CN109316501B - Method for extracting triterpenoid saponin from Gyroscope seed meal - Google Patents

Method for extracting triterpenoid saponin from Gyroscope seed meal Download PDF

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CN109316501B
CN109316501B CN201811457822.7A CN201811457822A CN109316501B CN 109316501 B CN109316501 B CN 109316501B CN 201811457822 A CN201811457822 A CN 201811457822A CN 109316501 B CN109316501 B CN 109316501B
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seed meal
gyroscope
saponin
gyroscopic
triterpenoid saponin
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许晓岗
童丽丽
冯凯
赵子荀
许中秋
张鑫洁
刘政
吴秀萍
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Nanjing Forestry University
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Abstract

The invention relates to a method for extracting triterpene saponin from Gyroscope seed meal, which comprises the steps of sequentially adding cellulase, beta-glucosidase and citric acid buffer solution into dried Gyroscope seed meal with the granularity of 20-120 meshes, then placing the dried Gyroscope seed meal on a shaking bed, reacting for 0.5-2.5h under the conditions of 45-55 ℃ and 100-200r/min, filtering the reaction solution to obtain a dissolution liquid of the triterpene saponin, then adding macroporous adsorption resin for adsorption for 1-5h, and filtering to obtain the macroporous adsorption resin adsorbed with the triterpene saponin; adding ethanol solution for desorption, and removing ethanol solution to obtain pure triterpene saponin. The invention provides a method for extracting triterpenoid saponin from the Gyroscope seed meal by taking the Gyroscope seed meal generated after oil pressing as a raw material.

