An extract of malpighia glabra callus cell culture and uses thereof

Abstract

The application discloses a kind of Malpighia glabra callus cell culture extract and application, culture preparation method is as follows: Malpighia glabra callus induction and culture, Malpighia glabra suspension cell culture, extract is prepared from culture and is extracted;The extract of Malpighia glabra callus cell culture prepared by the application can be applied in the preparation of cosmetics with antioxidant capacity or anti-inflammatory soothing effect.The application can be dedifferentiated into callus by tissue culture technology, and the explant of Malpighia glabra is reproduced by plant cell culture engineering, in the culture process, the basic nutrients in culture medium are converted into bioactive substances such as flavonoids and polyphenols by plant cells through their own metabolism;The technology can not be limited by the growth of Malpighia glabra, and there is no risk of heavy metal and pesticide residue in the production process.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an extract of a callus cell culture of Begonia variegata and its applications. Background Technology

[0002] Malus transitoria (Batalin) CKSchneid., belonging to the genus Malus in the family Rosaceae of the class Dicotyledonous plants in the phylum Angiosperm, is currently mainly distributed in the western Tibetan areas of Sichuan, southeastern Tibet, and the Beishan and Mahe River forest areas of eastern Qinghai in my country. It is endemic to the Hengduan Mountains region of my country and is an extremely valuable plant germplasm resource. According to the Flora of China, Malus transitoria is a shrub to small tree, reaching up to 8 meters in height, with slender, cylindrical branches and leaves. It flowers in May and fruits in September, with nearly umbel-shaped inflorescences bearing 3-6 flowers. The fruit, when ripe, becomes a small spherical shape.

[0003] Malus spectabilis has high economic and medicinal value. In Tibetan-populated areas of Qinghai, its leaves are often used to make Tibetan tea. Its fruits are frequently used as a high-quality raw material for health drinks, jams, canned goods, and pharmaceuticals. Chemical studies have revealed that both the fruits and leaves of Malus spectabilis contain various active substances, including polyphenols, flavonoids, amino acids, and vitamins. These bioactive components endow Malus spectabilis with antioxidant, anti-inflammatory, and blood pressure-lowering effects.

[0004] Currently, the development of active substances in Malus variegata focuses on traditional plant active ingredient extraction techniques. For example, invention patent CN102701938A provides a method for extracting and purifying phloretin from Malus toringo and Malus variegata, which separates phloretin by using a water-ethyl acetate system extraction and silica gel column elution technology.

[0005] Invention patent CN117959223A discloses a skin care product containing Russian sago extract and its preparation method. The Russian sago extract is obtained by mixing treated Russian sago pulp, iron oxide, and an extract (at least one of water, ethanol, acetone, and ethyl acetate), extracting under ultrasound to remove impurities, and collecting the extract.

[0006] Invention patent CN108850331A describes a tea paste made from flowering crabapple leaves and its preparation process, which involves the deep processing of flowering crabapple leaves. It uses a combination of water extraction and alcohol extraction to extract the highly soluble active ingredients from the flowering crabapple leaves.

[0007] Invention patent CN119081987A describes an apple nanovesicle, its preparation method, and its application. The method involves collecting young apple fruits 14–60 days after flowering as raw materials and extracting nanovesicles using direct centrifugation or sucrose density gradient centrifugation.

[0008] While these methods can obtain the natural active ingredients from Begonia florida, their drawbacks are becoming increasingly apparent. For example, the source is heavily dependent on plant growth, the extract is prone to heavy metal and pesticide residues, and the extraction solvent can cause environmental pollution.

[0009] Therefore, developing a technology that can efficiently obtain the active ingredients of Begonia florida without relying on natural plants or being limited by the growth cycle is of great significance for protecting plant germplasm resources and expanding high-end skin care raw materials.

[0010] Plant cell engineering is a bioengineering technology based on the totipotency of plant cells. It uses techniques such as tissue culture and genetic modification to reproduce and improve varieties at the cellular level. It is divided into two major stages: upstream engineering (cell culture, genetic manipulation, and preservation) and downstream engineering (large-scale production).

