Gleditsia sinensis saponin with anti-glycosylation activity and preparation method thereof

Abstract

The invention discloses gleditsia sinensis saponin with anti-glycosylation activity and a preparation method of the gleditsia sinensis saponin. The method comprises the following steps: washing, drying, crushing, degreasing and carrying out other pretreatment on gleditsia sinensis shells to obtain gleditsia sinensis powder; uniformly mixing gleditsia sinensis powder with a deep-eutectic solvent, extracting in an ultrasonic field, and centrifuging to obtain an extracting solution; dialyzing, concentrating and freeze-drying the extracting solution to obtain a gleditsia sinensis saponin crude extract, re-dissolving the crude extract in distilled water, and purifying by using macroporous resin and reversed-phase column chromatography in sequence to obtain purified gleditsia sinensis saponin; the method for preparing the gleditsia sinensis saponin is simple, convenient, rapid, green and environment-friendly, industrial production is facilitated, and the obtained saponin is high in purity and good in quality. In addition, the gleditsia sinensis saponin prepared by the method has the activity of inhibiting the formation of advanced glycosylation end products.

Description

Technical Field

[0001] The invention relates to the technical field of forest source active matter extraction, in particular to a nasturtium saponin with anti-glycosylation activity and a preparation method thereof. Background Art

[0002] Gleditsia sinensis, also known as soapberry, black rhinoceros, etc., is a plant of the Leguminosae family, a deciduous tree or small tree. Gleditsia sinensis has a very high application value. In addition to its important role in water conservation, ecological protection and tourism development, products such as soapberry rice and soapberry polysaccharide gum have been widely used in many fields such as food and daily chemicals.

[0003] Gleditsia husk is the main residue after the use of Gleditsia seeds, which contains rich pentacyclic triterpenoid saponins. Gleditsia saponin is a natural non-ionic surfactant with good foaming, emulsification, volume expansion, dispersion and other surface active effects. It also has anti-inflammatory, analgesic, anti-cancer, dehumidification and insecticidal active functions. It has great application prospects in many fields such as daily chemicals, medicine, pesticides, biology and foam fire extinguishing.

[0004] At present, the extraction methods of saponin from licorice are mainly hot water extraction and organic solvent extraction. It is difficult to purify saponin from hot water extraction, especially because the pectin content in licorice is high, and the viscosity of the extract from hot water extraction is large, which is not conducive to the separation and purification of saponin; the organic solvent method for extracting saponin is complex, environmentally unfriendly, and has high production costs. The prior art discloses a variety of saponin preparation methods, but there are also certain defects. For example, the Chinese patent with publication number CN103073599A discloses a method for extracting saponin from licorice, which uses water extraction, spray drying after concentration to obtain saponin from licorice, but due to the complex components in the licorice shell, the water extract contains a large amount of sugars, proteins, pigments and other substances, which easily causes the product purity to be low, and the method has low extraction efficiency and high energy consumption. The Chinese patent with publication number CN1696145A discloses a method for extracting saponin from licorice, which uses anhydrous ethanol to extract saponin, the extraction time is 3-9 hours, and acetone is used to wash the saponin. The method has a long extraction time and uses a large amount of organic reagents, which has high requirements for equipment and has residual hidden dangers. Chinese patent with publication number CN115286681A discloses a method for extracting tea saponin, which uses a variety of biological enzymes to assist extraction, which not only increases production costs, but the use of complex enzymes may also degrade saponin and change the properties and structure of saponin. Chinese patent with publication number CN113024629A discloses a method for preparing tea saponin, which uses organic solvents such as n-butanol, ethyl acetate and benzene in the process of extracting and purifying saponin, which has high requirements on equipment and poses safety hazards. The Chinese patent with publication number CN115322241A discloses a method for extracting tea saponin, including defatting and decolorizing camellia seed cake at 80-90 ℃, using a low eutectic solvent with hydrogen bond donors such as glucose, citric acid, acetic acid, oxalic acid or glycerol, extracting under the combination of high-voltage pulse electric field and ultrasonic multi-field, purifying the extract using flocculants and three-stage membrane filtration, and spray drying (air outlet temperature 80-90 ℃) to obtain saponin powder. This process is complex and requires high equipment. At the same time, the high defatting and drying temperatures are likely to affect the quality of saponin. The product is yellow or light brown. In addition, the use of flocculants for purification will cause saponin loss and low yield. It should also be pointed out that saponin is mainly composed of pentacyclic triterpenoid di-chain saponins, and tea saponin is a single-chain saponin. There are significant differences in properties and structures, which are suitable for the green and efficient extraction of saponin, especially the preparation method of saponin with anti-glycosylation function is still lacking.

