Preparation method and application of gynostemma pentaphylla exosome

Abstract

The invention discloses a preparation method and application of a gynostemma pentaphylla exosome. According to the method, a high-activity callus is obtained by optimizing a callus induction culture medium, and a suspension culture system is established to realize biomass accumulation with the dry cell weight of 19.8 + / -0.7 g / L. A sequential progressive composite induction strategy (methyl jasmonate is added after chitosan is pre-induced for 24 hours) is innovatively adopted, so that the saponin content is remarkably increased to 102.6 + / -4.8 mg / g DW. Exosomes (P-Exos) are extracted from induced cells through an enzymolysis-magnetic bead affinity purification technology (WGA modified magnetic bead affinity capture after mild enzymolysis of a cracking buffer solution), the yield reaches 1.45 + / -0.08 mg / g cells, and the extraction efficiency is remarkably improved compared with that of a traditional gradient centrifugation method (C-Exos). Moreover, the gynostemma pentaphyllum exosome prepared by the invention can effectively improve the synthesis of skin collagen, has a better effect, can be applied to medicines for promoting skin wound repair or can be added into anti-aging cosmetics as a functional component, and has a very high application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of exosome preparation, and specifically relates to a preparation method and application of Gynostemma pentaphyllum exosomes. Background Art

[0002] Gynostemma pentaphyllum is a traditional medicinal plant rich in saponins, flavonoids, and other bioactive secondary metabolites. These products possess antioxidant, anti-inflammatory, immunomodulatory, and anti-aging properties, and are widely used in the development of health foods and functional cosmetics. However, traditional methods for extracting Gynostemma pentaphyllum suffer from low active ingredient content, low extraction efficiency, and complex composition that is difficult to standardize, limiting its further development and application in high-value-added products.

[0003] In recent years, plant-derived exosomes (PDEs) have become an emerging research hotspot as natural drug delivery systems and carriers of active substances due to their small particle size, strong biocompatibility, and rich content of functional small molecules. Previous studies have demonstrated that plant exosomes have great potential for transdermal absorption, targeted delivery, and tissue repair, making them particularly suitable for cosmetics and skin repair products. However, the preparation of plant exosomes currently faces bottlenecks such as low yield, unstable active ingredients, and cumbersome extraction techniques, hindering their industrialization. In the study of Gynostemma pentaphyllum, although some literature has reported on callus tissue culture and saponin enrichment methods, a stable and efficient cell culture system for large-scale saponin enrichment has not yet been established. Furthermore, a method for efficient preparation and functional evaluation of exosomes based on suspension culture cells is lacking. In particular, research on the promotion of skin collagen synthesis by Gynostemma pentaphyllum exosomes, as well as the related mechanism and application verification, remains underdeveloped.

[0004] Therefore, there is an urgent need to develop an efficient and controllable method for preparing Gynostemma pentaphyllum exosomes, while improving the extraction efficiency and enrichment of active ingredients, and systematically verifying their functional effects in improving skin health. This will not only help enhance the utilization value of Gynostemma pentaphyllum resources, but also provide technical support for the development of natural functional cosmetic raw materials. Summary of the Invention

[0005] One of the objectives of the present invention is to provide a method for preparing Gynostemma pentaphyllum exosomes.

[0006] A second object of the present invention is to provide the use of the above-mentioned Gynostemma pentaphyllum exosomes in skin collagen synthesis.

[0007] In order to achieve the above object, the technical solution of the present invention is as follows:

[0008] A method for preparing Gynostemma pentaphyllum exosomes, comprising the following steps:

[0009] (1) Callus induction: Using Gynostemma pentaphyllum stem segments as explants, sterilize and remove the incisions at both ends to expose fresh tissue, inoculate on solid culture medium, and culture in the dark at 25°C to obtain yellow-white loose callus tissue; subculture three times, each cycle lasting 21 days;

[0010] (2) Suspension cell culture of Gynostemma pentaphyllum callus: mince the callus after three subcultures into 0.5-1.0 mm particle size, inoculate the callus into liquid culture medium at 15% inoculum, place in a constant temperature shaker at 25±1°C, and perform suspension culture at 110 r / min in the dark;

[0011] (3) Gynostemma pentaphyllum saponin induction: On the sixth day of suspension culture, 25 mg / L chitosan was added for 24 h, followed by 100 μM methyl jasmonate for a further 72 h.

