Extraction method of solanum nigrum fruit exosome and application of solanum nigrum fruit exosome in preparation of medicine for treating liver cancer

Abstract

The invention discloses an extraction method of solanum nigrum fruit exosomes and application of the solanum nigrum fruit exosomes in preparation of medicines for treating liver cancer, the solanum nigrum fruit exosomes are extracted by adopting a differential centrifugation method and are identified, so that the defects of low effective rate, large side effect, easiness in recurrence and metastasis and the like of the existing treatment means for liver cancer are overcome. The invention also relates to an application of the solanum nigrum fruit exosome obtained by the extraction method of the solanum nigrum fruit exosome, which is an application in preparation of drugs for treating liver cancer, and specifically comprises the following steps: (1) inhibiting proliferation of liver cancer cells; 2, inhibiting liver cancer cell invasion and metastasis;

Description

Technical Field

[0001] The invention belongs to the technical field of liver cancer drugs and relates to a method for extracting exosomes from Solanum nigrum fruit and an application thereof in preparing drugs for treating liver cancer. Background Art

[0002] Currently, targeted therapy, interventional therapy and immunotherapy are the main treatments for liver cancer. However, due to low efficacy, significant side effects and easy recurrence and metastasis, the clinical treatment of liver cancer still faces huge challenges.

[0003] Solanum nigrum, also known as night nettle or black nightshade, is a species of Solanaceae plant, widely distributed throughout China as well as Asia, Europe, America and other places. Solanum nigrum has a long history of application in the treatment of tumors. Its above-ground parts including stems, leaves, flowers and fruits can be used as medicine, especially those harvested in summer and autumn are the most commonly used. Solanum nigrum tastes bitter and sweet, and is cold in nature. It enters the liver, stomach and large intestine meridians. It has multiple pharmacological effects such as clearing away heat and detoxification, diuresis and swelling, anti-inflammatory and analgesic, and anti-cancer. It is often used to treat various diseases caused by intense heat and toxicity. Modern pharmacological studies have shown that the various chemical components contained in Solanum nigrum, including alkaloids, saponins, steroidal compounds and flavonoids, have multiple pharmacological effects such as anti-cancer and immunomodulatory.

[0004] Recent studies have shown that exosomes from plants have the characteristics of low cytotoxicity, good tissue compatibility, strong stability, and cross-species regulation. However, exosomes from different plant sources are highly heterogeneous and have different biological effects.

[0005] Therefore, actively searching for plant exosomes that act on liver cancer and extracting and studying their effective ingredients can provide a theoretical basis for the treatment of liver cancer with plant-derived exosomes. Summary of the invention

[0006] In view of the problem that exosomes from different plant sources in the prior art are highly heterogeneous and have different biological effects, the present invention provides a method for extracting exosomes from Solanum nigrum fruit and its application in the preparation of drugs for treating liver cancer, so as to solve the disadvantages of existing treatments for liver cancer, such as low efficacy, large side effects and easy recurrence and metastasis.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] The method for extracting exosomes from Solanum nigrum fruit is a method for extracting exosomes from Solanum nigrum fruit by differential centrifugation, comprising the following steps:

[0009] 1) Remove the stems of Solanum nigrum fruit, add PBS equivalent to twice the weight of Solanum nigrum fruit, soak and wash for 30 minutes, and then wash with deionized water for 5 times, 10 minutes each time;

[0010] 2) Put the cleaned Solanum nigrum fruit into a homogenizer and run for 3-5 minutes, homogenize continuously for 3 times, filter impurities through 200, 300, and 450 mesh filters, take the supernatant, set the centrifuge parameters to 1000g, 10min; 3000g, 20min; 10000g, 40min for gradient centrifugation, and take the supernatant;

[0011] 3) After filtering through a 0.45 μm filter membrane, set the centrifuge parameters to 150,000 g, ultra-high-speed centrifugation for 90 min to obtain the precipitate, resuspend the precipitate in PBS, vortex thoroughly, and store in a -80°C refrigerator;

