Preparation method of spina gleditsiae saponin hydrolysate with high acetylcholine enzyme inhibition activity

Abstract

The invention discloses a preparation method of spina gleditsiae saponin hydrolysate with high acetylcholinesterase inhibition activity, and relates to a method for preparing short-chain spina gleditsiae saponin from spina gleditsiae saponin by enzyme hydrolysis, and the acetylcholinesterase inhibition activity is improved by specific enzyme hydrolysis in a manner of reducing saponin glycosyl chains. The adopted hydrolase can effectively convert double-chain saponin of spina gleditsiae into single-chain saponin, and meanwhile, the hydrolysis rate of the hydrolase on total saponin of spina gleditsiae is quantified. Specific enzyme hydrolysis is adopted, different from traditional acid hydrolysis or alkaline hydrolysis, the method has the advantages of being high in hydrolysis efficiency, mild in reaction condition, high in specificity and the like.

Description

Technical Field

[0001] The invention relates to the technical field of natural plant chemistry for preparing acetylcholinesterase inhibitors by extracting, enriching, purifying and modifying natural active products, and specifically relates to a technology for green and efficient hydrolysis of double-chain saponins from saponins of Gleditsia sinensis into single-chain saponins by using biological enzymes. Background Art

[0002] Saponins are abundant secondary metabolites in plants. In plant and animal cells, saponins accumulate in a non-functional form. When encountering pathogens, saponins are converted into active antibiotics under the action of plant enzymes. Saponins are widely distributed in plants, and are distributed in the roots, stems, leaves, and flowers of plants. The content and types in different parts vary greatly. However, the structure of saponins always contains two different parts, namely the polar triterpenoid or steroidal aglycone and the polar oligosaccharide (glycosyl) part. Saponins are divided into monosaccharide glycosides (a single sugar chain is connected to the C-3 of the aglycone) and disaccharide glycosides (one sugar chain is connected to the C-3 of the aglycone, and the second sugar chain is connected to the C-28, C-27, C-26 or C-22 of the aglycone).

[0003] Saponins are non-volatile compounds. The presence of polar (sugar chains) and non-polar (aglycone) groups makes them surface-active compounds with wetting, emulsifying and foaming properties. Saponins have surface-active properties in aqueous solution systems. The connection between fat-soluble aglycones and water-soluble oligosaccharide chains gives saponins the characteristics of amphiphilic molecular processing surfactants, which leads to interactions between saponins and cell membranes. Due to this interaction, saponins exhibit a variety of biological effects on organisms, such as membrane permeation, hemolysis, insecticidal, and antifungal activity.

[0004] Studies have shown that saponins have repellent or deterrent effects, can cause insect molting defects or cause cytotoxicity. The unique structural properties of saponins and the screening of saponin insecticidal properties in previous studies indicate that they have great potential as biopesticides. Saponins have significant insecticidal potential for insects such as aphids, beetles, weevils, leafhoppers, worms, and moths, reducing the food intake of pests, causing indigestion, weight loss, stunted development, and reduced reproduction rates. The main mechanisms of saponins on insects are: affecting the insect digestive system, strengthening the membrane permeability of insects, and preventing the formation of steroid compounds in insects. Saponins affect the food intake, survival, growth rate, and reproduction of certain insects, fungi, bacteria, viruses, and mollusks. The mode of action of saponins on bacteria, fungi, or cancer cells is attributed to their membrane decomposition activity. Studies have shown that saponins can form saponin-sterol complexes with sterols on biofilms. The aggregation of these complexes will induce biofilm perforation and eventually lead to cell death as the membrane permeates. The saponin structure is related to the membrane permeability and biological activity of saponins, so saponins have potential for application in antibacterial and insecticidal applications. Acetylcholinesterase, as an enzyme that regulates the normal life activities of insects, plays an important role in the insect life cycle. If this biological enzyme can be effectively interfered with, biological pests can be effectively reduced.

[0005] The monoadhesive saponins with sugar groups at C-3 are more biologically active than the monoadhesive saponins with sugar groups at C-28. Most of the saponins of Gleditsia sinensis are oleanolic acid type saponins. Oleanane type triterpenoid saponins are classified as triterpenoid glycosides, with two sugar chains connected to the aglycone, no free carboxyl groups, and are double-chain saponins. Therefore, specific hydrolysis of the sugar groups at C-28 to hydrolyze them into single-chain saponins can effectively improve their biological activity. However, the hydrolysis of saponins mostly stays within the scope of broad-spectrum hydrolysis such as acid hydrolysis or alkaline hydrolysis, and there is no targeted hydrolysis of saponins. Biological enzymes have the function of acting on specific biological targets. Therefore, biological enzymes are used to hydrolyze specific fragments of saponins, and while acting on specific positions, the original structure of saponins will not be destroyed. Directed modification of the saponin structure enhances the insecticidal activity of saponins.

