Natural anti-sugar composition, preparation method thereof and application of natural anti-sugar composition in anti-sugar product

Abstract

The invention relates to the technical field of anti-sugar products, in particular to a natural anti-sugar composition, a preparation method of the natural anti-sugar composition and application of the natural anti-sugar composition in the anti-sugar products. The preparation method comprises the following steps: performing freeze drying on rambutan shells, and grinding the rambutan shells into powder to obtain rambutan shell powder; under the condition of heating in a water bath, extracting the rambutan shell powder by using a deep eutectic solvent solution to obtain a rambutan shell crude extracting solution; the eutectic solvent in the rambutan shell crude extract is removed, and a natural anti-sugar composition extract is obtained; and freeze-drying the natural anti-sugar composition extracting solution to obtain the natural anti-sugar composition. According to the preparation method, the rambutan shells are used as raw materials, the price is low, and positive contributions are made to comprehensive utilization of the rambutan shells and increase of the additional value of deep-processed products while the cost of the raw materials is reduced. The prepared natural anti-sugar composition not only has an excellent anti-sugar function, but also has anti-inflammatory, anti-oxidation and anti-depression functions, and the product is a natural extract, is free of additives or other chemical synthetic raw materials, and is high in use safety.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-sugar products, and in particular to a natural anti-sugar composition, a preparation method thereof, and application in anti-sugar products. Background Art

[0002] With the development of social economy and the improvement of people's living standards, diabetes has become a major public health problem facing the world. According to the report of the International Diabetes Federation (IDF), about 462 million adults in the world suffer from diabetes, and it is expected to rise to 700 million by 2045. The prevalence of diabetes in my country is also showing a rapid growth trend. According to the latest epidemiological survey, the prevalence of diabetes in Chinese adults is 10.9%, and the prevalence of prediabetes is 35.7%. Diabetes not only brings serious physical and mental health problems to patients, but also brings a huge burden to social and economic development. The occurrence of diabetes is related to many factors. Unreasonable diet, lack of exercise, obesity, etc. can increase the risk of diabetes. Anti-sugar research has become an important means to prevent and manage diabetes. Compared with anti-sugar drugs, natural anti-sugar substances can be obtained from fruits, vegetables and grains, and have the advantages of good biocompatibility and low side effects. However, its active ingredient content is low, which affects the clinical application and effect of natural anti-sugar substances. Summary of the invention

[0003] In order to solve or partially solve the problems existing in the related art, the present invention provides a natural anti-sugar composition and a preparation method thereof and application in anti-sugar products.

[0004] The present invention provides a method for preparing a natural anti-sugar composition, which is characterized by comprising: Step a), freeze-drying the rambutan husk and grinding it into powder to obtain rambutan husk powder; Step b), extracting the rambutan shell powder using a deep eutectic solvent solution under water bath heating to obtain a rambutan shell crude extract; Step c), removing the low eutectic solvent in the crude extract of rambutan shell to obtain a natural anti-sugar composition extract; Step d), freeze-drying the natural anti-sugar composition extract to obtain the natural anti-sugar composition.

[0005] Furthermore, the freeze-drying temperature in step a) is -70 to -50°C, and the time is 36 to 60 hours; and / or, the freeze-drying temperature in step d) is -70 to -50°C, and the time is 36 to 60 hours.

[0006] Furthermore, the water content of the low eutectic solvent solution is 39-42%; the low eutectic solvent in the low eutectic solvent solution includes: choline chloride and glycerol in a molar ratio of 1:2; or, choline chloride: citric acid in a molar ratio of 1:1.

[0007] Furthermore, in the step b), the water bath heating temperature is 60-80° C., and the time is 30-50 min; the solid-liquid ratio of the rambutan shell powder to the low eutectic solvent is 1 g: (13-18) mL.

[0008] Furthermore, step c) specifically comprises: removing the deep eutectic solvent in the crude extract of rambutan shell by a macroporous resin adsorption method or an organic solvent two-step extraction method to obtain a refined extract of rambutan shell; The organic solvent in the rambutan shell extract is removed to obtain a natural anti-sugar composition extract.

[0009] Furthermore, the macroporous resin adsorption method uses AB-8 macroporous adsorption resin; the loading concentration is 2.0 mg / mL, the loading volume is 500 mL, the loading flow rate is 5.0 mL / min; and the eluent is 70% ethanol.

[0010] Furthermore, the organic solvent two-step extraction method is specifically: Extracting the rambutan shell crude extract once using a first organic solvent, collecting the first organic solvent phase and the DES phase; Performing secondary extraction on the DES phase using a second organic solvent, and collecting the second organic solvent phase; combining the first organic solvent phase and the second organic solvent phase; The first organic solvent is ethyl acetate or acetone, and the second organic solvent is n-butanol.

[0011] The present invention also provides a natural anti-sugar composition, which is prepared according to any one of the methods described above.

[0012] Further, it includes: delphinidin-3-O-glucoside, gallic acid, epigallocatechin, chlorogenic acid, rutin, ellagic acid, isoquercetin, kaempferol, quercetin, proanthocyanidin C1, neohesperidin, zebutin, isoquercetin and corilagin.

[0013] The present invention also provides an application of the natural anti-sugar composition in anti-sugar products.

[0014] The natural anti-sugar composition and preparation method thereof provided by the present invention can have the following beneficial effects: (1) The preparation method uses rambutan shell as raw material, which is low in price. While reducing the cost of raw materials, it also makes a positive contribution to the comprehensive utilization of rambutan shell and increasing the added value of deep-processing products.

