A method for culturing cherry cells and cherry cell exosomes
By optimizing cherry cell culture methods and utilizing salicylic acid, methyl jasmonate, and UV-B light, combined with specific precursor substances and centrifugation, the flavonoid and polyphenol content in cherry cell cultures was increased. This solved the problem of unstable yield and quality in traditional methods and provided a highly efficient cosmetic raw material.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- LUDONG UNIVERSITY
- Filing Date
- 2024-12-31
- Publication Date
- 2026-06-30
AI Technical Summary
Traditional cherry cultivation and extraction methods are unable to meet the growing market demand, and the yield and quality stability are low. They are also unable to efficiently produce cherry cell cultures rich in flavonoids and polyphenols, thus failing to meet the application needs of cosmetics and other fields.
A specific combination of salicylic acid, methyl jasmonate, and UV-B light was used, along with phenylalanine and ascorbic acid as precursors. Cherry cells were cultured in MS medium, and exosomes were extracted by differential and ultracentrifugation. Culture conditions were optimized to increase the content of flavonoids and polyphenols.
It significantly increased the content of flavonoids and polyphenols in cherry cell cultures, with the total flavonoid content reaching 110 mg/g and the total polyphenol content reaching 138 mg/g. Exosomes have broad application prospects in the cosmetics field.
Smart Images
Figure CN122303123A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of cell culture technology, specifically relating to a cherry cell culture method and cherry cell exosomes. Background Technology
[0002] Early cherry (Prunus pseudocerasus), a popular fruit variety, has garnered attention not only for its delicious taste but also for its rich nutritional value and medicinal potential. Cherry fruit is rich in flavonoids and polyphenols, whose secondary metabolites possess significant antioxidant, anti-inflammatory, and antibacterial bioactivities, thus showing broad application prospects in medicine, health products, and cosmetics. However, traditional cultivation and extraction methods struggle to meet the growing market demand, and yield and quality stability are low due to environmental and climatic factors. Therefore, developing efficient and stable cherry cell culture technology has become a research hotspot.
[0003] Cell cultures offer numerous advantages as cosmetic raw materials. Firstly, they can be produced efficiently in controlled environments, ensuring a stable and consistent supply of raw materials. Secondly, cell cultures are unaffected by environmental pollution such as pesticides and heavy metals, ensuring the purity and safety of cosmetic raw materials. Furthermore, cell cultures are rich in natural active ingredients, such as antioxidants, flavonoids, and polyphenols, which can enhance the efficacy of cosmetics. Cell culture technology is not dependent on land or water resources, making it more environmentally friendly and sustainable. By adjusting culture conditions, specific ingredients can be customized to meet the needs of different cosmetic products.
[0004] Furthermore, the extraction of exosomes from *Prunus mume* suspension cells and their application in the cosmetics field demonstrates the application value of cell culture technology. Exosomes, as nanoparticles with important biological activity, exhibit good stability and biocompatibility. After disrupting cells with ultrasound, exosomes are extracted using differential centrifugation and ultracentrifugation, and their application in cosmetics can effectively exert antioxidant and anti-aging effects.
[0005] Therefore, developing stable and efficient cherry cell culture methods and obtaining cherry cell cultures with high levels of active ingredients are technical problems that need to be solved. Summary of the Invention
[0006] To address the shortcomings of the existing technology, this invention provides a cherry cell culture method and cherry cell exosomes. The culture method of this invention can increase the content of active ingredients, especially flavonoids and polyphenols, in cherry cultures, and the obtained cell cultures, especially exosomes, have good application prospects in the cosmetics field.
[0007] The specific technical solution is as follows:
[0008] One objective of this invention is to provide a method for culturing cherry cells, comprising the following steps:
[0009] S1. Obtain cherry cell lines;
[0010] S2. Prepare the culture medium by adding salicylic acid and methyl jasmonate;
[0011] S3. The cherry cell line obtained in step S1 is cultured under the following conditions: cultured at 20-30℃ and irradiated with UV-B light during the culture process.
[0012] Furthermore, in step S2, the amount of salicylic acid added to the culture medium is preferably 50–180 μM, more preferably 100–150 μM; the amount of methyl jasmonic acid added is 25–100 μM, more preferably 50–75 μM. Experiments have confirmed that adding salicylic acid and methyl jasmonic acid as inducers can significantly increase the flavonoid and polyphenol content in cherry cell cultures.
