A preparation method of a aronia oleracea fruit composition and its application in preparing an oral preparation for preventing nodules

Abstract

The present invention provides a method for preparing a composition of Aronia nigra fruit and its application in preparing an oral preparation for preventing nodules. First, matrine and coumaric acid are subjected to ultrasonic treatment, vacuum evaporation and drying to obtain a matrine-coumaric acid low eutectic solvent, then components including Aronia nigra fruit, dandelion root, burdock root, rose and auxiliary medicinal materials are pretreated to obtain plant raw material powder, the matrine-coumaric acid low eutectic solvent is diluted with water to obtain an extractant, which is mixed with the plant raw material powder, and then high-voltage pulse electric field extraction is performed to obtain the Aronia nigra fruit composition. The Aronia nigra fruit composition obtained by the method has a high content of active ingredients such as flavonoids, and the oral solution prepared using the Aronia nigra fruit composition as a raw material has the effect of preventing nodules.

Description

Technical Field

[0001] The invention relates to the technical field of composition preparation, and in particular to a method for preparing a aronia melanocarpa fruit composition and application of the composition in preparing an oral preparation for preventing nodules. Background Art

[0002] Thyroid nodules are lesions caused by abnormal growth of thyroid cells. The main treatment is surgical removal, but patients need to take thyroid hormones for a long time after removal. Traditional Chinese medicine treats thyroid nodules mainly through syndrome differentiation and internal administration of Chinese medicine and external treatment of Chinese medicine, such as using anti-tumor external application of Chinese medicine bags for auxiliary treatment, or combining acupuncture, moxibustion, scraping, cupping, etc., to achieve the effects of soothing the liver and regulating qi, promoting blood circulation and removing blood stasis, strengthening the spleen and resolving phlegm, and softening and dispersing nodules.

[0003] The fruit of Aronia nigra is rich in polysaccharides, anthocyanins, flavonoids, proanthocyanidins, phenolic acids and other active ingredients, and has high food and medicinal value; Dandelion is bitter, sweet and cold in nature, and has the effects of clearing away heat and detoxifying, reducing swelling and dispersing nodules, and promoting diuresis and relieving stranguria; Burdock root is bitter, cold and non-toxic, and can be used to treat wind-toxic carbuncle and heat-toxic swelling; and rose is rich in isoflavones, polyphenols and various trace elements, and has the effects of strengthening the liver and stomach, relieving depression and calming the nerves; the combined extraction of Aronia nigra, dandelion root, burdock root and rose can be made into an oral preparation to achieve the effects of promoting blood circulation, removing blood stasis, softening and dispersing nodules.

[0004] For the extraction of plant compositions, choosing a suitable extraction process is critical for the extraction of active ingredients. Although the traditional water extraction method is simple to operate, it faces the problems of low extraction efficiency and high impurity content; the organic solvent extraction method can improve efficiency, but the solvent used may have safety and environmental protection issues, and additional separation and detection steps may be required in subsequent processing, which increases costs. In addition, the traditional heating and stirring and water bath extraction methods still have the problems of low extraction efficiency, time-consuming and energy-consuming. The plant extract obtained in the end has poor stability, the active substances are easy to decompose, and the efficacy is not strong, which is difficult to meet market demand. Therefore, it is necessary to develop a green, efficient, and low-cost extraction process to ensure the extraction rate of active ingredients in black fruit glandular rib aronia fruit, dandelion root, burdock root, and rose flower, as well as the stability and efficacy of subsequent products. Summary of the invention

[0005] The purpose of the present invention is to provide a preparation method of a aronia oleracea fruit composition and its application in the preparation of an oral preparation for preventing nodules. An aqueous solution diluted with a matrine-coumaric acid low eutectic solvent is used as an extractant, and a pulsed electric field enhancement process is adopted to perform green and efficient extraction of plant raw materials. The finally prepared oral preparation has a good effect of soothing thyroid nodules.

[0006] The first aspect of the present invention provides a method for preparing a composition of Aronia nigra fruit, comprising the following steps:

[0007] S1: adding matrine and coumaric acid to a solvent to dissolve, and then performing ultrasonic treatment to obtain a first mixed solution; performing vacuum evaporation treatment on the first mixed solution until no distillate is evaporated to obtain a second mixed solution; drying the second mixed solution to a constant weight to obtain a matrine-coumaric acid low eutectic solvent; the matrine-coumaric acid low eutectic solvent is composed of matrine molecules and coumaric acid molecules, the carbonyl group in the matrine molecule and the hydroxyl group in the coumaric acid molecule have hydrogen bond interaction, and the distance between the amino group in the matrine molecule and the hydroxyl group in the coumaric acid molecule is 0.8Å-2Å;

[0008] S2: Pre-treating the components including Aronia nigra fruit, dandelion root, burdock root, rose flower and auxiliary medicinal materials to obtain plant raw material powder, wherein the auxiliary medicinal materials include at least three of Sophora japonica flower, Cordyceps militaris, Cassia seed, orange peel, Poria cocos, Licorice root, Pueraria root and jujube;

[0009] S3: diluting the matrine-coumaric acid low eutectic solvent with water to obtain an extractant, mixing the plant raw material powder with the extractant and performing high-voltage pulse electric field extraction to obtain an Aronia nigromaculata fruit composition.

[0010] Deep Eutectic Solvents (DES) are a type of mixture formed by hydrogen bond donors (HBD) and hydrogen bond acceptors (HBA) through hydrogen bonding. Compared with traditional organic solvents, deep eutectic solvents have many advantages such as high designability, green sustainability, low-cost synthesis, low toxicity, and high stability, and can achieve selective extraction of plant active ingredients.