[0015] (2) The preparation method adopts a low eutectic solvent extraction method, which is easy to operate, environmentally friendly, and recyclable for reuse, and has a high extraction rate of polyphenols. The extract is rich in natural anti-sugar compounds such as delphinidin-3-O-glucoside, gallic acid, epigallocatechin-3-ol, chlorogenic acid, rutin, ellagic acid, isoquercetin, kaempferol, quercetin, proanthocyanidin C1, neohesperidin, zeaxanthin, isoquercetin, and corilagin. The extract contains many types of natural anti-sugar compounds with high content.

[0016] (3) According to the biological activity test, the natural anti-sugar composition obtained by this method is α -Amylase IC 50 About 4.85~5.89mg / mL, α -Glucosidase IC 50 It is about 77.789~121.987μg / mL, which has good anti-sugar performance. In addition, the natural anti-sugar composition also has good anti-inflammatory and antioxidant properties. Moreover, the isosakuranoside in the natural anti-sugar composition also has anti-depressant properties. Therefore, the natural anti-sugar composition not only has excellent anti-sugar function, but also has anti-inflammatory, antioxidant and anti-depressant functions. The product is a natural extract without additives or other chemical synthetic raw materials, and is highly safe to use.

[0017] It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention in conjunction with the accompanying drawings, wherein like reference numerals generally represent like components throughout the exemplary embodiments of the present invention.

[0019] Figure 1 is a bar graph of polyphenol extraction amounts in crude rambutan shell extracts corresponding to different DES in the embodiments of the present invention; Figure 2 It is the effect of DES water content on the extraction amount of rambutan shell polyphenols in the single factor experiment of the embodiment of the present invention; Figure 3 The effect of the material-liquid ratio on the extraction amount of rambutan shell polyphenols in the single factor experiment of the embodiment of the present invention; Figure 4 The effect of water bath temperature on the extraction amount of rambutan shell polyphenols in the single factor experiment of the embodiment of the present invention; Figure 5 The effect of water bath time on the extraction amount of rambutan shell polyphenols in the single factor experiment of the embodiment of the present invention; Figure 6The effect of the amount of pectinase added on the amount of polyphenols extracted from rambutan shell in the single factor experiment of the embodiment of the present invention; Figure 7 The effect of the amount of cellulase added on the amount of polyphenols extracted from rambutan shell in the single factor experiment of the embodiment of the present invention; Figure 8 The effect of ultrasound time on the extraction amount of rambutan shell polyphenols in the single factor experiment of the embodiment of the present invention; Fig. 9 The effect of ultrasonic power on the extraction amount of rambutan shell polyphenols in the single factor experiment of the embodiment of the present invention; Fig.10 The response surface optimization experiment of the embodiment of the present invention is the effect of the interaction between the water content of the DES solution and the water bath temperature on the extraction amount of rambutan shell polyphenols; Fig.11 The invention relates to the effect of the interaction between the water content of the DES solution and the water bath time on the extraction amount of rambutan shell polyphenols in the response surface optimization experiment of the embodiment of the present invention; Fig.12 The invention discloses the effect of the interaction between water bath time and water bath temperature on the extraction amount of rambutan shell polyphenols in the response surface optimization experiment of the invention embodiment; Fig.13 The invention relates to the effects of different concentrations of ethanol as an eluent on the extraction of rambutan shell polyphenols in the process optimization of the macroporous resin removal method in the embodiment of the present invention; Fig.14 The effects of different desolventizing processes on the recovery rate of rambutan shell polyphenols in the embodiments of the present invention; Fig.15 It is a heat map of related clustering labels of the correlation analysis in the embodiment of the present invention.

[0020] Fig.15 * indicates significant difference at P<0.05, and ** indicates extremely significant difference at P<0.01. DETAILED DESCRIPTION

[0021] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] The terms used in the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The singular forms "a", "the" and "the" used in the present invention and the appended claims are also intended to include plural forms unless the context clearly indicates other meanings. It should also be understood that the term "and / or" used herein refers to and includes any or all possible combinations of one or more associated listed items.

[0023] It should be understood that although the terms "first", "second", "third", etc. may be used to describe various information in the present invention, such information should not be limited to these terms. These terms are only used to distinguish the same type of information from each other. For example, without departing from the scope of the present invention, the first information may also be referred to as the second information, and similarly, the second information may also be referred to as the first information. Thus, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. In the description of the present invention, the meaning of "multiple" is two or more, unless otherwise clearly and specifically defined.

[0024] The accompanying drawings of this application ( Figure 1-9 and Figure 13-14 ) and in the following tables (Tables 1, 6, 8 and 9), the same lowercase letters indicate no significant difference (P>0.05), and different lowercase letters indicate significant difference (P<0.05).

[0025] The embodiment of the present invention provides a method for preparing a natural anti-sugar composition, characterized by comprising: Step a), freeze-drying the rambutan husk and grinding it into powder to obtain rambutan husk powder; Step b), extracting the rambutan shell powder using a deep eutectic solvent solution under water bath heating to obtain a rambutan shell crude extract; Step c), removing the low eutectic solvent in the crude extract of rambutan shell to obtain a natural anti-sugar composition extract; Step d), freeze-drying the natural anti-sugar composition extract to obtain the natural anti-sugar composition.

[0026] The applicant has found that rambutan shells are rich in polyphenols, and many polyphenol compounds show anti-sugar effects, so the applicant first considers using rambutan shells as raw materials. In addition, rambutan shells account for a large proportion of the entire fruit, and using rambutan shells as raw materials to extract natural anti-sugar compositions also achieves waste resource recycling and reduces raw material costs.

