Preparation method and application of pericarpium citri reticulatae extract with pressure relieving effect

Abstract

The invention discloses a preparation method and application of a pericarpium citri reticulatae extract with a pressure relieving effect. The pericarpium citri reticulatae and the deep eutectic solvent are mixed and then subjected to ultrasonic extraction, macroporous resin adsorption and elution purification are performed, the pericarpium citri reticulatae extract rich in flavonol compounds is obtained, and the extract has a good pressure relieving effect. Wherein the eutectic solvent is prepared from a hydrogen bond donor, a hydrogen bond acceptor and an auxiliary solvent, has the characteristics of low viscosity, good selectivity and strong dissolving capacity, and is suitable for efficiently extracting flavonol compounds, such as kaempferol and other active substances with a pressure relieving function, from the dried orange peel. The preparation method of the pericarpium citri reticulatae extract provided by the invention has the characteristics of simplicity, convenience, low price, low volatility, strong dissolving capacity and the like, and provides more effective methods and ways for extracting more pericarpium citri reticulatae extracts with the effect of relieving pressure.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant product extraction and preparation, and more specifically, relates to a preparation method and application of a tangerine peel extract having a stress-relieving effect. Background Art

[0002] Tangerine peel (Citrus reticulata Blanco) is the dried mature peel of the Rutaceae plant and is a common Chinese medicinal material. Modern pharmacological studies have shown that the active substances in tangerine peel have the traditional effects of regulating qi and strengthening the spleen, drying dampness and resolving phlegm, and also have multiple pharmacological effects such as anti-oxidation, anti-inflammatory, antiviral, antibacterial, cardiovascular protection, digestion promotion, lipid-lowering, sedation and tranquilization. The active ingredients in tangerine peel include flavonoids, organic acids, sugars, vitamins, aromatic oils, etc. Among them, flavonoid compounds are a very important class of active ingredients in tangerine peel, mainly including flavonoids, isoflavones, flavanones, flavonols, etc. For a long time, people have focused on hesperidin and nobiletin as the active ingredients in tangerine peel that relieve stress, because their antioxidant capacity can reduce the accumulation of free radicals in the body, reduce oxidative stress reactions, and inhibit oxidative stress and inflammatory reactions in the nervous system. However, studies have shown that the 3-hydroxyl and 4-hydroxyl groups in flavonol molecules help provide stronger antioxidant capacity. Flavonols are generally more antioxidant than flavonoids and can better break the process of stress response and oxidative stress cycle. However, there are currently few methods specifically for extracting flavonols from tangerine peel. Due to the low content of flavonol compounds in tangerine peel, the application of tangerine peel flavonols with stronger antioxidant capacity in relieving stress and in the preparation of health care and medicinal products is limited.

[0003] The existing methods for extracting the effective components of dried tangerine peel usually adopt: solution extraction method, ultrasonic extraction method, microwave method, etc. Among them, traditional organic solvents such as n-hexane, ethyl acetate, chloroform, acetone, and methanol are widely used due to their high solubility and strong extraction ability. However, these solvents usually have the disadvantages of broad extraction targets, weak pertinence, high toxicity, high volatility, and easy environmental pollution. As a new type of solvent, low eutectic solvents have the advantages of low vapor pressure and can be designed for directional extraction of target objects. The prior art discloses a method for extracting flavonoids from dried tangerine peel based on a low eutectic solvent system, but flavonoids contain multiple subclasses (such as flavones, flavonols, isoflavones, flavanones, etc.), each of which has different structures, polarities and solubility. Therefore, the extraction effect of low eutectic solvent systems on flavonoids is often not universal. Therefore, the proportion of "flavonol compounds" in the extracts of "flavonoid compounds" extracted by the prior art is not high. Since flavonols are easily oxidized by oxygen in the air, traditional solvent extraction methods are difficult to avoid these problems, resulting in a low extraction rate of flavonols, which limits its effectiveness in practical applications. Therefore, it is necessary to develop more new methods to separate complex compound systems and improve the extraction and separation efficiency of flavonols in tangerine peel and the content of active ingredients. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the shortcomings of the existing method for extracting tangerine peel extract rich in flavonol compounds, as well as the disadvantages of low extraction rate and low content of extracted active ingredients, and to provide a method for preparing tangerine peel extract rich in flavonol compounds.

[0005] The invention aims to provide a method for improving the extraction rate of flavonol compounds in tangerine peel extract.

[0006] Another object of the present invention is to provide a tangerine peel extract.

