Camellia nitidissima suspension cell culture method

Abstract

The embodiment of the invention provides a golden camellia suspension cell culture method which comprises the following steps: golden camellia leaves are collected and inoculated into a WPM liquid culture medium containing hormone for culture, primary callus is formed through induction, and the hormone comprises naphthylacetic acid and 6-benayl aminopurine; and inoculating the primary callus into a WPM liquid culture medium containing auxin and cytokinin, and culturing to obtain the golden camellia suspension cells.

Description

Technical Field

[0001] This manual relates to the field of plant cell culture and preparation of bioactive substances, and in particular to a method for culturing suspension cells of Camellia chrysantha. Background Technology

[0002] Golden camellia tea (scientific name: Camellia petelotii Camellia chrysantha (also known as Golden Camellia) is an evergreen shrub belonging to the genus Camellia in the family Theaceae. Its tissues are rich in flavonoids (e.g., proanthocyanidins B2, catechins), polyphenols, and triterpenoid saponins, which have significant applications in functional foods and medicines. Currently, it faces challenges such as resource scarcity and low efficiency in artificial cultivation, making it difficult to meet the large-scale demand for active ingredients from Camellia chrysantha. Although cell culture technology exists, it still faces problems such as explant processing issues, inefficient callus culture, and significant obstacles to industrialization, thus hindering the standardized production of high-purity flavonoid extracts.

[0003] Therefore, it is desirable to provide a method for culturing suspension cells of Camellia chrysantha to solve the problem of producing active ingredients in Camellia chrysantha due to resource scarcity, inefficiency of traditional culture techniques, and inaccurate metabolic regulation, so as to achieve efficient, stable, and environmentally friendly large-scale preparation. Summary of the Invention

[0004] This specification provides one or more embodiments of a method for culturing Camellia chrysantha suspension cells, comprising the following steps: collecting Camellia chrysantha tea leaves, inoculating them into WPM liquid medium containing hormones for culture, inducing the formation of primary callus tissue, wherein the hormones include naphthaleneacetic acid and 6-benzylaminopurine; inoculating the primary callus tissue into WPM liquid medium containing auxin and cytokinin for culture, thereby obtaining Camellia chrysantha suspension cells.

[0005] In some embodiments, the concentration of naphthaleneacetic acid in the hormone-containing WPM liquid culture medium is 1-5 mg / L, and the concentration of 6-benzylaminopurine is 0.1-1 mg / L.

[0006] In some embodiments, the hormone-containing WPM liquid culture medium further contains an anti-browning substance, wherein the anti-browning substance is polyvinylpyrrolidone.

[0007] In some embodiments, before inoculating the golden chrysanthemum tea leaves into a hormone-containing WPM liquid culture medium, the method further includes: spraying the golden chrysanthemum tea leaves with a solution containing salicylic acid, wherein the concentration of salicylic acid in the solution is 0.05-0.2 mmol / L; and treating the golden chrysanthemum tea leaves with a composite solution containing a mercury disinfectant and a surfactant.

[0008] In some embodiments, the culture conditions in the hormone-containing WPM liquid medium include: a temperature of 25±2°C, dark culture for 6-8 days followed by alternating light and dark culture; the light-dark alternating culture has a light cycle of 13-15 hours of light followed by 9-11 hours of darkness, and a light intensity of 1500-2500 lux.

[0009] In some embodiments, in the WPM liquid culture medium containing auxin and cytokinin, the auxin is 2,4-dichlorophenoxyacetic acid, and the concentration of the auxin is 1-3 mg / L; the cytokinin is kinetin, and the concentration of the cytokinin is 0.2-1 mg / L.

[0010] In some embodiments, the culture conditions in the WPM liquid medium containing auxin and cytokinin include: a shaking speed of 100-150 rpm, a temperature of 20-28°C, a light cycle of 12-18 hours followed by 6-12 hours of darkness, and a light intensity of 800-1200 lux.

[0011] In some embodiments, the method further includes: subjecting the Camellia chrysantha suspension cells to at least one of the following treatments: cold treatment at 2-6°C and methyl jasmonate treatment, to further induce the accumulation of secondary metabolites in the cells, thereby obtaining the target Camellia chrysantha suspension cells.

[0012] In some embodiments, the 2-6°C cold treatment time is 12-36 hours, the methyl jasmonate treatment time is 24-72 hours, and the concentration of methyl jasmonate in the methyl jasmonate treatment is 50-150 μM.

[0013] This specification provides one or more embodiments of a Camellia chrysantha cell culture, which includes Camellia chrysantha suspension cells or target Camellia chrysantha suspension cells prepared by any of the above-described Camellia chrysantha suspension cell culture methods. The dried product of the Camellia chrysantha cell culture contains the following secondary metabolite contents: 10%-20% flavonoids and 15%-25% polyphenols.

[0014] In some embodiments, the Camellia chrysantha cell culture PAL , ANR1 Gene expression is upregulated. Attached Figure Description

[0015] This specification will be further illustrated by way of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are not limiting, wherein: Figure 1 This is a schematic diagram of solid-state cultured callus from Camellia chrysantha, as shown in some embodiments of this specification. Figure 2This is a schematic diagram of a Camellia chrysantha cell culture according to some embodiments of this specification; Figure 3 This is a schematic diagram illustrating the induced expression of key genes for the synthesis of total flavonoids and total polyphenols in Camellia chrysantha cell cultures under different treatment conditions, as shown in some embodiments of this specification (A: PAL Gene; B: ANR1 Gene; C: DFR Gene; D: different treatment conditions); Figure 4 This is a schematic diagram illustrating the differences in total flavonoid and total polyphenol content in dried products of Camellia chrysantha cell cultures under different treatment conditions, as shown in some embodiments of this specification (A: total flavonoids; B: total polyphenols; C: different treatment conditions). Figure 5 These are sample extracted ion chromatograms (EICs) shown in some embodiments of this specification. Figure 6 These are HE staining images of mouse aortic tissue according to some examples in this specification (A: blank control group; B: model group; C: low-dose Camellia chrysantha group; D: medium-dose Camellia chrysantha group; E: high-dose Camellia chrysantha group; F: positive control group). Figure 7 This is a schematic diagram illustrating the expression of eNOS protein in mouse aortic tissue according to some examples in this specification (A: blank control group; B: model group; C: low-dose Camellia japonica group; D: medium-dose Camellia japonica group; E: high-dose Camellia japonica group; F: positive control group). Figure 8 This is a schematic diagram illustrating the metabolism of flavonoids and polyphenols in Camellia chrysantha, based on some embodiments of this specification. Detailed Implementation

[0016] To more clearly illustrate the technical solutions of the embodiments in this specification, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are merely some examples or embodiments of this specification. For those skilled in the art, these drawings can be applied to other similar scenarios without creative effort.