[0006] Saponins have become a research hotspot due to their good water solubility, high safety, strong antibacterial effect, broad antibacterial spectrum, and good water solubility. However, there are relatively few studies on saponins, and their mechanism of action remains in the study of the destruction of biofilms. Therefore, it is of great significance to explore the antibacterial mechanism of saponins. Summary of the invention

[0007] The technical problem to be solved by the invention is to overcome the non-directional hydrolysis of the original hydrolysis technology, provide a green and efficient directional hydrolysis method, and convert the low-activity double-chain saponins of Gleditsia sinensis into high-activity single-chain saponins of Gleditsia sinensis. The method is simple in process and low in production cost. At the same time, the method develops a method for characterizing the hydrolysis rate of Gleditsia sinensis saponins by carboxylesterase, which is simple and efficient.

[0008] In order to solve the above technical problems, the basic concept of the technical solution adopted by the present invention is: A method for preparing a saponin hydrolyzate of a saponin having high acetylcholinesterase inhibitory activity, specifically a method for preparing a total saponin of a saponin having a saponin having a high acetylcholinesterase inhibitory activity and a low-sugar sapogenin thereof, which mainly comprises the following steps: S1: Extraction and preparation of total saponins from Gleditsia sinensis: Gleditsia sinensis powder was used as raw material, extracted once with 60% ethanol by ultrasonic method, extracted 3-5 times with concentrated liquid crystal water-saturated n-butanol, combined the extracts and concentrated under reduced pressure, dissolved in water 1-3 times, concentrated under reduced pressure, freeze-dried, and then Soxhlet extraction with petroleum ether was used to remove lipids, and dried to prepare total saponins from Gleditsia sinensis dry powder.

[0009] S2: Elution and fraction collection of total saponins from Gleditsia sinensis: The saponins obtained in S1 were used as raw materials, dissolved in water and then loaded using the D101 wet method. After loading, they were eluted with 10%-90% ethanol, and different fractions were collected and concentrated under reduced pressure. The fractions were redissolved in water for 1-3 times, concentrated under reduced pressure, and freeze-dried to obtain dry powders of different eluted fractions.

[0010] S3: Green directional hydrolysis of saponins from the thorns of Gleditsia sinensis: Carboxylesterase is used to hydrolyze the eluted fraction of total saponins from the thorns of Gleditsia sinensis with the highest purity, and the ester group at the C28 position of oleanolic acid type saponins is directional hydrolyzed into carboxylic acid and oligosaccharides.

[0011] S4: A method for determining the hydrolysis rate of saponin carboxylesterase by using a saponin carboxylesterase: After the hydrolysis, the saponin system is concentrated under reduced pressure and then anhydrous ethanol is added to precipitate oligosaccharides. The total sugar content in the precipitate is determined by the phenol-sulfuric acid method, and the hydrolysis rate is calculated based on the total sugar mass.

[0012] S5: Acetylcholinesterase inhibitory activity: The acetylcholinesterase activity of the Gleditsia sinensis thorn extract, total saponins, each elution fraction, and hydrolyzed saponins were inhibited, with Huperzine A as a positive control.

[0013] The moisture content of the Gleditsia sinensis powder should be less than 6%, and it can be used for extraction after passing through a 60-mesh sieve. The extraction solvent is 60% ethanol, the extraction temperature is 40 min, the liquid-to-solid ratio is 20 mL / g, and the ultrasonic power is 360 W.

[0014] The suction filtration and reduced pressure evaporation pressure are maintained at -0.01Mpa ~ -1Mpa, the temperature for removing ethanol by rotary evaporation is 45°C, the temperature for removing n-butanol is 65°C, the freeze-drying temperature is -30°C, and the freeze-drying time is 3-4 days.

[0015] The D101 macroporous resin was pre-treated by soaking in ethanol for 24 h and rinsing with deionized water to remove the ethanol smell. The elution flow rate of total saponins from Gleditsia sinensis was 5 mL / min and the elution volume was 1-2 column volumes.

[0016] The dosage of carboxylesterase in the hydrolysis of saponins from Gleditsia sinensis was 150 U, the working environment was pH=7, and the temperature was 37 ℃.

[0017] The concentrated volume of the hydrolyzate of Gleditsia sinensis thorns is 1 / 20 of the original volume, and the amount of ethanol used for the alcohol precipitation of oligosaccharides is 50 times the volume of the concentrated solution.