[0011] Matrine is an active ingredient extracted from the Chinese medicine Sophora flavescens, which has a wide range of pharmacological effects, including anti-tumor, anti-inflammatory, analgesic, anti-fibrosis, antiviral, anti-arrhythmic, improving immune function, etc., and can slow down or prevent the growth of nodules by affecting multiple cell signaling pathways, reducing the production of inflammatory mediators and inhibiting the fibrosis process. Coumaric acid is a natural compound present in a variety of plants, with multiple pharmacological effects, including anti-oxidation, anti-inflammatory, immunomodulation, anti-tumor, anti-platelet aggregation, cardiovascular protection, prevention and improvement of diabetes and neuroprotection, etc. Its anti-inflammatory and antioxidant properties can reduce the inflammatory response and oxidative stress in the pathological process, thereby helping to prevent the development of thyroid nodules. The present invention uses an aqueous solution of a matrine-coumaric acid low eutectic solvent as an extractant for plant raw materials, which can improve the extraction efficiency of active ingredients in plant raw materials on the one hand, and on the other hand, the pharmacological effects of matrine and coumaric acid themselves can be used to coordinate plant active ingredients to better play the effects of anti-oxidation, anti-inflammatory, immunomodulation, etc., effectively slowing down or preventing the growth of thyroid nodules.

[0012] As a further improvement of the above scheme, in the process of synthesizing the matrine-coumaric acid deep eutectic solvent in step S1, the molar ratio of matrine to coumaric acid is 1:(0.4-5), preferably 1:(0.5-3). When the molar ratio is 1:1, the obtained matrine-coumaric acid deep eutectic solvent has a higher extraction efficiency after dilution. The synthesis of the matrine-coumaric acid deep eutectic solvent is preferably carried out in a protective gas atmosphere, which refers to an anti-oxidative protective atmosphere, such as an atmosphere formed by one or more inert gases such as helium, argon, nitrogen, and carbon dioxide.

[0013] It should be noted that, in the present invention, "molar ratio" means "ratio of the amount of a substance".

[0014] As a further improvement of the above scheme, the solvent in step S1 is at least one of ultrapure water, methanol, ethanol, n-butanol, and chloroform, wherein ultrapure water is selected as the solvent to not only ensure the synthesis of the low eutectic solvent, but also reduce environmental pollution and improve the safety of the production process. There is no special requirement for the amount of the solvent, as long as it can dissolve and mix matrine and coumaric acid.

[0015] As a further improvement of the above scheme, the ultrasonic treatment time in step S1 is preferably 15 min-12 h, the temperature is preferably 30° C.-70° C., and the power is preferably 100 W-800 W.

[0016] As a further improvement of the above scheme, the vacuum evaporation treatment temperature in step S1 is preferably 40° C.-75° C., and the pressure is preferably minus 0.04 MPa-minus 0.09 MPa.

[0017] As a further improvement of the above scheme, the drying in step S1 uses a vacuum constant temperature drying oven, and the temperature cannot be too high, preferably between 40°C and 70°C.

[0018] As a further improvement of the above scheme, the concentration of the extractant in step S1 is 2wt%-50wt%, preferably 10wt%-40wt%. When the concentration of the extractant is 30wt%, a better extraction effect is achieved.

[0019] As a further improvement of the above scheme, the mixed solid-liquid ratio of the plant raw material powder and the extractant in step S3 is 1g:(20-60mL), the high-voltage pulse electric field treatment time is 2min-60min, the electric field strength is 5kV / cm-60kV / cm, and the number of pulses is 4-20.

[0020] As a further improvement of the above scheme, the extractant and the plant raw material powder in step S3 are extracted by high-voltage pulse electric field and then purified by centrifugal filtration. The centrifugal filtration treatment is carried out according to a conventional method, preferably with a centrifugal speed of 4000r / min-12000r / min, a centrifugal time of 5min-20min, and preferably a 0.45um organic phase filter membrane.

[0021] The second aspect of the present invention provides an Aronia melanocarpa fruit composition obtained by the preparation method described in the first aspect.

[0022] The third aspect of the present invention provides the use of the Aronia nigra berry composition described in the second aspect in the preparation of an oral preparation for preventing nodules, comprising using the Aronia nigra berry composition as a raw material to prepare an oral solution, powder or tablet for preventing nodules, and the oral preparation can be a food, a health product or a medicine.

[0023] It is understandable that in order to increase the content of active ingredients in the oral preparation, the same plant raw material powder can be extracted multiple times, or the same extracting solution can be used to extract multiple batches of plant raw materials, and then the multiple extracts are mixed with auxiliary ingredients to obtain an oral preparation. Among them, the oral preparation made from the multiple extracts of Aronia nigra fruit obtained by extracting 3-8 batches of plant raw material powder using the same extracting solution has a good effect in preventing nodules.

[0024] As a further improvement of the above scheme, the oral preparation is used to prevent thyroid nodules.

[0025] As a further improvement of the above scheme, the oral solution is prepared by the following method: according to weight, 50-90 parts of the black fruit glandular rib fruit composition, 1-10 parts of maltodextrin, 1-5 parts of trehalose, 1-3 parts of sweeteners, 1-3 parts of flavoring agents, and 1-3 parts of food thickeners are taken and stirred for 30-90 minutes, and then filled into bottles sterilized at high temperature, and sterilized again after filling, so as to obtain an oral solution for preventing nodules;

[0026] The sweetener is at least one of xylitol, sorbitol, mannitol, maltitol and sodium cyclamate;

[0027] The flavoring agent is at least one of malic acid, taurine, propolis, potassium carbonate, glycyrrhizic acid and stevioside;

[0028] The food thickener is at least one of gum arabic, agar, sodium alginate, carrageenan, sodium carboxymethyl cellulose and sodium carboxymethyl starch.

[0029] As a further improvement of the above scheme, the oral powder is prepared by the following method: the oral solution for preventing nodules is heated and passed through a spray dryer, the spray pressure of the spray dryer is 10kPa-20kPa, the air volume is 0.4m 3 / min-1.5m 3 / min, the solution flow rate is 50mL / h-300mL / h, the inlet temperature is 85℃-115℃, and the outlet temperature is 75℃-95℃ to obtain an oral powder for preventing nodules.