[0013] Furthermore, in step S3: it is preferable to provide 1-3 hours of UV-B light exposure daily. Experiments have confirmed that UV-B light exposure is beneficial for the accumulation of flavonoids and polyphenols in cherry cell cultures.
[0014] Furthermore, in step S3: the preferred culture time is 10–16 days, at which time the flavonoid and polyphenol content in the cells reaches a relatively high level. After multiple experiments, it was found that the peak of flavonoid and polyphenol synthesis occurs on day 14 of culture; therefore, the optimal harvest time is day 14, when the flavonoid and polyphenol content in the cells reaches its highest level.
[0015] Further, in step S2: preferably, the precursor phenylalanine and the co-precursor ascorbic acid are added to the culture medium. The amount of phenylalanine added is 0.5–1.5 mM, more preferably 0.8–1.2 mM; the amount of ascorbic acid added is 0.1–0.8 mM, more preferably 0.3–0.6 mM. Phenylalanine (Phe) is a key precursor in the flavonoid synthesis pathway. Phe enters the phenolic synthesis pathway through the phenylalanine ammonia-lyase pathway, increasing the synthesis rate of flavonoids and polyphenols. Ascorbic acid (AsA), as a co-precursor, can improve the intracellular antioxidant state, indirectly promoting the synthesis and stability of phenolic compounds. Experimental data confirm that phenylalanine and ascorbic acid have a synergistic effect; under the preferred ratio of the present invention, the flavonoid and polyphenol content in cherry cell cultures can be significantly increased simultaneously. It should be noted that the ratio of the two is important; an improper ratio may reduce the flavonoid and polyphenol content.
[0016] Furthermore, in step S2: MS (Murashige & Skoog) liquid medium is used as the basal medium. Specifically, in the medium: preferably, 20–40 g / L sucrose is used as the carbon source, and the nitrogen sources are set at 2.0–3.0 g / L potassium nitrate and 0.3–0.7 g / L ammonium nitrate; other macro-elements, micro-elements, and vitamins are in the standard MS medium formulation to ensure normal cell proliferation and metabolism. Such a medium setting is conducive to the accumulation of flavonoids and polyphenols.
[0017] Furthermore, in step S3: it is preferable to carry out the culture on a shaker at a speed of 80 to 120 rpm.
[0018] Furthermore, in step S1, the method for obtaining cherry cell lines includes: inducing and culturing cherry explant materials to obtain callus tissue; culturing the callus tissue to obtain suspension cell lines.
[0019] Specifically, in step S1: the cherry explant material is at least one of the leaves, stem segments and buds of cherry seedlings.
[0020] Specifically, in step S1: MS medium is preferably used to induce the culture of cherry explant materials. More specifically, the MS medium used for induction culture contains 0.5–1.5 mg / L 6-benzyladenine (6-BA), 1.5–2.5 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), 20–40 g / L sucrose, and 5–9 g / L agar, and its pH is adjusted to 5.6–6.0.
[0021] Specifically, in step S1, the preferred induction culture conditions are: culture temperature 20-30℃, light exposure 10-14h per day, and light intensity 30-50μmol·m-2·s-1.
[0022] Specifically, in step S1: MS liquid culture medium is preferably used to suspend the callus tissue for culture. The MS liquid culture medium used to culture the callus tissue preferably contains 0.1-0.3 mg / L 6-benzyladenine (6-BA) and 0.5-1.5 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D) to promote the growth of suspension cells.
[0023] Specifically, in step S1: during the culture of callus tissue, it is preferable to place the suspension culture in a shaker and culture it at a speed of 80-120 rpm, with the culture temperature preferably being 20-30°C.
[0024] Furthermore, in step S1: the cherry is the American Early cherry.
[0025] A second objective of this invention is to provide a cherry cell culture obtained using the aforementioned cherry cell culture method. The cherry culture comprises cultured suspension cells.
[0026] A third objective of this invention is to provide a cherry cell exosome, which is extracted from the aforementioned cherry cell culture.
[0027] Specifically, the cherry cell exosomes are obtained by first extracting the fragmented suspension cells by differential centrifugation and then purifying them by ultracentrifugation.
[0028] The crushing process is preferably performed using ultrasonic crushing.