[0027] In the above preparation method: Step a) is used to freeze-dry the rambutan husk and grind it into powder to obtain dry, powdered rambutan husk powder. This step is used to remove moisture from the husk to make it into a powder form that is easy to handle and extract. Freeze-drying can better retain the active ingredients in the rambutan husk and avoid the destruction of active substances caused by high-temperature treatment. The powder form increases the surface area in contact with the solvent, which is beneficial to improving the efficiency of subsequent DES extraction. In this step, the freeze-drying temperature is preferably -70~-50℃, and the time is preferably 36~60h. Most preferably, the freeze-drying temperature is -60℃ and the time is 48h; Step b) is a step of extracting the rambutan shell powder using a low eutectic solvent solution to extract the polyphenolic anti-sugar compounds in the shell. The low eutectic solvent DES (Deep Eutectic Solvent) is usually a low eutectic mixture composed of a hydrogen bond acceptor (HBA, such as a quaternary ammonium salt) and a hydrogen bond donor (HBD, such as compounds such as amides, carboxylic acids and polyols) in a certain molar ratio. Compared with traditional organic solvents, the low eutectic solvent DES has lower toxicity, higher solubility and lower cost. The applicant has found through research that compared with traditional water extraction and alcohol extraction methods, the extraction amount of anti-sugar compounds using the DES extraction method is higher. In addition, it is found that DES does not need to be supplemented with other methods, such as ultrasound, enzymatic hydrolysis, etc., to achieve a higher extraction amount. Taking the above factors into consideration, the present application adopts the DES extraction method in the extraction stage.

[0028] The low eutectic solvent solution used in this step is an aqueous solution of a low eutectic solvent. Preferably, the water content of the low eutectic solvent solution is 39-42%, and most preferably 41.3%. The low eutectic solvent includes: choline chloride and glycerol in a molar ratio of 1:2; or choline chloride: citric acid in a molar ratio of 1:1. As a preferred embodiment of this embodiment, the above-mentioned low eutectic solvent solution is prepared according to the following method: The hydrogen bond acceptor choline chloride and the hydrogen bond donor (glycerol or citric acid) are mixed in a preset molar ratio and heated to obtain a uniform, colorless and transparent low eutectic solvent liquid; the low eutectic solvent liquid is diluted with water to obtain a low eutectic solvent solution. The aforementioned heating time is preferably 70-85°C, and the time is preferably 80-120 minutes. Most preferably, the heating time is 80°C and the time is selected to be 80 minutes.

[0029] In step b), the DES extraction is carried out under the condition of water bath heating, which can provide a stable temperature, avoid local overheating, ensure the mildness of the extraction process and the stability of the effective ingredients, effectively extract the polyphenolic anti-sugar substances in the rambutan shell, and improve the purity and content of the extract. In this step, the water bath heating temperature is preferably 60~80℃, and the time is preferably 30~50min; the solid-liquid ratio of rambutan shell powder and low eutectic solvent is preferably 1g: (13~18)mL. Most preferably, the water bath heating temperature is 70℃, the time is 40min; the solid-liquid ratio of rambutan shell powder and low eutectic solvent is 1g: 15mL.

[0030] The above step c) of removing the low eutectic solvent from the crude extract of rambutan shells, on the one hand, helps to obtain a natural anti-sugar composition extract with higher purity; on the other hand, it facilitates the subsequent freeze-drying process. For the specific method of removing the low eutectic solvent, the present application preferably adopts a macroporous resin adsorption method or an organic solvent two-step extraction method. The following further describes the two methods: The macroporous resin adsorption method preferably uses AB-8 macroporous adsorption resin. The resin is preferably activated before use. The activation pretreatment can be: The AB-8 macroporous adsorption resin was activated with anhydrous ethanol for 24 h, and then soaked in 4% hydrochloric acid solution and 4% sodium hydroxide solution for 12 h, and finally washed with ultrapure water until the pH was neutral.

[0031] During the adsorption process, the sample concentration is preferably 2.0 mg / mL, the sample volume is preferably 500 mL, and the sample flow rate is preferably 5.0 mL / min; during the elution process, the eluent is 70% ethanol. The eluent flow rate is preferably 2 mL / min.

[0032] The above-mentioned two-step organic solvent extraction method is preferably: Extracting the rambutan shell crude extract once using a first organic solvent, collecting the first organic solvent phase and the DES phase; Performing secondary extraction on the DES phase using a second organic solvent, and collecting the second organic solvent phase; The first organic solvent phase and the second organic solvent phase are combined.

[0033] Through a two-step extraction method with organic solvents, highly non-polar and highly polar polyphenolic natural anti-sugar substances are selectively separated and recovered by ethyl acetate and n-butanol, respectively, and then the two organic solvents can be recovered by rotary distillation.

[0034] More preferably, the first organic solvent is ethyl acetate or acetone, and the second organic solvent is n-butanol.

[0035] After the low eutectic solvent is removed by the above method, new organic solvents will be added to the extract system, such as ethanol, an eluent introduced by the macroporous resin adsorption method, or two organic solvent extractants introduced by the organic solvent two-step extraction method. Therefore, after the step of removing the low eutectic solvent, it is also necessary to remove the organic solvent from the obtained rambutan shell extract, thereby obtaining a natural anti-sugar composition extract. For example: after using the macroporous resin adsorption method to remove the low eutectic solvent, it is also necessary to remove the eluent (such as ethanol); after the organic solvent two-step extraction method removes the low eutectic solvent, it is also necessary to remove the extractant (such as ethyl acetate, acetone and n-butanol), and finally obtain the natural anti-sugar composition extract.

[0036] Step d) is a step of freeze-drying the natural anti-sugar composition extract, thereby obtaining a solid natural anti-sugar composition that is easy to store and transport. Freeze-drying can remove moisture without destroying the active ingredients, thereby maintaining the original activity of the extract. The obtained solid product is small in size and light in weight, which is easy to store and transport; the stability of the product is improved and is not easily contaminated by microorganisms. In this step, the freeze-drying temperature is preferably -70~-50°C, and the time is preferably 36~60h. Most preferably, the freeze-drying temperature is -60°C and the time is 48h.