[0007] Another object of the present invention is to provide application of tangerine peel extract.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0009] The present invention provides a method for improving the extraction rate of flavonol compounds in tangerine peel extract, comprising the following steps:

[0010] (1) a hydrogen bond donor, a hydrogen bond acceptor, and an auxiliary solvent are mixed and heated to prepare a low eutectic solvent; the hydrogen bond donor is 4-amino-3-hydroxybutyric acid; the hydrogen bond acceptor is selected from one of methyl pyrrolidone, choline chloride, and betaine; and the auxiliary solvent is selected from one of propylene glycol and glycerol;

[0011] (2) mixing tangerine peel with a low eutectic solvent in a mass ratio of 1:(10-30), performing ultrasonic extraction at an extraction temperature of 40-60° C., and centrifuging after extraction to obtain a tangerine peel crude extract;

[0012] (3) taking a crude tangerine peel extract and purifying it with a macroporous adsorption resin, the obtained extract is a tangerine peel extract rich in flavonoid compounds.

[0013] The invention adopts a specific low eutectic solvent to extract tangerine peel under ultrasonic conditions, and then further purifies the tangerine peel extract rich in flavonol compounds by adsorption and elution with a macroporous resin. The method greatly improves the extraction efficiency and content of flavonol compounds in tangerine peel, the extraction rate of flavonol compounds is 0.14%, the proportion of flavonols in total flavonoids is 8.6%, the total flavonoid content is 15.8 mg / g, the proportion of kaempferol in total flavonoids is 5.28%, and the effect is significantly higher than that of traditional water extraction method and organic solvent extraction method, and the tangerine peel extract rich in flavonol compounds can be quickly extracted and prepared. The low eutectic solvent used in the present invention is more inclined to selectively dissolve and extract flavonol compounds, among which 4-amino-3-hydroxybutyric acid can form strong hydrogen bonds with flavonol compounds containing multiple hydroxyl groups and carbonyl groups (such as quercetin and kaempferol), and has a strong affinity for flavonol compounds; while methyl pyrrolidone and propylene glycol both show strong polarity, and show stronger affinity for flavonol compounds containing multiple hydroxyl groups or carbonyl groups. Propylene glycol can adjust the overall polarity, viscosity and relative volatility of DES, and improve the extraction rate and efficiency. Therefore, the method provided by the present invention can better extract flavonol compounds in tangerine peel, and its extraction efficiency is high. The method provided by the present invention is simple, convenient and efficient to prepare, and the raw materials used are low in price, low in volatility and strong in solubility, which provides more effective methods and approaches for extracting tangerine peel extracts rich in flavonol compounds with stress relief effects.

[0014] Furthermore, the molar ratio of the hydrogen bond donor, the hydrogen bond acceptor and the auxiliary solvent is 1:(1-3):(1-5).

[0015] More preferably, the molar ratio of the hydrogen bond donor, the hydrogen bond acceptor and the auxiliary solvent is 1:1:2.

[0016] Preferably, the auxiliary solvent is propylene glycol, and the hydrogen bond donor is 4-amino-3-hydroxybutyric acid.

[0017] Preferably, the mass ratio of tangerine peel to the low eutectic solvent is 1:(10-20).

[0018] Furthermore, the preparation method of the low eutectic solvent is: mixing the hydrogen bond donor, the hydrogen bond acceptor and the auxiliary solvent, heating and stirring at 50-60° C., and adding 10-30% (v / v) water and mixing after the mixture is uniformly dissolved.

[0019] Preferably, the conditions for ultrasonic extraction are: power 300-500W, time 30-60min.

[0020] Preferably, purification is performed after ultrasonic extraction to increase the concentration of flavonol compounds. The purification can be performed by commonly used methods in the art, such as macroporous adsorption resin, extraction, column chromatography separation, membrane separation, precipitation method, etc.

[0021] More preferably, the macroporous adsorption resin is selected from any one of HPD100 macroporous epoxy resin, NKA-II macroporous epoxy resin, NKA macroporous epoxy resin and HP-20 macroporous epoxy resin, and can be universally replaced.

[0022] Preferably, the adsorption conditions using the macroporous adsorption resin are: time 60-120 min, temperature 30-50° C., and sample solution flow rate 0.5-2.5 mL / min.

[0023] Preferably, the conditions for elution using the macroporous adsorption resin are: the eluent is 70%-90% ethanol solution, the flow rate is 0.5-2.5 mL / min, and the amount of eluent is 100 mL.

[0024] Preferably, the tangerine peel is tangerine peel powder, which is obtained by passing through a 20-60 mesh sieve.

[0025] More preferably, after washing the tangerine peel, blanch it at 75-85°C for 2-3 minutes, quickly take it out and place it in 20-30°C water to cool it, chop it, dry it, and then grind the sample to pass through a 20-60 mesh sieve to obtain it.

[0026] Further preferably, the tangerine peel is selected from high-quality tangerine peel that is free of mildew and rot.