[0030] As a further improvement of the above scheme, the oral tablets are prepared by the following method: magnesium stearate is added to the oral powder for preventing nodules, and the mixture is stirred for 10-30 minutes and then compressed using a tablet press to obtain oral tablets for preventing nodules, wherein the tablet press equipment parameters are as follows: filling depth is 75% of the mold volume, pre-compression pressure is 10-30 MPa, main compression pressure is 50-200 MPa, holding time is 5-30 s, and demolding pressure is 5-20 MPa.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) The present invention uses matrine and coumaric acid to form a matrine-coumaric acid low eutectic solvent, and then uses its water dilution as an extractant to extract plant raw materials. On the one hand, the extraction efficiency of active ingredients in plant raw materials can be improved. The flavonoid extraction efficiency is 2.1 times that of water extraction and more than 1.6 times that of ethanol extraction. On the other hand, the pharmacological effects of matrine and coumaric acid themselves can synergize the plant active ingredients to better exert antioxidant, anti-inflammatory, immunomodulatory and other effects, and effectively slow down or prevent the growth of thyroid nodules. In addition, the matrine-coumaric acid low eutectic solvent has a high solubility and good reusability for flavonoid compounds. It still does not reach saturation after five extractions, which is beneficial to saving resources.

[0033] (2) High-voltage pulsed electric field is selected for the extraction of plant raw material components. Compared with the traditional water bath heating and stirring method, it has the advantages of short time and high efficiency. The effect of 15 minutes of extraction is far better than the effect of 2 hours of extraction by the traditional water bath heating and stirring method, and is more suitable for large-scale production;

[0034] (3) Matrine-coumaric acid low eutectic solvent selectively extracts anthocyanins when extracting flavonoids. The content of anthocyanins in the extract accounts for a relatively high proportion. Practice and quantum chemical calculations have also verified that matrine-coumaric acid low eutectic solvent has better specific recognition ability for anthocyanins.

[0035] (4) The oral solution prepared by the matrine-coumaric acid low eutectic solvent extraction process has better antioxidant effect than the oral solution prepared by the traditional process, and has better therapeutic effects in relieving nodular goiter, regulating the level of thyroid function indicators in serum, inhibiting the proliferation of thyroid cells and promoting thyroid cell apoptosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] 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 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.

[0037] Figure 1 The quantum chemical characterization diagram of the matrine-coumaric acid deep eutectic solvent prepared in Example 1 of the present invention, wherein (a) is a structural optimization diagram of the matrine-coumaric acid deep eutectic solvent, (b) is an ESP potential analysis diagram of the matrine-coumaric acid deep eutectic solvent, and (c) is an IRI analysis diagram of the matrine-coumaric acid deep eutectic solvent;

[0038] Figure 2The quantum chemical characterization diagrams of the matrine-coumaric acid deep eutectic solvent and the active substance anthocyanin prepared in Example 1 of the present invention, wherein (a) is a structure optimization diagram, (b) is an ESP potential analysis diagram, and (c) is an IRI analysis diagram;

[0039] Figure 3 The present invention is about the influence of different extractants and re-extraction times on the flavonoids concentration in the re-extracts of Aronia nigra fruit;

[0040] Figure 4 is the DPPH free radical scavenging rate of different Pumei root oral liquids obtained in the present invention;

[0041] Figure 5 ABTS of different Pumei root oral liquids obtained in the present invention + Free radical scavenging rate. DETAILED DESCRIPTION

[0042] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0043] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass described in the embodiment description of the present application can be a mass unit known in the chemical industry such as μg, mg, g, kg, etc.

[0044] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0045] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0046] Matrine: 98% analytical grade, purchased from Shanghai MacLean Biochemical Technology Co., Ltd.;

[0047] Coumaric acid: 97% analytical grade, purchased from Shanghai MacLean Biochemical Technology Co., Ltd.;

[0048] Aronia nigra fruit: purchased from Jixiangyuan Ginseng and Antler Specialty Products Distribution Center in Qitamu Town, Jiutai District;

[0049] Dandelion root and rose flower: both purchased from Bozhou Fushuotang Biotechnology Co., Ltd.;

[0050] Burdock root: purchased from Anhui Kangweifu Pharmaceutical Co., Ltd.;

[0051] Sophora japonica flower, Poria cocos, and Licorice root were all purchased from Bozhou Yuanshengtang Pharmaceutical Co., Ltd.

[0052] Example 1

[0053] S1: In a nitrogen protective gas atmosphere, matrine and coumaric acid are added to ultrapure water at a molar ratio of 1:1 to dissolve, stirred at room temperature for 3 hours, and then reacted under ultrasonic conditions of 45°C and 500W for 8 hours to obtain a first mixed solution of matrine and coumaric acid; under vacuum conditions of 55°C and -0.06MPa, the first mixed solution is vacuum evaporated until no distillate is evaporated to obtain a second mixed solution; the second mixed solution is placed in a vacuum constant temperature drying oven and dried at 55°C until the weight is constant to obtain a matrine coumaric acid low eutectic solvent;

[0054] S2: According to weight, 30 parts of Aronia nigra fruits, 15 parts of dandelion roots, 15 parts of burdock roots, 25 parts of rose flowers, 5 parts of Sophora japonica flowers, 5 parts of Poria cocos and 5 parts of Licorice were crushed, passed through an 80-mesh sieve, and dried in a constant temperature drying oven at 40°C for 12 hours to obtain plant raw material powder after pretreatment;

[0055] S3: Dilute the matrine-coumaric acid low eutectic solvent with ultrapure water to obtain an extractant with a concentration of 5wt%, mix the plant raw material powder and the extractant at a solid-liquid ratio of 1g:40mL, and then perform high-voltage pulsed electric field assisted extraction, the extraction time is 15min, the electric field strength is 20kV / cm, and the number of pulses is 10. After the extraction, centrifugal filtration is performed at a centrifugal speed of 10000r / min and a centrifugal time of 10min. The supernatant is filtered with a 0.45um organic phase filter membrane to remove impurities, and a black fruit glandular rib aronia fruit composition is obtained.

[0056] For example, Figure 1 These are the quantum chemical characterization diagrams of the matrine-coumaric acid low eutectic solvent, where (a) is the structure optimization diagram, (b) is the ESP potential analysis diagram, and (c) is the IRI analysis diagram.

