Separation method of protein and secondary metabolite in plant exosome, electronic equipment and medium

Abstract

The invention discloses a centrifugal time and centrifugal force calculation method, which can efficiently separate protein and secondary metabolite components in exosomes by combining the centrifugal time and centrifugal force calculated by the method with an ultrafiltration centrifugal technology, and is suitable for analysis and application of plant exosomes. And a foundation is provided for further research of the plant exosome and application of the plant exosome in the fields of biotechnology and traditional Chinese medicine research and development.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a method for separating proteins and secondary metabolites in plant exosomes, an electronic device, and a medium. Background Art

[0002] Plant exosomes are small vesicles secreted by plant cells into the extracellular environment through exocytosis, usually with a diameter between 30 and 150 nanometers. As a medium for intercellular communication, exosomes can carry a variety of bioactive molecules, including proteins, lipids, RNA, and secondary metabolites. These components play important roles in plant growth and development, environmental stress responses, immune responses, and intercellular signal transduction. Therefore, the research on exosomes has gradually become a hot topic in plant biology and agricultural technology in recent years. Existing studies have shown that plant exosomes contain a variety of functional proteins and secondary metabolites, which not only play important roles in plant physiology but also have potential biotechnological application values. For example, specific proteins in plant exosomes can regulate cellular immune responses, while some of the secondary metabolites have antioxidant, anti-inflammatory, and other effects. There is no report in the current literature on the effective separation of proteins and secondary metabolites in plant exosomes, which poses an obstacle to the development and research of plant exosomes. Therefore, the separation and extraction of exosomes have become an important research direction in plant biology, agricultural biotechnology, and traditional Chinese medicine research.

[0003] Despite the growing importance of exosomes in botanical research and biotechnology, existing technologies still have significant limitations in the separation of proteins and secondary metabolites in exosomes (M.Cao, N.Diao, X.Cai, X.Chen, Y.Xiao, C.Guo, D.Chen, X.Zhang, Mater. Horiz. 2023.). Generally, existing exosome separation technologies mainly rely on methods such as ultracentrifugation, differential centrifugation, density gradient centrifugation, and ultrafiltration. Although exosomes can be effectively separated from plants, it is difficult to further efficiently and precisely separate proteins and secondary metabolites in exosomes (H.A. Dad, T.-W. Gu, A.-Q. Zhu, L.-Q. Huang, L.-H. Peng, Molecular Therapy 2021, 29, 13-31.). This limitation hinders the in-depth study of the functions and components of plant exosomes and also limits the practical applications of exosomes in the fields of plant pathology, ecology, agricultural applications, etc. There are reports in the literature on the use of immunomagnetic beads to separate plant-derived exosomes. By using immunomagnetic beads, plant-derived exosomes can be separated under the action of a magnetic field. This method can identify specific proteins on the surface of exosomes. This method can control the magnetic field to achieve the total or selective separation of exosomes, but it is relatively expensive and the operating conditions are relatively complex. Existing separation methods, especially the separation of proteins and secondary metabolites, usually rely on differences in physical or chemical properties. However, due to the insignificant differences in molecular weight and structure between proteins and secondary metabolites in exosomes, traditional separation technologies face technical bottlenecks in separating these components in such small vesicles. For example, although density gradient centrifugation can perform partial separation based on molecular density differences, the separation effect is often not ideal for small-molecular-weight secondary metabolites and larger proteins. Although ultrafiltration can fractionate according to molecular size, its rejection rate for secondary metabolites is low, easily leading to component mixing. In addition, for different plant species, the types and proportions of proteins and secondary metabolites contained in exosomes vary greatly, making it difficult for separation technologies with poor generality to be applicable to a wide range of plant species. The exosomes of different plants not only differ in composition but also in biological activity. For example, the exosome components of herbaceous plants and fruit plants are quite different. The exosomes of herbaceous plants focus more on signal transduction functions, while fruit plants are richer in compounds with functions such as antioxidant. This difference further increases the difficulty of separating plant exosomes. Summary of the Invention

[0004] The present invention aims to at least solve one of the above technical problems existing in the prior art. To this end, the object of the present invention is to provide a method for separating proteins and secondary metabolites in plant exosomes.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] The first aspect of the present invention provides a calculation method for centrifugation time and centrifugal force. The centrifugation time and centrifugal force calculated by the calculation method are used to separate proteins and secondary metabolites from plant exosomes by ultrafiltration centrifugation. The calculation method includes:

[0007] Obtain the numerical value Me of the molecular weight of proteins in the plant exosome sample;

[0008] Obtain the numerical value Pe of the protein content in the plant exosome sample;

[0009] Obtain the numerical value Rn of the cut-off molecular weight of the ultrafiltration centrifuge tube, where Me - Rn is the smallest positive number greater than 0;

[0010] Calculate the centrifugation time. The numerical calculation formula for the centrifugation time is:

[0011] t = 1.2 * [(Me - Rn) + 10 * Pe] + 30;

[0012] Calculate the centrifugal force. The numerical calculation formula for the centrifugal force is:

[0013] N = [(Me - Rn) * 1000 + Me * 5000 + 10000] / MePe;

[0014] When the unit of the molecular weight of the protein is KDa, the unit of the protein content is mg / mL, and the unit of the cut-off molecular weight of the ultrafiltration centrifuge tube is KDa, the unit of the calculated centrifugation time is minutes, and the unit of the centrifugal force is g.