[0017] As indicated in this specification and claims, unless the context clearly indicates otherwise, the words "a," "an," "an," and / or "the" are not specifically singular and may include the plural. Generally speaking, the terms "comprising" and "including" only indicate the inclusion of expressly identified steps and elements, which do not constitute an exclusive list.

[0018] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this specification pertains.

[0019] This manual aims to provide a complete process culture system for Camellia chrysantha callus to overcome the problems of traditional cell culture techniques, such as difficulties in explant processing, low efficiency in callus induction, asynchronous gene expression, large fluctuations in composition, uncontrollable processes, and high production costs. By using laboratory-cultured cells based on this scheme to replace wild or artificially cultivated Camellia chrysantha, it is possible to quickly and stably extract flavonoids, polyphenols, and other active ingredients that are beneficial to the human body. In addition, it further provides methods for detecting active ingredients and evaluating antioxidant efficacy of Camellia chrysantha cell cultures to achieve the detection and evaluation of corresponding components.

[0020] This specification provides one or more embodiments of a method for culturing Camellia chrysantha suspension cells, comprising the following steps: collecting Camellia chrysantha tea leaves, inoculating them into a WPM (Woody Plant Medium) liquid culture medium containing hormones for culture, inducing the formation of primary callus tissue, wherein the hormones include naphthaleneacetic acid (NAA) and 6-benzylaminopurine (6-BA); and inoculating the primary callus tissue into a WPM liquid culture medium containing auxin and cytokinin for culture to obtain Camellia chrysantha suspension cells.

[0021] In some embodiments, the concentration of naphthaleneacetic acid (NAA) in the hormone-containing WPM liquid culture medium can be 1-5 mg / L, and the concentration of 6-benzylaminopurine (6-BPA) can be 0.1-1 mg / L. In some embodiments, the concentration of NAA in the hormone-containing WPM liquid culture medium can be 1, 2, 3, 4, or 5 mg / L, and the concentration of 6-BPA can be 0.1, 0.3, 0.5, 0.7, 0.9, or 1 mg / L. In some embodiments, the concentration of NAA in the hormone-containing WPM liquid culture medium can be 3 mg / L, and the concentration of 6-BPA can be 0.5 mg / L.

[0022] In some embodiments, the hormone-containing WPM liquid culture medium may also contain an anti-browning substance and a carbon source. For example, the anti-browning substance may be polyvinylpyrrolidone (PVP), and the carbon source may be sucrose.

[0023] In some embodiments, the concentration of polyvinylpyrrolidone in the hormone-containing WPM liquid medium can be 0.05%-0.3% (w / w), and the concentration of sucrose can be 20-40 g / L. In some embodiments, the concentration of polyvinylpyrrolidone in the hormone-containing WPM liquid medium can be 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, or 0.3%, and the concentration of sucrose can be 20, 25, 30, 35, or 40 g / L. In some embodiments, the concentration of polyvinylpyrrolidone in the hormone-containing WPM liquid medium can be 0.1%, and the concentration of sucrose can be 30 g / L.

[0024] In some embodiments, before inoculating the golden chrysanthemum tea leaves into a hormone-containing WPM liquid culture medium, the method may further include: spraying the golden chrysanthemum tea leaves with a solution containing salicylic acid (SA) 1-5 days before collection, wherein the concentration of salicylic acid in the solution may be 0.05-0.2 mmol / L; sequentially using neutral detergent for ultrasonic cleaning, rinsing with running water, soaking in ethanol, treating with a compound solution containing mercury disinfectant and surfactant, and rinsing the golden chrysanthemum tea leaves with sterile water.

[0025] In some embodiments, a salicylic acid solution is sprayed onto the golden chrysanthemum tea leaves 1, 2, 3, 4, and 5 days before collection. In some embodiments, a salicylic acid solution is sprayed onto the golden chrysanthemum tea leaves 3 days before collection.

[0026] In some embodiments, the concentration of salicylic acid in the solution can be 0.05, 0.1, 0.15, or 0.2 mmol / L. The concentration of salicylic acid in the solution can be 0.1 mmol / L.

[0027] In some embodiments, the concentration of the neutral detergent can be 0.5%-2% (v / v), and the ultrasonic cleaning frequency can be 30-50 kHz. In some embodiments, the concentration of the neutral detergent can be 0.5%, 1%, 1.5%, or 2%, and the ultrasonic cleaning frequency can be 30, 35, 40, 45, or 50 kHz. In some embodiments, the concentration of the neutral detergent can be 1%, and the ultrasonic cleaning frequency can be 40 kHz.

[0028] In some embodiments, the mercury-containing disinfectant in the composite solution may be NaClO. In some embodiments, the concentration of NaClO may be 0.05%-0.2% (w / w). In some embodiments, the concentration of NaClO may be 0.05%, 0.1%, 0.15%, or 0.2%. In some embodiments, the concentration of NaClO may be 0.1%.

[0029] In some embodiments, the surfactant in the composite solution may be Tween-20 (polysorbate-20). In some embodiments, the concentration of Tween-20 may be 0.01%-0.1% (w / w). In some embodiments, the concentration of Tween-20 may be 0.01%, 0.03%, 0.05%, 0.07%, 0.09%, or 0.1%. In some embodiments, the concentration of Tween-20 may be 0.05%.

[0030] In some embodiments, the treatment time of the composite solution can be 3-10 minutes. In some embodiments, the treatment time of the composite solution can be 3, 4, 5, 6, 7, 8, 9, or 10 minutes. In some embodiments, the treatment time of the composite solution can be 6 minutes.