[0057] like Figure 1 As shown in (a), the geometry optimization and frequency calculation of the matrine-coumaric acid low eutectic solvent have converged, and no imaginary frequency was found, indicating that the current structure is at the local minimum point of the potential energy surface and can exist stably. The hydroxyl group on the carboxyl group of coumaric acid interacts with the carbonyl group of matrine through hydrogen bonding, and the distance between the hydroxyl group and the carbonyl group is 1.67121Å, which is much shorter than the van der Waals radius.

[0058] like Figure 1 As shown in (b), the formation mechanism of the matrine-coumaric acid eutectic solvent was qualitatively analyzed by ESP, and the depth of color represents different electric potentials. For matrine, the carbonyl region is electronegative, and a large area surrounding the nitrogen atom is electronegative; for coumaric acid, the hydroxyl region is electronegative and the carbonyl region is electronegative. After the formation of the eutectic solvent, the electronegative region of matrine and the electronegative region of coumaric acid attract each other to form a stable eutectic solvent.

[0059] The interaction region indicator function (IRI) is used to characterize the van der Waals force, hydrogen bond, and steric repulsion of molecules, such as Figure 1 As shown in (c), different color blocks represent different intermolecular forces. The small discs in the ring structure represent steric hindrance. The stronger the steric hindrance, the more difficult it is for the atoms in the corresponding two regions to attract each other. The small discs outside the ring structure represent interactions such as attraction, steric hindrance, or strong attraction, such as hydrogen bonds; the large block-shaped transitional areas represent weak van der Waals interactions. There are large block-shaped interaction force areas between matrine and coumaric acid, indicating that there is a van der Waals force between them, and there are small discs between the hydroxyl and carbonyl groups. According to existing research, it can be fully demonstrated that there is a strong hydrogen bond interaction force between these two sites.

[0060] In Examples 2-7, the feed ratio of matrine to coumaric acid in step S1 and the concentration of the extractant in step S3 were changed as shown in Table 1, and the other conditions were the same as in Example 1, to obtain Aronia nigra fruit compositions L2-L7.

[0061]

[0062] Comparative Example 1

[0063] S1: matrine and coumaric acid are added into purified water at a molar ratio of 1:1 and stirred evenly to obtain a matrine-coumaric acid mixed solution with a total concentration of matrine and coumaric acid of 30 wt%;

[0064] S2-S3: using matrine-coumaric acid mixed solution as an extractant, and the remaining step parameters are the same as those in Example 1 to obtain the Aronia nigra fruit composition DL1.

[0065] Comparative Example 2

[0066] The extractant in step S3 was replaced with ultrapure water, and the remaining step parameters were the same as in Example 1 to prepare the Aronia nigra fruit composition DL2.

[0067] Comparative Examples 3-6

[0068] The extractant in step S3 was replaced with 25wt%, 50wt%, 75wt% ethanol aqueous solution and pure ethanol respectively, and the remaining step parameters were the same as in Example 1 to prepare Aronia melanocarpa fruit compositions DL3-DL6.

[0069] Comparative Example 7

[0070] The coumaric acid in step S1 was replaced by myristic acid, and the remaining step parameters were the same as those in Example 4 to obtain the Aronia melanocarpa fruit composition DL7.

[0071] Comparative Examples 8-10

[0072] The extractant in step S3 was replaced with ultrapure water, the extraction method was replaced by water bath heating and stirring instead of high-voltage pulse electric field, the heating temperature was set to 50°C, the magnetic stirring speed was 600rpm, the extraction time was set to 30min, 60min, and 120min respectively, and the remaining step parameters were the same as in Example 1, to obtain the black fruit glandular rib aronia fruit compositions DL8-DL10.

[0073] Test Example 1

[0074] The Aronia nigra fruit compositions prepared in Examples 1-7 and Comparative Examples 1-10 were tested and calculated according to the following method to obtain the total flavonoids and anthocyanin yields of different extraction processes.

[0075] (1) Total flavonoid yield test method

[0076] Drawing of rutin standard curve: Weigh 21.0 mg of rutin standard accurately, dissolve it in 50% ethanol, and dilute it to a 100 mL volumetric flask to obtain a 0.210 mg / mL rutin standard solution. Pipette 2.0 mL, 2.5 mL, 3.0 mL, 3.5 mL, and 4.5 mL of rutin standard solution into 5 25 mL colorimetric tubes, then add 1.5 mL of 5% NaNO2 solution in turn, shake well, let stand for 5 min, add 1.5 mL of 10% Al(OH)3 solution, shake well and let stand for 5 min, add 15 mL of 10% NaOH solution, let stand for 10 min, and finally add 50% ethanol solution to dilute to 25 mL, and measure the absorbance at 510 nm. Draw a standard curve with absorbance as the ordinate and rutin mass concentration as the abscissa.

[0077] 1.0 mL was transferred from the Aronia nigra fruit composition as the test solution, diluted with 50% ethanol, and fixed to volume in a 10 mL volumetric flask. 1.0 mL of the diluted solution to be tested was transferred to a 25 mL volumetric flask, and 1.5 mL of 5% NaNO2 solution was added in sequence, shaken, and allowed to stand for 5 min; 1.5 mL of 10% Al(OH)3 solution was added, allowed to stand for 5 min; 15 mL of 10% NaOH solution was added, allowed to stand for 10 min, and the absorbance was measured at 510 nm.

[0078] The 50% ethanol solution was diluted to 25 mL and the absorbance was measured at 510 nm. The 50% ethanol solution was used as the blank control solution and the total flavonoid extraction rate was calculated according to Formula I.

[0079] Formula I: .

[0080] Wherein, Y is the yield of active ingredient, in mg / g; C is the concentration of active ingredient, in mg / mL; V is the volume of sample solution, in mL; x is the dilution factor; M is the mass of plant raw material powder, in g.

[0081] (2) Anthocyanin yield test method

[0082] Preparation of standard solution: Take 0.05g of anthocyanin sample, dissolve it in 500mL of distilled water, and dilute to obtain an anthocyanin solution with a concentration of 100 ug / mL; take 20mL, 40mL, 60mL, and 80mL of anthocyanin solution and dilute to 100mL with water to obtain anthocyanin solutions with concentrations of 20 ug / mL, 40 ug / mL, 60 ug / mL, and 80 ug / mL, respectively. Measure the absorbance at 530 nm, and make a standard curve with the concentration of anthocyanin solution as the X-axis and the absorbance as the Y-axis.