[0012] The morphology of the extracted Solanum nigrum fruit exosomes was observed by projection electron microscopy, and the nanoparticle tracking analysis (NTA) technology was used to track and analyze the Brownian motion nanoparticles extracted in the previous step. The hydrodynamic radius of the particles was calculated by the two-dimensional Stokes-Einstein equation, and the components in the exosomes were analyzed by metabolomics:

[0013] Under electron microscopy, it can be observed that the extracted Solanum nigrum fruit exosomes are spherical, cup-shaped lipid bilayer membrane microvesicles, and the results of nanoparticle tracking analysis (NTA) technology show that its hydrodynamic particle size is 160±10nm;

[0014] Metabolomics analysis was used to determine the fingerprint of the extract and the main target active ingredients, and the changes in the dissolution of the main target ingredients were analyzed to establish a fingerprint, indicating that the main components of Solanum nigrum fruit exosomes are:

[0015] Lipids and lipid molecules 28.14%; alkaloids and their derivatives 1.63%; benzene ring compounds about 7.91%; lignans, neolignans and related compounds 2.56%; nucleosides, nucleotides and analogs 3.26%; organic acids and derivatives 15.81%; organic nitrogen compounds 2.09%; organic oxygen compounds 10.47%; organic heterocyclic compounds 13.72%; alkanol compounds and derivatives 13.26%; others 1.16%.

[0016] The present invention also relates to the application of Solanum nigrum fruit exosomes obtained by the above-mentioned Solanum nigrum fruit exosome extraction method in the preparation of drugs for treating liver cancer, specifically: 1. inhibiting the proliferation of liver cancer cells; 2. inhibiting the invasion and metastasis of liver cancer cells.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] 1. The method for extracting Solanum nigrum exosomes described in the present invention can prepare exosomes with strong stability and high purity by using centrifugation-sucrose density gradient ultracentrifugation, which can improve the production quality of exosomes compared with the traditional centrifugation method.

[0019] 2. The application of the extracted Solanum nigrum fruit exosomes described in the present invention can inhibit the proliferation, invasion and metastasis of liver cancer cells in vitro, and has a good therapeutic effect.

[0020] 3. The Solanum nigrum fruit exosome extraction method of the present invention contains a large number of metabolic components with poor water solubility, easy to be quickly cleared by the human body and poor biocompatibility compared to the chemical extract of Solanum nigrum fruit, and the medicinal active ingredients are much higher than the chemical extract of Solanum nigrum fruit. The lipid bilayer of Solanum nigrum fruit exosomes can effectively and stably deliver active ingredients or small molecule RNA to liver cancer cells, has low immunogenicity, enhanced cellular uptake, good biocompatibility, higher gastrointestinal stability, and through special endocytosis mechanisms and cross-species targeting of specific tissues, effectively improves bioavailability and efficiently exerts anti-cancer effects. The Solanum nigrum exosomes extracted by the present invention have good compatibility with liver cancer cells and can be used as a drug delivery system to improve the efficacy of liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions implemented in the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0022] Figure 1 This is a diagram illustrating the characterization results of the extracted Solanum nigrum fruit exosomes in the examples of the present invention;

[0023] Figure 2 This is a diagram showing the effect of Solanum nigrum fruit exosomes on the proliferation of liver cancer cells in the experimental example of the present invention;

[0024] Figure 3 This is a diagram showing the effect of Solanum nigrum fruit exosomes on the migration of liver cancer cells in the experimental example of the present invention;

[0025] Figure 4 This figure shows the effect of Solanum nigrum fruit exosomes on the invasion of liver cancer cells in the experimental example of the present invention. DETAILED DESCRIPTION

[0026] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified. The quantitative tests in the following examples are all repeated three times.

[0027] Example:

[0028] A method for extracting exosomes from Solanum nigrum fruit, which uses differential centrifugation to extract exosomes from Solanum nigrum fruit and identifies the exosomes, comprises the following steps:

[0029] 1) Remove the stems of Solanum nigrum fruit, add appropriate amount of PBS to soak and wash for 30 minutes, and then wash with deionized water 5 times, 10 minutes each time;

[0030] 2) Put the cleaned Solanum nigrum fruit into a homogenizer, run for 3-5 minutes, homogenize continuously for 3 times, filter out impurities through 200, 300, and 450 mesh filters, and take the supernatant;

[0031] 3) Centrifuge the supernatant in the previous step at 4°C, 1000g for 10 min and collect the supernatant;

[0032] 4) Centrifuge the supernatant from the previous step at 4°C, 3000g for 20 min, and collect the supernatant;

[0033] 5) The supernatant in the above step was centrifuged at 4°C and 10,000 g for 30 min, and the supernatant was discarded to obtain Solanum nigrum fruit exosomes.