[0027] As the most preferred embodiment, the method comprises the following steps:

[0028] (1) respectively taking a hydrogen bond acceptor, a hydrogen bond donor, and an auxiliary solvent, adding them to a stirring state at 60° C., and after dissolving, adding 20% ​​(v / v) water and mixing to obtain a low eutectic solvent;

[0029] (2) selecting high-quality dried tangerine peel without mildew or rot, washing with clean water to remove mud and foreign matter on the surface of the dried tangerine peel; blanching the washed dried tangerine peel in hot water at 75-85° C. for 2-3 minutes, quickly taking it out and cooling it in water at 20-30° C.; chopping the dried tangerine peel; grinding the sample after drying, and sifting it through a 20-60 mesh sieve to obtain dried tangerine peel powder;

[0030] (3) Mixing tangerine peel powder with a low eutectic solvent and subjecting the mixture to ultrasonic treatment to obtain a tangerine peel crude extract.

[0031] (4) The crude extract of dried tangerine peel is passed through a macroporous resin under the adsorption conditions of 60-120 min, 30-50° C., a sample flow rate of 0.5-2.5 mL / min, and an elution condition of 70%-90% ethanol solution as an eluent, a flow rate of 0.5-2.5 mL / min, an eluent of 100 mL, and a pH of 2-4 to obtain a dried tangerine peel extract rich in flavonol compounds.

[0032] The invention also provides application of the extraction method in increasing the content of flavonol compounds in tangerine peel or in preparing tangerine peel products with high content of flavonol compounds.

[0033] The invention provides a tangerine peel extract rich in flavonol compounds, which is prepared by the method.

[0034] The invention also provides application of the extract in preparing a product for relieving stress.

[0035] In addition, the invention also provides a product for relieving stress, which contains the tangerine peel extract.

[0036] The present invention has the following beneficial effects:

[0037] The method provided by the present invention greatly improves the extraction efficiency and content of flavonol compounds in dried tangerine peel, not only can the total flavonoid content be improved, but also the extraction rate of flavonol compounds can be improved, the proportion of flavonols in total flavonoids and the proportion of kaempferol in total flavonoids, the content of active ingredients in dried tangerine peel is improved, and the effect is significantly higher than the traditional water extraction method, and the dried tangerine peel extract can be quickly extracted and prepared. And through the screening of low eutectic reagents and the optimization of extraction method conditions, a best extraction method for extracting dried tangerine peel is obtained, which can increase the effective active ingredients of dried tangerine peel extract and improve its antioxidant capacity. The method has the characteristics of simple preparation, low price, environmental friendliness, low volatility, strong solubility, high stability and safety, and provides more effective methods and approaches for extracting dried tangerine peel extracts rich in substances with stress relief functions, and provides a certain reference basis for the development of dried tangerine peel stress relief products.

[0038] At the same time, the present invention provides a tangerine peel extract rich in flavonol compounds. Compared with the tangerine peel extract prepared by the traditional extraction method, the tangerine peel extract rich in flavonol compounds has a stronger antioxidant capacity, can better reduce the accumulation of free radicals in the body, break the cycle of oxidative stress and stress response, and may be more effective in balancing neuroinflammation. It can be better used in the preparation of stress-relieving products. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the extraction effect of flavonol compounds in tangerine peel extract.

[0040] Figure 2 These are the extraction results of tangerine peel using different methods.

[0041] Figure 3 The content of flavonol compounds in different tangerine peel extracts. DETAILED DESCRIPTION

[0042] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0043] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0044] Example 1 Preparation of Tangerine Peel Extract

[0045] 1. Preparation of eutectic reagents (NADESs)

[0046] Different hydrogen bond acceptors and hydrogen bond donors were selected, and auxiliary solvents were added (to adjust the overall polarity, viscosity and relative volatility of DES, and improve the extraction rate and efficiency) to prepare NADESs. The specific ratios are shown in Table 1 below. Hydrogen bond acceptors, hydrogen bond donors, and auxiliary solvents were mixed at 60°C and stirred continuously until the liquid was evenly dissolved. Then, 20% (v / v) water was added to the NADESs and mixed evenly to obtain NADESs.

[0047] Table 1 Different eutectic solvent formulations

[0048]

[0049] 2. Pretreatment of macroporous resin

[0050] Soak in 95% ethanol for 12 hours, soak until fully swollen, then wash with water until odorless and the washing liquid is clear. Soak in 2% sodium hydroxide for 5 hours, filter the resin, rinse with distilled water until the washing liquid is neutral, soak in 2% hydrochloric acid for 5 hours, vacuum filter the resin again, rinse with distilled water until the washing liquid is neutral, put the treated resin in a beaker and soak in distilled water for storage.