Description

Method for extracting triterpenoid saponin from Gyroscope seed meal
Technical Field
The invention relates to a method for extracting triterpenoid saponin from Gyroscope seed meal, belonging to the technical field of natural medicine extraction.
Background
Gyroscope fruit (Melliodendron xylocarpum hand-Mazz.) is similar to Gyroscope, so the fruit is named Gyroscope fruit, is a plant of Gyroscope of Styracaceae (Melliodendron), is also called as jasmine, fructus benincasae and fructus bruceae in folk, produces Yunnan southeast, Sichuan southeast, Guizhou, Guangxi (except southeast), Hunan and Guangdong middle parts, and is a unique plant in China, namely North, Jiangxi and Fujian. Gyroscope trees are beautiful in shape, the trunk is straight, flowers are firstly planted and then leaves are planted, the flowers are full of trees and flower like snow when the flowers are full of flowers, and the Gyroscope trees can be used as garden ornamental trees and street trees. The tree species wood is yellow-white and compact in material, is a good tree species, and is suitable for manufacturing furniture, tools and the like. The Gyroscope seed is rich in oil, the main oil production part is kernel, the oil content is up to 49.6%, the Gyroscope seed is edible and is a good oil plant, and the seed also contains a large amount of various compounds such as triterpenoid saponin and the like. After the Gyroscope seeds are used for oil extraction, the generated Gyroscope seed meal (Gyroscope seed oil extraction meal) is usually discarded, and the Gyroscope seed meal also contains various compounds such as triterpenoid saponins, and the triterpenoid saponins have wide pharmacological effects and important biological activities, and particularly have remarkable effects in the aspects of antibiosis, anti-inflammation, liver protection, anticancer, body immune regulation and the like, so that the problem of resource waste exists.
At present, the main methods for extracting triterpenoid saponin include a water extraction method, an ultrasonic extraction method, an organic solvent method, a chemical purification method and the like, but the main methods all have the problems of complex process, high requirement on conditions, high energy consumption and the like, and the utilization rate of triterpenoid saponin compound natural products and the like in benzoin plants such as gyroscopic fruit and the like is restricted.
Disclosure of Invention
The invention aims to solve the defects of the prior art, provides the method for extracting the triterpenoid saponin from the Gyroscope seed meal, fully utilizes the Gyroscope seed meal generated after oil pressing, and has the advantages of simple method for extracting the triterpenoid saponin, low energy consumption and high extraction efficiency.
Technical scheme
A method for extracting triterpenoid saponin from Gyroscope seed meal comprises the following steps:
(1) dissolution: sequentially adding cellulase, beta-glucosidase and citric acid-sodium citrate buffer solution into dried 2g of Gyroscope seed meal with the granularity of 20-120 meshes, then placing the mixture on a shaking bed, reacting for 0.5-2.5h under the conditions of 45-55 ℃ and 100-200r/min, and filtering the reaction liquid to obtain a dissolving liquid of the triterpenoid saponin;
(2) adsorption: adding 2g of macroporous adsorption resin into the triterpene saponin dissolving liquid in the step (1), adsorbing for 1-5h at the temperature of 45-55 ℃ and under the condition of 100-;
(3) desorbing: and (3) adding an ethanol solution into the macroporous adsorption resin adsorbing the triterpene saponin in the step (2) for desorption, and then removing the ethanol solution to obtain a pure product of the triterpene saponin.
Further, in the step (1), the granularity of the Gyroscope seed meal is 80-100 meshes.
Further, in the step (1), the dosage of the cellulase and the dosage of the beta-glucosidase are both 1-2.5%.
Further, in the step (1), the citric acid-sodium citrate buffer solution is formed by mixing 9.2mL of 0.1mol/L citric acid solution and 10.8mL of 0.1mol/L sodium citrate solution.
Further, in the step (1), the solid-to-liquid ratio of the gyroscopic fruit seed meal to the citric acid buffer solution is 1:5-30, and more preferably 1: 25.
Further, in the step (1), the reaction temperature is 50 ℃, the rotation speed is 150r/min, and the reaction time is 1 h.
Further, in the step (3), the volume concentration of the ethanol solution is 80-100%, and more preferably 90%. When the volume fraction of the ethanol is continuously increased, the desorption rate of the triterpenoid saponin is increased, when the volume fraction reaches 90%, the desorption rate of the triterpenoid saponin is stable, and from the economic point of view, the ethanol with the volume fraction of 90% is used for desorbing the triterpenoid saponin optimally.
The invention has the beneficial effects that: the invention provides a method for extracting triterpenoid saponin from the Gyroscope seed meal by taking the Gyroscope seed meal generated after oil pressing as a raw material.
Drawings
FIG. 1 is a dissolution standard curve of triterpene saponin;
FIG. 2 is a UV spectrum of triterpene saponin extracted in example 1;
FIG. 3 is a graph showing the radical scavenging activity of triterpene saponin extracted in example 1.
Detailed Description
The invention is further described with reference to the following figures and specific examples.
In the following examples, Gyroscope seed, produced in Guangxi Jinxiu province, with a triterpene saponin content of 16%, was collected from Guangxi Jinxiu Daoyoshan forest farm; cellulase, having a trademark of Euter L-100, and filter paper enzyme activity of 188.54U, provided by Hunan Euter Biochemical Co., Ltd; beta-glucosidase, with the brand number of BG-188, the enzyme activity of the beta-glucosidase is 237.54U, provided by Beijing Novoxil; macroporous adsorption resin, the mark of which is AB-8, is provided by biological engineering (Shanghai) corporation. But are not limited thereto.
The composition analysis of the oil-pressed Gyroscope seed meal is shown in Table 1:
TABLE 1 Gyroscope seed meal principal ingredients
Figure BDA0001888081930000021
Figure BDA0001888081930000031
Example 1
A method for extracting triterpenoid saponin from Gyroscope seed meal comprises the following steps:
(1) dissolution: sequentially adding cellulase (1%), beta-glucosidase (1%) and 50mL of citric acid-sodium citrate (formed by mixing 9.2mL of 0.1mol/L citric acid solution and 10.8mL of 0.1mol/L sodium citrate solution) buffer solution into 2g of dried gyro fruit seed meal (the granularity of the gyro fruit seed meal is 80-100 meshes), wherein the solid-to-liquid ratio of the gyro fruit seed meal to the citric acid buffer solution is 1:25, then placing the gyro fruit seed meal on a shaking bed, reacting for 1h under the conditions of 50 ℃ and 150r/min, and filtering the reaction solution to obtain a triterpene saponin dissolving solution;
(2) adsorption: adding 2g of macroporous adsorption resin into the triterpene saponin dissolving liquid in the step (1), adsorbing for 4h at 50 ℃ at 150r/min, and filtering to obtain macroporous adsorption resin with triterpene saponin adsorbed thereon;
