: the total polyphenol content in the intermediate layer aqueous phase II was 44.27 mg GAE / g dw, and the total polyphenol content in the camphor tree seed kernel plant active ingredients obtained in Example 2 was 314.54 mg GAE / g dw. The total polyphenol content of the camphor tree seed kernel plant active ingredients obtained in Example 2 was 7.11 times that before enrichment, which indicates that the macroporous resin effectively enriched the plant active ingredients in the intermediate layer aqueous phase II.

[0082] (III) Color of the intermediate aqueous phase II before and after macroporous resin enrichment and purification and protein content of freeze-dried samples

[0083] Fig.10 is the aqueous solution diagram before and after macroporous resin enrichment. Fig.10 It can be seen that after macroporous resin enrichment and purification, the color of the intermediate aqueous phase II was significantly improved. The protein content in the solids of the intermediate aqueous phase II after freeze-drying before and after macroporous resin enrichment and purification was 45.12% and 68.97%, respectively, which shows that the macroporous resin effectively enriched the active substances in the intermediate aqueous phase II and improved the quality of the aqueous phase protein.

[0084] (IV) Reusability of macroporous resin

[0085] The reusability of macroporous resin is of great significance to the regeneration performance and use value of the resin. The macroporous resin recovered after elution can be reused after soaking in 1M NaOH, 1M HCl and distilled water. Fig. 9 The adsorption and desorption performance of HJ-18 resin in 10 adsorption-desorption cycles is shown in Figure 2. Fig. 9 It can be seen that after 10 cycles of adsorption and desorption, the adsorption / desorption rate is still higher than 80%, indicating that the HJ-18 macroporous resin has stable chemical properties and excellent regeneration performance and can be reused after recovery.

[0086] Experimental study on optimization of adsorption conditions of macroporous resin

[0087] 1. Materials and Reagents

[0088] Camphor tree seed kernel: Fresh mature camphor tree fruit was collected from Qingshan Lake Campus of Nanchang University. 95% ethanol. Folin-Ciocalteu reagent. Anhydrous sodium carbonate, sodium nitrite, sodium hydroxide, etc. were analytical grade. Methanol and acetonitrile were chromatographic grade. Experimental water: purified water, which was obtained by the laboratory. HJ-18 macroporous resin was purchased from Tianjin Haoju Resin Technology Co., Ltd.

[0089] 2. Instruments and equipment

[0090] Colloid mill; glass chromatography column; constant temperature water bath shaker; rotary evaporator; freeze dryer; UV spectrophotometer; Agilent 1260 high performance liquid chromatograph.

[0091] 3. Experimental methods and results

[0092] 3.1 Screening of macroporous resin

[0093] Weigh equal amounts of macroporous resins of different types into a conical flask, add equal amounts of aqueous solution, seal and place in a constant temperature shaker at 150r / min, oscillate and adsorb at room temperature for 1 hour, take a sample from the supernatant, and determine the polyphenol content in the aqueous solution; rinse the macroporous resins of different types after sufficient adsorption equilibrium with distilled water until no impurities remain on the surface, dry the water with filter paper, place in a conical flask, add equal amounts of ethanol solution with a volume fraction of 80%, take a sample from the desorption solution, and determine the total polyphenol content. Macroporous resins are screened by comparing the adsorption and desorption capabilities of different types of macroporous resins on plant active ingredients in aqueous solutions.

[0094] Depend on Figure 1 It can be seen that HJ-18 macroporous resin exhibits the best adsorption (62.91%) and desorption (93.87%) performance for the active ingredients of camphor tree seed kernels, so HJ-18 macroporous resin is selected as the best resin material.

[0095] 3.2 Screening of static desorption ethanol solution concentration

[0096] The HJ-18 macroporous resin that has reached sufficient adsorption equilibrium is rinsed with distilled water until no impurities remain on the surface, and the water is absorbed with filter paper, placed in a conical flask, and 50 mL of ethanol solution of different concentrations (20%, 40%, 60%, 80% and 95%) is added. The resin is sealed and placed in a constant temperature shaker at 150 r / min. The resin is shaken and desorbed at room temperature for 1 to 7 hours. Samples are taken from the supernatant to determine the total polyphenol content. The desorption amount and desorption rate are calculated according to the following formula to investigate the effects of eluent concentration and desorption time on the desorption performance of the macroporous resin.