[0011] There are currently no reports on the technology of plant cell engineering for Malus spectabilis, nor on the application of Malus spectabilis plant cell engineering products in cosmetics. Summary of the Invention

[0012] The purpose of this invention is to provide an extract from a Malus spectabilis callus cell culture and its applications. This invention utilizes tissue culture technology to dedifferentiate Malus spectabilis explants into callus tissue, and then uses plant cell culture engineering to mass-produce plant cells. During the culture process, the plant cells convert the basic nutrients in the culture medium into bioactive substances such as flavonoids and polyphenols through their own metabolism. This technology is not limited by the growth of Malus spectabilis and eliminates the risk of heavy metal and pesticide residues during production.

[0013] The technical solution of the present invention:

[0014] An extract from a culture of callus cells of Begonia variegata, prepared by the following steps:

[0015] S1. Induction and culture of callus from *Malus variegated*:

[0016] After surface disinfection of the Malus spectabilis explants, they were inoculated onto induction solid medium and cultured in the dark to induce callus tissue; then the induced callus tissue was transferred to proliferation solid medium for subculture.

[0017] The induction solid culture medium includes B5 basal medium, 30 g / L sucrose, 0.01–0.05 mg / L 6-BA, and 1.0–2.0 mg / L 2,4-D;

[0018] The proliferative solid culture medium includes B5 basal medium, 30 g / L sucrose, 0.01–0.1 mg / L 6-BA, and 0.5–2.0 mg / L 2,4-D;

[0019] S2, suspension cell culture of Begonia variegatedis:

[0020] The callus tissue obtained by subculturing the proliferation culture in step S1 1 to 3 times was cut into small pieces and inoculated into liquid culture medium at an inoculation rate of 3 to 10% (w / v) and cultured with shaking to obtain the callus tissue cell culture of Malus spectabilis.

[0021] The liquid culture medium includes B5 basal medium, 30 g / L sucrose, 0–2.0 mg / L 6-BA, and 0–2.0 mg / L 2,4-D.

[0022] S3. Extract preparation:

[0023] The Malus spectabilis callus cell culture obtained in step S2 is subjected to high-pressure homogenization, centrifugation, and filtration, or directly centrifuged and filtered without high-pressure homogenization, to obtain an extract of Malus spectabilis callus cell culture.

[0024] In the aforementioned extract of Malus spectabilis callus cell culture, the surface disinfection in step S1 is as follows: after rinsing the Malus spectabilis explants with running water, they are disinfected with 75% ethanol for 30 seconds, treated with a mixed treatment agent for 8 minutes, and finally rinsed clean with sterile water; the mixed treatment agent is an 11% sodium hypochlorite aqueous solution and 1% Tween 20.

[0025] In the aforementioned extract of Begonia variegata callus cell culture, in step S2, the callus is cut into 2-5 mm pieces. 3 Small pieces; the shaking culture conditions are a temperature of 25℃, a shaking speed of 110 rpm, and a culture time of 7 to 15 days.

[0026] In the extract of the aforementioned Malus spectabilis callus cell culture, in step S1, the induction solid medium was cultured in the dark for 21 days; the proliferation solid medium was used for subculture for 21 days.

[0027] In the extract of the aforementioned Begonia variegata callus cell culture, the shaking culture time in step S2 is 7–15 days.

[0028] In the extract of the aforementioned Begonia variegata callus cell culture, in step S3, the high-pressure homogenization pressure is 1000 bar, the homogenization time is 2 min, and the number of homogenization cycles is 5; the centrifugation conditions are 10000 rpm for 15 min; and the filtration is carried out using a 0.22 μm filter membrane.

[0029] The application of an extract from a callus cell culture of Begonia variegata in the preparation of cosmetics with antioxidant properties.

[0030] The application of an extract from a culture of callus cells of Begonia variegata in the preparation of cosmetics with anti-inflammatory and soothing effects.