[0051] 3. Preparation of Tangerine Peel Extract

[0052] (1) taking 40 mesh tangerine peel powder and a deep eutectic solvent in a mass ratio of 1:20, respectively, and performing ultrasonic extraction at an extraction temperature of 60° C., an ultrasonic power of 400 W, and a time of 40 min to obtain a tangerine peel crude extract;

[0053] (2) Take the crude extract of tangerine peel, use HPD100 macroporous epoxy resin, under the adsorption conditions of time 90min, temperature 40°C, sample flow rate 1.5mL / min, elution conditions of eluent 80% ethanol solution, flow rate 1.5mL / min, eluent dosage 100mL, pH=3, and obtain the extract as tangerine peel extract.

[0054] Example 2 Extraction efficiency of flavonol compounds in tangerine peel extract

[0055] The extraction effect of flavonols in the dried tangerine peel extract prepared in Example 1 was determined by high performance liquid chromatography. The dried tangerine peel extract to be tested was diluted 15 times with an acetonitrile aqueous solution, filtered through a 0.22 μm filter membrane, and 0.1% formic acid was added. The acetonitrile was gradually increased from 20% to 75% at a flow rate of 1 mL / min. The contents of flavonols, total flavonoids and kaempferol were detected with an ultraviolet detector at a wavelength of 360 nm, and the extraction rate was calculated.

[0056] Extraction rate Y = m1 / m2×100%

[0057] Where: m1 is the mass of monoterpenoids extracted;

[0058] m2 is the mass of raw materials.

[0059] The results are shown in Table 2 and Figure 1 As shown, it is shown that DESs-1 and DESs-4 groups have better extraction effects, among which the extraction efficiency of flavonol compounds in the dried tangerine peel extract obtained by extracting with 4-amino-3-hydroxybutyric acid, methyl pyrrolidone and propylene glycol as hydrogen bond acceptors, hydrogen bond donors and auxiliary solvents is the highest. Through analysis, it is shown that the low eutectic solvent prepared in Example 1 is more inclined to selectively dissolve and extract flavonol compounds, among which 4-amino-3-hydroxybutyric acid can form strong hydrogen bonds with flavonol compounds containing multiple hydroxyl groups and carbonyl groups (such as quercetin and kaempferol), and has a strong affinity for flavonol compounds; while methyl pyrrolidone and propylene glycol both show strong polarity, and show stronger affinity for flavonol compounds containing multiple hydroxyl groups or carbonyl groups. Propylene glycol as a solvent can increase the solubility of flavonol compounds, and can also enhance the dissolution and enrichment effects during the extraction process by changing the polarity of the solution. Therefore, the method provided in Example 1 can better extract flavonol compounds in tangerine peel, and its extraction efficiency is high.

[0060] Table 2 Extraction effect of flavonol compounds in tangerine peel extract

[0061]

[0062]

[0063] Example 3 Tangerine peel extract rich in flavonol compounds

[0064] 1. Preparation of NADESs: Mix 4-amino-3-hydroxybutyric acid, methylpyrrolidone, and propylene glycol (molar ratio 1:1:2), and stir continuously at 60°C until the liquid is evenly dissolved, then add 20% (v / v) water and mix evenly for later use.

[0065] 2. Pretreatment of macroporous resin: Soak in 95% ethanol for 12 hours, soak until fully swollen, then wash with water until odorless and the washing liquid is clear. Soak in 2% sodium hydroxide for 5 hours, filter the resin, rinse with distilled water until the washing liquid is neutral, soak in 2% hydrochloric acid for 5 hours, vacuum filter the resin again, rinse with distilled water until the washing liquid is neutral, put the treated resin in a beaker and soak in distilled water for storage.

[0066] 3. Extraction: (1) 40 mesh dried tangerine peel powder and a low eutectic solvent were mixed in a mass ratio of 1:15, and ultrasonic extraction was performed at an extraction temperature of 60°C, an ultrasonic power of 400 W, and a time of 40 min to obtain a crude dried tangerine peel extract; (2) The crude dried tangerine peel extract was taken and adsorbed using HPD100 macroporous epoxy resin under adsorption conditions of 90 min, a temperature of 40°C, a sample flow rate of 1.5 mL / min, an eluent of 80% ethanol solution, a flow rate of 1.5 mL / min, an eluent of 100 mL, and a pH of 3 to obtain a dried tangerine peel extract.

[0067] Example 4

[0068] The extraction preparation method is the same as that in Example 3, the only difference is that the molar ratio of 4-amino-3-hydroxybutyric acid, methylpyrrolidone and propylene glycol in the preparation of NADESs is 1:2:2.

[0069] Example 5

[0070] The extraction preparation method is the same as that in Example 3, the only difference being that the extraction temperature is 40°C.

[0071] Example 6

[0072] The extraction preparation method is the same as that in Example 3, the only difference being that the ultrasonic extraction power is 380W.