[0012] (4) Extraction of Gynostemma pentaphyllum exosomes: Gynostemma pentaphyllum cell precipitate after induction culture in step (3) was taken, and Gynostemma pentaphyllum exosomes were obtained by lysis, targeted enrichment, targeted elution and ultrafiltration concentration.

[0013] Preferably, the stem segment is 2-3 cm in length and has 1-2 axillary buds.

[0014] Preferably, the disinfection method is: soaking the explant in 75% ethanol for 30 seconds, rinsing with sterile water three times; then treating it in 0.1% HgCl2 solution for 8 minutes, rinsing with sterile water five times, and absorbing the moisture with sterile filter paper.

[0015] Preferably, the solid culture medium in step (1) is MS basic salt + 3 mg / L NAA + 0.5-0.6 mg / L 6-BA + 3% sucrose + 6.5 g / L agar, pH 5.8; the liquid culture medium is: MS basic salt + 3 mg / L NAA + 0.5 mg / L 6-BA + 3% sucrose.

[0016] Preferably, the specific steps of lysis, targeted enrichment, targeted elution and ultrafiltration concentration in step (4) are as follows:

[0017] Lysis: Add Tris-HCl lysis buffer containing 0.5% lysase and shake at 4°C for 30 minutes to release intracellular vesicles;

[0018] Targeted enrichment: Add WGA-modified magnetic beads and incubate at 4°C for 60 minutes to achieve targeted enrichment by utilizing the specific binding of exosome surface glycoproteins and wheat germ agglutinin;

[0019] Targeted elution: Wash the magnetic beads three times with 0.2 M N-acetylglucosamine solution to competitively dissociate the bound exosomes;

[0020] Ultrafiltration and concentration: The eluate was concentrated to a final volume of 5 mL using a 100 kDa ultrafiltration tube.

[0021] In addition, the Gynostemma pentaphyllum exosomes obtained according to the above preparation method also fall within the scope of protection of the present invention.

[0022] In addition, the present invention also protects the application of Gynostemma pentaphyllum exosomes in skin collagen synthesis. Among them, the Gynostemma pentaphyllum exosomes activate the TGF-β / Smad signaling pathway, increasing the phosphorylation level of Smad2 / 3 by 4.15 times; specifically, when used at a concentration of 50-100 μg / mL to treat human skin fibroblasts (HSF) for 72 hours, the COL1A1 gene expression increased by 4.73 times, and the collagen secretion reached 47.2±4.1 μg / 10 6 cell.

[0023] In addition, the above-mentioned Gynostemma pentaphyllum exosomes are used in the preparation of a drug for promoting skin wound repair. When the concentration of Gynostemma pentaphyllum exosomes in the drug is 50-100 μg / mL, the HSF cell scratch healing rate is increased to 89.4±6.7%.

[0024] In addition, the above-mentioned Gynostemma pentaphyllum exosomes are used in the preparation of anti-aging cosmetics, and the amount of Gynostemma pentaphyllum exosomes added to the cosmetics is not less than 0.1 mg / mL.

[0025] Advantages of the present invention:

[0026] (1) The present invention obtains highly active callus tissue by optimizing the callus induction medium (containing 3 mg / L NAA and 0.5-0.6 mg / L 6-BA), and establishes a suspension culture system to achieve biomass accumulation of 19.8±0.7 g / L of cell dry weight.

[0027] (2) Based on a deep understanding of the regulatory mechanism of plant secondary metabolism, an innovative "time-sequential compound induction" strategy was proposed (methyl jasmonate was added 24 hours after chitosan pre-induction), which significantly increased the saponin content to 102.6±4.8 mg / g DW (20.9 times higher than that of basal culture). The core of this strategy is that chitosan first acts as a stress / defense response elicitor, inducing cells to produce early stress responses (such as reactive oxygen species burst and defense gene activation), laying the foundation for the subsequent efficient synthesis of secondary metabolites; MeJA, as a core activator of the secondary metabolic pathway, more effectively activates the saponin biosynthesis pathway based on the "preparatory state" established by chitosan.