[0031] In some embodiments, the culture conditions in the hormone-containing WPM liquid medium include: a temperature of 25±2°C, dark culture for 6-8 days followed by light-dark alternating culture, during which the explants are periodically damaged to induce callus formation; the light-dark alternating culture has a light cycle of 13-15 hours of light followed by 9-11 hours of darkness, and a light intensity of 1500-2500 lux.

[0032] In some embodiments, the temperature can be 23, 24, 25, 26, or 27°C. In some embodiments, the temperature can be 25°C.

[0033] In some embodiments, the cells are cultured in the dark for 6, 7, or 8 days and then transferred to alternating light and dark culture. In some embodiments, the cells are cultured in the dark for 7 days and then transferred to alternating light and dark culture.

[0034] In some embodiments, the light-dark alternating culture period can be 13, 14, or 15 hours of light followed by 11, 10, or 9 hours of darkness, and the light intensity can be 1500, 1750, 2000, 2250, or 2500 lux. In some embodiments, the light-dark alternating culture period can be 14 hours of light followed by 10 hours of darkness, and the light intensity can be 2000 lux.

[0035] In some embodiments, in the WPM liquid culture medium containing auxin and cytokinin, the auxin can be 2,4-dichlorophenoxyacetic acid (2,4-D), and the concentration of auxin can be 1-3 mg / L; the cytokinin can be kinetin (KT), and the concentration of cytokinin can be 0.2-1 mg / L.

[0036] In some embodiments, the concentration of auxin can be 1, 1.5, 2, 2.5, or 3 mg / L, and the concentration of cytokinin can be 0.2, 0.3, 0.5, 0.7, 0.9, or 1 mg / L. In some embodiments, the concentration of auxin can be 2 mg / L, and the concentration of cytokinin can be 0.5 mg / L.

[0037] In some embodiments, the culture conditions in WPM liquid medium containing auxin and cytokinin include: a shaking speed of 100-150 rpm, a temperature of 20-28°C, a light cycle of 12-18 hours followed by 6-12 hours of darkness, and a light intensity of 800-1200 lux.

[0038] In some embodiments, the oscillation speed can be 100, 110, 120, 130, 140, or 150 rpm. In some embodiments, the oscillation speed can be 120 rpm.

[0039] In some embodiments, the temperature can be 20, 21, 23, 25, 27, or 28°C. In some embodiments, the temperature can be 25°C.

[0040] In some embodiments, the illumination period can be 12, 14, 16, or 18 hours followed by 12, 10, 8, or 6 hours of darkness, and the light intensity can be 800, 900, 1000, 1100, or 1200 lux. In some embodiments, the illumination period can be 16 hours followed by 8 hours of darkness, and the light intensity can be 1000 lux.

[0041] In some embodiments, the method further includes: treating the Camellia chrysantha suspension cells by at least one of the following methods: cold treatment at 2-6°C and treatment with methyl jasmonate (MeJA), to further induce the accumulation of secondary metabolites in the cells, thereby obtaining the target Camellia chrysantha suspension cells.

[0042] In some embodiments, the 2-6°C cold treatment temperature can be 2-6°C, the time can be 12-36 hours, the methyl jasmonate treatment time can be 24-72 hours, and the concentration of methyl jasmonate in the methyl jasmonate treatment can be 50-150 μM. In some embodiments, the 2-6°C cold treatment temperature can be 2, 3, 4, 5, or 6°C, the time can be 12, 18, 24, 30, or 36 hours, the methyl jasmonate treatment time can be 24, 36, 48, 60, or 72 hours, and the concentration of methyl jasmonate in the methyl jasmonate treatment can be 50, 75, 100, 125, or 150 μM. In some embodiments, the 2-6°C cold treatment temperature can be 4°C, the time can be 24 hours, the methyl jasmonate treatment time can be 48 hours, and the concentration of methyl jasmonate in the methyl jasmonate treatment can be 100 μM.

[0043] In some embodiments, the time interval between the 2-6°C cold treatment and the methyl jasmonate treatment does not exceed 2 hours.

[0044] This specification provides one or more embodiments of a Camellia chrysantha cell culture, which includes Camellia chrysantha suspension cells or target Camellia chrysantha suspension cells prepared by a Camellia chrysantha suspension cell culture method. The dried product of the Camellia chrysantha cell culture contains the following secondary metabolite contents: 10%-20% flavonoids and 15%-25% polyphenols.

[0045] In some embodiments, the flavonoid content may be 10%, 12%, 14%, 16%, 18%, or 20%, and the polyphenol content may be 15%, 17%, 19%, 21%, 23%, or 25%.

[0046] In some embodiments, in Camellia chrysantha cell cultures PAL , ANR1 Gene expression is upregulated.

[0047] The terms "expression level" and "gene expression level" used in this article refer to the relative expression level of genes.

[0048] PAL The gene encodes phenylalanine ammonia-lyase (PAL), a key enzyme in the phenylpropanoid metabolic pathway in plants. PAL catalyzes the conversion of phenylalanine to cinnamic acid, which then participates in the synthesis of various secondary metabolites, including flavonoids.

[0049] ANR1 The gene encodes anthocyanidin reductase 1 (ANR1), which catalyzes the synthesis of proanthocyanidins from anthocyanins and plays an important role in plant nutrient metabolism.

[0050] In some embodiments of this specification, the technical bottlenecks of high browning rate and long induction period of Camellia chrysantha callus were overcome through synergistic optimization of explant pretreatment, disinfection, and culture medium formulation. The composite induction process achieved precise regulation of metabolic pathways, solving the industrialization problem of unstable content of active ingredients in suspension cell lines. In addition, a complete technical system from explant treatment, callus induction, suspension culture to targeted regulation of active ingredients was established, filling the gap in the field of Camellia chrysantha cell engineering.

[0051] The present specification will be described in detail below with reference to embodiments. These embodiments are merely illustrative and not intended to limit the scope of the specification. Those skilled in the art should understand that modifications or substitutions to the details and form of the technical solutions in this specification can be made without departing from the spirit and scope thereof, but such modifications and substitutions are all within the protection scope of this specification. Example

[0052] Example 1: Induction of Camellia chrysantha callus

[0053] Select the 2nd-3rd pairs of leaves (≤50% expansion) at the top of the current year's semi-lignified young shoots of Camellia chrysantha, and spray them with 0.1 mmol / L salicylic acid (SA) solution 3 days before harvest to enhance stress resistance.