[0073] Example 7

[0074] The extraction and preparation method is the same as in Example 3, the only difference being that the ultrasonic extraction power is 450W.

[0075] Example 8

[0076] The only difference between the extraction and preparation method and Example 3 is that the ultrasonic extraction time is 45 minutes.

[0077] Example 9

[0078] The extraction and preparation method is the same as in Example 3, the only difference being that the ultrasonic extraction time is 35 min.

[0079] Example 10

[0080] The extraction preparation method is the same as that in Example 3, the only difference being that the macroporous resin is NKA-II type macroporous epoxy resin.

[0081] Embodiment 11

[0082] The extraction preparation method is the same as in Example 3, the only difference being that the extraction temperature is 50°C.

[0083] Example 12

[0084] The extraction preparation method is the same as in Example 3, the only difference being that the extraction temperature is 70°C.

[0085] Comparative Example 1 Traditional water extraction

[0086] Take 10g of tangerine peel powder, add 200mL of distilled water, set the ultrasonic power to 400W, and perform ultrasonic extraction for 40min. After the extraction is completed, the extraction temperature is 50℃, filter, and the filtrate is concentrated to 60mL under reduced pressure, and 60mL of anhydrous ethanol is added, and refrigerated at 4℃ overnight. Take the supernatant and filter, and the filtrate is concentrated under reduced pressure to remove 90% of the solvent. The remaining extract is freeze-dried for 24h, and then dried in a vacuum drying oven to constant weight. The extraction rate is calculated by dividing the dry weight by the feed amount.

[0087] Comparative Example 2 Traditional deep eutectic solvent extraction

[0088] Choline chloride and lactic acid were taken in a molar ratio of 1:4, a low eutectic solvent with a water content of 30% was prepared, and the solid-liquid ratio with tangerine peel powder was 1:100 g / mL, and ultrasonic extraction was performed at 50°C for 30 minutes. The mass ratio of tangerine peel to low eutectic solvent was 1:20, and extraction was performed at an ultrasonic power of 400 W, a time of 40 minutes, and an extraction temperature of 60°C to obtain tangerine peel extract.

[0089] Comparative Example 3: Traditional organic solvent extraction

[0090] Tangerine peel powder and petroleum ether were mixed in a mass ratio of 1:20, and ultrasonic extraction was performed at an extraction temperature of 60°C, a power of 400 W, and a time of 40 min to obtain tangerine peel extract.

[0091] Test Example 1 Effects of Different Methods on Tangerine Peel Extract

[0092] 1. Extraction temperature

[0093] The DESs-1 group eutectic extraction method in Example 1 and the traditional water extraction method in Comparative Example 1 were respectively used to perform ultrasonic extraction (power 400 W, time 40 min) on tangerine peel at a temperature of 60° C., with other method conditions being the same, and then the contents of flavonol compounds and total flavonoids were determined as in Example 1.

[0094] The extraction results of dried tangerine peel by different methods are as follows Figure 2 As shown, the content of flavonol compounds extracted by low eutectic reagent is significantly higher than that extracted by water. Comparing the effects of low eutectic reagent and traditional water extraction on the flavonol compounds extracted from tangerine peel, it was found that at 60°C, the total flavonoid content, flavonol content and the proportion of flavonols in total flavonoids in the extract of tangerine peel extracted by traditional water extraction were significantly lower than the extraction effect of low eutectic reagent method.

[0095] 2. Quality ratio

[0096] By adjusting the mass ratio of tangerine peel to the low eutectic solvent, the extraction effects of the DESs-1 group in Example 1 and Example 3 were compared (the detection method was the same as in Example 1). The results showed that when the mass ratio of tangerine peel powder to the low eutectic solvent used in Example 1 was 1:15-20, the flavonol compound content and extraction rate of the tangerine peel extract obtained were optimal.

[0097] 3. Comparison of different extraction conditions and formulas

[0098] The contents of flavonol compounds and total flavonoids in the tangerine peel extracts prepared in Examples 3-4, 7-12 and Comparative Examples 1-3 were determined, and the results are shown in Tables 3 and 4. Figure 3 As shown, the extraction conditions affect the content of flavonol compounds extracted from tangerine peel, and the proportion of flavonols in total flavonoids is also different, and its effect is better than the traditional extraction method of the comparative example. Optimizing the extraction conditions on the extraction method of Example 1 can further increase the total flavonoid content and flavonol content of tangerine peel extract and the proportion of flavonols in total flavonoids.