[0015] In the present invention, "plant exosomes" refer to tiny vesicles secreted by plant cells into the extracellular environment through exocytosis and can be prepared from plant tissues. In the present invention, "secondary metabolites" refer to a class of small molecular organic compounds that are non-essential for the normal operation of plant growth and development produced by secondary metabolism. In the present invention, "when the unit of the molecular weight of the protein is KDa, the unit of the protein content is mg / mL, and the unit of the cut-off molecular weight of the ultrafiltration centrifuge tube is KDa, the unit of the calculated centrifugation time is minutes, and the unit of the centrifugal force is g", it can be understood that those skilled in the art can obtain calculation formulas different from those of the present application according to conventional unit conversions and the calculation formulas provided in the present application, and these calculation formulas are also within the protection scope of the present application.

[0016] In some embodiments, the molecular weight of the protein is determined by SDS-PAGE method.

[0017] In some embodiments, the protein content is determined by BCA method.

[0018] In some embodiments, the range of Me is 10 - 100, and the range of Pe is 1 - 5.

[0019] In some embodiments, Rn is selected from the cut-off molecular weight values of available ultrafiltration centrifugal tubes, including but not limited to 3, 10, 30, 50, 100. It can be understood that the selection of Rn needs to consider the cut-off molecular weight of the available ultrafiltration centrifugal tubes, and only the cut-off molecular weight values of common ultrafiltration centrifugal tubes are listed here.

[0020] In some embodiments, the plant exosomes include herbaceous plant exosomes and / or fruit plant exosomes.

[0021] In some embodiments, the herbaceous plant exosomes include centella asiatica exosomes; and / or

[0022] The fruit plant exosomes include rosa roxburghii tratt exosomes, seabuckthorn exosomes, and evodia rutaecarpa exosomes.

[0023] The second aspect of the present invention provides a method for separating proteins and secondary metabolites from plant exosomes, including

[0024] Calculating the centrifugation time and centrifugal force through the calculation method described in the first aspect;

[0025] Separating proteins and secondary metabolites from plant exosomes by ultrafiltration centrifugation according to the centrifugation time and centrifugal force.

[0026] The third aspect of the present invention provides a device for calculating centrifugation time and centrifugal force, including:

[0027] An acquisition module: acquiring the numerical value Me of the molecular weight of proteins in a plant exosome sample, the numerical value Pe of the protein content in the plant exosome sample, and the numerical value Rn of the cut-off molecular weight of an ultrafiltration centrifugal tube;

[0028] A calculation module: obtaining the numerical value of the centrifugation time and the numerical value of the centrifugal force based on the numerical value Me of the molecular weight of proteins in the plant exosome sample, the numerical value Pe of the protein content in the plant exosome sample, and the numerical value Rn of the cut-off molecular weight of the ultrafiltration centrifugal tube acquired by the acquisition module.

[0029] In some embodiments, Rn satisfies that Me - Rn is the smallest positive number greater than 0;

[0030] The calculation formula for the numerical value of the centrifugation time is:

[0031] t = 1.2 * [(Me - Rn) + 10 * Pe] + 30;

[0032] The calculation formula for the numerical value of the centrifugal force is:

[0033] N = [(Me - Rn) * 1000 + Me * 5000 + 10000] / MePe。

[0034] The present invention provides an electronic device in four aspects, including:

[0035] A memory for storing a computer program;

[0036] A processor for executing the computer program to implement the calculation method of centrifugation time and centrifugal force as described in the first aspect.

[0037] The present invention provides a computer-readable storage medium in a fifth aspect. The computer-readable storage medium is used to store a computer program, and when the computer program is executed by a processor, it implements the calculation method of centrifugation time and centrifugal force as described in the first aspect.

[0038] The beneficial effects of the present invention are as follows: The centrifugation time and centrifugal force calculated by the centrifugation time and centrifugal force calculation method provided by the present invention, combined with the ultrafiltration centrifugation technology, can efficiently separate the protein and secondary metabolite components in exosomes, and are applicable to the analysis and application of plant exosomes, providing a basis for the further research of plant exosomes and their applications in the fields of biotechnology and traditional Chinese medicine research and development. Description of the Drawings

[0039] Figure 1 For the separation effect in Example 2, the left figure is the protein concentration, and the right figure is the content of asiatic acid.