[0029] The differential centrifugation steps include: centrifuging at 300-700g for 5-15 minutes to remove unbroken cells and large particulate impurities; centrifuging the supernatant at 1000-3000g for 10-30 minutes to remove cell debris and small particulate impurities; and centrifuging the supernatant at 5000-15000g for 20-40 minutes to further purify the exosome suspension.
[0030] The ultracentrifugation step includes: taking 80,000 to 120,000 g of the supernatant obtained by differential centrifugation and centrifuging for 60 to 80 minutes to obtain exosome precipitate, then resuspending the exosome in PBS solution, and performing ultracentrifugation again under the above conditions to obtain purified exosomes.
[0031] The beneficial effects of this invention are as follows:
[0032] This invention improves the cherry cell culture method, increasing production efficiency and enhancing the content of active ingredients, especially flavonoids and polyphenols, in cherry cell cultures. Using the culture method of this invention, the total flavonoid content in the cell culture can reach 110 mg / g, the total polyphenol content can reach 138 mg / g, and the dry weight can reach 2.8 g / L. The cherry cell cultures obtained by this invention, especially the exosomes extracted from them, have broad application potential in the cosmetics field. Attached Figure Description
[0033] Figure 1 This is a photograph of cherry exosomes obtained in Example 2 of the present invention. Detailed Implementation
[0034] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.
[0035] Example 1
[0036] The steps for culturing Early Cherry cells are as follows:
[0037] S1. Obtain cherry cell lines;
[0038] (1) Induction of cherry callus:
[0039] a. Select healthy, disease-free seedlings of early-maturing cherry (Prunus mume) from the Taishan region, including leaves, stem segments, and buds, as explant materials. Sterilize the cherry explant materials. Use MS medium as the basic medium, add 1.0 mg / L 6-benzyladenine (6-BA) and 2.0 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), then add 30 g / L sucrose and 7 g / L agar, and adjust the pH to 5.8. Inoculate the treated cherry explants onto the prepared medium, ensuring that the wound side of the leaves, stem segments, or buds is facing down and in contact with the medium surface.
[0040] b. After inoculation, the explants were cultured at 25±2℃ and light intensity of 40μmol·m-2·s-1, with 12 hours of light and 12 hours of darkness per day. Within 2 to 4 weeks after inoculation, the wound site of the cherry explants began to swell and gradually formed callus tissue. Transplantation was performed every 2 to 3 weeks to ensure the continuous proliferation of callus tissue.
[0041] (2) Establishment and screening of high-efficiency cherry suspension cell lines:
[0042] a. Cut the callus obtained in step S1(1) into small pieces with a diameter of 1-2 mm and transfer them to an Erlenmeyer flask containing liquid MS medium; add 0.2 mg / L 6-BA and 1.0 mg / L 2,4-D to the medium to promote the growth of suspension cells;
[0043] b. The suspension culture was placed in a constant-temperature shaker at 100 rpm and the temperature was controlled at 25±2℃. The cultured suspension cells were observed every 3 days, and the cell status was monitored to see if small suspension cell clusters were formed. After 2 weeks of culture, suspension cell lines with high cell proliferation rates were screened. A certain amount of suspension culture medium was taken, and the cell density was measured using a hemocytometer or an automated cell counter. The cell density change was recorded every 7 days, and a cell proliferation curve was plotted. The morphology of the suspension cells was observed using an inverted microscope. Suspension cell lines with uniform morphology, intact cell walls, and few cell clusters were selected as excellent candidates.
[0044] c. The cell viability of different suspension cell cultures was assessed using the MTT assay. A certain volume of suspension cell culture was collected by centrifugation and washed twice with sterile PBS. MTT reagent was added and incubated for 4 hours, followed by the addition of DMSO to dissolve the resulting purple formazan crystals. The OD value was measured at 570 nm using a microplate reader. A higher OD value indicates stronger cell viability. The cell culture with the highest MTT value was selected and confirmed as the suspension cell line with the best cell viability.
[0045] d. The selected suspension cell lines were transferred to new liquid MS medium at a mass ratio of 1:5 and cultured on a shaker at 100 rpm. Subculture was performed every 2 to 3 weeks, and the cells were passaged 3 times to ensure that the proliferation capacity and activity of the selected cell lines remained stable in multiple passages.
[0046] e. Use flow cytometry to perform cell cycle analysis on the selected suspension cell lines to determine the proportion of cells in the S phase; a high proportion of S phase cells indicates that the cells have high proliferative activity; observe the selected high-efficiency cell lines under a microscope to confirm that the cells have uniform morphology, moderate size, and compact texture.