[0083] The Aronia nigra fruit composition was diluted 20 times with ultrapure water and then tested on an instrument. The ultraviolet absorption of the diluted solution was measured at 530 nm. The sample concentration was calculated by comparing with the standard curve, and the anthocyanin yield was calculated according to formula I.

[0084] The test results of the total flavonoids yield and anthocyanin yield in the Aronia nigra fruit compositions obtained by the extraction methods of Examples 1-7 and Comparative Examples 1-10 are shown in Table 2.

[0085]

[0086] The results show that with the increase of the extractant concentration, the total flavonoids and anthocyanins first gradually increase and then tend to stabilize or even slightly decrease. This is because with the increase of the extractant concentration, the conductivity of the solvent is enhanced, the number of electroporations increases, the degree of cell wall damage increases, and the number of matrine-coumaric acid low eutectic solvent molecules that interact with the plant raw material powder increases. However, when the concentration of the extractant is too high, the viscosity of the system is large, which affects the mass transfer efficiency, and the cost increases while the yield decreases. Therefore, 30wt% is selected as the optimal extraction concentration.

[0087] It can be seen from the test results of total flavonoids and anthocyanins in Examples 4 and 6-7 that when the molar ratio of matrine to coumaric acid in the deep eutectic solvent is 1:1, the extraction effect is best.

[0088] From the test results of Example 4 and Comparative Example 1, it can be found that the mixed solution of matrine and coumaric acid also has the effect of promoting the extraction of total flavonoids and anthocyanins, but the extraction efficiency can be significantly enhanced after the formation of a deep eutectic solvent. This may be attributed to the fact that after the formation of a deep eutectic solvent, the intermolecular forces between matrine and coumaric acid, such as hydrogen bonds and van der Waals forces, are much stronger than those of the physical blending group, so the cell walls of the plant raw materials can be more effectively destroyed, and the extraction efficiency of total flavonoids and anthocyanins in the plant raw materials can be effectively improved, which has positive significance for the deep development and utilization of plant resources.

[0089] From Comparative Examples 2-6, it can be seen that with the increase of ethanol concentration, the total flavonoids and anthocyanins first increase and then decrease; when 50wt% ethanol is selected for extraction, the yield is the highest. This is because the addition of ethanol changes the polarity of the original system. When the concentration reaches 50wt%, the polarity of the system is most consistent with the polarity of the flavonoid active substances, and the extraction efficiency is the highest.

[0090] It can be seen from the results of Example 4 and Comparative Example 7 that changing the composition of the low eutectic solvent will affect the extraction efficiency. The extraction effect of the matrine-coumaric acid low eutectic solvent dilution is better than that of the matrine-myristic acid low eutectic solvent dilution. This may be because the chain length of myristic acid is longer, which leads to a weakened penetration of the solvent during the extraction process and reduces the extraction efficiency.

[0091] It can be seen from Comparative Examples 2 and 8-10 that the selection of high-voltage pulsed electric field for the extraction of plant raw material powder has the advantages of short-time and high efficiency compared to the traditional water bath heating and stirring method. When the extraction is carried out for 15 minutes, the extraction effect is far better than that of the traditional water bath heating and stirring method for 120 minutes, and is more suitable for large-scale production.

[0092] It can be seen from Example 4 and Comparative Examples 2-6 that the selection of matrine-coumaric acid low eutectic solvent as an extractant can solve the pain point problem of poor extraction efficiency of traditional solvents. Its extraction efficiency is 2.1 times that of water extraction and more than 1.6 times that of ethanol extraction. The use of matrine-coumaric acid low eutectic solvent can reduce production costs and reduce the dependence of Chinese medicinal materials extraction on petrochemical organic solvents.

[0093] By comparing the proportion of anthocyanins in total flavonoids in Examples 1-7, it can be found that the matrine-coumaric acid low eutectic solvent selectively extracts anthocyanins when extracting flavonoid compounds, and the anthocyanic acid content accounts for a relatively high proportion, indicating that the matrine-coumaric acid low eutectic solvent has better specific recognition ability for anthocyanins.

[0094] The possible mechanism of high extraction efficiency of flavonoids including anthocyanins in the matrine-coumaric acid deep eutectic solvent system was studied by quantum chemical calculation. Taking anthocyanins as the calculation model, the force form and strength of the matrine-coumaric acid deep eutectic solvent on it were analyzed. The quantum chemical characterization results of the interaction between the matrine-coumaric acid deep eutectic solvent and anthocyanins are shown in Figure 2. Figure 2 As shown, (a) is the structure optimization diagram, (b) is the ESP potential analysis diagram, and (c) is the IRI analysis diagram.

[0095] Matrine-coumaric acid deep eutectic solvent and optimized structure of anthocyanins Figure 2 As shown in (a), both the geometry optimization and frequency calculation converged, and no imaginary frequency was found, indicating that the current structure is at the local minimum of the potential energy surface and can exist stably. The distance between the carbonyl group of coumaric acid and the hydroxyl group of anthocyanin is much shorter than the van der Waals radius, which indicates that there is a hydrogen bond interaction between the matrine coumaric acid deep eutectic solvent and anthocyanin. Figure 2 As shown in (b), the carbonyl regions of coumaric acid and anthocyanin are electronegative, while the hydroxyl groups are electronegative. When forming a stable structure, the electronegative region of the matrine-coumaric acid deep eutectic solvent and the electronegative region of anthocyanin attract each other, and the overall electrostatic potential is close to 0, thus forming a stable structure. The interaction region indicator function analysis of matrine-coumaric acid deep eutectic solvent and anthocyanin is shown in Figure 2. Figure 2 As shown in (c), there are large-scale interaction regions between the matrine-coumaric acid low eutectic solvent and anthocyanins, indicating that there are van der Waals forces between them. At the same time, there are small discs between the carbonyl group in the matrine-coumaric acid low eutectic solvent and the hydroxyl group of anthocyanins, which means that there are strong hydrogen bond interactions between them at these two sites, which contributes to the formation and stability of the entire system. ESP, structural optimization, and IRI can explain why the anthocyanins and total flavonoids in the matrine-coumaric acid low eutectic solvent system have higher yields.