[0018] Beneficial effects: The present invention uses carboxylesterase for the first time to carry out green directional hydrolysis of total saponins from Gleditsia sinensis, targeting the ester group at the C28 position of oleanolic acid type saponins in Gleditsia sinensis, aiming to hydrolyze the ester group into carboxylic acid and remove the sugar fragment at the C28 position in the double-chain saponins. Different from the uncertainty of traditional acid / base hydrolysis, the carboxylesterase hydrolysis of the present invention has the specificity of hydrolysis of ester groups.

[0019] (2) For the first time, the hydrolysis rate of saponins from the thorns of Gleditsia sinensis was quantified by precipitation of oligosaccharides with anhydrous ethanol, and single-chain saponins rich in the thorns of Gleditsia sinensis were isolated.

[0020] (3) The in vitro inhibitory activity of acetylcholinesterase verified that the hydrolyzed single-chain saponin had more efficient acetylcholinesterase inhibitory activity, which is of great significance for subsequent studies on the influence of saponin structure on insecticidal effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The process flow of total saponin extraction, fraction separation, hydrolysis and enzyme inhibition from Gleditsia sinensis spines.

[0022] Figure 2 Saponin quantification standard curve.

[0023] Figure 3 Standard curve for total sugar quantification.

[0024] Figure 4 Purity chart of saponins in various components of Gleditsia sinensis.

[0025] Figure 5 Inhibitory activity of huperzine A on acetylcholinesterase.

[0026] Figure 6 The inhibition rate of saponins from various components of Gleditsia sinensis on acetylcholinesterase. Figure 7 Structural formula of double-chain oleanolic acid-type saponin from Gleditsia sinensis. DETAILED DESCRIPTION

[0027] In order to further explain the technical problems covered by the present invention, the invention is explained and illustrated in combination with the following figures and examples. It should be noted that the specific examples described are only used to explain the scope covered by the present invention and are not used to limit the present application. The methods involved in the present invention are conventional methods unless otherwise specified, and the materials involved are conventional materials unless otherwise specified. Example

[0028] Using Gleditsia sinensis as raw material, the total saponins of Gleditsia sinensis were prepared and enriched. Extraction of Gleditsia sinensis saponins: Take 1 kg of Gleditsia sinensis, crush it with a small grinder and pass it through a 60-mesh sieve. Gleditsia sinensis with a mesh size larger than 60 are crushed repeatedly until all of them are crushed to a size smaller than 60 mesh. Use 20 L 60% ethanol, ultrasonically extract at 50 °C at 360W for 40 min, and filter under reduced pressure to obtain the Gleditsia sinensis extract. Concentrate under reduced pressure to remove ethanol, add water to dissolve 1-3 times, and concentrate under reduced pressure again to remove most of the water. Add water-saturated n-butanol and extract repeatedly for 3-5 times, rotary evaporate n-butanol, add water to dissolve 1-3 times, rotary evaporate to remove most of the water, and freeze-dry at -30 °C for 3-4 d. The dried product is extracted with petroleum ether to remove lipids, and the total saponin powder of Gleditsia sinensis is obtained after drying. Example

[0029] (1) Elution and preparation of saponin fractions from Gleditsia sinensis Take a certain amount of total saponins from Gleditsia sinensis spines, prepare a 20 mg / mL solution with deionized water, wet load, and elute with 10%, 30%, 50%, 70%, and 90% ethanol for 1-2 column volumes, respectively. After collecting different fractions, concentrate under reduced pressure to remove ethanol, and freeze-dry at -30°C for 3-4 days to remove remaining water.

[0030] (2) Purity determination of different elution fractions The vanillin-sulfuric acid method was used to quantify the purity of total saponins and each fraction of Gleditsia sinensis: 0.5 mL of sample solution and 8% vanillin-ethanol solution were taken respectively, mixed evenly, and then 4 mL of 77% sulfuric acid solution was added. After mixing evenly, the mixture was incubated in a 60 ℃ water bath for 15 min, and then the reaction was terminated in an ice water bath for 10 min. After standing at room temperature for 5 min, the absorbance was measured at 544 nm. Oleanolic acid was used as the standard, and the standard curve was calculated as follows: y=1.4276x+0.0167 (R 2 =0.998), the purity of various saponins was calculated based on this standard curve. Figure 2 . Example