[0047] f. Take the stable proliferating cherry suspension cell line obtained through screening, centrifuge the culture at 1000 rpm for 5 min, remove the culture medium, and collect the cell pellet.
[0048] S2. Preparation of culture medium: MS liquid medium is used as the base medium as the basic nutrient support system; in the medium: the carbon source is 30 g / L sucrose; the nitrogen source is 2.5 g / L potassium nitrate and 0.5 g / L ammonium nitrate, and other macro-elements, micro-elements and vitamins are in the standard MS medium formula; 1 mM phenylalanine (precursor) and 0.5 mM ascorbic acid (auxiliary precursor) are added to the medium; 150 μM salicylic acid and 75 μM methyl jasmonic acid are added to the medium.
[0049] S3. The cherry cell line obtained in step S1 was cultured: the cell inoculum was 10 g / L based on the culture medium, and the cells were cultured in a shaker at 100 rpm at 25 ± 2 ℃, with 2 h of UV-B light per day for 14 days. The cell pellet was collected by low-speed centrifugation (3000 g, 10 min) to obtain cherry suspension cell culture.
[0050] Example 2
[0051] Exosomes were extracted from cherry suspension cell cultures obtained in Example 1-1, using the following steps:
[0052] S1. Cell collection and disruption: The suspended cell culture was disrupted using an ultrasonic cell disruptor. The parameters were set to power 300W, time 30min, and cycle interval 10s. The operation was performed in an ice bath at 4℃.
[0053] S2. Preliminary extraction: Differential centrifugation was used, and the following steps were performed in sequence: centrifugation at 500g for 10 min to remove undisturbed cells and large particulate impurities; centrifugation at 2000g for 20 min was taken from the supernatant to remove cell debris and small particulate impurities; centrifugation at 10000g for 30 min was taken from the supernatant to further purify the exosome suspension;
[0054] S3. Exosome purification: High-purity exosome precipitate was obtained by ultracentrifugation at 100,000g for 70 min. The exosome precipitate was resuspended in PBS solution and ultracentrifuged again (100,000g, 70 min) to finally obtain purified exosomes, as shown in the image below. Figure 1 As shown.
[0055] Test 1
[0056] The effects of salicylic acid, methyl jasmonic acid, UV-B light exposure, and culture temperature and time on the culture of early cherry cells were tested. The results are shown in Table 1. In Table 1, each treatment group was treated according to Example 1, except for the dosage of salicylic acid and methyl jasmonic acid, and the UV-B light exposure time. The dosage of salicylic acid and methyl jasmonic acid, and the UV-B light exposure time for each treatment group are shown in Table 1. The total flavonoid and total polyphenol contents in the obtained cherry cell cultures were also tested.
[0057] Table 1. Effects of salicylic acid, methyl jasmonate, and UV-B irradiation time.
[0058]
[0059] Table 1 shows that under the conditions of Example 1 (YT-9-5I), cherry suspension cell culture can significantly increase the content of flavonoids and polyphenols, with total flavonoids reaching 110 mg / g and total polyphenols reaching 138 mg / g, while the dry weight is 12.8 g / L, showing very high production efficiency.
[0060] Test 2
[0061] Nitrogen sources were screened, and the effect of nitrogen source configuration in the culture medium on the culture of early cherry was tested. The results are shown in Table 2. The cherry suspension cell lines obtained in step S1 of the example were used for culturing in each treatment group in Table 2. The obtained cherry cell cultures were lyophilized, and the total flavonoid and total polyphenol contents were tested. The culture method is as follows:
[0062] S2. Preparation of culture medium: MS liquid medium is used as the base medium as the basic nutrient support system; in the culture medium: the carbon source is 30 g / L sucrose; the nitrogen source is prepared as shown in Table 2; other macro-elements, micro-elements and vitamins are in the standard MS medium formula.
[0063] S3. The cherry cell line obtained in step S1 is cultured: the cell inoculum is 10 g / L based on the culture medium, and the cells are cultured in a shaker at 100 rpm at 25 ± 2 ℃, with 2 h of UV-B light per day for 14 days to obtain cherry suspension cell culture.
