Preparation method of gleditsia sinensis polyphenol with anti-glycosylation activity

Abstract

The invention relates to a preparation method of gleditsia sinensis polyphenol with anti-glycosylation activity, and belongs to the technical field of development and application of natural plant products. According to the method, mature fruit pods of a leguminous plant Gleditsia sinensis Lam. Are used as raw materials, and the high-purity Gleditsia sinensis Lam. Extract is obtained through extraction, separation and purification. The preparation method specifically comprises the following steps: performing drying, crushing and degreasing pretreatment on mature fruit pods to prepare gleditsia sinensis powder; mixing the obtained gleditsia sinensis powder with an ethanol solution, and extracting under an ultrasonic-assisted condition; carrying out centrifugal treatment on the extract to separate solid from liquid, and collecting supernate; concentrating the extracting solution, fixing the volume, and carrying out acid hydrolysis reaction; and purifying the acid hydrolysis product through a macroporous adsorption resin column to finally obtain purified gleditsia sinensis polyphenol. The product obtained by the method has remarkable anti-glycosylation activity, and is suitable for development and utilization in the fields of medicines, functional foods, cosmetics and the like.

Description

Technical Field

[0001] This invention relates to the field of plant natural product development and application technology, specifically to a method for preparing saponins with anti-glycation activity. Background Technology

[0002] Soapberry ( Gleditsia sinensis Soapberry (Laminaria japonica), also known as soapberry, is a multi-purpose tree species with both ecological and economic value. It is widely distributed in my country, with a planting area exceeding 1 million mu (approximately 66,667 hectares). This species integrates edible, medicinal, chemical raw material, and afforestation uses, demonstrating significant resource utilization potential. Traditional Chinese medicine believes that soapberry is pungent, salty, and warm in nature, with slight toxicity. It mainly enters the lung and large intestine meridians, possessing effects such as resolving phlegm, opening orifices, dispersing nodules, and reducing swelling. Its fruit, seeds, and thorns have a long history of clinical application in traditional Chinese medicine, and also show broad application prospects in industrial processing, food development, and ecological restoration. Modern pharmacological studies have shown that soapberry is rich in saponins, polyphenols, and other active ingredients, which can promote phlegm expulsion, reduce lung inflammation, and have significant anti-inflammatory and anti-allergic activities.

[0003] Compared to saponins, polyphenols, due to the presence of multiple phenolic hydroxyl groups in their molecular structure, generally exhibit more significant biological activity, particularly in anti-glycation activity. However, because saponins and polyphenols share some similarities in polarity, they are often extracted simultaneously during the extraction of saponins from saponins, thus reducing the purity of the polyphenol product and affecting its applications. Previous studies have shown that acid hydrolysis can effectively break the O-glycosidic bonds in saponins, with the cleavage products typically appearing as water-insoluble precipitates, thereby improving the purity of phenolic substances. However, no literature has yet reported the application of acid hydrolysis technology to the treatment of saponin-rich polyphenol extracts to achieve high-purity separation of polyphenols. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing saponins with anti-glycation activity. This invention addresses the problem of high saponin content during saponin extraction by employing acid hydrolysis to prepare high-purity saponins. Since there is limited literature on saponin polyphenol purification, this invention aims to prepare saponins of a certain purity through acid hydrolysis, followed by purification with macroporous resin to obtain even higher-purity saponins, thereby achieving high-value utilization of saponins.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: A method for preparing saponins with anti-glycation activity, characterized by the following steps: 1) Soap pod pretreatment: Wash, dry, crush, sieve, and degrease the soap pods to obtain degreased soap pod powder; 2) Extraction of crude saponin polyphenols: Take the defatted saponin powder from step 1) and mix it with ethanol solution at a certain solid-liquid ratio, extract it under ultrasonic assistance, centrifuge, and concentrate it under reduced pressure until there is no ethanol to obtain crude saponin polyphenol extract. 3) Acid hydrolysis of saponin polyphenols: Take the crude extract of saponin polyphenols from step 2) and make up the volume, mix it with the acid solution in a certain proportion, bathe it in a water bath under appropriate temperature conditions, filter it, collect the filtrate, adjust the pH to neutral, and obtain acid-hydrolyzed saponin polyphenols. The method for preparing anti-glycation active soapberry polyphenols is characterized in that, in step 1), the soapberries are crushed and sieved to 80-200 mesh and degreased at 20-50 °C.

[0006] The method for preparing saponins with anti-glycation activity is characterized in that, in step 2), the concentration of the ethanol solution is 50-90%, the solid-liquid ratio is 1:10-50, the ultrasonic power is 100-500 W, and the extraction time is 10-40 min.

[0007] The method for preparing a saponin polyphenol with anti-glycation activity is characterized in that, in step 3), the acid solution used is at least one of acetic acid, malic acid, lactic acid, and hydrochloric acid, the concentration of the acid solution is 0~3 M, and the solution is placed in a water bath at 60~90 ℃ for 2~5 h.