(3) desorbing: and (3) adding 10mL of 90% ethanol solution into the macroporous adsorption resin adsorbing the triterpene saponin in the step (2) for desorption, and then removing the ethanol solution to obtain a pure product of the triterpene saponin. The ultraviolet spectrum of the product is shown in figure 2, and the product has maximum absorption at about 215nm of ultraviolet, which indicates that the product is triterpene saponin.
Measuring the concentration of the triterpenoid saponin in the dissolution liquid in the step (1) and calculating the dissolution rate:
the dissolution rate (%) of triterpene saponin is that the concentration of triterpene saponin is multiplied by the volume of the solution/the mass of the Gyroscope seed is multiplied by 100%.
The content of the triterpene saponin is measured by a vanillin concentrated sulfuric acid method, the absorbance of different mass concentrations is measured at the maximum absorption wavelength, the content of the triterpene saponin is taken as the abscissa, the absorbance is taken as the ordinate, and the standard curve is shown in figure 1, so that the concentration of the triterpene saponin presents a good linear relationship within the range of 0.7g/L, which shows that the content of the triterpene saponin can be accurately and rapidly measured by the vanillin-concentrated sulfuric acid method.
In this example, the dissolution rate of triterpene saponin in the triterpene saponin dissolution liquid was 4.05%.
Example 2
In the step (1), the granularity of the adopted gyroscopic fruit seed meal is 20-40 meshes, and the rest is the same as that in the example 1. And (3) measuring the concentration of the triterpenoid saponin in the dissolution liquid and calculating the dissolution rate, wherein the dissolution rate is 2.41%.
Example 3
In the step (1), the granularity of the adopted gyroscopic fruit seed meal is 40-60 meshes, and the rest is the same as that in the example 1. And (3) measuring the concentration of the triterpenoid saponin in the dissolution liquid and calculating the dissolution rate, wherein the dissolution rate is 3.1%.
Example 4
In the step (1), the granularity of the adopted gyroscopic fruit seed meal is 60-80 meshes, and the rest is the same as that in the example 1. And (3) measuring the concentration of the triterpenoid saponin in the dissolution liquid and calculating the dissolution rate, wherein the dissolution rate is 3.5%.
Example 5
In the step (1), the granularity of the Gyroscope seed meal is 100-120 meshes, and the rest is the same as that in the example 1. And (3) measuring the concentration of the triterpenoid saponin in the dissolution liquid and calculating the dissolution rate, wherein the dissolution rate is 4.07%.
As can be seen from the dissolution rates of the triterpene saponins in the dissolution liquids of examples 1 to 5, the material particle size is also one of the factors affecting the enzymolysis effect of the Gyroscope seed meal, and as the mesh number increases, that is, the material particle size becomes finer and finer, the dissolution rate of the triterpene saponins increases, the dissolution rate of 20 to 40 meshes is only 2.41%, and when the mesh number is 80 to 100 meshes, the dissolution rate begins to become gentle and is 3.8%.
Example 6
In the step (1), the solid-to-liquid ratio of the gyroscopic fruit seed meal to the citric acid buffer solution is 1:5, and the rest is the same as that in the example 1. And (3) measuring the concentration of the triterpenoid saponin in the dissolution liquid and calculating the dissolution rate, wherein the dissolution rate is 3.25%.
Example 7
In the step (1), the solid-to-liquid ratio of the gyroscopic fruit seed meal to the citric acid buffer solution is 1:10, and the rest is the same as that in the example 1. And (3) measuring the concentration of the triterpenoid saponin in the dissolution liquid and calculating the dissolution rate, wherein the dissolution rate is 3.38%.
Example 8
In the step (1), the solid-to-liquid ratio of the gyroscopic fruit seed meal to the citric acid buffer solution is 1:15, and the rest is the same as that in the example 1. And (3) measuring the concentration of the triterpenoid saponin in the dissolution liquid and calculating the dissolution rate, wherein the dissolution rate is 3.3%.
Example 9
In the step (1), the solid-to-liquid ratio of the gyroscopic fruit seed meal to the citric acid buffer solution is 1:20, and the rest is the same as that in the example 1. And (3) measuring the concentration of the triterpenoid saponin in the dissolution liquid and calculating the dissolution rate, wherein the dissolution rate is 3.26%.
Example 10
In the step (1), the solid-to-liquid ratio of the gyroscopic fruit seed meal to the citric acid buffer solution is 1:30, and the rest is the same as that in the example 1. And (3) measuring the concentration of the triterpenoid saponin in the dissolution liquid and calculating the dissolution rate, wherein the dissolution rate is 3.3%.
The dissolution rates of comparative examples 1 and 6 to 10 revealed that the dissolution rates of triterpene saponins were about 3.3% under the conditions of a solid-to-liquid ratio of 1:5, 1:10, 1:15, 1:20 and 1:30, but the dissolution rate of triterpene saponins was highest under the conditions of a solid-to-liquid ratio of 1: 25.
Comparative example 1
In the step (1), the procedure was the same as in example 1 except that cellulase and β -glucosidase were not added. And (3) measuring the concentration of the triterpenoid saponin in the dissolution liquid and calculating the dissolution rate, wherein the dissolution rate is 2.2%.
The anti-oxidation activity of the triterpenoid saponin is tested by a DPPH free radical scavenging method:
DPPH free radical is a stable free radical, purple in organic solvents, with greater absorption at 517nm, and when an antioxidant is added, some of the free radical is scavenged, causing a reduction in the wavelength absorption. The antioxidant activity of the substance was evaluated by decreasing the absorbance at this wavelength as the antioxidant activity increased.
The determination method comprises the following steps: the triterpene saponin extracted in example 1 is prepared into 10mg/ml triterpene saponin solution by using absolute ethyl alcohol, and is diluted into sample solution with a series of concentrations of 1mg/ml, 2mg/ml, 3mg/ml, 4mg/ml and 5mg/ml by using the absolute ethyl alcohol when in use. Adding 2mL of sample solution to be tested into 2mL of LDPPH, storing the solution in a water bath at 25 ℃ in a dark place for 30min, and measuring the absorbance of the sample solution at 517nm as A1(ii) a Taking 2mL of absolute ethyl alcohol solution to replace the sample to measure the absorbance A0(ii) a Adding 2mL of absolute ethyl alcohol solution into 2mL of sample to measure the background absorbance A of the sample2；
Clearance K (%) ═ a0－(A1﹣A2)]/A0×100％
The results are shown in FIG. 3. As can be seen from fig. 3, the triterpene saponin contained in the seed meal of gyroscopic fruit has an ability to scavenge free radicals, and its scavenging ability increases with the increase in concentration. Therefore, it can be concluded that the triterpenoid saponin in the Gyroscope seed meal has a certain oxidation resistance, and the oxidation resistance of the triterpenoid saponin increases with the increase of the concentration.