[0040] Figure 2 For the separation effect of the ultrafiltration centrifugal tube with a molecular weight cut-off of 30KDa in Example 3, the left figure is the protein concentration, and the right figure is the content of asiatic acid.

[0041] Figure 3 For the separation effect of the ultrafiltration centrifugal tube with a molecular weight cut-off of 100KDa in Example 3, the left figure is the protein concentration, and the right figure is the content of asiatic acid.

[0042] Figure 4 For the separation effect at a centrifugal force of 4000g in Example 4, the left figure is the protein concentration, and the right figure is the content of asiatic acid.

[0043] Figure 5 For the separation effect at a centrifugal force of 7000g in Example 4, the left figure is the protein concentration, and the right figure is the content of asiatic acid.

[0044] Figure 6 For the separation effect at a centrifugation time of 30 min in Example 5, the left figure is the protein concentration, and the right figure is the content of asiatic acid.

[0045] Figure 7For the separation effect at a centrifugation time of 80 min in Example 5, the left figure shows the protein concentration and the right figure shows the asiatic acid content.

[0046] Figure 8 For the separation effect in Example 6, the left figure shows the protein concentration and the right figure shows the vitamin C content.

[0047] Figure 9 For the separation effect in Example 7, the left figure shows the protein concentration and the right figure shows the vitamin C content. Detailed implementation manners

[0048] The content of the present invention will be further described in detail through specific examples below. The raw materials, reagents or devices used in the examples can be obtained from conventional commercial channels or by existing technical methods without special instructions. Unless otherwise specified, the test or measurement methods are conventional methods in the art.

[0049] In the embodiments of the present invention, exosomes of Centella asiatica, Ziziphus jujuba, Euodia rutaecarpa, Hippophae rhamnoides, and Rosa roxbunghii are used as research objects. As a herbaceous plant, the proteins and compounds rich in the exosomes of Centella asiatica have relatively uniform molecular weights and relatively clear biological characteristics, so they can provide reliable data support for the derivation of the formula. As a fruit plant, the protein and compound components of the exosomes of Hippophae rhamnoides are more complex and have a large molecular weight difference. Therefore, the experimental verification of Hippophae rhamnoides exosomes has important representative significance.

[0050] Exosome extraction:

[0051] 1) Take fresh plant raw materials, add 4°C phosphate buffer PBS and break them into juice to obtain crude extract A; the mass ratio of fresh plant raw materials to phosphate buffer PBS is 1:1.5; the time for breaking into juice is 2 minutes.

[0052] 2) Add PBS ice cubes to crude extract A and break them into juice for 6 minutes to obtain crude juice extract B; the mass ratio of crude extract A to PBS ice cubes is 2:1;

[0053] 3) After standing the crude juice extract, filter it through a 100-mesh gauze to obtain supernatant A (plant raw material crushing solution);

[0054] 4) Centrifuge supernatant A at 2000×g for 10 minutes at 4°C to obtain supernatant B;

[0055] 5) Centrifuge the supernatant B at 4000×g for 30 minutes at 4°C to obtain supernatant C;

[0056] 6) Centrifuge the supernatant C at 10000×g for 90 minutes at 4°C to obtain supernatant D;

[0057] 7) Centrifuge the supernatant D at 150000×g for 120 minutes at 4°C to obtain precipitate A;

[0058] 8) Take precipitate A and resuspend it in PBS at 4°C in a mass ratio of 1:50 to obtain the refined extract A;

[0059] 9) Prepare sucrose-PBS solutions with mass fractions of 60%, 45%, 30%, and 15%. Add them to the bottom of the centrifuge tube in a volume ratio of 1:1:1:1, and the height of each sucrose-PBS solution on the tube wall is greater than 1.2 cm; then add the refined extract A to the top of the solution, slowly place it in an ultra-high-speed centrifuge, and centrifuge at 150000×g for 120 minutes at 4°C. Take the solution in the 30%-45% sucrose solution layer to obtain the refined extract B;

[0060] 10) Take the refined extract B and centrifuge it at 150000×g for 120 minutes at 4°C to obtain exosomes (located between the 30% sucrose solution and the 45% sucrose solution).

[0061] The raw material of Centella asiatica is the whole herb, the raw material of Ziziphus jujuba is the mature fruit, the raw material of Evodia rutaecarpa is the mature fruit, the raw material of Hippophae rhamnoides is the mature fruit, and the raw material of Rosa roxburghii Tratt is the mature fruit.

[0062] The extracted exosomes are resuspended in PBS buffer for subsequent separation of proteins and secondary metabolites.

[0063] SDS-PAGE detection of molecular weight: Perform SDS-PAGE electrophoresis on Centella asiatica exosomes, Ziziphus jujuba exosomes, Evodia rutaecarpa exosomes, Hippophae rhamnoides exosomes, and Rosa roxburghii Tratt exosomes respectively to confirm the molecular weight value Me of the proteins in the exosomes, and the unit of molecular weight is KDa.