[0054] Explants sprayed with salicylic acid solution can effectively inhibit browning in subsequent culture and maintain high cell metabolic activity. The effects of different concentrations of salicylic acid solution on the browning rate and callus induction rate of explants are shown in Table 1.

[0055] Table 1 SA concentration (mmol / L) Browning rate of explants (%) Callus induction rate (%) 0 45.2±3.1 65.5±4.2 0.01 38.7±2.8 68.9±3.8 0.03 25.4±2.3 78.3±3.5 0.05 8.5±1.5 92.6±2.1 0.1 5.2±1.2 95.8±1.5 0.2 9.8±1.7 90.1±2.4 0.25 18.3±2.0 82.4±3.0 0.3 28.6±2.4 75.1±3.7 0.4 40.5±3.0 63.2±4.0 0.5 55.7±3.5 52.0±4.3

[0056] As shown in Table 1, compared with explants that were not sprayed with salicylic acid solution, when the concentration of salicylic acid solution was in the range of 0.05-0.2 mmol / L, the browning rate of explants was significantly reduced and the callus induction rate was significantly increased.

[0057] The following steps were performed sequentially to disinfect the explants: ultrasonic cleaning with 1% (v / v) neutral detergent for 5 min (40 kHz), rinsing with running water for 30 min, ultrasonic cleaning with 1% (v / v) neutral detergent for 5 min (40 kHz), rinsing with running water for 30 min, soaking in 75% ethanol for 40 s, rinsing with sterile water 5 times, treatment with a compound disinfectant solution of 1% sodium hypochlorite (NaClO) and 0.05% Tween-20 for 10 min, and rinsing with sterile water 7 times.

[0058] Using WPM as the basal medium, plant growth regulators of 3 mg / L NAA and 0.5 mg / L 6-BA, carbon source of 30 g / L sucrose, anti-browning agent of 0.1% PVP, solidifying agent of 6.5 g / L agar, and pH of 5.8, a solid medium was prepared. The formula of the Camellia chrysantha callus induction medium is shown in Table 2. The sterilized Camellia chrysantha explants were cut into 0.5 cm * 0.5 cm pieces and transferred to the above medium.

[0059] Table 2

[0060] After 7 days of dark culture, the cells were transferred to alternating light and dark culture (light intensity of 2000 lux, light cycle of 14 hours followed by 10 hours of darkness, temperature of 25±1℃) to induce callus formation.

[0061] During the induction period, explants were lesioned every week using a sterile scalpel to stimulate callus formation. After 8 weeks of induction, explants with significant callus formation were selected for subculture. The effects of different lesion treatments on callus induction in Camellia chrysantha are shown in Table 3.

[0062] Table 3

[0063] As shown in Table 3, compared to injury frequencies of once every 3 days and once every two weeks (or random injury or no injury), injury treatment with weekly injury resulted in a lower browning rate and a higher callus induction rate, as well as better growth (e.g., callus tissue was less prone to browning, had a loose texture, and a uniform growth rate). Meanwhile, superficial cutting or multiple cross-cuttings led to poor callus growth, thus requiring standardized injury treatment.

[0064] Using WPM salt as the basal culture medium, the optimal hormone ratio combination was screened out. The ease with which callus tissue would turn brown, its softness, and its growth rate were used as observation indicators to screen for combinations of callus masses that were brightly colored, loosely textured, and grew rapidly.

[0065] Figure 1 This is a schematic diagram of solid-state cultured callus tissue of Camellia chrysantha according to some embodiments of this specification.

[0066] A schematic diagram of callus tissue cultured from Camellia chrysantha in solid state is shown below. Figure 1 As shown in Table 4, the results of screening for the optimal hormone ratio in Camellia chrysantha callus culture are presented.

[0067] Table 4 serial number NAA (mg / L) BA (mg / L) growth status serial number NAA (mg / L) KT (mg / L) growth status 1 0.5 0.5 hard 13 0.5 0.5 Soft / slow 2 1 0.5 germination 14 1 0.5 germination 3 1.5 1 browning 15 1.5 1 hard 4 1 1 hard 16 1 1 Soft / slow 5 2 0.5 hard 17 2 0.5 Soft / slow 6 2 1 browning 18 2 1 Soft / slow serial number 2,4-D (mg / L) 6-BA (mg / L) growth status serial number 2,4-D (mg / L) KT (mg / L) growth status 7 0.5 0.5 browning 19 0.5 0.5 browning 8 1 1.5 germination 20 1 1.5 germination 9 1.5 1 browning 21 1.5 1 hard 10 1 1 browning 22 1 1 Soft / slow 11 2 0.5 hard 23 2 0.5 Soft / Fast 12 2 1 hard 24 2 1 browning

[0068] Table 4 shows that the optimal ratio of auxin (2,4-dichlorophenoxyacetic acid) to cytokinin (kinin) is 1-3 mg / L for auxin and 0.2-1 mg / L for cytokinin. Under these optimal conditions, the callus tissue is soft and grows rapidly.

[0069] Example 2: Establishment and Optimization of the Culture Process of Camellia chrysantha Suspension Culture Cell Line

[0070] After screening, it was found that the optimal conditions for callus growth were WPM medium supplemented with 2 mg / L 2,4-D, 0.5 mg / L KT, and 3% sucrose. Subsequent callus culture was carried out under these conditions.

[0071] Solid-state cultured Camellia chrysantha callus was inoculated into WPM medium containing 2 mg / L 2,4-D, 0.5 mg / L KT and 3% sucrose. The medium was shaken at 120 rpm and cultured at 25°C. The photoperiod was 16 hours followed by 8 hours of darkness, and the light intensity was 1000 lux.

[0072] A subculture cycle of 21 days was used, and after three subcultures, the most vibrant and round red Camellia chrysantha cells were selected to obtain a uniform Camellia chrysantha suspension cell system.

[0073] The optimal induction conditions for the synthesis of total flavonoids and total polyphenols in Camellia chrysantha cell cultures were screened in WPM medium with 2 mg / L 2,4-D, 0.5 mg / L KT and 3% sucrose, and an inoculum of 60 g / L.