[0099] Table 3 Contents of flavonol compounds in different tangerine peel extracts

[0100]

[0101]

[0102] Test Example 2: Scavenging effect of tangerine peel extract on free radicals

[0103] Since oxidative stress can cause mood disorders and sleep disorders, leading to insomnia, the DPPH free radical scavenging ability of the prepared tangerine peel extract was determined. Accurately weigh 4 mg DPPH and dissolve it in methanol to a 100 mL volumetric flask to obtain the DPPH solution. Take 0.1, 0.3, and 0.5 mL of the extract (fill up to 0.5 mL with methanol if the volume is insufficient) and 2 mL of DPPH solution in the same test tube and shake well. Place in the dark for 30 minutes, use the corresponding blank solution as a control, and measure its absorbance at 517 nm. The DPPH free radical inhibition rate is calculated as follows:

[0104] Inhibition rate % = [A0-(A b -A * )] / A0*100%

[0105] Where: A0 is the absorbance after reaction without adding sample inhibitor;

[0106] A b is the absorbance after adding sample inhibitor;

[0107] A * is the absorbance without adding inhibitor and DPPH.

[0108] The measurement results are shown in Table 4, which show that the scavenging rates of the tangerine peel extracts of Examples 3-4 and 6-12 on DPPH free radicals are significantly higher than those of Comparative Examples 1-3 of the traditional extraction method, indicating that the tangerine peel extracts prepared by the present invention have good antioxidant effects; among them, the tangerine peel extract obtained by Example 3 has the most obvious free radical scavenging effect.

[0109] Table 4 Scavenging effect of tangerine peel extract on free radicals

[0110]

[0111]

[0112] Test Example 3: Stress Relief Test

[0113] 1. Animal feeding and medication

[0114] C57BL / 6 male Kunming mice weighing 18-20 g were selected and randomly divided into the following groups: healthy control group, model control group, and tangerine peel extract group, with 10 mice in each group.

[0115] All four groups were fed with standard diets. After 14 days of feeding, except for the healthy control group, the other groups of mice were subjected to chronic stress modeling. The method was modified from the CUMS chronic stress modeling method. Seven stimulations were given seven days a week, and the stimulation methods were not repeated on two consecutive days. The stimulation methods were: S1: cold water swimming (4℃, 5min); S2: tail clamping (60s); S3: fasting and water deprivation (24h); S4: exposure to light (24h); S5: room temperature swimming (23℃, 15min); S6: tilted cage (30℃); S7: wet bedding (100mL).

[0116] The sucrose preference and locomotor activity were measured on the 1st and 29th days of modeling, and a successful model was defined when there were significant differences in sucrose preference and locomotor activity between the model group and the healthy control group.

[0117] The healthy control group and the model control group were gavaged with 10 mL / kg·d normal saline solution; the tangerine peel extract group was gavaged with 54.56 mg / kg·d tangerine peel extract (Example 3, Comparative Example 2) in normal saline solution. The mice in each group were gavaged for 4 consecutive weeks. During the experiment, the mice were free to drink water and eat. The food intake was recorded every day and the body weight was recorded once a week. After the last gavage, the mice's sucrose preference, spontaneous activity and behavioral indicators were measured.

[0118] 2. Determination of sucrose preference

[0119] Humans and animals will show characteristics such as anhedonia under long-term stress, and sucrose intake will decrease. The sucrose preference experiment is a classic experiment to detect the degree of pleasure. The preference experiment consists of two parts, namely the adaptation training part and the test part. After two weeks of adaptive feeding, sucrose water adaptation training was carried out 72 hours before the establishment of the UCMS model. During the training, two bottles of 1% (w / v) sucrose solution were placed in each cage of mice in the first 24 hours, and one of the bottles was replaced with pure distilled water in the next 24 hours. After the adaptation, fasting and water deprivation for 24 hours, and a 12-hour baseline test began. In the test, mice can only choose two weighed bottles, one bottle of 1% (w / v) sucrose solution and the other bottle of pure water. After fasting for 12 hours, the two bottles were taken away and weighed, and the total liquid consumption, sugar water consumption, pure water consumption, and sugar water preference index of the animals were calculated. The tests and comparisons were carried out on the first day and the 29th day, respectively.

[0120] Sugar water preference index % = sugar water consumption / (sugar water consumption + pure water consumption) × 100%.

[0121] The measurement results are shown in Table 5. It can be seen that compared with the model group, the percentage of sucrose consumption of mice in the tangerine peel extract group under stress on the 29th day increased, indicating that the tangerine peel extract can effectively increase the pleasure of mice to relieve stress, and the tangerine peel extract of Example 1 had a better improvement than that of Comparative Example 2 on the 29th day.