[0096] The Aronia nigra fruit composition obtained by one extraction was used as an extractant and mixed with the unextracted plant raw material powder, and high-voltage pulsed electric field-assisted extraction was performed to achieve multiple repeated extractions of the plant raw material powder with the same matrine-coumaric acid low eutectic solvent, verifying the reusability of the matrine-coumaric acid low eutectic solvent.

[0097] Example 8

[0098] The Aronia nigra fruit composition L4 prepared in Example 4 was used as an extractant, and the plant raw material powder obtained in step S2 of Example 1 was extracted 1-5 times in the manner of step S3 of Example 1 to complete single extraction of different batches of plant raw material powders with the same extractant, thereby obtaining Aronia nigra fruit extracts L8-1~L8-5.

[0099] Comparative Example 11

[0100] According to the method described in Example 8, the Aronia nigra fruit composition L4 was replaced by the Aronia nigra fruit composition DL1 to obtain Aronia nigra fruit multiple extracts DL11-1 to DL11-5.

[0101] Comparative Example 12

[0102] According to the method described in Example 8, the Aronia nigra fruit composition L4 was replaced by the Aronia nigra fruit composition DL2 to obtain Aronia nigra fruit multiple extracts DL12-1 to DL12-5.

[0103] The concentration of flavonoids in the aronia oleracea fruit extracts L8-1 to L8-5, DL11-1 to DL11-5 and DL12-1 to DL12-5 obtained in Example 8, Comparative Example 11 and Comparative Example 12 was detected. The results are as follows: Figure 3 shown.

[0104] The results showed that the flavonoids concentration in the multiple extracts of Aronia nigra fruit remained basically stable after repeated extraction with ultrapure water for two times, indicating that the solubility of ultrapure water was limited and saturation was reached after two repeated extractions; although the matrine-coumaric acid mixed solution extractant was not saturated after four repeated extractions, the increase in flavonoids in the multiple extracts of Aronia nigra fruit decreased significantly with the increase in the number of extractions; while the matrine-coumaric acid low eutectic solvent extractant was still not saturated after four more extractions, and the flavonoids concentration in the multiple extracts of Aronia nigra fruit increased steadily with the increase in the number of extractions, indicating that the matrine-coumaric acid low eutectic solvent has high solubility for the active ingredients in plant raw materials and has good reusability as a plant extractant.

[0105] Example 9

[0106] 83 parts of the black fruit glandular rib aronia fruit complex extract L8-5 obtained in Example 8 were taken by weight and stirred with 5 parts of maltodextrin, 5 parts of trehalose, 2 parts of mannitol, 2 parts of taurine and 3 parts of sodium alginate for 60 minutes, and then filled in bottles sterilized by high temperature. After filling, the bottles were sterilized again to obtain Pumei root oral liquid L9.

[0107] Comparative Example 13

[0108] The Aronia nigra fruit extract L8-5 in Example 9 was replaced by the Aronia nigra fruit extract DL11-5 obtained in Comparative Example 11, and the Pumei root oral liquid DL13 was obtained according to the method described in Example 9.

[0109] Comparative Example 14

[0110] The Aronia nigra fruit composition L8-5 in Example 9 was replaced with the Aronia nigra fruit complex extract DL12-5 obtained in Comparative Example 12, and the Pumei root oral liquid DL14 was obtained according to the method described in Example 9.

[0111] Example 10

[0112] The Pumei root oral liquid L9 obtained in Example 9 was heated and passed through a spray dryer with a spray pressure of 16 kPa and an air volume of 0.68 m 3 / min, the solution flow rate was 150mL / h, the inlet temperature was 102℃, and the outlet temperature was 90℃ to form the Pu rose root powder L10.

[0113] Embodiment 11

[0114] Add 3 wt% of magnesium stearate to the Pumei root powder L10, stir for 20 minutes and then use a tablet press to compress the tablets. The tablet press equipment parameters are: filling depth of 75% of the mold volume, pre-compression pressure of 20 MPa, main compression pressure of 120 MPa, holding time of 15 s, and demolding pressure of 10 MPa to obtain Pumei root tablets L11.

[0115] Test Example 2

[0116] Passed DPPH free radical scavenging test and ABTS + The free radical scavenging test was conducted to investigate the antioxidant properties of the oral preparation obtained by the preparation method of the present invention.

[0117] 1. DPPH free radical scavenging test

[0118] Pumeigen Oral Liquid L9, Pumeigen Oral Liquid DL13 and Pumeigen Oral Liquid DL14 were tested for DPPH free radical scavenging according to the following methods:

[0119] Set up sample tubes (T), sample background (T0), DPPH tubes (C) and solvent background (C0). The sample tube (T) of Pumei root oral liquid needs to be set up with 3 parallel tubes. Use water as the diluent to prepare Pumei root oral liquid of different concentrations (0.125mg / ml, 0.25mg / ml, 0.5mg / ml, 1mg / ml, 2mg / ml).

[0120] For example, taking 1 mg / ml Pumei root oral solution as an example, the following sample tube (T), sample background (T0), DPPH tube (C) and solvent background (C0) are set:

[0121] Sample tube (T): 1 mL of Pumei root oral solution (concentration 1 mg / mL) + 2 mL of water + 1 mL of DPPH ethanol solution (concentration 0.04 mg / mL);

[0122] Sample background tube (T0): 1 mL of Pumei root oral solution (concentration 1 mg / mL) + 2 mL of water + 1 mL of 95 vol% ethanol solution;

[0123] DPPH tube (C): 3 mL of water + 1 mL of DPPH ethanol solution (concentration 0.04 mg / mL);

[0124] Solvent background tube (C0): 3 mL water + 1 mL 95% vol ethanol solution;

[0125] The solutions in the above reaction tubes were transferred into cuvettes, and the absorbance was measured at 517 nm. The DPPH free radical scavenging rates of different Pumei root oral solutions were calculated by the following formula II. The specific results are as follows: Figure 4 shown.