[0034] Experimental Example 1: Cell colony formation assay

[0035] HepG2 and Hepa1-6 hepatoma cells in logarithmic growth phase were digested with trypsin and gently pipetted to suspend them. The centrifuge parameters were set to 1000g for 5 minutes. After centrifugation, culture medium or 1×10 5 Take a small amount of cell suspension and count the cell concentration with a cell counter to adjust the cell concentration to 50 cells / ml. Add 5 ml of the diluted cell suspension to a 6 mm culture dish and shake gently to evenly distribute the cells. Place the inoculated culture dish in a 37°C, 5% CO 2 Incubate in a cell culture incubator; replace the culture medium every 2-3 days. After 7 days, add 5 ml PBS to each well to wash the cells 3 times, add 2 ml formaldehyde to each culture dish, fix at room temperature for 15 minutes, aspirate the formaldehyde, add 5 ml PBS to wash once, add 2 ml, 0.1% crystal violet, stain at room temperature for 5 minutes, wash the excess dye with clean water, observe the cell clones after drying, and take pictures.

[0036] Figure 1 For the characteristics of the extracted Solanum nigrum fruit exosomes;

[0037] Using this research method, the extracted Solanum nigrum fruit exosomes were observed under a transmission electron microscope. The results showed that the Solanum nigrum fruit exosomes were spherical or quasi-spherical. Nanoparticle tracking technology was used to observe the exosome diameter. The results showed that the diameter of the extracted exosomes was distributed between 160±10nm, which is within the range of exosome size (30-200nm).

[0038] Figure 2 Effects of Solanum nigrum fruit exosomes on the proliferation of liver cancer cells.

[0039] like Figure 2 As shown, compared with normal cells, the cell colonies decreased after 24 hours of in vitro intervention with Solanum nigrum exosomes, which indicates that the extracted Solanum nigrum fruit exosomes can inhibit the proliferation of liver cancer cells.

[0040] Experimental Example 2: Scratch Test

[0041] HepG2 and Hepa1-6 hepatoma cells in the logarithmic growth phase were digested with trypsin and gently pipetted to suspend the cells. After the cells were evenly distributed, the cell concentration was adjusted to 1×10 4 / ml, add 5ml of cell suspension to a 6mm culture dish, wait for the cells to adhere, use a 200ul pipette tip to draw a straight line on the cell monolayer to form a "scratch"; gently aspirate the culture medium, add 3ml PBS to wash the cells twice to remove the detached cell debris, and add 5ml serum-free culture medium and 5ml of 1×10 5 After adding serum-free medium containing Solanum nigrum fruit exosomes, the culture dish was returned to the cell culture incubator at 37°C and 5% CO2 for incubation; photos of the scratched area were immediately taken under a microscope to record the initial state; and photos of the same scratched area were taken under a microscope at 24 hours, 48 ​​hours and 72 hours to record cell migration and healing.

[0042] Experimental Example 3: Cell Migration Assay

[0043] HepG2 and Hepa1-6 hepatoma cells in logarithmic growth phase were digested with trypsin and centrifuged at 1000 g for 5 min. After centrifugation, culture medium or 1×10 5 The cells were suspended and evenly distributed in the culture medium of Solanum nigrum fruit exosomes. The cell concentration was adjusted to 1×10 4 Place the Transwell chamber in a 24-well plate, add 200ul of the prepared cell suspension to the upper chamber of the Transwell chamber, make sure there are no bubbles, and gently shake to evenly distribute the cells; place the 24-well plate in a 37°C, 5% CO 2After incubation in a cell culture incubator for 24 hours, the 24-well plate was removed, the culture medium in the upper and lower chambers was aspirated, and 200ul of sterile PBS was added to each well to gently wash the Transwell chamber to remove the cells that did not migrate or invade; 200ul of 4% paraformaldehyde was added to fix the cells in the Transwell chamber for 10 minutes, and 20ul of PBS was added to wash the Transwell chamber to remove the fixative; the cells at the bottom of the membrane were observed and photographed under a microscope; the number of migrated or invaded cells in each Transwell chamber was counted, the results were recorded, and the cell migration or invasion rate was calculated.