[0031] Compared with the prior art, the beneficial effects of this application are as follows:

[0032] 1. This invention is the first to successfully establish a system for inducing, proliferating and culturing suspension cells of callus from Malus spectabilis, filling a technological gap in this field;

[0033] 2. This invention does not rely on the growth of natural flowering crabapple plants, is not limited by season, climate, or region, and can be industrialized year-round and in all weather conditions. It effectively protects rare plant germplasm resources, avoids pesticide pollution and heavy metal contamination, and realizes a green and sustainable production method.

[0034] 3. The method of this invention has a short production cycle (a batch can be harvested in 7-15 days). By optimizing the hormone ratio (such as 6-BA and 2,4-D) in the culture medium and the culture conditions, the cell biomass (fresh cell weight) and the accumulation of target active products (total flavonoids and total polyphenols) can be significantly increased.

[0035] Data from the examples show that, under optimal conditions (Example 1), the cell fresh weight can reach 3.72 g / mL, and the total flavonoid and total polyphenol contents are as high as 47.86 μg / mL and 49.38 μg / mL, respectively, which are much higher than those of the comparative example.

[0036] 4. The *Malus spectabilis* callus cell culture prepared by this invention exhibits excellent biological activity; experiments have shown that the IC50 of the DPPH free radical scavenging ability in Example 1 is [missing information]. 50 The value was 50.31 mg / mL, indicating good antioxidant capacity;

[0037] Example 1 showed that the inhibition rates of VEGF / VEGFR2 and TNF-α / TNFR1 binding were 54.76% and 61.35%, respectively, indicating that it can effectively block inflammatory signaling pathways and has significant anti-inflammatory and soothing effects. Attached Figure Description

[0038] Figure 1 This is a liquid chromatogram of the samples from Example 1 and Comparative Example 3 in Experiment 3 of the present invention at a detection wavelength of 285 nm. Detailed Implementation

[0039] The present invention will be further described below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the present invention.

[0040] Example 1.

[0041] An extract from a culture of callus cells of Begonia variegata, prepared by the following steps:

[0042] S1. Rinse the plant tissue of *Malus spectabilis* with running water for 2 hours, disinfect it with 75% ethanol for 30 seconds under sterile conditions, rinse with sterile water for 1 minute, treat with 11% sodium hypochlorite (containing 1% Tween 20) for 8 minutes, rinse with sterile water for 5 minutes, and blot dry with sterile filter paper for later use.

[0043] In a clean bench, the damaged part of the explant of Malus spectabilis was removed, and the explant was cut into 1 cm pieces. The pieces were then inoculated into induction solid medium (B5 basal medium with 30 g / L sucrose, 0.05 mg / L 6-BA, and 2.0 mg / L 2,4-D added) and cultured in the dark for 21 days to induce callus tissue.

[0044] Select healthy, light yellow, and loosely textured tissue samples and cut them into 0.5 cm pieces. 3 Small pieces were transferred to proliferation solid medium (B5 basal medium, with 30 g / L sucrose, 0.01 mg / L 6-BA, and 1.5 mg / L 2,4-D added), and the temperature was controlled at 23±2℃ and the humidity at 40±5%. After 21 days of dark culture, a large amount of callus tissue was obtained through subculture.

[0045] S2. Take the callus tissue that has grown vigorously after three subcultures, and cut it into 2-5 mm pieces using sterile forceps or a scalpel. 3 Small pieces were inoculated at a rate of 5% (w / v) into liquid culture medium (B5 basal medium with 30 g / L sucrose, 2.0 mg / L 6-BA, and 1.5 mg / L 2,4-D added), and cultured on a constant temperature shaker at 25 ℃ with shaking at 110 rpm / min for 10 days to obtain the callus cell culture of Malus spectabilis.