[0037] Another embodiment of the present invention further provides a natural anti-sugar composition, which is prepared according to the above method. The specific implementation method is the same as the above embodiment and will not be repeated here. The natural anti-sugar composition contains: delphinidin-3-O-galactoside, delphinidin-3-O-glucoside, delphinidin 3-O-rutinoside, gallic acid, cyanidin-3-O-galactoside, cyanidin-3-O-arabinoside, proanthocyanidin B1, caffeic acid, geraniol, epigallocatechin gallate, chlorogenic acid, proanthocyanidin B3, proanthocyanidin B2, proanthocyanidin B4, proanthocyanidin A2, apigenin, salicylamide, epicatechin, homovanillic acid, rutin, ellagic acid, isoquercetin, kaempferol, quercetin, peony pigment 3-O-glucoside, salicylic acid, proanthocyanidin C1, neohesperidin, quercetin, luteolin, zephyranthesin, isoceranoin, corilagin, dihydromyricetin, dihydroquercetin, etc. Among them, delphinidin-3-O-glucoside, gallic acid, epigallocatechin, chlorogenic acid, rutin, ellagic acid, isoquercetin, kaempferol, quercetin, proanthocyanidin C1, neohesperidin, zephyranthesin, isoceratoside, and corilagin have anti-sugar functions. In addition, it also contains compounds with anti-inflammatory and antioxidant functions. The isoceratoside contained therein also has anti-depressant properties. Therefore, the natural anti-sugar composition not only has excellent anti-sugar function, but also has anti-inflammatory, antioxidant and anti-depressant functions. The product is a natural extract, without additives or other chemical synthetic raw materials, and is highly safe to use.

[0038] From the above content, it can be seen that the natural anti-sugar composition and the preparation method thereof provided by the embodiments of the present invention have the following advantages: (1) The preparation method uses rambutan shell as raw material, which is low in price. While reducing the cost of raw materials, it also makes a positive contribution to the comprehensive utilization of rambutan shell and increasing the added value of deep-processing products.

[0039] (2) The preparation method adopts a low eutectic solvent extraction method, which is easy to operate, environmentally friendly, and recyclable for reuse, and has a high extraction rate of polyphenols. The extract is rich in natural anti-sugar compounds such as delphinidin-3-O-glucoside, gallic acid, epigallocatechin-3-ol, chlorogenic acid, rutin, ellagic acid, isoquercetin, kaempferol, quercetin, proanthocyanidin C1, neohesperidin, zeaxanthin, isoquercetin, and corilagin. The extract contains many types of natural anti-sugar compounds with high content.

[0040] (3) According to the biological activity test, the natural anti-sugar composition obtained by this method is α -Amylase IC 50 About 4.85~5.89mg / mL, α -Glucosidase IC 50 It is about 77.789~121.987μg / mL, which has good anti-sugar performance. In addition, the natural anti-sugar composition also has good anti-inflammatory and antioxidant properties. Moreover, the isosakuranoside in the natural anti-sugar composition also has anti-depressant properties. Therefore, the natural anti-sugar composition not only has excellent anti-sugar function, but also has anti-inflammatory, antioxidant and anti-depressant functions. The product is a natural extract without additives or other chemical synthetic raw materials, and is highly safe to use.

[0041] Another embodiment of the present invention also provides an application of the above-mentioned natural anti-sugar composition in an anti-sugar product. The anti-sugar product can specifically be a health product or a food additive. In addition, the natural anti-sugar composition can also be applied to anti-inflammatory products, antioxidant products or anti-depressant products.

[0042] The technical solution of the present invention is further described below in conjunction with specific embodiments: The sources of raw materials in the following examples and comparative examples are as follows: Rambutan: (purchased from Baoting County, Hainan Province, Baoyan No. 7); Choline chloride: (AR 98%, white granules, Aladdin); DL-lactic acid: (reagent specifications, white transparent liquid, Xilong Scientific); Glucose: (AR, white crystals, Xilong Science); Glycerol: (AR, clear viscous liquid, Xilong Scientific); Malic acid: (AR, colorless crystalline white powder, Guangzhou brand); Citric acid: (AR, colorless crystalline white powder, Guangzhou brand); AB-8 macroporous resin (AR, white particles, Hecheng New Materials); NKA-9 macroporous resin (AR, white particles, Hecheng New Materials); Polyamide powder (AR, white powder, Hecheng new material); D101 (BR, white particles, new materials); DM301 (AR 500g, white granules, new material); n-Butanol (AR, colorless transparent liquid with pungent odor, Xilong Science); Ethyl acetate (AR, colorless transparent liquid with fruity aroma, Xilong Science); Acetone (AR is a colorless, transparent liquid, volatile, flammable, and slightly fragrant. Xilong Science); The instruments used in the following examples and comparative examples are as follows: Ultrasonic machine (UH600, intelligent digital display ultrasonic instrument, Shanghai Ouhe Machinery Equipment Co., Ltd.); Crusher (FWB5, Tianjin Test Co., Ltd.); analytical balance (AL-204, Mettler Toledo); Constant temperature water bath (HH-4, Changzhou Wohua Instrument); Ultra-high performance liquid chromatography (Waters, USA); Constant flow pump (HL-1, Shanghai Qingpu Instrument Factory); Freeze dryer (LGJ-10, Beijing Songyuanxing Technology Development Co., Ltd.); Centrifuge (TDZ5-WS, multi-tube rack centrifuge, Hunan Xiangyi Instrument Development Co., Ltd.) Reciprocating constant temperature oscillator (GHA-S, Xuri Laboratory Instrument Factory, Zhiqian Town, Jintan District, Changzhou) The inventors analyzed the components of rambutan extracts and found that most of the polyphenolic compounds were related to anti-glycemic activity. Therefore, the polyphenol content and / or extraction amount were used as indicators to characterize the anti-glycemic compounds during the extraction stage.