[0064] BCA method for detecting protein content: Use the BCA protein quantification method to determine the protein content value Pe in the Centella asiatica exosome solution, Ziziphus jujuba exosome solution, Evodia rutaecarpa exosome solution, Hippophae rhamnoides exosome solution, and Rosa roxburghii Tratt exosome solution respectively, and the unit of protein content is mg / mL.

[0065] Derivation of the centrifuge time and centrifuge force calculation formula in Example 1

[0066] I. Derivation method of centrifuge time

[0067] Step 1:

[0068] Set the formula framework according to physical meaning

[0069] Molecular weight difference: Setting of Me - Rn

[0070] Me represents the value of the molecular weight of the protein in the exosome sample, and Rn represents the value of the cut-off molecular weight of the ultrafiltration centrifugal tube. The difference between the two, Me - Rn, reflects the difference in the size of exosome particles relative to the cut-off molecular weight of the ultrafiltration centrifugal tube.

[0071] Physically, usually when exosome particles with a larger molecular weight pass through a filter membrane with a smaller cut-off molecular weight, the larger molecular weight will be retained with a higher retention efficiency. Therefore, it is set that Me - Rn > 0. When exosome particles with an even larger molecular weight pass through a filter membrane with an even smaller cut-off molecular weight, larger centrifugal force and centrifugation time are often required. Therefore, the difference between the exosome molecular weight and the cut-off molecular weight of the ultrafiltration centrifugal tube is an important influencing factor, and Me - Rn is set as a part of the formula.

[0072] Pe represents the value of the protein concentration, that is, the protein content in the exosome solution. It reflects the density of the sample. The higher the concentration, the stronger the interaction between molecules, and the more difficult it is to separate during centrifugation.

[0073] Protein concentration and coefficient: 10*P e Set, where the equilibrium coefficient for deriving the formula is 10.

[0074] Physically, usually a higher protein concentration means more complex interactions between particles, which may lead to a decrease in centrifugation efficiency. The higher the protein concentration, the more significant the trend of increasing centrifugation time. By multiplying the protein concentration by an amplification factor, it can better match the experimental data and explain that a higher concentration requires a longer centrifugation time.

[0075] Step two:

[0076] (M e -R n ) + 10*P e Derivation of the linear relationship

[0077] By controlling the molecular weight, the cut-off molecular weight of the ultrafiltration centrifugal tube, and the protein concentration, verify the influence of (M e -R n ) + 10*P e on the centrifugation time t, and further derive the formula.

[0078] Prepare Centella asiatica exosome samples, Hippophae rhamnoides exosome samples, Ziziphus jujuba exosome samples, and Evodia rutaecarpa exosome samples. The molecular weight of the protein in the Centella asiatica exosome sample is 60KDa, the molecular weight of the protein in the Hippophae rhamnoides exosome sample is 35KDa, the molecular weight of the protein in the Ziziphus jujuba exosome sample is 22KDa, and the molecular weight of the protein in the Evodia rutaecarpa exosome sample is 50KDa.

[0079] Adjust the protein concentration of the samples, ranging from 1mg / mL to 5mg / mL. Specifically, they are 1, 2, 3, 4, 5mg / mL.

[0080] Use different ultrafiltration centrifugal tubes for separation. The molecular weight cut-off of the ultrafiltration centrifugal tubes is selected as 3KDa, 10KDa, 30KDa, 50KDa, 100KDa.

[0081] Centrifuge different protein samples to separate them, record the centrifugation time t when 90% separation is achieved, and use a centrifugal force of 3000g for centrifugation.

[0082] Focus on observing the effects of the molecular weight value Me, the molecular weight cut-off value Rn of the ultrafiltration centrifugal tube, and the protein concentration value Pe on the centrifugation time t.

[0083] Step Three:

[0084] Record Me, Pe, Rn of each group of experiments and the corresponding centrifugation time t, and analyze the formula (M e -R n ) + 10 * P e , and the data is as follows:

[0085] Table 1

[0086]

[0087]

[0088] Step Four: Establishment of the linear regression model

[0089] When performing linear regression analysis, the goal is to determine the linear relationship between the independent variable and the dependent variable, which is usually expressed as:

[0090] y = C * x + T0

[0091] y is the dependent variable centrifugation time, x is the independent variable (M e -R n ) + 10 * P e , C is the coefficient of the independent variable, and T0 is the intercept.

[0092] Use LinearRegression in scikit - learn of Python to create a linear regression model and fit the experimental data.

[0093] The fitting coefficient C is close to 1.2, and the intercept T0 is close to 30.