[0046] S3. Take 500 mL of Begonia variegata callus cell culture and homogenize it under high pressure at 1000 bar for 5 cycles. Centrifuge the broken homogenate at 10000 rpm for 15 min. Take the supernatant and filter it through a 0.22 μm filter membrane. Collect the filtrate to obtain the Begonia variegata callus cell culture extract.

[0047] Example 2.

[0048] An extract from a culture of callus cells of Begonia variegata is essentially the same as in Example 1, except that:

[0049] Step S1: The hormone concentration in the induction solid culture medium is 0.01 mg / L 6-BA and 1.0 mg / L 2,4-D;

[0050] The hormone concentrations in the proliferative solid culture medium were 0.1 mg / L 6-BA and 2.0 mg / L 2,4-D.

[0051] Example 3.

[0052] An extract from a culture of callus cells of Begonia variegata is essentially the same as in Example 1, except that:

[0053] Step S1: Take the vigorous callus tissue that has been subcultured once, inoculate it at a density of 10% (w / v), and culture it for 7 days.

[0054] Step S3: The Malus spectabilis callus cell culture obtained in step S2 is directly centrifuged and filtered to obtain the Malus spectabilis callus cell culture extract.

[0055] Example 4.

[0056] The application of an extract from a culture of callus cells of Begonia variegata in the preparation of cosmetics with antioxidant effects.

[0057] Example 5.

[0058] The application of an extract from a culture of callus cells of Begonia variegata in the preparation of cosmetics with anti-inflammatory and soothing effects.

[0059] Comparative Example 1.

[0060] Callus culture of Begonia variegatedis was performed in the same manner as in Example 1, except that:

[0061] Step S1: The hormone ratio in the induction solid culture medium is 0.01 mg / L 6-BA and 1.0 mg / L 2,4-D;

[0062] The hormone concentrations in the proliferative solid culture medium were 0.01 mg / L 6-BA and 0.01 mg / L 2,4-D.

[0063] Comparative Example 2.

[0064] Callus and suspension cell cultures of *Malus spectabilis* were performed in the same manner as in Example 1, except that:

[0065] Step S2: No hormones are added to the liquid culture medium.

[0066] Comparative Example 3.

[0067] An extract from the fruit of the flowering crabapple, prepared by the following steps:

[0068] Weigh 10g of Malus spectabilis fruit, freeze in liquid nitrogen for 10min, grind using a grinder, add pure water at a ratio of 1:15, extract by sonication, centrifuge at 10000 rpm for 15min, and filter the supernatant through a 0.22 μm filter membrane to obtain Malus spectabilis fruit extract.

[0069] Verification Experiment

[0070] Experiment 1: Statistical analysis of fresh weight of cells in callus culture of *Malus variegata*

[0071] Determination of fresh cell weight:

[0072] The harvested variegated crabapple liquid suspension cells were filtered for about 10 seconds. The filtered cells were then slightly dried with absorbent paper and weighed. The fresh weight was measured three times.

[0073] Table 1. Fresh cell weight content of examples and comparative examples

[0074]

[0075] Table 1 shows that the fresh weight of cells in Examples 1-3 was higher than that in Comparative Example 2; thus, it can be seen that the hormone concentrations of 6-BA and 2,4-D in the culture medium have a significant impact on the proliferation of suspended cells, and appropriate hormone concentrations and combinations can increase the content of Malus spectabilis cells in the culture system.

[0076] Note: The main difference between Comparative Example 1 and the Example is the hormone ratio; this ratio resulted in the induction solid culture medium only inducing a small amount of callus tissue. Comparative Example 1 only obtained a small amount of callus tissue and could not carry out subsequent suspension cell culture, so the fresh weight data of the suspension cells could not be measured. Therefore, there is no data for Comparative Example 1. The same applies to the subsequent experiments.

[0077] Experiment 2: Determination of total flavonoids and total polyphenols in callus cell cultures of *Malus variegata*

[0078] 2.1 Determination of total flavonoid content:

[0079] Weigh 14.5 mg of rutin standard into a 50 mL volumetric flask, add 20 mL of anhydrous ethanol and sonicate to dissolve, then dilute to volume with ultrapure water to prepare rutin working solution; then prepare 5% sodium nitrite solution, 10% aluminum nitrate solution, and sodium hydroxide (4.3 g / 100 mL) for the detection of total flavonoids in Malus spectabilis callus cell culture samples.