[0028] (3) Exosomes (P-Exos) were extracted from induced cells using an enzymatic hydrolysis-magnetic bead affinity purification technique (mild enzymatic hydrolysis in lysis buffer followed by affinity capture with WGA-modified magnetic beads), with a yield of 1.45±0.08 mg / g cell (equivalent to a yield of approximately 28.7 mg / L suspension culture), significantly improving the extraction efficiency compared to the traditional gradient centrifugation method (C-Exos). The contents of saponins (gypenosides XLIX and LI) and flavonoids (isoquercitrin and rutin) in P-Exos were 3-5.5 times that of C-Exos, respectively, with the active ingredient accounting for 72.5%. Furthermore, the particle size was smaller (85.3±3.2 nm), facilitating transdermal absorption.

[0029] (4) Experiments confirmed that the Gynostemma pentaphyllum exosomes activated the TGF-β / Smad signaling pathway (Smad2 / 3 phosphorylation level increased by 4.15 times), significantly enhanced COL1A1 gene expression (4.73 times) and collagen secretion (47.2±4.1μg / 10^6 cells) in human skin fibroblasts (HSF), and increased the wound healing rate to 89.4±6.7%. The present invention provides an innovative solution for the efficient preparation of natural active ingredients and the development of anti-aging products. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a diagram of the Gynostemma pentaphyllum callus obtained by culture in Example. DETAILED DESCRIPTION

[0031] The present invention will be further described below with reference to specific examples, and the advantages and features of the present invention will become more apparent as the description proceeds. However, the specific experimental methods involved in the following examples, unless otherwise specified, are all conventional methods or are performed under the conditions recommended by the manufacturer's instructions.

[0032] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The experimental methods in the following examples are all conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials used can be purchased from the market.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0034] The following is a detailed description of the method for preparing Gynostemma pentaphyllum exosomes. The method primarily includes the following steps: callus induction, Gynostemma pentaphyllum callus suspension cell culture, gypenosin induction, and Gynostemma pentaphyllum exosome extraction. Each step is described in detail below.

[0035] Example 1 Gynostemma pentaphyllum callus induction and establishment of high-efficiency cell lines

[0036] 1. Cut young stem segments of Gynostemma pentaphyllum (Thunb.) Makino (2-3 cm in length, with 1-2 axillary buds) as explants.

[0037] 2. Rinse the Gynostemma pentaphyllum explants with running water for 30 minutes to remove surface impurities and place them on a sterile operating table for later use; soak them in 75% ethanol for 30 seconds and rinse them with sterile water three times; treat them with 0.1% HgCl2 solution for 8 minutes and rinse them with sterile water five times; after absorbing the moisture with sterile filter paper, cut off the incisions at both ends to expose fresh tissue.

[0038] 3. Cut the sterilized stem segments into 0.5 cm long segments and inoculate them onto the surface of a solid culture medium (5-6 segments per dish) at 25°C in the dark. The medium should contain MS basal salts, 2 mg / L 2,4-D, 0.5 mg / L 6-BA, 3% sucrose, and 6.5 g / L agar as a solidifying agent, with a pH of approximately 5.8.

[0039] 4. After obtaining the primary callus, the subculture cycle was 21 days. After 3 consecutive subcultures, the type and dosage of growth regulators were adjusted (Table 1) to screen out Gynostemma pentaphyllum callus with fast biomass accumulation and loose morphology ( Figure 1 ).

[0040] Table 1 Effects of different growth regulator types and dosages on callus formation

[0041]

[0042] As can be seen from Table 1, a rapidly growing, yellow-white, soft Gynostemma pentaphyllum cell line can be obtained under the conditions of MS basic salt, 3 mg / L NAA, 0.5 mg / L 6-BA, 3% sucrose, 6.5 g / L agar, pH 5.8, and dark culture at 25°C.

[0043] Example 2 Establishment of Gynostemma pentaphyllum callus suspension cell culture system

[0044] 1. Chop the solid callus tissue of Gynostemma pentaphyllum that has been subcultured for three generations into a particle size of 0.5-1.0 mm. Inoculate the callus tissue at a 15% inoculum amount into a liquid culture medium containing MS basal salts + 3 mg / L NAA, 0.5 mg / L 6-BA, and 3% sucrose. Place the culture medium in a constant temperature shaker at 25±1°C and perform suspension culture at a speed of 110 r / min in the dark.