[0005] Therefore, it is necessary to develop a method for preparing nasturtium saponin, which is green and efficient, and the prepared nasturtium saponin has high purity, light color and good activity. Summary of the invention

[0006] The invention aims to provide a method for extracting saponin from soapberry, in particular to provide a method for extracting saponin with anti-glycosylation activity from soapberry in a green and efficient manner, so as to achieve high-value utilization of soapberry.

[0007] To achieve the above object, the present invention adopts the following steps: (1) washing the soapberry shells with the soapberry rice removed with clean water, drying them, and crushing them into soapberry powder, and fully defatting the soapberry powder; (2) The defatted saponin powder and the low eutectic solvent are fully mixed in a certain proportion, extracted under an ultrasonic field, and then centrifuged at high speed to obtain an extract; (3) The extract is dialyzed, concentrated and freeze-dried in sequence to obtain a crude extract of saponin. The crude extract is redissolved in distilled water, purified by macroporous resin and reverse phase column chromatography in sequence, and finally freeze-dried to obtain purified off-white saponin.

[0008] As a further solution of the present invention: the soap locust shell should be dried to a moisture content of ≤5%, crushed into 80-200 mesh soap locust powder, and the solvent used for degreasing is petroleum ether with a boiling range of 30-60°C, degreasing at 40-50°C for 4-12 hours and then drying to evaporate the residual solvent.

[0009] The low eutectic solvent hydrogen bond acceptor is choline chloride, and the hydrogen bond donor is at least one of lactic acid, malic acid, propylene glycol, and sorbitol. The mixture is fully mixed in a molar ratio of 1:1-2, heated and stirred at 80-120° C. for 0.5-1.5 h to obtain a low eutectic solvent.

[0010] The low eutectic solvent used for extracting saponin should contain a certain amount of water, the water content of which is 5-50%.

[0011] The solid-liquid ratio of the saponin powder to the low eutectic solvent is 1:5-50 g / mL, the ultrasonic power is 200-800 W, the extraction temperature is 30-60 ° C, the extraction time is 5-30 min, and the centrifugal speed is 8000-12000 rpm / min.

[0012] The molecular weight cut-off of the dialysis bag is 500-1500 Da, the dialysis temperature is 5-40°C, and the dialysis time is 48-96h.

[0013] The dialysate is concentrated to 1 / 6-1 / 2 of the original volume.

[0014] The saponin is fully dissolved in distilled water before being purified by macroporous resin, with a ratio of 1:4-20 (w / v).

[0015] The macroporous resin purification is first eluted with distilled water for 2-4 column volumes, and then eluted with an ethanol solution, the eluent is 90% ethanol solution, and eluted for 2-4 column volumes.

[0016] The reverse phase column chromatography uses a C18 silica gel column, and the mobile phase is a formic acid aqueous solution (0.2-1‰) and methanol gradient elution, and the saponin-rich fraction is collected, concentrated by rotary evaporation and freeze-dried to finally obtain off-white saponin. Beneficial Effects

[0017] The invention is simple to operate, and the conditions are easy to control. The yield of extracting saponin using the low eutectic solvent is more than 30% higher than that of the traditional extraction method. The saponin extraction method used in the invention is simple to operate, has mild conditions, is environmentally friendly, and has good industrial production prospects. The saponin prepared by the scheme has high purity, light color, good physical and chemical properties, and good anti-glycosylation activity. The invention provides a new reference approach for the processing and utilization of saponins. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 It is a purified saponin extracted using a deep eutectic solvent. Figure 2 It is a saponin obtained by hot extraction with ethanol solution. DETAILED DESCRIPTION

[0018] The following examples are some examples of the present invention and should not be considered as limiting the present invention. Example