[0080] Take 6 mL of sample solution, add 1.0 mL of sodium nitrite and 1.0 mL of aluminum nitrate (shake well after each step and let stand at room temperature for 6 min), then add 10 mL of sodium hydroxide, add distilled water to make up to the mark, shake well and let stand at room temperature for 15 min;

[0081] Take 200 μL into a 96-well plate and measure the absorbance at 500 nm using an ELISA reader. Finally, test a series of standard solutions with different rutin contents. Plot a standard curve with rutin content on the x-axis and absorbance on the y-axis. Substitute the absorbance of the test sample into the curve equation to calculate the total flavonoid content (unit: μg / mL).

[0082] 2.2 Determination of total polyphenol content:

[0083] Weigh 20 mg of gallic acid standard, add 10 mL of methanol and dilute to 100 mL with pure water, shake well, and store in the dark. Record this as gallic acid standard stock solution (200 mg / L). Use pure water to prepare a series of standard working solutions of 0 mg / L, 40 mg / L, 80 mg / L, 120 mg / L, 160 mg / L and 200 mg / L from the gallic acid standard stock solution.

[0084] Weigh 7 g of anhydrous sodium carbonate, dissolve it in pure water and bring the volume to 100 ml to prepare a 7% sodium carbonate solution;

[0085] For the test, take 0.2 mL of standard curve solution (or sample solution), add 1.8 mL of pure water, then add 0.2 mL of Folin-Ciocalteu reagent and shake to mix. After reacting for 5 min, add 2.0 mL of 7.5% Na2CO3, then add 0.8 mL of pure water. React in the dark for 1 h, and then measure the absorbance at 750 nm in a 96-well plate.

[0086] According to the experimental method for determining the total flavonoid content, the standard curve for rutin is y = 0.0059x + 0.0002, R0 2 =0.9994;

[0087] According to the experimental method for determining the total polyphenol content, the standard curve for gallic acid is y = 0.0021x + 0.0043, R0 2 =0.9989.

[0088] Table 2 Total flavonoid and total polyphenol content in examples and comparative examples

[0089]

[0090] Table 2 shows that the total flavonoid and total polyphenol content of Examples 1-3 is higher than that of Comparative Example 2. This indicates that the concentrations of 6-BA and 2,4-D hormones in the culture medium have a significant impact on the accumulation of active substances in the suspension cells of Malus spectabilis, and that appropriate hormone concentrations can promote the accumulation of active substances.

[0091] Experiment 3: Detection of Active Ingredients in Samples

[0092] The active ingredients of the callus cell culture extract of Malus spectabilis from Example 1 and the fruit extract of Malus spectabilis prepared in Comparative Example 3 were detected and analyzed.

[0093] 3.1 Sample preparation: Take 2.0 mL of sample, centrifuge at 12000 rpm for 15 min, and collect the supernatant to obtain the test sample;

[0094] 3.2 HPLC determination of active ingredients in the extract:

[0095] The samples from Example 1 and Comparative Example 3 were analyzed by HPLC using an Agilent Eclipse XDB-C18 column (250 mm × 4.6 mm, 5 μm). Gradient elution was performed using acetonitrile (A) and 0.1% formic acid water (B) as the mobile phase: 0–10 min, 10% A; 10–15 min, 10% → 15% A; 15–17 min, 15% A; 17–32 min, 15% → 17% A; 32–37 min, 17% A; 37–44 min, 17% → 20% A; 44–46 min, 20% A; 46–51 min, 20% → 30% A; 51–55 min, 30% A → 100% A; 55–58 min, 100% A; 58–60 min, 100% A → 30% A; 60–61 min, 10% A → 30% A; 60–61 min, 10% A → 30% A. min, 30%A→10%A; 61~70 min, 10%A; flow rate 1.0 mL / min, column temperature 30 ℃, detection wavelength 285 nm and 254 nm.