[0045] 2. Dynamic Monitoring and Sampling: 50 mL of culture medium was collected at a time, filtered through a 200-mesh nylon mesh, and centrifuged at 4000 rpm for 15 minutes to collect the cell pellet. The pellet was freeze-dried and weighed. Culture was continued for 12 days, with samples collected at 0, 2, 4, 6, 8, 10, and 12 days for testing of relevant parameters.

[0046] Table 2 Dynamic monitoring indicators of Gynostemma pentaphyllum callus

[0047]

[0048]

[0049] As can be seen from Table 2, (1) the fresh weight of cells increased from the initial 30.0±0.8 g / L to 198.3±3.6 g / L; (2) the dry weight of cells increased from 3.0±0.1 g / L to 19.8±0.7 g / L; (3) the cell viability decreased gradually from 96.2% to 82.1%; (4) the conductivity showed a trend of first decreasing and then increasing, from 980→320→450 μS / cm; (5) the pH value decreased slowly from 5.8 to 5.1; (6) the saponin content increased significantly, from 1.30±0.07 mg / g DW to a peak of 4.90±0.25 mg / g DW on the 10th day.

[0050] Example 3 Establishment of a Saponin Induction System for Gynostemma Pentaphyllum Suspension Cell Culture

[0051] Continuing with the steps of Example 2, on the 6th day of suspension culture (cell dry weight of about 13.5 g / L, mid-logarithmic growth phase), chitosan mother solution (final concentration 25 mg / L) was first added and induced for 24 hours; after 24 hours, methyl jasmonate (MeJA) mother solution (final concentration 100 μM) was added and the induction treatment was continued for 72 hours (i.e., a total induction time of 96 hours); simultaneously, during the experiment, a control group was set:

[0052] a) blank solvent control group;

[0053] b) Group with only MeJA (100 μM);

[0054] c) group with chitosan (25 mg / L) added only;

[0055] d) Group with simultaneous addition of MeJA (100 μM) and chitosan (25 mg / L);

[0056] e) Sequential induction (chitosan followed by MeJA, the method of the present invention).

[0057] Table 3 Effects of different treatments on the content of gypenosin

[0058] serial number Induction treatment group Induction treatment group (mg / g DW) Increase multiple a Blank solvent control 4.90±0.25 1.00x b MeJA only (100 μM) 52.3±2.8 10.7x C Chitosan only (25 mg / L) 34.6±1.9 7.06x d Add MeJA+chitosan at the same time 78.5±3.5 16.0x e Sequential induction (Chitosan followed by MeJA) 102.6±4.8 20.9x

[0059] The UV method was used to determine the total saponin content and evaluate the induction effect. The specific results are shown in Table 3. It can be seen from Table 3 that the highest content of gypenosin can be obtained by the time-sequential induction method, that is, first adding 25 mg / L chitosan for 24 hours and then adding MeJA at a final concentration of 100 μM.

[0060] Example 4 Efficient Isolation of Gynostemma Pentaphyllum Exosomes

[0061] Test materials and pretreatment: Take the Gynostemma pentaphyllum suspension culture induced in Example 3, centrifuge it at 4000×g for 20 min at 4°C, discard the supernatant, collect the cell pellet, and wash the pellet three times with pre-cooled PBS (0.1 M, pH 7.4) to obtain a clean cell pellet (wet weight of about 150 g / L).

[0062] The extraction process of P-Exos in the experimental group of the present invention (enzymatic hydrolysis-affinity magnetic bead coupling method) is as follows: (1) gentle cell disruption: add 10 times the volume of lysis buffer (0.1 M Tris-HCl, 0.5% lysozyme, pH 7.4) to the cell pellet, and shake and lyse at 4°C for 30 min (150 rpm); (2) exosome affinity capture: add WGA-modified magnetic beads (particle size 1 μm, loading capacity 15 mg / g), incubate at 4°C for 60 min, and magnetically separate and discard the supernatant; (3) targeted elution: wash the magnetic beads three times with 0.2 M N-acetylglucosamine solution, and collect the eluate; (4) concentration and purification: the eluate is concentrated to 5 mL through a 100 kDa ultrafiltration tube.