[0097]

[0098] Where: Q dis the desorption amount, mg / g; D is the desorption rate, %; C d is the concentration of polyphenols in the desorption solution, mg / mL; C0 is the initial polyphenol concentration in the intermediate aqueous phase II, mg / mL; C e is the polyphenol concentration at adsorption equilibrium, mg / mL; V d is the volume of desorption liquid, mL; V i is the volume of the intermediate layer aqueous phase II, mL; W is the wet weight of the macroporous adsorption resin, g.

[0099] Depend on Figure 3 It can be seen that when 80% ethanol solution is used as the desorbent, the desorption amount of the active ingredients of camphor tree seed kernel by HJ-18 macroporous resin is the largest, and the desorption amount Q d It is 93.87 mg / g, and the desorption rate D is 93.26%.

[0100] 3.3 Dynamic adsorption sample flow rate and sample volume screening

[0101] Take the pretreated HJ-18 macroporous resin, rinse it with distilled water until the effluent is clear, place it in an appropriate amount of distilled water, wet-pack it, and balance the pressure inside and outside the column with distilled water. Pass the intermediate layer aqueous phase II solution through the chromatography column at a sample flow rate of 0.75, 1.5, 3.0, and 4.5 BV / h, sample it every 0.5 BV, determine the polyphenol content in the effluent, determine the optimal sample flow rate and sample volume, obtain the optimal adsorption conditions, and draw a dynamic adsorption curve.

[0102] Depend on Figure 5 It can be seen that when the adsorption reaches the leakage point, the corresponding loading volumes are 5.68, 4.79, 3.47 and 1.66 BV at loading flow rates of 0.75, 1.5, 3.0 and 4.5 BV / h, respectively. Since the loading volumes at loading flow rates of 0.75 BV / h and 1.5 BV / h are not much different, in order to improve production efficiency, the optimal loading flow rate is determined to be 1.5 BV / h and the loading volume is 4.79 BV.

[0103] 3.4 Dynamic desorption flow rate and desorption liquid volume screening

[0104] After adsorption under the optimal loading conditions, the obtained adsorption saturated HJ-18 resin was subjected to desorption experiment and rinsed with distilled water until the effluent was clear. An 80% ethanol solution was passed through the chromatography column at flow rates of 1.5, 3.0, and 4.5 BV / h, and samples were taken at intervals of 0.5 BV to determine the polyphenol content in the effluent, determine the optimal desorption flow rate and desorption liquid volume, obtain the optimal desorption conditions, and draw a dynamic desorption curve.

[0105] Depend on Figure 6It can be seen that with 80% ethanol solution as the desorbent, the desorption capacity increases as the desorption flow rate decreases, but there is no significant difference in the dynamic desorption curves when the desorption flow rate is 1.5BV / h and 3.0BV / h. Considering that a smaller flow rate will reduce production efficiency and increase production costs, the optimal desorption flow rate is determined to be 3.0BV / h and the desorption liquid volume is 8BV.

[0106] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for separating and purifying plant active ingredients in camphor tree seed kernels, characterized in that: The following steps are involved: Step S1: fresh camphor tree seed kernels are mixed with water, and then ultrafinely ground by a colloid mill to obtain slurry I, and then centrifugally separated to obtain an upper emulsified phase I, a middle aqueous phase I, and a lower slag phase I; The middle layer water phase I and the lower layer slag phase I are again ultrafinely ground by colloid mill wet method to obtain slurry II, and the upper layer emulsified phase II, the middle layer water phase II and the lower layer slag phase II are obtained by centrifugal separation; Collect the middle aqueous phase II and refrigerate for later use; Step S2: using HJ-18 macroporous resin to perform static or dynamic adsorption on the active ingredients of the camphor tree seed kernel in the intermediate layer water phase II, wherein during static adsorption, whether the adsorption equilibrium is reached is determined by the adsorption amount of the polyphenols by the resin; during dynamic adsorption, the intermediate layer water phase II flows through the resin filler at a flow rate of 1.5 BV / h according to 5 to 10 column volumes, so that the resin is fully adsorbed, and the water phase after dynamically flowing through the macroporous resin is collected; Step S3: if static adsorption is adopted in step S2, the resin is recovered by filtration or centrifugation after adsorption equilibrium, and the aqueous phase after static adsorption of the macroporous resin is collected; the resin is statically desorbed by 80% ethanol solution, and the desorption amount of polyphenols in the supernatant is used to determine whether the desorption equilibrium is reached; if dynamic adsorption is adopted in step S2, the adsorption saturated HJ-18 resin is dynamically desorbed by 80% ethanol solution, and the elution flow rate is controlled to be 3.0 BV / h; Step S4: After static desorption equilibrium, the eluate is filtered or centrifuged to recover the eluate, and the eluate is subjected to reduced pressure rotary distillation to recover the ethanol, and after removing the eluent, the eluent is vacuum freeze-dried to obtain the plant active ingredients of the camphor tree seed kernel; after the dynamic desorption is completed, the eluate is recovered, and the eluate is subjected to reduced pressure distillation to remove the eluent, and after vacuum freeze-drying to obtain the enriched plant active ingredients of the camphor tree seed kernel.