[0043] The rambutan shell powder in the following embodiments is processed according to the following method: Take fresh rambutan fruits, remove the flesh and keep the skin, and perform freeze drying at a freeze drying temperature of -60°C for 48 hours, and use a pulverizer to grind the rambutan shell into a particle-free powder.

[0044] The polyphenol extraction amount in the following examples is calculated as follows: Polyphenol extraction capacity (TPC) = polyphenol content (mg GAE) / freeze-dried rambutan shell powder weight (g); polyphenol content is calculated in gallic acid equivalent (GAE).

[0045] [Effects of different extraction methods on polyphenol extraction yield] Example 1 - DES extraction (1) Choline chloride and glycerol were mixed in a molar ratio of 1:2, heated and stirred in a constant temperature water bath at 80°C for 80 minutes until a uniform colorless and transparent liquid was formed to obtain a low eutectic solvent, and the low eutectic solvent was diluted with water to obtain a DES solution with a water content of 41.3% to obtain an extraction solvent; (2) Mix the rambutan shell powder and the extraction solvent at a solid-liquid ratio of 1:15 (g / mL), and heat the mixture in a water bath at 70 °C for 40 min. (3) The mixture after water bath heating was centrifuged and the supernatant was collected to obtain a crude extract of rambutan shell. The amount of polyphenols extracted in the crude extract was calculated to be 176 mg / g.

[0046] Comparative Example 1 - Pure Water Extraction (1) Mix rambutan shell powder and pure water at a solid-liquid ratio of 1:15 (g / mL), vortex and let stand for one hour; (2) The treated mixture was centrifuged and the supernatant was collected to obtain a crude extract of rambutan shell. The amount of polyphenols in the crude extract was calculated to be 82.5 mg / g.

[0047] Comparative Example 2 – 80% ethanol extraction (1) Pure water and ethanol are mixed evenly in a volume ratio of 2:8 to obtain an extraction solvent; (2) Mix rambutan shell powder and extraction solvent at a solid-liquid ratio of 1:15 (g / mL), vortex and let stand for one hour; (3) The treated mixture was centrifuged and the supernatant was collected to obtain a crude extract of rambutan shell. The amount of polyphenols in the crude extract was calculated to be 126 mg / g.

[0048] Comparative Example 3 - Ultrasound combined with enzymatic hydrolysis to assist DES extraction (1) Prepare a pectinase solution with an enzyme activity value of 660 U / mL and a cellulase solution with an enzyme activity value of 840 U / mL, mix them, adjust the pH value to between 4.5 and 6.5, and perform enzymolysis at a temperature of 55°C for 1 h.

[0049] (2) Prepare a mixture of choline chloride and glycerol (molar volume ratio of 1:2) and heat it in a water bath at 80°C for 60 min.

[0050] (3) The enzymatic hydrolyzate and the prepared DES solution were mixed evenly in a volume ratio of 4:6 to obtain an extraction solvent; (4) Mix the rambutan shell with the extraction solvent at a solid-liquid ratio of 1:15 (g / mL), vortex and let stand for one hour; (5) The treated mixture was centrifuged and the supernatant was collected to obtain a crude extract of rambutan shell. The amount of polyphenols in the crude extract was calculated to be 176 mg / g.

[0051] It can be seen from the above Example 1 and Comparative Examples 1-3 that the amount of polyphenols extracted in the extract obtained by using DES is significantly higher than that extracted by pure water and 80% ethanol. The amount of polyphenols extracted in the extract obtained by the DES extraction method and the ultrasound combined with enzymatic hydrolysis assisted DES extraction method is similar, so the DES extraction method is used in this application.

[0052] The biological activity of the crude extracts of rambutan shells obtained in Example 1 and Comparative Examples 1-2 was tested, and the test results are listed in Table 1: Table 1 Biological activity test results

[0053] It can be seen from Table 1 that the crude extract of rambutan shell obtained by DES extraction has obvious advantages in anti-sugar and improving antioxidant capacity.

[0054]

DES Optimization

[0055] Table 2 Composition and ratio of deep eutectic solvent (DES)

[0056] Depend on Figure 1 It can be seen that when the composition and molar ratio of DES is: choline chloride: glycerol = 1:2, or, choline chloride: citric acid = 1:1, the extraction amount of polyphenols is higher, especially when choline chloride: glycerol = 1:2, the extraction amount of polyphenols is the highest. Therefore, in this application, DES preferably uses choline chloride: glycerol = 1:2, or, choline chloride: citric acid = 1:1; most preferably, choline chloride: glycerol = 1:2.

[0057]

Screening of optimal extraction process conditions

[0058] (2) Choline chloride and glycerol were mixed in a molar ratio of 1:2, heated and stirred in a constant temperature water bath at 80° C. until a uniform colorless and transparent liquid was formed to obtain a low eutectic solvent, and the low eutectic solvent was added to the mixed enzyme solution to dilute it into a DES solution with a water content of 40% to obtain an extraction solvent; (3) The rambutan shell powder was mixed with the extraction solvent at a solid-liquid ratio of 1:15 (g / mL) and treated in a water bath at 70°C for 40 min.

[0059] (4) The mixture after water bath treatment is subjected to ultrasonic treatment.

[0060] (5) The ultrasonically treated mixture was centrifuged at a speed of 15,000 r / min for 10 min.