[0044] Figure 3 Effects of Solanum nigrum fruit exosomes on the migration of hepatocellular carcinoma cells;

[0045] like Figure 3 As shown, compared with the normal group, the cell migration level was significantly decreased after 24 hours, 48 ​​hours and 72 hours of intervention with Solanum nigrum fruit exosomes, which indicates that Solanum nigrum fruit exosomes can inhibit the migration of liver cancer cells.

[0046] Figure 4 Effects of Solanum nigrum fruit exosomes on the invasion of hepatocellular carcinoma cells.

[0047] like Figure 4 As shown, the results of the Transwell experiment showed that compared with the normal group, the cell invasion rate was significantly decreased after intervention with Solanum nigrum fruit exosomes, which indicates that Solanum nigrum fruit exosomes can inhibit the invasion of liver cancer cells.

[0048] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for extracting exosomes from Solanum nigrum fruit, characterized in that: The invention discloses a method for extracting exosomes from Solanum nigrum fruit by using differential centrifugation, comprising the following steps: 1) Remove the stems of Solanum nigrum fruit, add PBS equivalent to twice the weight of Solanum nigrum fruit, soak and wash for 30 minutes, and then wash with deionized water for 5 times, 10 minutes each time; 2) Put the cleaned Solanum nigrum fruit into a homogenizer and run for 3-5 minutes, homogenize continuously for 3 times, filter impurities through 200, 300, and 450 mesh filters, take the supernatant, set the centrifuge parameters to 1000g, 10min; 3000g, 20min; 10000g, 40min for gradient centrifugation, and take the supernatant; 3) After filtering through a 0.45 μm filter membrane, set the centrifuge parameters to 150,000 g, ultra-high-speed centrifuge for 90 min to obtain the precipitate, resuspend the precipitate in PBS, vortex thoroughly, and store in a -80°C refrigerator; The morphology of the extracted Solanum nigrum fruit exosomes was observed by projection electron microscopy, and the nanoparticle tracking analysis technology was used to track and analyze the Brownian motion nanoparticles extracted in the previous step. The hydrodynamic radius of the particles was calculated by the two-dimensional Stokes-Einstein equation, and the components in the exosomes were analyzed by metabolomics: Under electron microscopy, it can be observed that the extracted Solanum nigrum fruit exosomes are spherical, cup-shaped lipid bilayer membrane microvesicles, and the results of nanoparticle tracking analysis technology show that its hydrodynamic particle size is 160±10nm; Metabolomics analysis was used to determine the fingerprint of the extract and the main target active ingredients, and the changes in the dissolution of the main target ingredients were analyzed to establish a fingerprint, indicating that the main components of Solanum nigrum fruit exosomes are: Lipids and lipid molecules 28.14%; alkaloids and their derivatives 1.63%; benzene ring compounds about 7.91%; lignans, neolignans and related compounds 2.56%; nucleosides, nucleotides and analogs 3.26%; organic acids and derivatives 15.81%; organic nitrogen compounds 2.09%; organic oxygen compounds 10.47%; organic heterocyclic compounds 13.72%; alkanol compounds and derivatives 13.26%; others 1.16%.

2. The use of the Solanum nigrum fruit exosomes obtained by the method for extracting Solanum nigrum fruit exosomes according to claim 1, characterized in that: The invention is used in preparing medicine for treating liver cancer.

3. The use of Solanum nigrum fruit exosomes according to claim 2, characterized in that: The exosomes of Solanum nigrum fruit are used to prepare the following drugs for treating liver cancer:

1. Inhibit the proliferation of liver cancer cells; 2. Inhibit the invasion and metastasis of liver cancer cells.

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