[0008] The present invention has the following beneficial effects: This invention removes saponin components mixed in polyphenols through acid hydrolysis, which has the advantages of simple operation, rapid efficiency, and easy control of conditions, while significantly improving anti-glycation activity. Attached Figure Description

[0009] Figure 1 The activity of polyphenols eluted with 20% ethanol in inhibiting glycosylation is shown. Figure 2 The activity of polyphenols eluted with 60% ethanol in inhibiting glycosylation is shown. Detailed Implementation

[0010] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0011] Example 1 The soap pods were washed, dried, pulverized through an 80-mesh sieve, and defatted at 45 °C. After defatting, they were dried again to obtain defatted soap pod powder. The soap pod powder was mixed with 80% ethanol solution at a material-to-liquid ratio of 1:30 g / mL and extracted with ultrasound at 100 W for 30 min. After extraction, the mixture was centrifuged at 8000 rpm for 5 min, and the supernatant was concentrated under reduced pressure until no ethanol remained, yielding a crude extract of soap pod polyphenols. The concentration of polyphenols in the crude extract was measured to be 346.9910 µg / mL, and the concentration of saponins was 22.4180 g / L.

[0012] 25 mL of crude saponin extract was precisely pipetted and mixed with 1 M hydrochloric acid solution at a 1:1 (v:v) ratio. The mixture was then placed in an 80°C water bath and magnetically stirred for 4 h. After filtration, the filtrate was collected, the pH was adjusted to 7, and the volume was brought to 100 mL to obtain acid-hydrolyzed saponin polyphenols. The polyphenol content was measured to be 3.0212 mg and the saponin content was 0.0070 g.

[0013] Example 2 450 g of soapberry powder was weighed and mixed with 80% ethanol solution at a material-to-liquid ratio of 1:30 g / mL. Extraction was performed under ultrasonic power of 100W for 30 min. After extraction, the mixture was centrifuged at 8000 rpm for 5 min. The supernatant was collected and concentrated under reduced pressure until no ethanol remained, yielding a crude extract of soapberry polyphenols. The volume was adjusted to 500 mL, and the polyphenol content was measured to be 3.5950 g and the saponin content to be 29.2848 g.

[0014] The crude extract of saponins was mixed with 6 M hydrochloric acid solution at a ratio of 1:1 (v:v), and then magnetically stirred in an 80 ℃ water bath for 3 h. After filtration, the filtrate was collected, the pH was adjusted to 7, and the volume was brought to 1000 mL to obtain acid-hydrolyzed saponins. The polyphenol content was measured to be 0.6756 g, and no saponins were detected.

[0015] Example 3 Saponins eluted with 2 mg, 3 mg, 4 mg, 5 mg, and 6 mg of 20% and 60% ethanol were weighed out, respectively. The saponins and 10 mg of bovine serum albumin (BSA) were dissolved in 10 mL of 0.01 M phosphate buffer (pH 7.4) containing 0.2% PC 300. 7.5 µL of methylglyoxal (MGO, 40%) was added to induce glycosylation. Aminoguanidine (AG) was used as a positive control, and neither saponins nor AG was added as a negative control. The BSA-MGO glycosylation model was incubated at 37°C for 96 h. Advanced glycation end products (AGEs) were detected at 0 h, 24 h, 48 h, 72 h, and 96 h. The anti-glycosylation activity of the obtained saponins was superior to that of the positive control (AG).

[0016] Comparative Example 1 Compared with Example 1, the only difference is that the acid used for acid hydrolysis is acetic acid, and the polyphenol content was measured to be 7.3809 mg and the saponin content was 0.4093 g.

[0017] Comparative Example 2 Compared with Example 1, the only difference is that the acid used for acid hydrolysis is malic acid, and the polyphenol content was measured to be 7.4729 mg and the saponin content was 0.4084 g.

[0018] Comparative Example 3 Compared with Example 1, the only difference is that the acid used for acid hydrolysis is lactic acid, and the polyphenol content was measured to be 7.7444 mg and the saponin content was 0.4101 g.

Claims

1. A method for preparing saponins with anti-glycation activity, characterized in that... The process includes the following steps: 1) Soap pod pretreatment: Wash, dry, crush, sieve, and degrease the soap pods to obtain degreased soap pod powder; 2) Extraction of crude saponin polyphenols: Take the defatted saponin powder from step 1) and mix it with ethanol solution at a certain solid-liquid ratio, and extract it under ultrasonic assistance; 3) Acid hydrolysis of saponins: Take the crude extract of saponins from step 2) and the acid solution in a certain proportion, and hydrolyze it under appropriate temperature conditions. After hydrolysis, adjust the pH to neutral, filter, and collect the filtrate. The hydrolysate is purified by macroporous resin to finally obtain the purified saponin extract.

2. The method for preparing saponins with anti-glycation activity as described in claim 1, characterized in that... In step 1), the soap pods are crushed and sieved to 80-200 mesh, and then degreased at 20-50 ℃.

3. The method for preparing saponins with anti-glycation activity as described in claim 1, characterized in that... In step 2), the concentration of the ethanol solution is 50-90%, the solid-liquid ratio is 1:10-50, the ultrasonic power is 100-500 W, and the extraction time is 10-40 min.

4. The method for preparing saponins with anti-glycation activity as described in claim 1, characterized in that... In step 3), the acid solution used is at least one of acetic acid, malic acid, lactic acid, and hydrochloric acid, with a concentration of 0-3 M. Hydrolysis is carried out at 60-90 °C for 2-5 h.

5. The method for preparing saponins with anti-glycation activity as described in claim 1, characterized in that... In step 3), the ethanol concentration of the eluent purified by macroporous resin is 20-80%.

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