[0122] Table 5 Sucrose preference index

[0123]

[0124] 3. Forced swimming test

[0125] Under normal circumstances, mice will struggle to escape when swimming due to their avoidance of water. However, under long-term stress, mice will show despair and frustration, which is specifically manifested by reduced swimming behavior and struggle, and a floating and immobile state. The forced swimming test (FST) is a classic experiment that reflects the degree of despair of mice under stress. On the 28th day after the establishment of the model, mice were placed in a cylindrical beaker filled with warm water, with a diameter of 14 cm, a height of 20 cm, a water depth of 12 cm, and a water temperature of 25±1°C. The mice were forced to swim, and the camera system was used to record the animal's immobility latency within 6 minutes and the duration of the immobility within the next 4 minutes. The immobility state refers to the animal giving up active struggle and the body is in a floating and non-twisting state. In the last 4 minutes, two operators recorded the duration of the immobility state when the mouse could not see the operator.

[0126] The results are shown in Table 6. It can be seen that compared with the model group, the immobility time of mice in the tangerine peel extract group during swimming under stress was shortened, indicating that the tangerine peel extract can relieve stress by reducing the sense of despair in mice, while the immobility time of the tangerine peel extract of Example 1 was lower than that of Comparative Example 2.

[0127] Table 6 Immobility time of mice in forced swimming test

[0128] C57 mice Immobility time (s) Blank Group 66.8±6.9 Model Group 118.9±10.9 Comparative Example 2 Tangerine Peel Extract Group 86.4±.5.1 Example 1 Tangerine peel extract group 69.6±.8.2

[0129] 4. Tail suspension test

[0130] The tail suspension test (TST) is a classic method that can quickly evaluate the anti-stress effect. The principle is to use the fact that mice attempt to escape after being suspended by their tails but cannot escape, so they give up struggling and enter a unique state of immobility. During the experiment, the immobility time of the animals is recorded to reflect the stress state. Anti-stress drugs can significantly shorten and change their state. On the 28th day after the modeling began, the operator used non-stick adhesive to suspend the tip of the mouse's tail 2 cm in the tail suspension box, so that its head was facing the bottom of the box 50 cm above the ground. The camera system was used to record the animal's immobility latency within 6 minutes and the immobility within the next 4 minutes. The immobility state means that the animal gives up active struggle and the body is in a suspended and non-twisting state. In the last 4 minutes, two operators recorded the duration of the immobility state when the mouse could not see the operator.

[0131] The results are shown in Table 7. It can be seen that compared with the model group, the immobility time of the mice in the tangerine peel extract group under stress was shortened, indicating that this extract can relieve stress by reducing the sense of despair in mice, while the immobility time of the tangerine peel extract of Example 1 was lower than that of Comparative Example 2.

[0132] Table 7 Immobility time of tail-suspended mice

[0133] C57 mice Immobility time (s) Blank Group 52.7±2.6 Model Group 137.3±5.4 Comparative Example 2 Tangerine Peel Extract Group 87±2.5 Example 1 Tangerine peel extract group 60.8±6.9

[0134] 5. Morris water maze test

[0135] On days 30-35, a six-day Morris water maze experiment was conducted, which was divided into a positioning cruise experiment and a space exploration experiment:

[0136] (1) Positioning cruise experiment: The pool is theoretically divided into four quadrants, and the platform is placed in the center of one of the quadrants. The model animal is placed in the water with its head facing the pool wall, and the placement position is randomly selected from one of the four starting positions of east, west, south, and north. Record the time (s) for the animal to find the underwater platform. In the first few training sessions, if this time exceeds 60s, guide the animal to the platform. Allow the animal to stay on the platform for 10s. After each session, remove the animal and wipe it dry. Each mouse is trained 4 times a day, with an interval of 15-20 minutes between two training sessions, and the training is continuous for 5 days. The time from entering the water to climbing onto the platform is recorded as the training latency.

[0137] (2) Spatial exploration experiment: On the second day after the last spatial exploration experiment, the platform was removed and a 60-second spatial exploration experiment was started. The animals were placed in the water from the opposite side of the original platform quadrant. The activities of the mice in the water maze within 60 seconds were recorded with a camera system, and statistical analysis was performed using the Noldus behavioral analysis system. The latency of learning to find the platform, the number of platform crossings, and the time spent by the animals in the target quadrant (the quadrant where the platform was originally placed) and the number of times they entered the quadrant were recorded to determine the memory storage and retrieval and reproduction capabilities of the mice, which was used as a detection indicator of spatial memory. At the same time, the average movement speed of the mice was recorded, from which individual differences in the swimming speed of the mice can be seen. There is also a certain relationship between the latency and swimming speed of the animals.

[0138] The results are shown in Tables 8 and 9. It can be seen that compared with the model group, the latency time of the spatial exploration experiment of the mice in the tangerine peel extract group was significantly shortened. As time went on, the shortening time of the tangerine peel extract in Example 1 was better than that in Comparative Example 2. Under stress, the number of times the mice entered the platform and the proportion of the platform quadrants in the Morris water maze increased, indicating that the tangerine peel extract can relieve stress by improving memory ability. Combined with the above behavioral experiments, it is shown that the tangerine peel extract in Example 1 is better than that in Comparative Example 2.