[0096] From the appendix Figure 1 The liquid chromatography results show that Example 1 contains chlorogenic acid, catechin, caffeic acid, rutin, isoquercitrin, quercetin and phlorizin.

[0097] Among them, catechin and rutin are the active ingredients that distinguish it from Comparative Example 3, while the contents of chlorogenic acid, caffeic acid, isoquercitrin and quercetin are higher than those of Comparative Example 3.

[0098] Experiment 4 Determination of antioxidant capacity

[0099] The DPPH scavenging ability was examined using Examples 1, 3, and Comparative Example 2 to determine its antioxidant performance.

[0100] The ability to scavenge DPPH free radicals was determined using a kit method, and the specific determination method is as follows:

[0101] 4.1 Preparation of 0.2 mmol / L DPPH solution: Weigh 8 mg DPPH, dissolve it in anhydrous ethanol and dilute to 100 mL in a volumetric flask;

[0102] 4.2 Preparation of test samples: The raw materials are diluted with distilled water or anhydrous ethanol. 3-5 concentration gradients are set for each test sample, and the DPPH scavenging rate of the sample is determined.

[0103] 4.3 Determination: Mix an equal volume of sample solution with DPPH solution and name it tube A1; mix an equal volume of anhydrous ethanol with DPPH solution and name it tube A2; mix an equal volume of sample solution with anhydrous ethanol and name it tube A3; perform three replicates for each sample.

[0104] After reacting in the dark for 30 min, the absorbance values ​​of tubes A1, A2, and A3 at 517 nm were measured using an ELISA reader.

[0105] 4.4 Result Calculation: Clearance Rate (%) = [(A2 + A3) - A1] / A2

[0106] Table 3. DPPH radical scavenging capabilities of different embodiments and comparative examples

[0107]

[0108] As shown in Table 3, Example 1's IC for DPPH removal 50 The value was 50.31 mg / mL, indicating that the callus cell culture of Malus spectabilis has a certain antioxidant capacity.

[0109] Experiment 5: Determination of anti-inflammatory and soothing effects

[0110] 5.1 Assay for VEGF and VEGFR2 binding inhibitory activity: The inhibitory effect of Begonia variegata callus cell culture on the binding of VEGF and VEGFR2 was evaluated using an ELISA kit (Bepsys, catalog number EP-141). The specific assay method is as follows:

[0111] 1) Use human VEGFR2 protein-coated plates and incubate overnight or for 16 hours at 4°C;

[0112] 2) After washing the coated plate three times, incubate it with blocking buffer at 37°C for 1.5 hours; after washing again, add the sample to be tested for pre-incubation;

[0113] 3) Add VEGF-Biotin to allow it to bind to the coated VEGFR2;

[0114] 4) After washing the plate three times, add streptavidin-horseradish peroxidase. After the reaction, add horseradish peroxidase substrate to carry out the colorimetric reaction.

[0115] 5) Measure OD using an ELISA reader 450 The absorbance readings and the inhibitory activity of the callus material on the binding of VEGF and VEGFR2 are shown in Table 4.

[0116] 5.2 Assay for inhibitory activity against human TNF-α and TNFR1 binding: The inhibitory effect of Begonia variegata callus cell culture on the binding of TNF-α and TNFR1 was evaluated using an ELISA kit (Bepsys, catalog number EP-143). The specific assay method is as follows:

[0117] 1) Use human TNFR1 protein-coated plates and incubate overnight or for 16 hours at 4°C;

[0118] 2) After washing the coated plate three times, incubate it with blocking buffer at 37°C for 1.5 hours; after washing again, add the sample to be tested for pre-incubation;

[0119] 3) Add TNF-α-Biotin to enable it to bind to the coated TNFR1;

[0120] 4) After washing the plate three times, add streptavidin-horseradish peroxidase, and then add horseradish peroxidase substrate after the reaction.