[0094] Therefore, the centrifugation time formula is derived as t = 1.2 * [(Me - Rn)+10 * Pe]+30

[0095] II. Centrifugal force derivation method

[0096] Step 1: Set the formula framework according to physical meaning

[0097] Setting of (Me - Rn)+Me

[0098] Physically, Me - Rn reflects the difference between the molecular weight of exosome particles in the sample and the molecular weight cut - off of the ultrafiltration centrifuge tube. Larger molecular weight particles (compared with the molecular weight cut - off of the ultrafiltration centrifuge tube) are more difficult to pass through the filter membrane during centrifugation and require a greater centrifugal force. Me - Rn needs to have a positive contribution to the centrifugal force formula. At the same time, the molecular weight Me itself also has an important impact on the centrifugal force. The larger the molecular weight, the heavier the particles, and the greater the required centrifugal force. Therefore, it also needs to have a positive contribution to the centrifugal force formula. By combining these two factors, the set formula of (Me - Rn)+Me can more accurately describe the influence of the centrifugal force on the change of molecular weight and the molecular weight cut - off of the ultrafiltration centrifuge tube.

[0099] Setting of dividing by MePe

[0100] Physically, dividing by the molecular weight Me is to balance the influence of particle mass on the centrifugal force, prevent larger particles from causing excessive centrifugal force, and can limit the excessive increase in centrifugal force brought about by too rapid growth of the molecular weight. The protein concentration Pe represents the density of the sample. The higher the concentration, the stronger the interaction between particles, the lower the fluidity, and the lower the efficiency of the centrifugal force. This means that in a high - concentration solution, larger forces are required for particle separation. Dividing by M e P e adjusts the influence of molecular weight and protein concentration on the centrifugal force. Large molecular weight and high concentration will inhibit the action of the centrifugal force, making the influence of other parts in the formula more balanced.

[0101] Therefore, set N = [(M e -R n )*C1+M e *C2+C3] / M e P e in the form.

[0102] Step 2: Derivation of C1, C2 and C3

[0103] Prepare Centella asiatica exosome samples, Hippophae rhamnoides exosome samples, Ziziphus jujuba exosome samples, and Evodia rutaecarpa exosome samples. The molecular weight of proteins in the Centella asiatica exosome samples is 60KDa, the molecular weight of proteins in the Hippophae rhamnoides exosome samples is 35KDa, the molecular weight of proteins in the Ziziphus jujuba exosome samples is 22KDa, and the molecular weight of proteins in the Evodia rutaecarpa exosome samples is 50KDa.

[0104] Adjust the protein concentration of the samples, with the range being from 1mg / mL to 5mg / mL. Specifically, they are 1, 2, 3, 4, 5mg / mL.

[0105] Use different ultrafiltration centrifugal tubes for separation. The cut-off molecular weights of the ultrafiltration centrifugal tubes are selected as 3KDa, 10KDa, 30KDa, 50KDa, and 100KDa.

[0106] Centrifuge different protein samples. The centrifugation time is calculated according to the centrifugation time formula t = 1.2*[(Me - Rn)+10*Pe]+30, and record the centrifugal force N when 90% separation is achieved.

[0107] Focus on observing the effects of the molecular weight Me, the cut-off molecular weight Rn of the ultrafiltration centrifugal tube, and the protein concentration Pe on the centrifugal force N.

[0108] Record Me, Pe, Rn, and the centrifugal force N when 90% separation is achieved for each group of experiments. The data is as follows:

[0109] Table 2

[0110] Me (KDa) Pe (mg / mL) Rn (KDa) Centrifugal force N (g) for 90% separation 35 1 3 6200 50 3 10 2000 60 5 30 1100 22 2 50 2100

[0111] Step three: Establishment of the linear regression model

[0112] Use LinearRegression in scikit - learn of Python to create a linear regression model and fit the experimental data.

[0113] The form of the formula is:

[0114] N = [(M e -R n )*C1+M e *C2+C3] / M e P e

[0115] Use the linear regression model to fit the coefficients C1, C2 and the constant C3 in the formula.

[0116] The fitted coefficient C1 is close to 1000, C2 is close to 5000, and C3 is close to 10000.

[0117] Therefore, the centrifugation time formula is derived as [(M e -R n )*1000 + M e *5000 + 10000] / M e P e 。

[0118] Example 2: Verification of the separation effect of Centella asiatica exosomes

[0119] In this example, the exosomes of the herb Centella asiatica were used as the research object, and the specific implementation steps are as follows:

[0120] SDS-PAGE was used to detect the molecular weight. The exosome sample of Centella asiatica was subjected to SDS-PAGE electrophoresis to confirm that the molecular weight of the protein in the exosome sample was 60KDa, that is, Me was 60.

[0121] The BCA method was used to detect the protein content. The protein content in the exosomes of Centella asiatica was determined to be 1mg / mL by the BCA protein quantification method, that is, Pe was 1.

[0122] Ultrafiltration separation: Based on the smallest positive number where Me - Rn > 0, when Rn was 50, 60 - 50 = 10, which was the smallest positive number; ensuring the separation of proteins and compounds.