[0139] Table 8 Latency time of spatial exploration experiment (s)

[0140]

[0141] Table 9 Positioning cruise experimental data

[0142]

[0143] 6. Determination of neurotransmitter levels

[0144] When humans and animals are under long-term stress, they will experience anxiety, depression and other psychological states. Studies have shown that these psychological changes are closely related to neurotransmitters such as 5-HT (5-hydroxytryptamine) and norepinephrine in the brain and blood. After behavioral assessment, mice were killed. Blood was collected from the mouse eyeballs, and the hippocampus and prefrontal cortex were dissected. Brain tissue homogenate (1:10 w / v) was prepared in cold saline in an ice bath. The tissue homogenate was centrifuged at 4000 rpm and 4°C for 10 minutes, and the supernatant was collected; the blood was centrifuged at 3500 rpm and 4°C for 15 minutes to obtain serum. ELISA kits were used to detect the levels of 5-hydroxytryptamine and norepinephrine in serum, hippocampus and cortex.

[0145] The measurement results are shown in Table 10. It can be seen that compared with the model group, the concentrations of 5-HT and NE in the serum, hippocampus and cortex of mice in the tangerine peel extract group of Example 1 under stress state increased significantly, and the effect was better than that of Comparative Example 2, indicating that the tangerine peel extract prepared by the present invention can relieve stress by improving physiological indicators such as neurotransmitter concentrations in the blood and brain of mice.

[0146] Table 10 5-HT and NE content in brain and serum

[0147]

[0148] In summary, the present invention studies and obtains an optimal extraction method for improving the extraction rate of flavonol compounds in tangerine peel extract by screening natural low eutectic reagents and optimizing the conditions of the extraction method. The content of flavonol compounds extracted and the extraction rate are higher than those of the traditional water extraction method and the traditional organic solvent extraction method. The present invention uses a specific natural low eutectic reagent with a strong affinity for flavonol compounds, which can better extract and prepare tangerine peel extracts with high flavonol compound content, improve the extraction efficiency of flavonol compounds in tangerine peel, and further enhance the stress relief effect of tangerine peel extracts, providing a certain reference basis for the development of tangerine peel stress relief products.

[0149] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for improving the extraction rate of flavonol compounds in tangerine peel extract, characterized in that: The following steps are involved: (1) a hydrogen bond donor, a hydrogen bond acceptor, and an auxiliary solvent are mixed and heated to prepare a low eutectic solvent; the hydrogen bond donor is 4-amino-3-hydroxybutyric acid; the hydrogen bond acceptor is selected from one of methyl pyrrolidone, choline chloride, and betaine; and the auxiliary solvent is selected from one of propylene glycol and glycerol; (2) mixing tangerine peel with a low eutectic solvent in a mass ratio of 1:(10-30), performing ultrasonic extraction at an extraction temperature of 40-60° C., and centrifuging after extraction to obtain a crude tangerine peel extract; (3) taking a crude extract of tangerine peel and purifying it with a macroporous adsorption resin, and the extract obtained after elution is the tangerine peel extract.

2. The method according to claim 1, characterized in that: The molar ratio of the hydrogen bond donor, the hydrogen bond acceptor and the auxiliary solvent is 1:(1-3):(1-5).

3. The method according to claim 1, characterized in that: The mass ratio of the tangerine peel to the low eutectic solvent is 1:(10-20).

4. The method according to claim 1, characterized in that: The preparation method of the low eutectic solvent is: mixing a hydrogen bond donor, a hydrogen bond acceptor and an auxiliary solvent, heating and stirring at 50-60° C., and adding 10-30% water and mixing after the mixture is evenly dissolved.

5. The method according to claim 1, characterized in that: The conditions of the ultrasonic extraction are: power 300-500W, time 30-60min.

6. The method according to claim 1, characterized in that: The macroporous adsorption resin is selected from one of HPD100 macroporous epoxy resin, NKA-II macroporous epoxy resin, NKA macroporous epoxy resin and HP-20 macroporous epoxy resin.

7. The method according to claim 1, characterized in that: The adsorption conditions of the macroporous adsorption resin are as follows: time 60-120 min, temperature 30-50° C., and sample solution flow rate 0.5-2.5 mL / min.

8. The method according to claim 1, characterized in that: The conditions for elution using the macroporous adsorption resin are as follows: the eluent is 70-90% ethanol solution, the flow rate is 0.5-2.5 mL / min, and the amount of the eluent is 100 mL.

9. A tangerine peel extract, characterized in that Prepared by the method according to any one of claims 1 to 8.

10. Use of the extract according to claim 9 in preparing a product for relieving stress.

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