[0019] Select seeded sapodilla shells, wash with water and dry them, the moisture content is 4.18%. The dried soapberry shell was crushed by a pulverizer and passed through a 100-mesh sieve. The soapberry powder was degreased at 40°C for 10 h using petroleum ether with a boiling range of 30-60°C. After degreasing, it was dried at 45°C to evaporate the residual solvent. A low eutectic solvent was prepared: the hydrogen bond acceptor was choline chloride and the hydrogen bond donor was propylene glycol. They were weighed and mixed in a molar ratio of 1:2, and stirred at 85°C for 1 h to obtain a clear and transparent low eutectic solvent. Distilled water was added to the prepared low eutectic solvent and mixed thoroughly to make the moisture content 20%. The solvent was used to extract soapberry saponins. The soapberry powder and the water-containing low eutectic solvent were fully mixed at a solid-liquid ratio of 1:10 g / mL, extracted at an ultrasonic power of 400 W and a temperature of 45°C for 20 min, and centrifuged at 8000 rpm / min for 10 min after the extraction was completed to obtain an upper extract supernatant. The supernatant was dialyzed at 30°C for 48 min using a dialysis bag with a molecular weight cutoff of 1000 Da. h, after dialysis, rotary evaporation was performed to concentrate the extract to 1 / 4 of the original volume, and freeze-dried to obtain a crude extract of saponin; the crude extract of saponin was dissolved in distilled water at a ratio of 1:5 (w / v), and purified on a macroporous resin column. After eluting with distilled water for 3 column volumes, 90% ethanol solution was added to elute for 3 column volumes, and 20 mL was collected in each tube. The saponin-rich fractions were collected, concentrated by rotary evaporation, and then freeze-dried. After redissolving in methanol solution, the fractions were purified using a reversed-phase C18 silica gel column. The mobile phase was a formic acid aqueous solution (1‰) and methanol gradient elution. The saponin-rich fractions were collected, concentrated by rotary evaporation, and then freeze-dried to obtain off-white purified saponin, see the attached manual. Figure 1 The extraction rate of saponin from Gleditsia sinensis using this method was 8.32%. The inhibition rate of saponin (1 mg / mL) extracted using this method on the glycosylation reaction of bovine serum albumin for 7 days could reach more than 20%. Example

[0020] Select seeded sapodilla shells, wash with water and dry them, the moisture content is 3.13%. The dried soapberry shell was crushed by a pulverizer and passed through an 80-mesh sieve. The soapberry powder was degreased at 45 °C for 8 h using petroleum ether with a boiling range of 30-60 °C. After degreasing, it was dried at 50 °C to evaporate the residual solvent. A low eutectic solvent was prepared: the hydrogen bond acceptor was choline chloride and the hydrogen bond donor was lactic acid. They were weighed and mixed in a 1:1 molar ratio, and stirred at 90 °C for 45 min to obtain a clear and transparent low eutectic solvent. Distilled water was added to the prepared low eutectic solvent and mixed thoroughly to make the moisture content 10%. The solvent was used to extract soapberry saponins. The soapberry powder and the water-containing low eutectic solvent were fully mixed at a solid-liquid ratio of 1:15 g / mL, extracted at an ultrasonic power of 500 W and a temperature of 40 °C for 25 min, and centrifuged at 10000 rpm / min for 5 min after the extraction was completed to obtain the supernatant. The supernatant was dialyzed at 25 °C for 72 min using a dialysis bag with a molecular weight cutoff of 1500 Da. h. After dialysis, the extract was concentrated by rotary evaporation to 1 / 5 of the original volume, and freeze-dried to obtain a crude extract of saponin. The crude extract of saponin was dissolved in distilled water at a ratio of 1:10 (w / v), purified on a macroporous resin column, eluted with distilled water for 2.5 column volumes, and then eluted with 90% ethanol solution for 2 column volumes. 15 mL was collected in each tube, and the saponin-rich fractions were collected, concentrated by rotary evaporation, and then freeze-dried. After redissolving in methanol solution, it was further purified by a reversed phase C18 silica gel column. The mobile phase was formic acid aqueous solution (0.5‰) and methanol. Gradient elution was performed, and the saponin-rich fractions were collected. The saponin-rich fractions were concentrated by rotary evaporation, and then freeze-dried to obtain off-white purified saponin. The extraction yield of saponin using this method was 7.85%. The saponin (1 mg / mL) extracted using this method had an inhibition rate of more than 20% when the glycosylation reaction of bovine serum albumin was 7 days.

[0021] Comparative Example Hot water extraction and organic solvent extraction are traditional methods for preparing saponins from Gleditsia sinensis. However, a large amount of saponins exist in the cell walls of plant tissues, and the efficiency of extraction is low by a single water extraction or organic solvent. At the same time, the components of Gleditsia sinensis shell are complex, containing polysaccharides, proteins and other substances, which will affect the purity, quality and biological activity of saponins.

[0022] The extraction method used in Comparative Example 1 is as follows: The seeded sapodilla shell was selected, washed with water and dried, with a moisture content of 4.82%. The dried sapodilla shell was crushed by a grinder and passed through a 100-mesh sieve. The sapodilla powder was degreased at 45 °C for 12 h using 30-60 °C boiling range petroleum ether. After degreasing, it was dried at 40 °C to evaporate the residual solvent. The sapodilla powder and distilled water were fully mixed at a solid-liquid ratio of 1:20 g / mL, and extracted at an ultrasonic power of 500 W and a temperature of 40 °C for 25 min. After the extraction was completed, it was centrifuged at 10000 rpm / min for 5 min to obtain the supernatant. The sapodilla saponin was extracted by hot water extraction after rotary concentration to 1 / 4 of the original volume and freeze-dried. The extraction yield of sapodilla saponin using this method was 3.87%.