[0074] Figure 2 This is a schematic diagram of a Camellia chrysantha cell culture according to some embodiments of this specification. The schematic diagram of the Camellia chrysantha cell culture is shown below. Figure 2 As shown.

[0075] Camellia chrysantha cell cultures were collected after 14 days of culture. Different conditions were set to treat the Camellia chrysantha cell cultures, and the expression levels of genes encoding key enzymes for the synthesis of flavonoids and polyphenols were detected by qPCR. The primer sequences for detecting the expression of key genes for the synthesis of flavonoids and polyphenols in Camellia chrysantha cell cultures are shown in Table 5.

[0076] Table 5

[0077] Figure 3 This is a schematic diagram illustrating the induced expression of key genes for the synthesis of total flavonoids and total polyphenols in Camellia chrysantha cell cultures under different treatment conditions, as shown in some embodiments of this specification (A: PAL Gene; B: ANR1 Gene; C: DFR Gene; D: different treatment conditions).

[0078] like Figure 3 As shown in D, this embodiment designed two control groups: no treatment (No. 1) and continued growth for 48 hours under no treatment (No. 2); and 12 treatment groups: 4℃ cold treatment for 6 hours (No. 3), 24 hours (No. 4) and 48 hours (No. 5); 254nm UV-B ultraviolet treatment for 6 hours (No. 6), 24 hours (No. 7) and 48 hours (No. 8); 4000lux strong light treatment for 6 hours (No. 9), 24 hours (No. 10) and 48 hours (No. 11); and 100μM methyl jasmonate treatment for 6 hours (No. 12), 24 hours (No. 13) and 48 hours (No. 14).

[0079] golden flower tea PAL The gene encodes phenylalanine ammonia-lyase, an enzyme that initiates phenylpropane metabolism. It catalyzes the synthesis of cinnamic acid from phenylalanine and promotes the synthesis of flavonoids and polyphenols. ANR1 The gene encodes anthocyanin reductase 1, an enzyme that catalyzes the synthesis of proanthocyanidins from anthocyanins. DFR The gene encodes dihydroflavonol 4-reductase, an enzyme that catalyzes the synthesis of colorless anthocyanins from dihydroflavonols, affecting the branching metabolism of anthocyanins and proanthocyanidins. This enzyme is found in Camellia chrysantha cell cultures. PAL Gene expression status, such as Figure 3 As shown in Figure A, the results indicate that cold treatment for 24 hours significantly induced expression (3.20±0.25), the highest value among all treatment groups, but decreased after 48 hours; UV treatment for 6 hours rapidly activated expression (2.50±0.30), followed by gradual inhibition; MeJA treatment for 48 hours continuously upregulated expression (1.60±0.22); and strong light treatment inhibited expression throughout. (The text then abruptly shifts to a discussion of Camellia chrysantha cell culture.) ANR1 Gene expression status, such as Figure 3 As shown in Figure B, the results indicate that MeJA treatment for 48 hours had the strongest induction effect (3.20±0.40), which increased in a time-dependent manner; cold treatment for 48 hours (0.30±0.10) and UV treatment for 48 hours (0.40±0.10) significantly inhibited expression; strong light treatment had no significant activation; and the expression in Camellia chrysantha cell culture was significantly reduced. DFR Gene expression status, such as Figure 3 As shown in C, the results indicate that strong light treatment for 24 hours (2.50±0.40) and ultraviolet treatment for 6 hours (2.20±0.35) can significantly induce expression, followed by MeJA treatment for 48 hours (1.80±0.30); cold treatment inhibits expression throughout the process.

[0080] Furthermore, based on the above qPCR results, this embodiment selected 4℃ cold treatment for 24 hours, followed by 100μM MeJA treatment for 48 hours as the combined treatment conditions, and compared the differences in total flavonoids and total polyphenols content in dried Camellia chrysantha cell cultures under different conditions.

[0081] Figure 8 This is a schematic diagram illustrating the metabolism of flavonoids and polyphenols in Camellia chrysantha, based on some embodiments of this specification.

[0082] Cold treatment can induce high expression of PAL, a key enzyme in the first step of the phenylpropane metabolic network, providing abundant precursor substances for the entire metabolic pathway. Methyl jasmonic acid can significantly activate the expression of DFR and ANR1, thereby promoting the synthesis of secondary metabolites abundant in Camellia chrysantha cultures, such as anthocyanins, proanthocyanidins, catechins, and epicatechins.

[0083] Figure 4 This is a schematic diagram illustrating the differences in total flavonoid and total polyphenol content in dried products of Camellia chrysantha cell cultures under different treatment conditions, as shown in some embodiments of this specification (A: total flavonoids; B: total polyphenols; C: different treatment conditions).

[0084] like Figure 4 As shown in the table, under the combined treatment conditions, the contents of total flavonoids and total polyphenols in the dried product of Camellia chrysantha cell culture were significantly higher than those in other treatment groups and the control group, at 15.6% (w / w, dry weight) and 17.8% (w / w, dry weight), respectively. The effect of the interval between cold treatment and methyl jasmonate treatment on the accumulation of metabolites is shown in Table 6.

[0085] Table 6:

[0086] Example 3: LC-MS / MS mass spectrometry analysis of Camellia chrysantha cell cultures According to the method in Example 2, a Camellia chrysantha cell culture with high total flavonoid and total polyphenol content was obtained.

[0087] The target compounds were separated chromatographically using an EXION LC System (SCIEX) ultra-high performance liquid chromatograph and a Waters UPLC column. Phase A of the HPLC system consisted of an aqueous solution containing 0.1% formic acid, and phase B consisted of acetonitrile. The column oven temperature was 40°C, the autosampler temperature was 4°C, and the injection volume was 2 μL.

[0088] Mass spectrometry analysis was performed using a SCIEX 6500 QTRAP+ triple quadrupole mass spectrometer equipped with an Ion Drive Turbo V ESI ion source in multiple reaction monitoring (MRM) mode. The ion source parameters were as follows: Ion Spray Voltage: +5500 / -4500V, Curtain Gas: 35psi, Temperature: 400℃, IonSource Gas 1: 60psi, Ion Source Gas 2: 60psi, DP: ±100V.

[0089] All mass spectrometry data acquisition and quantitative analysis of target compounds were performed using SCIEX Analyst WorkStation Software (Version 1.6.3). MS Conventer software was used to convert the raw mass spectra to TXT format, and a self-written R package combined with a self-built database was used to complete peak extraction, annotation, and other tasks.