[0061] The extraction conditions in the above method were taken as conventional quantities, and eight single-factor variables including water content of DES solution (30%, 40%, 50%, 60%, 70%), solid-liquid ratio (1:10, 1:15, 1:20, 1:25, 1:30, unit: g / mL), water bath temperature (40, 50, 60, 70, 80, unit: ℃), water bath time (20, 30, 40, 50, 60, unit: min), pectinase (660, 880, 1100, 1320, 1440, unit: U / mL), cellulase (630, 840, 1050, 1260, 1470, unit: U / mL), ultrasonic power (200, 300, 400, 500, 600, unit: W) and ultrasonic time (3, 6, 9, 12, 15, unit: min) were used to replace the corresponding conventional quantities in the experiment. The single factor experiment was used to explore the influence of various factors on the extraction of rambutan shell polyphenols. Figures 2 to 9 shown.

[0062] Depend on Figures 2 to 9 It can be seen that various factors affect the extraction amount of rambutan shell polyphenols. Figure 2 It can be seen that when the water content of the DES solution increases from 30% to 40%, the amount of polyphenols extracted increases, reaching the optimum at 40%, and then decreases. This shows that adding an appropriate amount of water can increase the amount of polyphenols extracted from rambutan shells by DES, but excessive water will reduce the amount of polyphenols extracted. Figure 3It can be seen that the amount of polyphenols extracted first increased and then decreased, reaching a maximum at 1:15 (g / mL). This shows that appropriately increasing the volume of the solvent is beneficial to increasing the contact area between the freeze-dried powder and the solvent, thereby facilitating the extraction of the target active substances, but this factor has limited contribution to the amount of polyphenols extracted. When the ratio of solvent to rambutan shell freeze-dried powder is greater than 1:15, it may exceed the threshold of the solid-liquid ratio. At this time, the amount of solvent has little effect on the amount of polyphenols extracted, resulting in the polyphenols extraction amount tending to be stable. Figure 4 It can be seen that as the water bath temperature increases, the amount of polyphenols extracted increases first and then decreases. This shows that at higher temperatures, the viscosity of DES decreases, the fluidity increases, and the interaction between the solvent and polyphenols is enhanced, which is conducive to better dissolution of polyphenols. When the temperature is further increased to above 70°C, the amount of polyphenols extracted decreases instead. This may be because when the temperature is too high, the degree of destruction of the active substances is greater than the positive effect brought about by the decrease in solvent viscosity. Figure 5 It can be seen that as the water bath time increases, the amount of polyphenols extracted increases first and then decreases, and reaches the maximum at 40 minutes. This shows that in the first 40 minutes, the amount of rambutan shell polyphenols extracted increases with the increase of extraction time, which may be due to this period of time being the "washing stage" and "slow extraction stage". When it reaches 40 minutes, the amount of polyphenols extracted has reached saturation. In addition, Figures 6 to 9 As shown in the figure, the changes in the levels of ultrasonic power, ultrasonic time, pectinase and cellulase did not cause significant changes in the amount of polyphenols extracted. This shows that DES has a strong shell-breaking ability, so these auxiliary conditions have no significant effect on the extraction of rambutan shell polyphenols. Therefore, the optimal process parameters of each important single factor are as follows: DES solution water content 40%, solid-liquid ratio 1:15 (g / mL), water bath temperature 70℃, water bath time 40min.

[0063] 2. Response surface optimization experiment According to the results of the single-factor experimental design, a Box-Behnken experimental design was conducted on the three factors of water content (%) of DES solution, water bath temperature (℃), and water bath time (min) to determine the optimal extraction process of rambutan shell polyphenols. The specific factor levels of the Box-Behnken experimental design are shown in Table 3.

[0064] Table 3 Factor levels of Box-Behnken experimental design

[0065] The Box-Behnken experimental design and test results are shown in Table 4. The variance analysis results of the response surface experimental design are shown in Table 5. As can be seen from Table 5, the regression model P <0.0001 (very significant), lack of fit P=0.2324>0.05 (not significant), the model's coefficient of determination R 2 =0.9571, indicating that the regression model is reliable and can be used to predict the polyphenol extraction of rambutan shell. In addition, the effects of the first-order term, the second-order term and the interaction term on the polyphenol extraction in the regression model reached a significant level except for AC and BC ( P <0.05).

[0066] Table 4 Box-Behnken experimental design and test results

[0067] Table 5 Results of variance analysis

[0068] In addition, the experimental data obtained according to the conditions in Table 3 were subjected to multinomial fitting regression using the Design-Expert software, and the resulting quadratic polynomial equation is as follows: Y=173.31-4.9X1+2.94X2-6.66X3-2.39X1X2+1.96X1X3+0.9350X2X3-5.06X1 2 -10.45X2 2 -4.73X3 2 Wherein, X1 is the water content of DES solution, X2 is the water bath temperature, X3 is the water bath time, and Y is the amount of polyphenols extracted from rambutan shell.

[0069] In addition, the 3D response surface diagram of the effects of the water content of DES solution (A) and water bath temperature (B), the water content of DES solution (A) and water bath time (C), and the interaction between water bath temperature (B) and water bath time (C) on the extraction of polyphenols from rambutan shell is shown in Figure 10~Figure 12 . Fig.10 It can be seen that the curvature is larger, indicating that the interaction between A and B is larger; Figures 11-12 It can be seen that the curvatures are small, indicating that the interactions between A and C, and between B and C are small. This result is consistent with the results shown in Table 5.

[0070] 3. Determination and verification of optimal process conditions The optimal extraction process conditions obtained by the response surface optimization experiment are: DES solution water content 41.3%, water bath temperature 71.3℃, water bath time 40.1min. Under this condition, the predicted extraction amount of rambutan shell polyphenols is 178.3mgGAE / g. In actual operation, the process optimization parameters are: DES solution water content 41.3%, water bath temperature 71.3℃, water bath time 40.1min, and the specific steps refer to the above Example 1. According to this extraction condition, the optimal extraction process fitted by the model is verified. The experiment is repeated three times, and the extraction amounts of rambutan shell polyphenols are 176.3mg GAE / g, 183.6 mg GAE / g, and 180.4 mg GAE / g respectively. This test result is close to the predicted value.