[0031] (1) Plotting the standard curve for determining total sugar content using the phenol-sulfuric acid method Prepare glucose solutions with concentrations of 0.01, 0.02, 0.05, 0.08, and 0.1 mg / mL using 1.00 mg / mL glucose standard solution. Accurately measure 2 mL of each of the above solutions, add 1 mL of 5% phenol aqueous solution (take 1 g of phenol in a beaker, add 20 mL of water and dissolve in an 80 ℃ water bath, mix well, and prepare and use immediately.), shake well, then add 5 mL of concentrated H2SO4, heat in a 40 ℃ water bath for 30 min, take out, cool to room temperature, and measure the absorbance at 490 nm. Use the absorbance value A as the ordinate and the concentration C (mg / mL) as the abscissa to draw a standard curve: y=6.4031x+0.0858(R 2 =0.9993), and the content of hydrolyzed oligosaccharides was calculated based on this standard curve.

[0032] (2) Determination of hydrolysis and hydrolysis rate of saponins from saponins from saponins from saponins Most of the saponins from Gleditsia sinensis are double-chain saponins of oleanolic acid type, with an ester group at the C28 position. Therefore, they can be hydrolyzed by carboxylesterase into single-chain saponins and oligosaccharides with carboxylic acid ( Figure 7 ). The hydrolysis conditions are as follows: weigh 150 U of carboxylesterase and hydrolyze at 37°C, pH=7 for 2 h. After the hydrolyzate is concentrated to 1 / 10, add 50 times the volume of anhydrous ethanol and place it at -4-4°C for alcohol precipitation for 24 h. Separate the oligosaccharides at 5000 r / min, and the supernatant is the hydrolyzed single-chain saponin. It is concentrated under reduced pressure and freeze-dried to obtain the hydrolyzed saponin powder.

[0033] The purity of the sugars in the centrifuged oligosaccharide mixture was determined by the phenol-sulfuric acid method ( ω %), and the hydrolysis rate of saponins by carboxylesterase was calculated by formula (1) φ %): φ % = (1) in m 0 is the alcohol precipitation mass, M It is the mass of saponin before hydrolysis.

[0034] The present invention uses alcohol precipitation for the first time to precipitate oligosaccharides hydrolyzed by acetylcholinesterase from saponins of ... Example

[0035] (1) Assay for acetylcholinesterase inhibitory activity The solvents used below are all 0.1 M, pH=8.0 phosphate buffer (PBS), and the experiment is carried out in a 96-well plate. 15 μL of 0.5 U acetylcholinesterase solution is added to 20 μL of the sample solution to be tested, and 145 μL of PBS solution is added to start the reaction; after incubation at 37°C for 20 min, 10 μL of 2mM DTNB (5,5'-dithiothio-(2-nitrobenzoic acid) solution and 10 μL of 15mM ATCI (acetylthiocholine iodide) solution are added, and the absorbance is measured at 405 nm after incubation at 37°C for another 20 min ( A sample The color of the sample itself is used as the blank control ( A b ), PBS was used as negative control ( A con ), Huperzine A (0.05-1 mg / mL) was used as a positive control ( A pos ). Inhibition rate η % is calculated by formula (2).

[0036] η %= ×100% (2) (2) Determination of acetylcholinesterase inhibition rate of different saponins 1 mg / mL total saponins from Gleditsia sinensis, eluted saponins from each fraction, and hydrolyzed saponin solutions were prepared respectively, and their acetylcholinesterase inhibitory activity was determined and analyzed, and the effects of purity and structure on enzyme activity were analyzed. The results showed that the acetylcholinesterase inhibitory activity was positively correlated with the purity of saponins, and the enzyme inhibition rate of hydrolyzed saponins was higher than that of unhydrolyzed saponins. Although the acetylcholinesterase inhibition rate of hydrolyzed saponins was lower than that of the positive control Huperzine A ( Figure 5 ), but it still has great research significance. The present invention shows that the hydrolyzed single-chain saponin enhances the acetylcholinesterase inhibitory activity, indicating that this research result can be used to inhibit insect endogenous enzymes and thus reduce the degree of pests and diseases.

[0037] It should be noted that: The above embodiments are only to illustrate the preferred examples of the present invention, and are not to be formally limited to the present invention. Studies have shown that the insecticidal activity of double-chain saponins is weaker than that of single-chain saponins, so the transformation of double-chain saponins and the definition and quantification of saponin hydrolysis rates have important research significance. This experiment preliminarily completed the hydrolysis and hydrolysis rate determination of oleanolic acid type double-chain saponins in Gleditsia sinensis thorns, as well as the inhibition activity determination of acetylcholinesterase of each saponin component, further proving that the activity of single-chain saponins is better than that of double-chain saponins, which has certain research value and significance for subsequent insecticidal research.