[0090] Figure 5 These are sample extracted ion chromatograms (EICs) shown in some embodiments of this specification.

[0091] Through comparative analysis, a total of 632 compounds were identified in this embodiment, including 245 shikimic acid and phenylpropionic acid compounds (including flavonoids), 129 terpenoids, 64 alkaloids, 45 amino acids and small peptides, and 149 other organic compounds.

[0092] In addition, this embodiment lists 33 of the most enriched compounds, mainly including the following compounds: proanthocyanidin B2, arginine, maltine, DL-glutamine, 1-oleoylglycerol phosphorylcholine, catechin, lysine, epicatechin, piperacillin, D-proline, isoquercetin, quercetin-4'-glucoside, camel pine oleate, choline, 4-hydroxyisoleucine, γ-aminobutyric acid, 5'-deoxy-5'-methionine, proanthocyanidin C1, DL-tryptophan, jujubeside, valine, genistein 7-O-rutin, hyperoside, luteolin-7-O-glucoside, trimethyllysine, neo-isoglycyrrhizin, physostigmine, 5-caffeoylshikimic acid, and acetylglutamine. Precise broad-target metabolomics data of Camellia chrysantha cultures are shown in Table 7.

[0093] Material noun Precise molecular weight Q1 Q3 ionmode rt relative content Proanthocyanidins B2 578.142 579.149 127.0384,287.0541 1 222.4 552576563 Arginine 174.112 175.119 70.0651,72.0808 1 49.249 333602485.9 maltine 165.115 166.122 121.0705,103.0571 1 157.746 320009935.5 DL-glutamine 146.069 147.076 130.0494,84.0441 1 52.951 290376275.9 1-Oleoylglycerol phosphorylcholine 522.356 522.355 184.073,104.107 1 485.309 244186513.3 Catechins 290.079 291.086 139.0385,123.0436 1 224.042 230805198.9 Lysine 146.106 147.113 84.0807,130.086 1 53.216 223882269.3 Catechin 290.079 289.076 109.03,123.045 -1 236.081 179708966.2 Piperacic acid 129.079 130.086 84.0803,56.0491 1 54.121 146425769.1 D-proline 115.063 116.07 116.07,70.0648 1 49.295 116579804.3 Isoquercetin 464.095 465.102 303.0849,85.0308 1 276.416 70041837.07 Quercetin-4'-glucoside 464.095 465.102 303.0853,85.0308 1 276.416 70041837.07 Camel alkali 188.095 189.102 171.092,189.102239 1 202.529 69511132.07 choline 104.108 104.106 60.0805,58.0653 1 47.466 65167324.69 4-Hydroxyisoleucine 147.09 148.096 74.025,84.0832 1 54.121 57588935.8 γ-aminobutyric acid 103.063 104.07 87.0437,69.0332 1 48.914 47291183.24 5'-Deoxy-5'-Methionine 297.09 298.096 136.0695,61.0105 1 224.539 45396870.7 Proanthocyanidins C1 866.206 867.213 247.0592,245.0435 1 231.326 38902007.6 DL-Tryptophan 204.09 205.097 132.0802,146.0595 1 202.602 34561673 jujubesides 434.121 433.114 271.0612,151.0038 -1 282.229 27224306.02 Valine 117.079 118.086 72.0804,55.054 1 53.951 26753295.98 Proanthocyanidins C1 866.206 867.213 247.0592,245.0435 1 231.326 38902007.6 DL-Tryptophan 204.09 205.097 132.0802,146.0595 1 202.602 34561673 jujubesides 434.121 433.114 271.0612,151.0038 -1 282.229 27224306.02 Valine 117.079 118.086 72.0804,55.054 1 53.951 26753295.98 genistein 7-O-rutin 578.164 579.172 271.06,85.0282 1 254.426 22179251.67 Hypericin 464.095 463.089 300.028,271.025 -1 276.823 21381467.37 Luteolin 7-O-glucoside 448.101 449.107 287.0839,449.107 1 270.552 19704602.28 Trimethyllysine 189.16 189.159 84.0804,130.086 1 46.4 18939164.72 Neo-glycyrrhizin 418.126 419.133 257.1045,137.031 1 300.323 17140007.52 Plantago asiatica glycoside 462.116 463.123 301.1061,286.0783 1 281.809 16688264.87 5-Caffeoylshikimic acid 336.085 335.077 179.0483,135.0533 -1 249.1 16071762.96 Acetylglutamine 188.08 189.086 130.0576,84.0465 1 47.229 15643944.3

[0094] In Table 7, Q1 represents the molecular weight of the parent ion after the substance is treated with an electrospray ionization source. Q3 represents the characteristic fragment ion. ionmode represents the ionization mode (-1 for negative ion mode, 1 for positive ion mode). rt represents the retention time.

[0095] Example 4: Protective effect of Camellia chrysantha cell culture on D-galactose-induced senescent vascular endothelial cell damage

[0096] This embodiment aims to verify the in vivo efficacy of cell cultures derived from Camellia chrysantha callus prepared by the method of this invention in delaying vascular aging, improving endothelial function, and resisting oxidative stress, providing experimental evidence for the application of this cell culture in the prevention of cardiovascular and cerebrovascular diseases. The experiment was conducted using a D-galactose (D-gal)-induced aging mouse model to systematically evaluate the effects of the cell culture on blood pressure, vascular structure, and molecular markers.

[0097] Preparation of Camellia chrysantha cell culture: The dried cell culture was prepared according to the method described in Example 2. The total flavonoid content of the dried product was 15.6%, and the total polyphenol content was 17.8% (identified by LC-MS / MS as containing 245 active ingredients including proanthocyanidins B2 and catechins). The dried cell culture was weighed and dissolved in physiological saline to prepare a solution of the required concentration for animal experiments.