[0123] Specific calculations were carried out according to the formula:

[0124] Centrifugal force:

[0125] 1.2*[(Me - Rn)+10*Pe]+30 = 1.2*[(60 - 50)+10*1]+30 = 5333g;

[0126] Centrifugation time:

[0127] [(M e -R n )*1000 + M e *5000 + 10000] / M e P e = [(60 - 50)*1000 + 60*5000 + 10000] / 60*1 = 54min.

[0128] First, the sample was placed in an ultrafiltration centrifuge tube with a molecular weight cut-off of 50KDa. The ultrafiltration tube was placed in an ALLEGRA X-15R CENTRIFUGE multi-functional bench-top refrigerated centrifuge for centrifugation. The centrifugation time and centrifugal force were calculated according to the formula, which were: the centrifugal force was 5333g and the centrifugation time was 54min.

[0129] Asiatic acid is a characteristic secondary metabolite in Centella asiatica exosomes. The separation effect of protein and secondary metabolites was verified by detecting the protein content and asiatic acid content.

[0130] Waters Arc HPLC was used to detect the peak area of the sample before separation, the precipitate after separation, the supernatant after separation, and the compound standard (asiatic acid), and the compound concentration of each sample was determined by the peak area method. The protein content was detected by the BCA method, and the protein content of the sample before separation, the precipitate after separation, the supernatant after separation, and FBS (protein content standard) was detected to determine the protein concentration of each sample. The test results are shown in Figure 2. Figure 1 As shown, the retention rate of precipitated protein after separation was about 82.4%, and the removal rate of Centella asiatica acid was about 94.4%.

[0131] from Figure 1 The results show that under the selected centrifugation conditions, the proteins in the exosomes of Centella asiatica were well retained, and the removal rate of asiatic acid was high, indicating that the centrifugation conditions can effectively separate proteins and secondary metabolites, and preliminarily verify the feasibility of the separation method. At the same time, the protein retention rate was about 82.4%, and the removal rate of asiatic acid was about 94.4%. This data also provides a reference benchmark for subsequent experiments, and can further explore how to improve the removal rate of secondary metabolites while ensuring the protein separation effect, or further optimize the separation conditions while improving the protein retention rate.

[0132] Example 3: Ultrafiltration centrifuge tube Rn setting verification experiment

[0133] In order to verify the scientificity of the selection of ultrafiltration centrifuge tubes for the separation technology, this example further uses different types of ultrafiltration centrifuge tubes for separation. Centella asiatica exosomes were selected as the experimental object. According to the experimental conditions of Example 2, the centrifugal force was 5333 g and the centrifugation time was 54 min. Ultrafiltration centrifuge tubes with a molecular weight cutoff of 30 KDa (the positive integer of Me-Rn is 30) and ultrafiltration centrifuge tubes with a molecular weight cutoff of 100 KDa (there is no positive integer of Me-Rn) were used, which did not meet the condition that Me-Rn was the smallest positive number > 0.

[0134] Proteins and secondary metabolites in exosomes were isolated sequentially.

[0135] Waters Arc HPLC was used to detect the peak area of the sample before separation, the precipitate after separation, the supernatant after separation, and the compound standard (asiatic acid), and the compound concentration of each sample was determined by the peak area method. The protein content was detected by the BCA method, and the protein content of the sample before separation, the precipitate after separation, the supernatant after separation, and FBS (protein content standard) was detected to determine the protein concentration of each sample. The test results are shown in Figure 2. Figure 2 and Figure 3 As shown,Figure 2 After separation, the retention rate of precipitated protein was about 81.7%, and the removal rate of asiatic acid was about 74.5%; Figure 3 After separation, the retention rate of precipitated protein was about 0, and the removal rate of Centella asiatica acid was about 99.5%.

[0136] From the perspective of separation efficiency, compared with the experimental scheme in this example, Example 2 can retain more protein content while also having a lower secondary metabolite content, so Example 2 is the best.

[0137] Example 4: Verification experiment of centrifugal force calculation

[0138] In order to verify the scientificity of the centrifugal force selection of the separation technology, this example further separated by different centrifugal forces. Centella asiatica exosomes were selected as the experimental object. According to the experimental conditions of Example 2, the centrifugation time was 54 minutes, and an ultrafiltration centrifuge tube with a molecular weight cutoff of 50KDa was used. The centrifugal force was changed to 4000g and 7000g, and the proteins and secondary metabolites in the exosomes were separated in turn.