Claims

1. A method for preparing a hydrolyzate of saponin from Gleditsia sinensis having high acetylcholinesterase inhibitory activity, comprising the following specific steps: S1: Extraction and preparation of total saponins from Gleditsia sinensis: Gleditsia sinensis was used as the raw material, crushed and extracted with 60% ethanol under ultrasound, the extraction number was 1, the extract was filtered, concentrated under reduced pressure to remove ethanol, and then extracted with water-saturated n-butanol 3-5 times. The upper n-butanol phase was concentrated under reduced pressure to remove n-butanol, redissolved in water 1-3 times, concentrated under reduced pressure, and freeze-dried at -30 °C to remove the remaining water. The dried product was defatted with petroleum ether to obtain the total saponins from Gleditsia sinensis.

2. S2: Elution of total saponins from Gleditsia sinensis: Using the total saponins from Gleditsia sinensis obtained in S1 as raw materials, D101 macroporous resin was used to elute the total saponins to obtain saponins of higher purity. 10%-90% ethanol was used to elute the saponins from Gleditsia sinensis, and different eluents were collected, rotary evaporated, and freeze-dried to obtain different elution fractions of total saponins from Gleditsia sinensis. 3.S3: A green directional hydrolysis method for saponins from the thorns of Gleditsia sinensis: Carboxylesterase is used to hydrolyze the saponins from the eluted fractions of S2 with the highest purity, and the C28 ester group in the saponins from the thorns of Gleditsia sinensis is directional hydrolyzed to hydrolyze the ester group into the C28 carboxyl group and oligosaccharide fragments. 4.S4: A method for determining the hydrolysis rate of saponin by carboxylesterase: After the hydrolyzed single-chain saponin is concentrated under reduced pressure, anhydrous ethanol is added to precipitate oligosaccharides, and the hydrolysis rate of saponin by carboxylesterase is calculated based on the mass of oligosaccharides. 5.S5: An acetylcholinesterase inhibition rate determination: The acetylcholinesterase inhibition rates of Gleditsia sinensis extract, Gleditsia sinensis total saponins, Gleditsia sinensis total saponins elution fractions, and Gleditsia sinensis single-chain saponin hydrolyzate were determined and evaluated.

6. The method for preparing total saponins from Gleditsia sinensis and highly active low-glucosyl sapogenins thereof according to claim 1, characterized in that: S1 Gleditsia sinensis powder should be sieved through a 60-mesh sieve and the moisture content should not exceed 6% before it can be used for subsequent extraction. The extraction temperature is 50°C, the extraction time is 40 min, and the liquid-to-solid ratio is 20 mL / g. The temperature for removing ethanol by vacuum filtration is 45°C, and the temperature for removing n-butanol is 65°C, and the pressure is controlled at -0.01 Mpa ~ -0.1 Mpa.

7. The method for preparing total saponins from Gleditsia sinensis and highly active low-glucosyl sapogenins thereof according to claim 1, characterized in that: The D101 macroporous resin described in S2 should be pretreated in advance. The purchased resin was soaked in anhydrous ethanol for 24 hours to remove broken particles, and then repeatedly rinsed with deionized water until there was no ethanol. Wet column packing and wet loading were used, the elution phase flow rate was 5 mL / min, and 10%, 30%, 50%, 70%, and 90% elution phases were collected.

8. The method for preparing total saponins from Gleditsia sinensis and highly active low-glucosyl sapogenins thereof according to claim 1, characterized in that: The dosage of carboxylesterase in S3 was 150 U, the hydrolysis environment was pH = 7.0, and the temperature was 37 ℃.

9. The method for preparing total saponins from Gleditsia sinensis and highly active low-glucosyl sapogenins thereof according to claim 1, characterized in that: The steps of hydrolyzing saponins with carboxylesterase in S3 and S4 are: enzymatic hydrolysis → centrifugation → rotary evaporation concentration → alcohol precipitation of oligosaccharides → centrifugal separation of oligosaccharides → collection of supernatant → concentration of supernatant → hydrolysis of saponins.

10. The method for preparing total saponins from Gleditsia sinensis and highly active low-glucosyl sapogenins thereof according to claim 1, characterized in that: In S4, the saponins with the highest purity in the eluted fraction of total saponins from Gleditsia sinensis were hydrolyzed. Oligosaccharides were precipitated using 50 times the volume of the concentrate in ethanol, and the centrifuge was 5000 r / min. The hydrolysis rate was the total mass of sugars produced divided by the mass of saponins before hydrolysis.

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