[0098] Animal models and experimental design: Select 7-8 week old animals without specific pathogens. Free (SPF) grade C57BL / 6 male mice were randomly divided into groups of 10 mice each after one week of adaptive culture. The groups included the following groups: (1) Blank control group: daily gavage with distilled water; (2) Model group: daily subcutaneous injection of 120 mg / kg D-gal to simulate aging, and gavage with distilled water; (3) Camellia chrysantha low-dose group: D-gal treatment was combined with gavage with Camellia chrysantha cell culture water extract (9 mg / kg dry, i.e., 9 mg dry product per kilogram of body weight); (4) Camellia chrysantha medium-dose group: gavage with Camellia chrysantha cell culture water extract (45 mg / kg dry); (5) Camellia chrysantha high-dose group: gavage with Camellia chrysantha cell culture water extract (90 mg / kg dry); (6) Positive drug group: gavage with captopril (7 mg / kg, i.e., 7 mg captopril per kilogram of body weight). All groups were administered 10 mL / kg via gavage once daily for 90 days. Weights were taken weekly and dosages adjusted accordingly.

[0099] Detection indicators and methods: (1) Blood pressure measurement: After the last administration, the systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial pressure (MBP) of mice in the conscious state were measured using a non-invasive tail artery blood pressure measurement system. The above indicators were measured three times for each mouse and the average value was taken; (2) Vascular ultrasound imaging: After anesthetizing the mice, the morphology of the carotid artery was observed using a high-frequency ultrasound system, and the thickness of the vascular wall and the diastolic amplitude of the mice were measured; (3) Serum index detection: After anesthetizing the mice, blood was collected from the eyeballs, the serum was separated by centrifugation, and the content of nitric oxide (NO), hydrogen peroxide (H2O2) and total antioxidant capacity (T-AOC) were detected using a kit; (4) Histopathological analysis: The aortic tissue of the mice was taken, fixed with 4% paraformaldehyde solution, embedded in paraffin, and sectioned for HE staining to observe the intima and media structure. Immunohistochemistry was used to detect the protein expression of endothelial nitric oxide synthase (eNOS) in aortic tissue (slices with eNOS primary antibody were incubated overnight at 4°C before DAB staining).

[0100] Analysis of experimental results:

[0101] (1) Regulation of blood pressure in aging mice by Camellia chrysantha cell culture: After D-gal induction, the systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MBP) of the model group mice were significantly increased (SBP: 134.8±6.2 mmHg, compared with SBP of 102.5±2.9 mmHg in the blank control group, p<0.001). After intervention with Camellia chrysantha cell culture, blood pressure decreased in a dose-dependent manner: SBP in the low-dose Camellia chrysantha group decreased to 125.6±2.4 mmHg (p<0.01), in the medium-dose group it decreased to 119.5±0.6 mmHg (p<0.01), and in the high-dose group it further decreased to 113.8±3.4 mmHg (p<0.01), with the effect comparable to that of the positive control group (112.7±2.4 mmHg). The results indicate that Camellia chrysantha cell culture can effectively reverse age-related hypertension.

[0102] (2) Improvement of vascular structure and function: Vascular ultrasound showed that the carotid wall thickness in the model group was significantly increased (0.150±0.030 mm, compared with 0.042±0.003 mm in the blank control group, p<0.001), and the diastolic amplitude was significantly reduced (0.040±0.010 mm, compared with 0.120±0.010 mm in the blank control group, p<0.001). The carotid wall thickness in the high-dose Camellia chrysantha group recovered to 0.060±0.010 mm (p<0.01), and the diastolic amplitude increased to 0.094±0.007 mm (p<0.01), suggesting that Camellia chrysantha cell culture improves vasomotor function by inhibiting vascular remodeling.

[0103] (3) Changes in serum oxidative stress indicators: The serum H2O2 content in the model group increased to 67±8.46μmol / L (compared to 21±5.38μmol / L in the blank control group, p<0.001), the NO level decreased to 1.3±0.3μmol / L (10.0±1.2μmol / L in the blank group, p<0.001), and the T-AOC decreased to 0.520±0.156μmol / mL (1.409±0.313μmol / mL in the blank group, p<0.001). After intervention with Camellia chrysantha cell culture, the high-dose group showed that H2O2 decreased to 31±2.62 μmol / L (p<0.01), NO content increased to 5.1±0.6 μmol / L (p<0.01), and T-AOC increased to 0.786±0.277 μmol / mL (p<0.01), confirming that Camellia chrysantha cell culture enhances the body's antioxidant defense and alleviates oxidative damage.

[0104] Figure 6 These are HE staining images of mouse aortic tissue according to some examples in this specification (A: blank control group; B: model group; C: low-dose Camellia japonica group; D: medium-dose Camellia japonica group; E: high-dose Camellia japonica group; F: positive control group).

[0105] Figure 7 This is a schematic diagram of eNOS protein expression in mouse aortic tissue according to some examples in this specification (A: blank control group; B: model group; C: low-dose Camellia japonica group; D: medium-dose Camellia japonica group; E: high-dose Camellia japonica group; F: positive control group).

[0106] (4) Pathological evaluation of aortic tissue: HE staining showed that in the model group, the endothelial cells of the aortic intima were disordered and partially detached, the media structure was significantly thickened and smooth muscle cells were hyperplastic and hypertrophic, while the pathological damage in the Camellia chrysantha treatment group was significantly improved. The intima structure of the high-dose Camellia chrysantha group was basically restored to its original integrity, and the thickness of the media structure was close to normal, indicating that Camellia chrysantha cell culture effectively protected the integrity of blood vessels. Immunohistochemical results further showed that the expression of eNOS protein in the model group was significantly inhibited, while the expression of eNOS-positive cells in the medium-dose and high-dose Camellia chrysantha groups was significantly upregulated (p<0.01), suggesting that Camellia chrysantha cell culture promotes NO synthesis and improves endothelial function by activating the eNOS pathway.

[0107] Conclusion: This embodiment demonstrates through in vivo experiments that Camellia chrysantha cell culture can significantly improve D-gal-induced vascular endothelial damage in aging mice. The mechanisms include: (1) downregulating blood pressure, reversing vascular wall thickening and diastolic dysfunction; (2) increasing serum NO levels and total antioxidant capacity, and inhibiting H2O2 accumulation; and (3) repairing endothelial function by upregulating eNOS expression. The above results verify the potential application value of Camellia chrysantha cell culture in anti-vascular aging and prevention of related diseases, and provide data support for its standardized development.