[0126] Formula II: .

[0127] from Figure 4 It can be seen that the Pumeigen oral liquid L9 and Pumeigen oral liquid DL13 prepared by the present invention have good scavenging effects on DPPH free radicals, while the DPPH free radical scavenging effect of Pumeigen oral liquid DL14 is poor, among which the scavenging effect of Pumeigen oral liquid L9 is the best.

[0128] 2. ABTS + Free Radical Scavenging Test

[0129] Pumeigen oral liquid L9, Pumeigen oral liquid DL13 and Pumeigen oral liquid DL14 were subjected to ABTS according to the following method + Free Radical Scavenging Test:

[0130] Set up the sample tube (A S )、Sample background(A b), sample blank tube (A0), sample tubes of each tested concentration of each sample (A S ) need to set up 3 parallel tubes, use water as diluent, and prepare different concentrations of Pu Mei root oral solution (0.0625mg / ml, 0.125mg / ml, 0.25mg / ml, 0.5mg / ml, 1mg / ml). At the same time, the sample blank tube (A0) also needs to set up 3 parallel tubes. S ) and sample background (A b ) were added with 0.2 mL of sample solution of the same concentration, and 0.2 mL of PBS buffer was added to the blank sample tube (A0). S ) and the sample blank tube (A0) were added with 0.8 mL ABTS + Working solution, sample background (A b ) Add 0.8 mL PBS buffer. Incubate in dark for 6 min, transfer the solution in each reaction tube into a 1 cm cuvette, measure the absorbance at 734 nm, and calculate ABTS according to formula III. + Free radical scavenging rate, the results are as follows Figure 5 shown.

[0131] Formula III: .

[0132] The results showed that the Pumei root oral liquid L9 and Pumei root oral liquid DL13 prepared by the present invention had an effect on ABTS + It has a good scavenging effect on free radicals. The ABTS of Pumeigen oral liquid DL14 + The free radical scavenging effect was poor, among which Pumeigen oral liquid L9 had poor free radical scavenging effect on ABTS + It has the best free radical scavenging effect.

[0133] Test Example 3

[0134] The effect of the oral preparation obtained by the preparation method of the present invention on the thyroid mass, functional indicators, and thyroid cell proliferation and apoptosis in rats with nodular goiter model was investigated.

[0135] 1. Experimental Methods

[0136] (1) Modeling and grouping

[0137] Sixty SPF grade 7-8 week old male rats were selected and housed in an SPF grade animal room, with fluorescent lighting, 12h light and dark cycle, free access to water and food, temperature 20-26℃, air supply 6-10 times / h, humidity 40%-70%. After all rats were adaptively fed for 5 days, 10 rats were randomly selected as the control group, and the remaining rats were used to construct the nodular goiter model.

[0138] The model was established by intragastric administration of 0.1% PTU solution (prepared with saline) with normal saline at a dose of 1 mL / kg for 8 consecutive weeks. After the model was established, all rats were found to have irregular changes on both sides of the thyroid gland by color Doppler ultrasound, showing multiple nodules of different sizes and different echo intensities of the nodules, which indicated that the model was successful. The rats with successful modeling were randomly divided into the model group, the levothyroxine sodium tablet group, the Pumeigen oral liquid L9 group, the Pumeigen oral liquid DL13 group and the Pumeigen oral liquid DL14 group, with 10 rats in each group.

[0139] (2) Experimental drug administration

[0140] From the 9th week onwards, each group was gavaged with different drugs at 10 mL / kg body weight. The rats in the levothyroxine sodium tablets group were gavaged with 1.5 mg / L levothyroxine sodium tablets suspension aqueous solution; the rats in the control group and the model group were gavaged with double distilled water; the Pumeigen oral liquid L9 group, the Pumeigen oral liquid DL13 group, and the Pumeigen oral liquid DL14 group were gavaged with the corresponding dose concentration of 100 g / L Pumeigen oral liquid (the dilution solvent was water) for 6 consecutive weeks. The number of mice in each group and the dosage are shown in Table 3.

[0141]

[0142] (3) Collection of materials

[0143] After continuous oral administration for 6 weeks, rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital 3 mL / kg. After successful anesthesia, blood was collected from the abdominal aorta. After the collected blood was allowed to stand for 2 hours, the speed was 3000 r / min, the centrifugal radius was 10 cm, and the low-temperature centrifugation was performed for 10 minutes. The serum was collected in an EP tube and stored in a -80℃ refrigerator for later use. After blood was collected, the rats were fixed on a rat platform, the neck skin was cut, and the layers were separated to completely expose the trachea. Red oval tissue was visible on both sides of the trachea, which was the thyroid gland. After complete separation, the surrounding tissues were removed, rinsed with cold saline, and dried with filter paper. The thyroid gland was weighed on a balance and the relative mass of the thyroid gland was calculated. Relative mass of the thyroid gland = thyroid gland mass / rat body mass. A part of the left thyroid gland of each rat was taken and immersed in 4% paraformaldehyde buffer for making tissue sections; the remaining thyroid tissue was placed in a -80℃ refrigerator for Western blotting detection.

[0144] (4) Observation indicators

[0145] Enzyme-linked immunosorbent assay (ELISA) was used to detect serum TSH, FT3, and FT4 levels: serum TSH, FT3, and FT4 levels were detected strictly according to the instructions of the ELISA kit.

[0146] Apoptosis of rat thyroid tissue cells: After dewaxing, the tissue sections were stained with TUNEL according to the TUNEL kit method. Observed under a fluorescence microscope, the excitation wavelength was 450-500 nm, and the emission wavelength was 515-565 nm. The apoptotic cells stimulated green fluorescence, which was observed and photographed in time. After scanning and photographing the whole slide, the average optical density value was calculated using Image-Pro Plus 7.0 software.