2. The method for separating and purifying plant active ingredients in camphor tree seed kernels according to claim 1, characterized in that: In the step S1, the ratio of the fresh camphor tree seed kernel to water is 1:(3-5); the ultrafine grinding time is 4-6 minutes, the centrifugal separation speed is 4000-6000rpm, and the time is 15-30 minutes.

3. The method for separating and purifying plant active ingredients in camphor tree seed kernels according to claim 1, characterized in that: In step S2, the HJ-18 macroporous resin is pretreated before static or dynamic adsorption. The pretreatment method is as follows: the macroporous resin is soaked in 95% ethanol with a volume of 3 to 4 times the volume of the resin, and after the resin is fully swollen for 12 to 24 hours, the resin is rinsed with distilled water until there is no alcohol smell, then soaked in 1M HCl solution for 6 to 12 hours, rinsed with purified water until the resin is neutral, and then soaked in 1M NaOH solution for 6 to 12 hours, and rinsed with purified water until the resin is neutral, so as to obtain the treated macroporous resin, and the resin is soaked in purified water for standby use.

4. The method for separating and purifying plant active ingredients in camphor tree seed kernels according to claim 3, characterized in that: The specific process of static adsorption in step S2 is: adding the macroporous resin into the intermediate layer aqueous phase II, sealing and placing in a constant temperature shaker at 150 r / min, and oscillating and adsorbing at room temperature until adsorption equilibrium.

5. The method for separating and purifying plant active ingredients in camphor tree seed kernels according to claim 4, characterized in that: The adsorption amount in step S2 is calculated according to the following formula: Where: Q e is the adsorption amount, mg / g; C0 is the initial polyphenol concentration in the intermediate aqueous phase II, mg / mL; C e is the polyphenol concentration at adsorption equilibrium, mg / mL; V i is the volume of the intermediate layer aqueous phase II, mL; W is the wet weight of the macroporous adsorption resin, g.

6. The method for separating and purifying plant active ingredients in camphor tree seed kernels according to claim 3, characterized in that: The specific process of dynamic adsorption in step S2 is: the pre-treated macroporous resin is wet loaded into the chromatography column, after being balanced with distilled water, the intermediate layer aqueous phase II is passed through the chromatography column at a flow rate of 1.5 BV / h and the effluent is collected; the specific process of dynamic desorption in step S3 is: firstly, 3 to 5 BV of distilled water is passed through the resin filler at a flow rate of 3.0 BV / h to remove protein and pigment, and the effluent is cooled to 0.1% by volume at OD 280nm When the pH is 0, 5 to 10 BV of 80% ethanol solution eluent is passed through the resin packing at a flow rate of 3.0 BV / h.

7. The method for separating and purifying plant active ingredients in camphor tree seed kernels according to claim 5, characterized in that: The specific process of desorption in step S3 is as follows: the macroporous resin material obtained by filtration or centrifugation is rinsed with distilled water to remove surface proteins and pigments, and then the water is absorbed with filter paper, and 80% ethanol solution is added as an eluent according to a solid-liquid ratio of 1: (5-10) (w / v), and the mixture is sealed and placed in a constant temperature shaker at 150 r / min, and desorbed by shaking at room temperature, and then allowed to stand and settle.

8. The method for separating and purifying plant active ingredients in camphor tree seed kernels according to claim 7, characterized in that: The desorption amount in step S3 is calculated according to the following formula: Where: Q d is the desorption amount, mg / g; C d is the concentration of polyphenols in the desorption solution, mg / mL; V d is the volume of desorption liquid, mL; W is the wet weight of macroporous adsorption resin, g.

9. An extract of plant active ingredients from camphor tree seed kernels, characterized in that: The invention is obtained according to the method for separating and purifying plant active ingredients in camphor tree seed kernels according to any one of claims 1 to 8.

10. A protein in camphor tree seed kernel, characterized in that: The method for separating and purifying plant active ingredients in camphor tree seed kernels according to any one of claims 1 to 8 is prepared by obtaining an aqueous phase after static adsorption by a macroporous resin or an aqueous phase after dynamic flow through a macroporous resin, and then drying it.

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