[0064] Table 2 Effects of potassium nitrate and ammonium nitrate
[0065]
[0066] The results in Table 2 show that the combination of 2.5 g / L potassium nitrate and 0.5 g / L ammonium nitrate can improve nitrogen utilization efficiency during metabolism and promote the accumulation of phenolic compounds.
[0067] Test 3
[0068] Precursor substances were screened, and the effects of the precursor phenylalanine and the cofactor precursor ascorbic acid on the culture of early cherry cells were tested. The results are shown in Table 3. The cherry suspension cell lines obtained in step S1 of the example were used for culture in each treatment group in Table 3. The obtained cherry cell cultures were lyophilized, and the total flavonoid and total polyphenol contents were tested. The culture method is as follows:
[0069] S2. Preparation of culture medium: MS liquid medium is used as the base medium as the basic nutrient support system; in the culture medium: the carbon source is 30 g / L sucrose; the nitrogen source is 2.5 g / L potassium nitrate and 0.5 g / L ammonium nitrate, and other macro-elements, micro-elements and vitamins are in the standard MS culture medium formula; the amount of phenylalanine and ascorbic acid added to the culture medium is shown in Table 3.
[0070] S3. The cherry cell line obtained in step S1 is cultured: the cell inoculum is 10 g / L based on the culture medium, and the cells are cultured in a shaker at 100 rpm at 25 ± 2 ℃, with 2 h of UV-B light per day for 14 days to obtain cherry suspension cell culture.
[0071] Table 3 Effects of phenylalanine and ascorbic acid
[0072]
[0073]
[0074] To further improve the synthesis efficiency of flavonoids and polyphenols, adding precursor substances is a key step in increasing the accumulation of cellular metabolites. After screening, the following precursor substances and their concentrations were considered the optimal combination: Phenylalanine (Phe): Phenylalanine is a key precursor in the flavonoid synthesis pathway. Adding 1 mM phenylalanine to the culture medium significantly increased the accumulation of phenolic metabolites. Phe enters the phenolic synthesis pathway via the phenylalanine ammonia-lyase pathway, increasing the synthesis rate of flavonoids and polyphenols. Ascorbic acid (AsA): As an auxiliary precursor, adding 0.5 mM ascorbic acid can improve intracellular antioxidant status, indirectly promoting the synthesis and stability of phenolic compounds.
[0075] Table 3 shows that phenylalanine and ascorbic acid have a synergistic effect, and with the optimal combination of YT-9-5, they can significantly increase the content of flavonoids and polyphenols in cherry cell cultures simultaneously. It is important to note that the ratio of the two is crucial; an improper ratio may actually reduce the content of flavonoids and polyphenols.
[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A method for culturing cherry cells, characterized in that, Includes the following steps: S1. Obtain cherry cell lines; S2. Prepare the culture medium by adding salicylic acid and methyl jasmonate; S3. The cherry cell line obtained in step S1 is cultured under the following conditions: cultured at 20-30℃ and irradiated with UV-B light during the culture process.
2. The cherry cell culture method according to claim 1, characterized in that, In step S2, the amount of salicylic acid added to the culture medium is 50–180 μM; the amount of methyl jasmonic acid added is 25–100 μM.
3. The cherry cell culture method according to claim 1, characterized in that, In step S3: Expose yourself to UV-B light for 1 to 3 hours daily; The incubation period is 10 to 16 days.
4. The cherry cell culture method according to any one of claims 1 to 3, characterized in that, In step S2: Phenylalanine and ascorbic acid are added to the culture medium; The amount of phenylalanine added is 0.5–1.5 mM; the amount of ascorbic acid added is 0.1–0.8 mM.
5. The cherry cell culture method according to any one of claims 1 to 3, characterized in that, In step S2: MS liquid medium is used as the basal medium.
6. The cherry cell culture method according to any one of claims 1 to 3, characterized in that, In step S1: cherry explant material is induced and cultured to obtain callus tissue; the callus tissue is cultured to obtain a suspension cell line.
7. The cherry cell culture method according to any one of claims 1 to 3, characterized in that, In step S1: the cherries mentioned are early-maturing cherries.
8. A cherry cell culture obtained using the cherry cell culture method as described in any one of claims 1 to 7.
9. A cherry cell exosome, extracted from the cherry cell culture as described in claim 8.
10. The cherry cell exosomes according to claim 9, characterized in that, The cells were obtained by first extracting the fragmented suspended cells using differential centrifugation, and then purifying them using ultracentrifugation.