Claims (8)

1. A method for extracting triterpenoid saponin from Gyroscope seed meal is characterized by comprising the following steps:
(1) dissolution: sequentially adding cellulase, beta-glucosidase and citric acid-sodium citrate buffer solution into dried 2g of Gyroscope seed meal with the granularity of 20-120 meshes, then placing the mixture on a shaking bed, reacting for 0.5-2.5h under the conditions of 45-55 ℃ and 100-200r/min, and filtering the reaction liquid to obtain a dissolving liquid of the triterpenoid saponin;
(2) adsorption: adding 2g of macroporous adsorption resin into the triterpene saponin dissolving liquid in the step (1), adsorbing for 1-5h at the temperature of 45-55 ℃ and under the condition of 100-;
(3) desorbing: and (3) adding an ethanol solution into the macroporous adsorption resin adsorbing the triterpene saponin in the step (2) for desorption, and then removing the ethanol solution to obtain a pure product of the triterpene saponin.
2. The method for extracting triterpenoid saponin from gyroscopic fruit seed meal as claimed in claim 1, wherein in step (1), the particle size of the gyroscopic fruit seed meal is 80-100 meshes.
3. The method for extracting triterpenoid saponin from the gyroscopic fruit seed meal as claimed in claim 1, wherein in the step (1), the dosage of the cellulase and the beta-glucosidase in the step (1) is 1-2.5% of the dosage of the gyroscopic fruit seed meal.
4. The method for extracting triterpenoid saponin from the Gyroscope seed meal as claimed in claim 1, wherein in the step (1), the citric acid-sodium citrate buffer solution is prepared by mixing 9.2mL of 0.1mol/L citric acid solution and 10.8mL of 0.1mol/L sodium citrate solution.
5. The method for extracting triterpenoid saponin from gyroscopic fruit seed meal as claimed in claim 1, wherein in the step (1), the solid-to-liquid ratio of the gyroscopic fruit seed meal to the citric acid-sodium citrate buffer solution is 1: 5-30.
6. The method for extracting triterpenoid saponin from gyroscopic fruit seed meal as claimed in claim 5, wherein the solid-to-liquid ratio of the gyroscopic fruit seed meal to the citric acid-sodium citrate buffer solution is 1: 25.
7. The method for extracting triterpenoid saponin from the gyroscopic fruit seed meal as claimed in claim 1, wherein in the step (1), the reaction temperature is 50 ℃, the rotation speed is 150r/min, and the reaction time is 1 h.
8. The method for extracting triterpenoid saponin from gyroscopic fruit seed meal as claimed in any one of claims 1 to 7, wherein the ethanol solution in step (3) has a volume concentration of 80-100%.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102048885A (en) * 2011-01-10 2011-05-11 广州市中医医院 Extraction process for lychee seed saponin
CN103961388A (en) * 2014-05-22 2014-08-06 江苏博益生物科技有限公司 Method for preparing total saponins of radix cynanchi bungei
CN104971084A (en) * 2015-07-21 2015-10-14 安徽师范大学 Purifying method of gypenoside

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102048885A (en) * 2011-01-10 2011-05-11 广州市中医医院 Extraction process for lychee seed saponin
CN103961388A (en) * 2014-05-22 2014-08-06 江苏博益生物科技有限公司 Method for preparing total saponins of radix cynanchi bungei
CN104971084A (en) * 2015-07-21 2015-10-14 安徽师范大学 Purifying method of gypenoside

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