[0023] The extraction method used in Comparative Example 2 is as follows: Select the seeded sapodilla shell, wash it with water and dry it, the moisture content is 3.71%. Use a grinder to crush the dried sapodilla shell through an 80-mesh sieve, use 30-60 ℃ boiling range petroleum ether to defatted the sapodilla powder at 40 ℃ for 10 h, and then dry it at 50 ℃ to evaporate the residual solvent; sapodilla powder and 80% ethanol solution are fully mixed at a solid-liquid ratio of 1:15 g / mL, and extracted at an ultrasonic power of 500W and a temperature of 40 ℃ for 25 min. After the extraction is completed, centrifuge at 10000 rpm / min for 5 min to obtain the supernatant; rotate and concentrate to 1 / 4 of the original volume and freeze-dry to obtain sapodilla saponins extracted with ethanol solution, see the attached manual Figure 2 The extraction rate of saponin from Gleditsia sinensis using this method was 5.91%. The inhibition rate of saponin (1 mg / mL) extracted using this method on the glycosylation reaction of bovine serum albumin for 7 days was 11.49%.

[0024] In summary, the extraction rate of saponin from Gleditsia sinensis by the deep eutectic solvent method is higher and the anti-glycosylation activity is stronger, which is the feature of the present invention. The saponin prepared by the present invention is expected to be used as a green and safe additive for functional foods, daily chemical products, etc. Therefore, the present invention is of great significance to the refined utilization of Gleditsia sinensis.

[0025] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A saponin with anti-glycosylation activity and a preparation method thereof, characterized by the following steps: The soapberry shells from which the soapberry rice has been removed are washed with clean water, dried, and crushed into 80-200 mesh soapberry powder, which is fully defatted; the defatted soapberry powder is fully mixed with a low eutectic solvent in a certain proportion, extracted under an ultrasonic field, and centrifuged at high speed to obtain an extract; the extract is dialyzed, concentrated, and freeze-dried in sequence to obtain a crude extract of soapberry saponin, which is redissolved in distilled water, and purified by macroporous resin and reverse phase column chromatography in sequence to obtain purified soapberry saponin. The soapberry saponin prepared by the above method has high purity, light color, and has biological activity of inhibiting glycosylation.

2. The nasturtium saponin having anti-glycosylation activity and the preparation method thereof as claimed in claim 1, characterized in that: Before extraction, the soapberry shell should be fully crushed to 80-200 mesh, and the crushed soapberry powder should be defatted using low-boiling petroleum ether (30-60 ℃).

3. The nasturtium saponin having anti-glycosylation activity and the preparation method thereof as claimed in claim 1, characterized in that: In the low eutectic solvent used, the hydrogen bond acceptor is choline chloride, the hydrogen bond donor is at least one of lactic acid, malic acid, propylene glycol, and sorbitol, the low eutectic solvent is mixed in a molar ratio of 1:1-2, heated and stirred at 80-120°C for a certain time to obtain a transparent low eutectic solvent, a certain amount of distilled water is added to the low eutectic solvent, and the low eutectic solvent (water content 5-50%) after vortex oscillation and mixing can be used for the extraction of saponin from licorice; defatted saponin powder and the water-containing low eutectic solvent are fully mixed at a certain material-liquid ratio (1 / 5-50 g / mL), extracted for 5-30 min at an ultrasonic power of 200-800 W and an extraction temperature of 30-60°C, and after the extraction is completed, high-speed centrifugation is performed to obtain an upper saponin-rich solution.

4. The nasturtium saponin having anti-glycosylation activity and the preparation method thereof as claimed in claim 1, characterized in that: The extract is dialyzed using a dialysis bag with a molecular weight cutoff (500-1500 Da) (dialysis temperature is 5-40°C, dialysis time is 48-96 h), and after the dialysis is completed, the solution in the bag is further concentrated by rotary evaporation (concentrated to 1 / 6-1 / 2 of the original volume) and freeze-dried to obtain a crude extract of saponin. The crude extract is fully dissolved by adding distilled water (1:4-20, w / v) in a certain proportion, and then purified by macroporous resin column chromatography. After eluting with distilled water for 2-4 column volumes, eluting with ethanol solution for 2-4 column volumes, the eluate rich in saponin is concentrated by rotary evaporation and freeze-dried, and then further purified by semi-preparative liquid phase; the chromatography column is a reverse phase C18 silica gel column, the mobile phase is formic acid aqueous solution (0.2-1‰) and methanol, gradient elution is used, the saponin-rich fraction is collected, rotary evaporation is concentrated, and then freeze-dried to obtain off-white purified saponin.

5. The method for preparing Gleditsia saponin according to any one of claims 1 to 4.

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