[0063] At the same time, other extraction methods were set up for comparison during the experiment. Specifically, the extraction process of C-Exos in the control group was as follows:

[0064] The cell pellet was added with 10 volumes of PBS, disrupted by ultrasonication on ice (200 W, 5 s / 10 s interval, total time 5 min), centrifuged at 10,000 × g for 30 min at 4°C to remove debris, centrifuged at 10,000 × g for 30 min at 4°C to remove debris, ultracentrifuged at 100,000 × g for 2 h, and the pellet was resuspended in PBS.

[0065] The exosome yield, effective ingredients, active substances, and particle size of the experimental and control groups were compared. The specific results are as follows:

[0066] 1. Exosome yield: The exosome yield of the experimental group P-Exos was 1.45±0.08 mg / g cell, and the exosome yield of the control group C-Exos was 0.63±0.08 mg / g cell;

[0067] 2. Analysis of active ingredients in exosomes: UPLC-Q-Exactive HF-X mass spectrometry was used to analyze exosome components. For saponins, the contents of gypenosides XLIX and LI in P-Exos were 1.25 ± 0.08 μg / mg and 0.93 ± 0.06 μg / mg, respectively, which were 3.0-5.5 times higher than those in C-Exos (0.31 ± 0.03 μg / mg and 0.17 ± 0.02 μg / mg). For flavonoids, the contents of isoquercetin and rutin in P-Exos reached 0.68 ± 0.05 μg / mg, 2.8 times that of C-Exos.

[0068] 3. Based on the peak area normalization method, the proportion of active substances in exosomes was calculated. The secondary metabolites in P-Exos accounted for 38.7±2.1% of the total components, of which the active ingredients (saponins + flavonoids) accounted for 72.5%. The secondary metabolites in C-Exos accounted for only 19.2±1.8%, and the active ingredients accounted for 51.3%.

[0069] 4.NTA detected the exosome particle size. The average particle size of P-Exos was 85.3±3.2nm, which was smaller than that of C-Exos (120.5±5.1nm).

[0070] Example 5 Application of Gynostemma pentaphyllum exosomes in enhancing skin collagen synthesis

[0071] 1. Human skin fibroblast (HSF) cell culture: HSF cells were seeded in DMEM medium containing 10% FBS and cultured at 37°C and 5% CO2 until the logarithmic growth phase;

[0072] 2. Experimental groups: A blank control group (PBS treatment), a traditional exosome extraction group (C-Exos, 50 μg / mL), a low-concentration cell culture exosome group (P-Exos-L, 50 μg / mL), and a high-concentration group (P-Exos-H, 100 μg / mL) were set up, with 6 replicates in each group.

[0073] 3. Intervention treatment: Replace the culture medium containing the corresponding concentration of exosomes every 24 hours for 72 hours;

[0074] 4. Index detection (Table 4):

[0075] (1) COL1A1 gene expression: qRT-PCR detection of mRNA levels (primer sequences:

[0076] F: 5'-CAGGGTACTCGATGGATTCC-3′,

[0077] R: 5′-GTGCCATCTCTCTGCTCATT-3′);

[0078] (2) Collagen content: Type I collagen secretion was measured by ELISA (Human COL1A1 ELISA Kit);

[0079] (3) Cell migration ability: scratch test to observe the healing rate for 24 hours;

[0080] (4) Signaling pathway analysis: Western blot was used to detect the phosphorylation levels of proteins related to the TGF-β / Smad pathway.

[0081] Table 4 Effects of different treatments on skin collagen synthesis related indicators

[0082]

[0083] * indicates p<0.05 compared with the blank group, ** indicates p<0.01

[0084] As can be seen from Table 4, the Gynostemma pentaphyllum exosomes obtained by the improved extraction method (especially at high concentrations) strongly activated the TGF-β / Smad pathway, enhancing collagen synthesis and cell repair capacity in a dose-dependent manner. The effect was significantly better than that of traditional exosomes, and they have outstanding application potential in the fields of anti-aging and wound repair.