[0139] Waters Arc HPLC was used to detect the peak area of the sample before separation, the precipitate after separation, the supernatant after separation, and the compound standard (asiatic acid), and the compound concentration of each sample was determined by the peak area method. The protein content was detected by the BCA method, and the protein content of the sample before separation, the precipitate after separation, the supernatant after separation, and FBS (protein content standard) was detected to determine the protein concentration of each sample. The test results are shown in Figure 2. Figure 4 and Figure 5 shown. Figure 4 After separation, the retention rate of precipitated protein was about 84.1%, and the removal rate of asiatic acid was about 72.8%; Figure 5 After separation, the retention rate of precipitated protein was about 57.7%, and the removal rate of Centella asiatica acid was about 92.4%.

[0140] Example 5: Verification experiment of centrifugal time calculation

[0141] In order to verify the scientificity of the centrifugation time selection of the separation technology, this example further separated by different centrifugation times. Centella asiatica exosomes were selected as the experimental object. According to the experimental conditions of Example 2, the centrifugal force was 5333g, and an ultrafiltration centrifuge tube with a molecular weight cutoff of 50KDa was used. The centrifugation time was changed to 30min and 80min, and the proteins and secondary metabolites in the exosomes were separated in turn.

[0142] Waters Arc HPLC was used to detect the peak area of the sample before separation, the precipitate after separation, the supernatant after separation, and the compound standard (asiatic acid), and the compound concentration of each sample was determined by the peak area method. The protein content was detected by the BCA method, and the protein content of the sample before separation, the precipitate after separation, the supernatant after separation, and FBS (protein content standard) was detected to determine the protein concentration of each sample. The test results are shown in Figure 2. Figure 6 and Figure 7 shown. Figure 6 After separation, the retention rate of precipitated protein was about 87.8%, and the removal rate of asiatic acid was about 66.1%; Figure 7 After separation, the retention rate of precipitated protein was about 66.7%, and the removal rate of Centella asiatica acid was about 95.0%.

[0143] Example 6: Separation of proteins and secondary metabolites based on seabuckthorn exosomes

[0144] This example uses the exosomes of Hippophae rhamnoides as the research object, and the specific implementation steps are as follows:

[0145] SDS-PAGE detection of molecular weight: SDS-PAGE electrophoresis was performed on the seabuckthorn exosome samples to confirm that the molecular weight of the protein in the exosome samples was 35KDa, that is, Me was 35.

[0146] BCA method for detecting protein content: The protein content in seabuckthorn exosome samples was determined to be 1 mg / mL using the BCA protein quantification method, that is, Pe was 1.

[0147] Ultrafiltration separation: Based on the minimum positive number of Me-Rn>0, ultrafiltration centrifuge tubes with a molecular weight cutoff of 30KDa were used for separation to ensure that proteins and compounds were graded.

[0148] According to the formula, the centrifugal force is 5400g and the centrifugal time is 48min.

[0149] Firstly, the sample was placed in an ultrafiltration tube with a molecular weight cutoff of 30 KDa, and the ultrafiltration tube was placed in an ALLEGRA X-15RCENTRIFUGE multifunctional desktop refrigerated centrifuge for centrifugation. The centrifugal time and centrifugal force were calculated according to the formula, which were: centrifugal force was 5400 g and centrifugal time was 48 min.

[0150] Vitamin C is a characteristic secondary metabolite in seabuckthorn exosomes. The separation effect of protein and secondary metabolites was verified by detecting the protein content and vitamin C content.

[0151] Waters Arc HPLC was used to detect the peak area of the sample before separation, the precipitate after separation, the supernatant after separation, and the compound standard (vitamin C), and the compound concentration of each sample was determined by the peak area method. The protein content was detected by the BCA method, and the protein content of the sample before separation, the precipitate after separation, the supernatant after separation, and FBS (protein content standard) was detected to determine the protein concentration of each sample. The test results are as follows: Figure 8 As shown, after separation, the precipitated protein retention rate was about 83.2%, and the vitamin C removal rate was about 93.5%.

[0152] Example 7: Separation of proteins and compounds based on roxburghii exosomes

[0153] In this example, exosomes from Ribes burejense were used as the research object.

[0154] SDS-PAGE molecular weight detection: SDS-PAGE electrophoresis was performed on the Rosa roxburghii exosome samples to confirm that the molecular weight Me of the protein in the exosome samples was 45KDa, that is, Me was 45.

[0155] BCA method for detecting protein content: The BCA protein quantification method was used to determine the protein content in the roxburghii exosome sample to be 1 mg / mL, that is, Pe was 1.

[0156] Ultrafiltration separation: Based on the minimum positive number of Me-Rn>0, ultrafiltration centrifuge tubes with a molecular weight cutoff of 30KDa were used for separation to ensure that proteins and compounds were graded.

[0157] According to the formula, the centrifugal force is 5600g and the centrifugal time is 25min.

[0158] Firstly, the sample was placed in an ultrafiltration tube with a molecular weight cutoff of 30 KDa, and the ultrafiltration tube was placed in an ALLEGRA X-15RCENTRIFUGE multifunctional desktop refrigerated centrifuge for centrifugation. The centrifugal time and centrifugal force were calculated according to the formula, which were: centrifugal force 5600 g and centrifugal time 25 min.