[0071]

Composition analysis of rambutan shell crude extract

[0072] Example 1: The crude extract of rambutan husk is subjected to AB-8 macroporous resin to remove DES (the specific method is detailed in Example 2 below). The obtained extract is freeze-dried into powder and then prepared into a 1 mg / mL solution with anhydrous ethanol for later use.

[0073] Comparative Example 1: The crude extract of rambutan husk was freeze-dried into powder and then prepared into a 1 mg / mL solution with ethanol for later use.

[0074] Comparative Example 2: The crude extract of rambutan husk was freeze-dried into powder and then prepared into a 1 mg / mL solution with ethanol for later use.

[0075] The above test solution was analyzed by ultra-high performance liquid chromatography, and the analysis conditions were as follows: The column was C18 (30m×0.25mm, 0.25µm), and the mobile phase solvent was composed of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). The elution gradient was as follows: 0min, 15%B; 0~5min, 15%B; 5~23min, 15~25%B; 23~36min, 25~35%B; 36~40min, 35~50%B; 40~43min, 50%B; 43~49min, 50~80%B; 49~52min, 80%B; 52.01min, 15%B; 52.01~60min, 15%B. The detection wavelength was 280nm, the flow rate was 0.7mL / min, and the injection volume was 10μL.

[0076] Standard sample preparation: Prepare a mother solution with a concentration of 0.2 mol / L using dimethyl sulfoxide (DMSO, AR), dilute the mother solution with 70% ethanol, and prepare a series of standard solutions. The mixed standard concentration range is 0~1500µmol / L. After testing, the contents of the various components in the rambutan shell extract freeze-dried powder obtained in Example 1 and Comparative Examples 1~2 are listed in Table 6, in terms of the weight of each component per gram of freeze-dried powder (μg): Table 6 Analysis of ingredients in rambutan shell extract freeze-dried powder

[0077] It can be seen from Table 6 that the extract obtained by the method of the embodiment of the present application contains more types of polyphenol compounds, and the content of functional compounds with anti-inflammatory and anti-sugar properties, such as ellagic acid, delphinidin-3-O-glucoside, delphinidin 3-O-rutinoside, and quercetin, is higher.

[0078]

Solvent removal process

[0079] Accurately weigh 1.0 g of the five activated macroporous resins into a 50 mL conical flask, add 25 mL of rambutan shell polyphenol crude extract, place in a constant temperature shaker, and adsorb for 24 h at 30°C and 120 r / min. During this period, 25 μL of the supernatant was taken every 1 h to determine its polyphenol concentration, and the adsorption rate, resolution rate and recovery rate were calculated. The results are listed in Table 7: Table 7 Adsorption rate, resolution rate and recovery rate of macroporous resin Resin Type Adsorption rate (%) Resolution rate (%) Recovery rate (%) AB-8 36.48 45.92 16.75 D-101 35.97 50.62 18.21 Polyamide 60.01 20.13 16.28 NKA-9 36.37 46.68 24.04 DM-301 60.02 27.13 16.98 Taking into account the adsorption rate, resolution rate, recovery rate and cost, the present application prefers AB-8 resin in the step of desolvation of the macroporous resin.

[0080] 2. Concentration of eluent ethanol Activation of macroporous resin: AB-8 resin was activated according to the above method.

[0081] 50.0 g of activated macroporous resin was wet loaded into a glass chromatography column (Ф 0.6 cm×50 cm). The crude extract was loaded at a concentration of 2.0 mg / mL, a loading volume of 500 mL, and an loading flow rate of 5.0 mL / min for adsorption. Impurities were washed away with a large amount of deionized water, and every 50 mL of effluent was collected until there was no significant difference in the polyphenol concentration of the effluent, indicating that the adsorption reached saturation; then 60%, 70%, 80%, 90% and 100% ethanol were used for elution at a flow rate of 2 mL / min, and every 50 mL of the eluate was collected and the polyphenol content was determined until the polyphenol content was extremely low and basically unchanged, then the elution could be stopped. The effect of elution with different concentrations of ethanol on the polyphenol recovery amount (TPC) is shown in the figure. Fig.13 As shown. Fig.13 It can be seen that 70% ethanol is the best eluent.

[0082] 2. Comparison of different desolventization processes: The crude extract of rambutan shell obtained in Example 1 was subjected to solvent removal according to the methods of Examples 2 to 4: Example 2 Macroporous resin removal method Resin activation: Same as above.

[0083] 50.0 g of activated macroporous resin was wet loaded into a glass chromatography column (Ф 0.6 cm×50 cm). The crude extract was loaded at a concentration of 2.0 mg / mL, a loading volume of 500 mL, and a loading flow rate of 5.0 mL / min for adsorption. A large amount of deionized water was used to wash away impurities, and every 50 mL of the effluent was collected until there was no significant difference in the polyphenol concentration of the effluent, indicating that the adsorption reached saturation; then 70% ethanol was used for elution at a flow rate of 2 mL / min, and every 50 mL of the eluate was collected and the polyphenol content was determined until the polyphenol content was extremely low and basically unchanged, and the elution was stopped to obtain the rambutan shell extract.

[0084] Example 3 Two-step removal of organic reagents of ethyl acetate + n-butanol (1) Mix ethyl acetate (1 mL) with 1 mL of rambutan husk crude extract. Vortex the two phases for 30 s and then centrifuge at 10,000 rpm for 3 min. Collect the ethyl acetate phase and retain the DES phase.

[0085] (2) Dilute the retained DES phase with pure water to 5 mL, and then extract it three times with 5 mL of n-butanol. Collect the n-butanol phase and combine it with the ethyl acetate phase to obtain the rambutan shell extract.