[0121] 5) Measure OD using an ELISA reader 450 The absorbance readings were used to calculate the inhibitory activity of the Begonia variegata callus cell culture on the binding of TNF-α and TNFR1. The results are shown in Table 4.

[0122] Table 4. Inhibitory activity assay of VEGF-VEGFR2 binding and TNF-α-TNFR1 binding in Example 1.

[0123]

[0124] As shown in Table 4, Example 1 showed an inhibitory activity of 54.76% on the binding of VEGF and VEGFR2 and 61.35% on the binding of TNF-α and TNFR1. This indicates that the extract from the callus cell culture of Malus spectabilis can inhibit the binding of inflammatory factors and receptors, thereby blocking the transmission and amplification of inflammatory signals and having a certain anti-inflammatory and soothing effect.

Claims

1. An extract from a culture of callus cells of Begonia variegata, characterized in that, The preparation method steps are as follows: S1. Induction and culture of callus from *Malus variegated*: After surface disinfection of the Malus spectabilis explants, they were inoculated onto induction solid medium and cultured in the dark to induce callus tissue; then the induced callus tissue was transferred to proliferation solid medium for subculture. The induction solid culture medium includes B5 basal medium, 30 g / L sucrose, 0.01–0.05 mg / L 6-BA, and 1.0–2.0 mg / L 2,4-D; The proliferative solid culture medium includes B5 basal medium, 30 g / L sucrose, 0.01–0.1 mg / L 6-BA, and 0.5–2.0 mg / L 2,4-D; S2, suspension cell culture of Begonia variegatedis: The callus tissue obtained by subculturing the proliferation culture in step S1 1 to 3 times was cut into small pieces and inoculated into liquid culture medium at an inoculation rate of 3 to 10% (w / v) and cultured with shaking to obtain the callus tissue cell culture of Malus spectabilis. The liquid culture medium includes B5 basal medium, 30 g / L sucrose, 0–2.0 mg / L 6-BA, and 0–2.0 mg / L 2,4-D. S3. Extract preparation: The Malus spectabilis callus cell culture obtained in step S2 is subjected to high-pressure homogenization, centrifugation, and filtration, or directly centrifuged and filtered without high-pressure homogenization, to obtain an extract of Malus spectabilis callus cell culture.

2. An extract from a callus cell culture of *Malus spectabilis* according to claim 1, characterized in that, The surface disinfection described in step S1 is as follows: After rinsing the Malus spectabilis explant with running water, it is disinfected with 75% ethanol for 30 seconds, treated with a mixed treatment agent for 8 minutes, and finally rinsed clean with sterile water; the mixed treatment agent is an 11% sodium hypochlorite aqueous solution and 1% Tween 20.

3. An extract from a callus cell culture of Begonia variegata according to claim 1, characterized in that: In step S2, the callus tissue is cut into 2-5 mm pieces. 3 Small pieces; the shaking culture conditions are a temperature of 25°C and a shaking speed of 110 rpm.

4. An extract from a callus cell culture of Begonia variegata according to claim 1, characterized in that: In step S1, the induction solid medium was cultured in the dark for 21 days; the proliferation solid medium was cultured for 21 days.

5. An extract from a callus cell culture of *Malus spectabilis* according to claim 1, characterized in that: In step S2, the shaking culture time is 7–15 days.

6. An extract from a callus cell culture of *Malus spectabilis* according to claim 1, characterized in that: In step S3, the high-pressure homogenization pressure is 1000 bar, the homogenization time is 2 min, and the number of homogenization cycles is 5; the centrifugation conditions are 10000 rpm for 15 min; and the filtration uses a 0.22 μm filter membrane.

7. The use of the extract according to any one of claims 1-6 in the preparation of cosmetics with antioxidant effects.

8. The use of the extract according to any one of claims 1-6 in the preparation of cosmetics with anti-inflammatory and soothing effects.

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