[0159] Vitamin C is a characteristic secondary metabolite in Rosa roxburghii exosomes. The separation effect of protein and secondary metabolites was verified by detecting the protein content and vitamin C content.

[0160] Waters Arc HPLC was used to detect the peak area of the sample before separation, the precipitate after separation, the supernatant after separation, and the compound standard (vitamin C), and the compound concentration of each sample was determined by the peak area method. The protein content was detected by the BCA method, and the protein content of the sample before separation, the precipitate after separation, the supernatant after separation, and FBS (protein content standard) was detected to determine the protein concentration of each sample. The test results are as follows: Figure 9As shown, the retention rate of the precipitated protein after separation was approximately 85.9%, and the removal rate of vitamin C was approximately 92.7%.

[0161] In the embodiments of the present invention, relevant separation formulas were derived based on the separation experimental data of exosomes from representative plant types (Centella asiatica exosomes, Hippophae rhamnoides exosomes, Ziziphus jujuba exosomes, Evodia rutaecarpa exosomes), and were verified using Centella asiatica exosomes, Hippophae rhamnoides exosomes, and Rosa roxburghii Tratt exosomes.

[0162] The experimental results showed that the calculation methods of centrifugal force and centrifugation time in the present invention had wide applicability for the separation of proteins and secondary metabolites in different types of plant exosomes. Through experiments on two types of representative plant exosomes, namely herbaceous plant exosomes and fruit plant exosomes, the scientificity and practicality of the calculation method of the present invention were further verified, providing a solid experimental basis for the popularization and application of this technology.

[0163] The experimental results also showed that the separation technology proposed in the present invention could be widely applied to different types of plant exosomes, achieving the precise separation of proteins and secondary metabolites, and providing an effective technical means for the research and application of plant exosomes.

[0164] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Calculation method of centrifugation time and centrifugal force, the calculation method comprising: Obtaining the numerical value Me of the molecular weight of the protein in the plant exosome sample; Obtaining the numerical value Pe of the protein content in the plant exosome sample; Obtaining the numerical value Rn of the cut-off molecular weight of the ultrafiltration centrifuge tube, where Rn satisfies that Me - Rn is the smallest positive number greater than 0; Calculating the centrifugation time, the numerical calculation formula for the centrifugation time is: t = 1.2 * [(Me - Rn) + 10 * Pe] + 30; Calculating the centrifugal force, the numerical calculation formula for the centrifugal force is: N = [(Me - Rn) * 1000 + Me * 5000 + 10000] / MePe; When the unit of the molecular weight of the protein is KDa, the unit of the protein content is mg / mL, and the unit of the cut-off molecular weight of the ultrafiltration centrifuge tube is KDa, the unit of the calculated centrifugation time is minutes and the unit of the centrifugal force is g.

2. The calculation method according to claim 1, characterized in that The molecular weight of the protein is determined by SDS-PAGE method.

3. The calculation method according to claim 1, characterized in that, The protein content is determined by BCA method.

4. The calculation method according to claim 1, wherein The range of Me is 10 - 100, and the range of Pe is 1 - 5.

5. The calculation method according to claim 1, wherein The plant exosomes include herbaceous plant exosomes and / or fruit plant exosomes.

6. The calculation method according to claim 1, wherein The herbaceous plant exosomes include centella asiatica exosomes; and / or The fruit plant exosomes include jujube exosomes, evodia rutaecarpa exosomes, seabuckthorn exosomes, and rosa roxburghii tratt exosomes.

7. A method for separating proteins and secondary metabolites from plant exosomes, comprising Calculating the centrifugation time and centrifugal force by the calculation method according to any one of claims 1 - 6; Separating proteins and secondary metabolites in plant exosomes by ultrafiltration centrifugation according to the centrifugation time and centrifugal force.

8. A centrifugation time and centrifugal force calculation device, comprising: An acquisition module: acquiring the numerical value Me of the molecular weight of the protein in the plant exosome sample, the numerical value Pe of the protein content in the plant exosome sample, and the numerical value Rn of the cut-off molecular weight of the ultrafiltration centrifuge tube; A calculation module: obtaining the numerical value of the centrifugation time and the numerical value of the centrifugal force based on the numerical value Me of the molecular weight of the protein in the plant exosome sample, the numerical value Pe of the protein content in the plant exosome sample, and the numerical value Rn of the cut-off molecular weight of the ultrafiltration centrifuge tube acquired by the acquisition module.

9. An electronic device, comprising: A memory for storing a computer program; A processor for executing the computer program to implement the calculation method of centrifugation time and centrifugal force according to any one of claims 1 to 7.

10. A computer-readable storage medium for storing a computer program, where the computer program, when executed by a processor, implements the calculation method of centrifugation time and centrifugal force according to any one of claims 1 to 7.

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