[0086] Example 4 Acetone + n-butanol organic reagent two-step removal method (1) Mix acetone (1 mL) with 1 mL of rambutan husk crude extract. Vortex the biphasic system for 30 s and then centrifuge at 10,000 rpm for 3 min. After phase separation, collect the acetone phase and retain the DES phase.

[0087] (2) Dilute the retained DES phase to 5 mL with pure water, and then extract it three times with 5 mL of n-butanol. Collect the n-butanol and combine it with the acetone phase to obtain the rambutan shell extract.

[0089] The recovery rate of polyphenol compounds after solvent removal was calculated according to the following formula: ER = ×100% where m R and m0 represent the mass of polyphenols in rambutan husk refined extract and the mass of polyphenols in rambutan husk crude extract, respectively.

[0090] The recovery results are as follows Fig.14 As shown. Fig.14 It can be seen that compared with the macroporous resin removal method, the organic reagent two-step removal method has a higher polyphenol recovery rate. However, considering that the macroporous resin removal method is green and pollution-free, the process does not involve the introduction of any organic reagents, and the macroporous resin can be recycled, saving costs and protecting the environment. Therefore, this application considers the above three methods as the preferred solutions for the desolventization process.

[0091]

Analysis of ingredients of natural anti-sugar composition

[0092] The components of the natural anti-sugar composition solution are listed in Table 8. The content of each component in Table 8 is the mass (µg) of the corresponding compound in each mL of the natural anti-sugar composition solution.

[0093] Table 8 Analysis of ingredients of natural anti-sugar composition

[0094] The results of the biological activity test of the natural anti-sugar composition solution are listed in Table 9.

[0095] Table 9 Biological activity analysis of natural anti-sugar composition

[0096] As can be seen from Table 9, the natural anti-sugar compositions obtained in Examples 3 and 4 showed better antioxidant activity than Example 2, but there was no significant difference in inhibiting the activity of α-amylase (α-Amylase) and they were both better than Example 2, but they were lower than Example 2 in inhibiting the activity of α-glucosidase (α-Glucosidase).

[0097]

Correlation Analysis

[0098] Analysis method: Correlation clustering labeling heat map Components in natural anti-sugar composition α -glucosidase inhibitory activity Fig.15 shown. Fig.15 In the table, DPPH represents the activity of scavenging DPPH free radicals, and FRAP represents the total reducing power of iron ions. α -Glucosidase indicates α -glucosidase inhibitory capacity (IC 50 ). The natural anti-sugar composition is rich in 17 anti-sugar compounds, the specific names and their α -glucosidase inhibitory activity (IC 50 The correlation coefficients of the compounds are listed in Table 10. The larger the absolute value of the correlation coefficient, the stronger the α -glucosidase inhibitory activity.

[0099] Table 10 Natural anti-sugar compounds and correlation coefficients

[0100] Combining Table 10 and Table 8, it can be seen that the natural anti-sugar composition obtained according to the method provided in the present application has more types of anti-sugar components and higher content.

[0101] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or improvements to the technology in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A method for preparing a natural anti-sugar composition, characterized in that: include: Step a), freeze-drying the rambutan husk and grinding it into powder to obtain rambutan husk powder; Step b), extracting the rambutan shell powder using a deep eutectic solvent solution under water bath heating to obtain a rambutan shell crude extract; Step c), removing the low eutectic solvent in the crude extract of rambutan shell to obtain a natural anti-sugar composition extract; Step d), freeze-drying the natural anti-sugar composition extract to obtain the natural anti-sugar composition.

2. The preparation method according to claim 1, characterized in that: The freeze-drying temperature in step a) is -70 to -50°C for 36 to 60 hours; and / or the freeze-drying temperature in step d) is -70 to -50°C for 36 to 60 hours.

3. The preparation method according to claim 1, characterized in that: The water content of the low eutectic solvent solution is 39-42%; the low eutectic solvent in the low eutectic solvent solution includes: choline chloride and glycerol in a molar ratio of 1:2; or, choline chloride: citric acid in a molar ratio of 1:

1.

4. The preparation method according to claim 3, characterized in that: In the step b), the water bath heating temperature is 60-80° C., and the heating time is 30-50 min; the solid-liquid ratio of the rambutan shell powder to the low eutectic solvent is 1 g: (13-18) mL.

5. The preparation method according to claim 1, characterized in that: Step c) is specifically: A macroporous resin adsorption method or an organic solvent two-step extraction method is used to remove the low eutectic solvent in the rambutan shell crude extract to obtain the rambutan shell refined extract; The organic solvent in the rambutan shell extract is removed to obtain a natural anti-sugar composition extract.

6. The preparation method according to claim 5, characterized in that The macroporous resin adsorption method adopts AB-8 macroporous adsorption resin; the loading concentration is 2.0 mg / mL, the loading volume is 500 mL, the loading flow rate is 5.0 mL / min; and the eluent is 70% ethanol.

7. The preparation method according to claim 5, characterized in that: The organic solvent two-step extraction method is specifically: Extracting the rambutan shell crude extract once using a first organic solvent, collecting the first organic solvent phase and the DES phase; Performing secondary extraction on the DES phase using a second organic solvent, and collecting the second organic solvent phase; combining the first organic solvent phase and the second organic solvent phase; The first organic solvent is ethyl acetate or acetone, and the second organic solvent is n-butanol.

8. A natural anti-sugar composition, characterized in that: It is prepared according to the method according to any one of claims 1 to 7.

9. The natural anti-sugar composition according to claim 8, characterized in that: They include: delphinidin-3-O-glucoside, gallic acid, epigallocatechin-3-ol, chlorogenic acid, rutin, ellagic acid, isoquercetin, kaempferol, quercetin, proanthocyanidin C1, neohesperidin, zebutin, isoquercetin and corilagin.

10. Use of the natural anti-sugar composition according to claim 8 in anti-sugar products.

Images