[0085] Therefore, the Gynostemma pentaphyllum exosomes prepared by the present invention can effectively enhance the synthesis of skin collagen with good effect, can be applied to drugs that promote skin wound repair, or added as a functional ingredient to anti-aging cosmetics, so it has high application value.

[0086] The embodiments described above are only preferred embodiments of the present invention and are only used to explain the present invention, not to limit the scope of implementation of the present invention. For those skilled in the art, it is of course possible to easily make other implementation methods by replacing or changing the technical content disclosed in this specification. Therefore, all changes and improvements made on the principles of the present invention should be included in the scope of the patent application of the present invention.

Claims

1. A method for preparing Gynostemma pentaphyllum exosomes, characterized in that: The preparation method comprises the following steps: (1) Callus induction: Using Gynostemma pentaphyllum stem segments as explants, sterilize and remove the incisions at both ends to expose fresh tissue, inoculate on solid culture medium, and culture in the dark at 25°C to obtain yellow-white loose callus tissue; subculture three times, each cycle lasting 21 days; (2) Suspension cell culture of Gynostemma pentaphyllum callus: mince the callus after three subcultures into 0.5-1.0 mm particle size, inoculate the callus into liquid culture medium at 15% inoculum, place in a constant temperature shaker at 25±1°C, and perform suspension culture at 110 r / min in the dark; (3) Gynostemma pentaphyllum saponin induction: On the sixth day of suspension culture, 25 mg / L chitosan was added for 24 h, followed by 100 μM methyl jasmonate for a further 72 h. (4) Extraction of Gynostemma pentaphyllum exosomes: Gynostemma pentaphyllum cell precipitate after induction culture in step (3) was taken, and Gynostemma pentaphyllum exosomes were obtained by lysis, targeted enrichment, targeted elution and ultrafiltration concentration.

2. The preparation method according to claim 1, characterized in that The stem segment is 2-3 cm long and has 1-2 axillary buds.

3. The preparation method according to claim 1, characterized in that The disinfection method is as follows: soaking the explants in 75% ethanol for 30 seconds, rinsing with sterile water for 3 times; then treating them in 0.1% HgCl2 solution for 8 minutes, rinsing with sterile water for 5 times, and absorbing the moisture with sterile filter paper.

4. The preparation method according to claim 1, characterized in that The solid culture medium in step (1) is MS basic salt + 3 mg / L NAA + 0.5-0.6 mg / L 6-BA + 3% sucrose + 6.5 g / L agar, pH 5.8; the liquid culture medium is: MS basic salt + 3 mg / L NAA + 0.5 mg / L 6-BA + 3% sucrose.

5. The preparation method according to claim 1, characterized in that The specific steps of lysis, capture, targeted elution and ultrafiltration concentration in step (4) are as follows: Lysis: Add Tris-HCl lysis buffer containing 0.5% lysase and shake at 4°C for 30 minutes to release intracellular vesicles; Targeted enrichment: Add WGA-modified magnetic beads and incubate at 4°C for 60 minutes to achieve targeted enrichment by utilizing the specific binding of exosome surface glycoproteins and wheat germ agglutinin; Targeted elution: Wash the magnetic beads three times with 0.2 M N-acetylglucosamine solution to competitively dissociate the bound exosomes; Ultrafiltration and concentration: The eluate was concentrated to a final volume of 5 mL using a 100 kDa ultrafiltration tube.

6. Gynostemma pentaphyllum exosomes obtained according to the preparation method according to any one of claims 1 to 5.

7. Use of the Gynostemma pentaphyllum exosomes according to claim 6 in promoting skin collagen synthesis.

8. The use of the Gynostemma pentaphyllum exosomes according to claim 6 in the preparation of a medicament for promoting skin wound repair, characterized in that: The concentration of Gynostemma pentaphyllum exosomes in the medicine is 50-100 μg / mL.

9. The use of the Gynostemma pentaphyllum exosomes according to claim 6 in the preparation of anti-aging cosmetics, characterized in that: The amount of Gynostemma pentaphyllum exosomes added to the cosmetic is not less than 0.1 mg / mL.

Images