[0108] The basic concepts have been described above. Obviously, for those skilled in the art, the detailed disclosure above is merely illustrative and does not constitute a limitation of this specification. Although not explicitly stated herein, those skilled in the art may make various modifications, improvements, and corrections to this specification. Such modifications, improvements, and corrections are suggested in this specification and therefore remain within the spirit and scope of the exemplary embodiments described herein.

[0109] Furthermore, this specification uses specific terms to describe embodiments thereof. For example, "an embodiment," "one embodiment," and / or "some embodiments" refer to a particular feature, structure, or characteristic associated with at least one embodiment of this specification. Therefore, it should be emphasized and noted that references to "an embodiment," "one embodiment," or "an alternative embodiment" in different locations throughout this specification do not necessarily refer to the same embodiment. Moreover, certain features, structures, or characteristics in one or more embodiments of this specification can be appropriately combined.

[0110] Furthermore, unless expressly stated in the claims, the order of processing elements and sequences, the use of numbers and letters, or other names described in this specification are not intended to limit the order of the processes and methods described herein. Although some inventive embodiments that are currently considered useful have been discussed by way of various examples in the foregoing disclosure, it should be understood that such details are for illustrative purposes only, and the appended claims are not limited to the disclosed embodiments. Rather, the claims are intended to cover all modifications and equivalent combinations that conform to the substance and scope of the embodiments described herein.

[0111] Similarly, it should be noted that, in order to simplify the description disclosed herein and thus aid in the understanding of one or more embodiments of the invention, the foregoing description of embodiments in this specification may sometimes combine multiple features into a single embodiment, drawing, or description thereof. However, this method of disclosure does not imply that the subject matter of this specification requires more features than those mentioned in the claims. In fact, the embodiments contain fewer features than all the features of a single embodiment disclosed above.

[0112] In some embodiments, numbers describing the quantity of components and attributes are used. It should be understood that such numbers used in the description of embodiments are modified in some examples with the terms "approximately," "approximately," or "generally." Unless otherwise stated, "approximately," "approximately," or "generally" indicates that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may be changed depending on the characteristics required by individual embodiments. In some embodiments, numerical parameters should take into account specified significant digits and employ a general method of digit reservation. Although the numerical ranges and parameters used to confirm their breadth of range in some embodiments of this specification are approximate values, in specific embodiments, such values ​​are set as precisely as feasible.

[0113] For each patent, patent application, patent application publication, and other material such as articles, books, specifications, publications, and documents referenced in this specification, the entire contents of which are incorporated herein by reference. This excludes historical application documents that are inconsistent with or conflict with the content of this specification, as well as documents that limit the broadest scope of the claims in this specification (currently or subsequently appended to this specification). It should be noted that in the event of any inconsistency or conflict between the descriptions, definitions, and / or terminology used in the supplementary materials to this specification and the content of this specification, the descriptions, definitions, and / or terminology used in this specification shall prevail.

[0114] Finally, it should be understood that the embodiments described in this specification are merely illustrative of the principles of the embodiments described herein. Other variations may also fall within the scope of this specification. Therefore, alternative configurations of the embodiments described herein are intended to be illustrative rather than limiting, and should be considered consistent with the teachings of this specification. Accordingly, the embodiments described herein are not limited to those explicitly introduced and described herein.

Claims

1. A method for culturing suspension cells of Camellia chrysantha, characterized in that, Includes the following steps: Golden flower tea leaves were collected and inoculated into WPM liquid medium containing hormones for culture to induce the formation of primary callus tissue. The hormones included naphthaleneacetic acid and 6-benzylaminopurine. The primary callus tissue was inoculated into WPM liquid medium containing auxin and cytokinin and cultured to obtain Camellia chrysantha suspension cells.

2. The method according to claim 1, characterized in that, The hormone-containing WPM liquid culture medium also contains an anti-browning substance, namely polyvinylpyrrolidone.

3. The method according to claim 2, characterized in that, Before inoculating the golden chrysanthemum tea leaves into the hormone-containing WPM liquid culture medium, the method further includes: A solution containing salicylic acid is sprayed onto the golden chrysanthemum tea leaves, wherein the concentration of salicylic acid in the solution is 0.05-0.2 mmol / L; The golden chrysanthemum tea leaves were treated with a compound solution containing mercury disinfectant and surfactant.

4. The method according to claim 1, characterized in that, The culture conditions in the hormone-containing WPM liquid medium include: a temperature of 25±2°C, dark culture for 6-8 days, followed by light-dark alternating culture; the light-dark alternating culture has a light cycle of 13-15 hours of light followed by 9-11 hours of darkness, and a light intensity of 1500-2500 lux.

5. The method according to claim 1, characterized in that, In the WPM liquid culture medium containing auxin and cytokinin, the auxin is 2,4-dichlorophenoxyacetic acid, and the concentration of the auxin is 1-3 mg / L; the cytokinin is kinetin, and the concentration of the cytokinin is 0.2-1 mg / L.

6. The method according to claim 1, characterized in that, The culture conditions in the WPM liquid medium containing auxin and cytokinin include: a shaking speed of 100-150 rpm, a temperature of 20-28°C, a light cycle of 12-18 hours followed by 6-12 hours of darkness, and a light intensity of 800-1200 lux.

7. The method according to claim 1, characterized in that, The method further includes: The *Camellia chrysantha* suspension cells were further induced to accumulate secondary metabolites by at least one of the following treatments: cold treatment at 2-6°C and treatment with methyl jasmonate, to obtain the target *Camellia chrysantha* suspension cells.

8. The method according to claim 7, characterized in that, The 2-6℃ cold treatment time is 12-36 hours, the methyl jasmonate treatment time is 24-72 hours, and the concentration of methyl jasmonate in the methyl jasmonate treatment is 50-150 μM.

9. A Camellia chrysantha cell culture, characterized in that, The Camellia chrysantha cell culture comprises Camellia chrysantha suspension cells or target Camellia chrysantha suspension cells prepared by any of the methods described in claims 1 to 8, wherein the dried product of the Camellia chrysantha cell culture contains the following secondary metabolite contents: 10%-20% flavonoids and 15%-25% polyphenols.

10. The Camellia chrysantha cell culture according to claim 9, characterized in that, The Camellia chrysantha cell culture PAL , ANR1 Gene expression is upregulated.

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