[0147] Western blotting was used to detect the expression level of VEGF protein: total protein was extracted from thyroid tissue, and the total protein concentration was measured by BCA assay kit. After separation by 10% SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis, equal amounts of protein were transferred to PVDF membranes, blocked with 5% bovine serum albumin (BSA), and then GAPDH and VEGF antibodies (both at a volume dilution ratio of 1:1000) were added to react overnight at 4°C; washed 3 times with PBST, incubated with corresponding goat anti-rabbit IgG secondary antibodies (1:5000) at room temperature for 60 min, washed 3 times with PBST, and the gray value was measured after color development.

[0148] 2. Experimental Results

[0149] (1) Thyroid mass and relative mass

[0150] The thyroid mass and relative mass of rats in each group are shown in Table 4. Compared with the control group, the thyroid mass and relative mass of rats in the model group were significantly increased; compared with the model group, the thyroid mass and relative mass of rats in the levothyroxine sodium tablets group and the Pumeigen oral liquid group were significantly reduced, among which the relative mass of the thyroid of rats in the Pumeigen oral liquid L9 group decreased the most.

[0151]

[0152] (2) Rat serum thyroid function indicators

[0153] The contents of serum thyroid function indexes of rats in each group are shown in Table 5. Compared with the control group, the serum TSH level of rats in the model group was significantly increased, and the levels of FT3 and FT4 were significantly decreased; compared with the model group, the serum TSH level of rats in the levothyroxine sodium tablets group and the Pumeigen oral liquid group was significantly decreased, and the levels of FT3 and FT4 were significantly increased. The serum TSH level of sick mice taking Pumeigen oral liquid DL13 was closest to that of the control group, but the improvement effect of FT3 and FT4 levels was poor. Overall, the Pumeigen oral liquid L9 group had the best improvement effect on the three indicators, indicating that Pumeigen oral liquid L9 has the best effect on improving the thyroid function of rats with nodular thyroid.

[0154]

[0155] (3) Apoptosis of thyroid tissue cells in rats

[0156] The apoptosis of thyroid cells in rats in each group is shown in Table 6. The results of TUNEL staining showed that compared with the control group, the average optical density of thyroid tissue in the model group was significantly reduced; compared with the model group, the average optical density of thyroid tissue in the three groups of rats taking Pumeigen oral liquid was significantly increased, among which the Pumeigen oral liquid L9 group had the highest value, indicating that compared with Pumeigen oral liquid DL13 and DL14, Pumeigen oral liquid L9 can more effectively promote thyroid cell apoptosis in rats with nodular goiter.

[0157]

[0158] (4) VEGF protein expression in thyroid tissue

[0159] The expression of VEGF protein in the thyroid tissue of rats in each group is shown in Table 7. The results showed that compared with the control group, the expression of VEGF protein in the thyroid tissue of rats in the model group was increased; compared with the model group, the expression of VEGF protein in the thyroid tissue of rats in the levothyroxine sodium tablets group and the Pumeigen oral liquid group was decreased, among which the expression of VEGF protein in the Pumeigen oral liquid L9 group was the lowest.

[0160]

[0161] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A method for preparing a composition of Aronia nigra fruit, characterized in that: The following steps are involved: S1: adding matrine and coumaric acid to a solvent to dissolve, and then performing ultrasonic treatment to obtain a first mixed solution; performing vacuum evaporation treatment on the first mixed solution until no distillate is evaporated to obtain a second mixed solution; drying the second mixed solution to a constant weight to obtain a matrine-coumaric acid low eutectic solvent; the matrine-coumaric acid low eutectic solvent is composed of matrine molecules and coumaric acid molecules, the carbonyl group in the matrine molecule and the hydroxyl group in the coumaric acid molecule have hydrogen bond interaction, and the distance between the amino group in the matrine molecule and the hydroxyl group in the coumaric acid molecule is 0.8Å-2Å; S2: Pre-treating the components including Aronia nigra fruit, dandelion root, burdock root, rose flower and auxiliary medicinal materials to obtain plant raw material powder, wherein the auxiliary medicinal materials include at least three of Sophora japonica flower, Cordyceps militaris, Cassia seed, orange peel, Poria cocos, Licorice root, Pueraria root and jujube; S3: diluting the matrine-coumaric acid low eutectic solvent with water to obtain an extractant, mixing the plant raw material powder with the extractant and performing high-voltage pulse electric field extraction to obtain an Aronia nigromaculata fruit composition.

2. The preparation method according to claim 1, characterized in that: In step S1, the molar ratio of matrine to coumaric acid is 1:(0.4-5), and the solvent is at least one of ultrapure water, methanol, ethanol, n-butanol, and chloroform.

3. The preparation method according to claim 1, characterized in that: The ultrasonic treatment time in step S1 is 15min-12h, the temperature is 30°C-70°C, and the power is 100W-800W.

4. The preparation method according to claim 1, characterized in that: The vacuum evaporation treatment temperature in step S1 is 40°C-75°C, and the pressure is -0.04MPa--0.09MPa; the drying uses a vacuum constant temperature drying oven at a temperature of 40°C-70°C.

5. The preparation method according to claim 1, characterized in that: The components of the plant raw material powder in step S2 include 10wt%-30wt% of Aronia nigra fruit, 10wt%-20wt% of dandelion root, 10wt%-20wt% of burdock root, 10wt%-30wt% of rose, 5% Sophora japonica flower, 5% Poria cocos and 5% Licorice.

6. The preparation method according to claim 1, characterized in that: The concentration of the extractant in step S3 is 2wt%-50wt%.

7. The preparation method according to claim 1, characterized in that: The solid-liquid ratio of the plant raw material powder and the extractant in step S3 is 1g:(20-60mL).

8. The preparation method according to claim 1, characterized in that: The high-voltage pulse electric field treatment time in step S3 is 2 min-60 min, the electric field strength is 5 kV / cm-60 kV / cm, and the number of pulses is 4-20.

9. Use of the Aronia nigra fruit composition obtained by the preparation method according to any one of claims 1 to 8 in the preparation of an oral preparation for preventing nodules, characterized in that: The aronia melanocarpa fruit composition is used as a raw material to prepare an oral solution for preventing thyroid nodules.

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