Methods and applications for increasing the content of chlorobenzan alkaloid in Zanthoxylum nitidum tissue culture materials

Abstract

This invention relates to a method for increasing the content of chlorobenzanine in tissue culture materials of Zanthoxylum nitidum, belonging to the field of biotechnology. Currently, traditional methods for extracting chlorobenzanine from Zanthoxylum nitidum suffer from problems such as long production cycles and unstable content. To address this, the method provided by this invention includes explant selection, shoot induction culture, cluster shoot culture, callus induction culture, and directed regulation culture. The directed regulation culture involves transferring cluster shoots or callus tissue to red light conditions for 1–2 months, with a light intensity of 150–250 μmol・m⁻²・s⁻¹ and a photoperiod of 10–14 hours / day, to activate the expression of genes involved in the chlorobenzanine synthesis pathway, effectively increasing the chlorobenzanine content.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and more specifically, this invention relates to a method and application for increasing the content of chlorobenzan alkaloid in Zanthoxylum nitidum tissue culture materials. Background Technology

[0002] As one of the "Ten Delicacies of Guangxi" in Guangxi Zhuang Autonomous Region, Zanthoxylum nitidum is used as medicine with its root. It has the effects of promoting blood circulation, removing blood stasis, regulating qi and relieving pain. Its indicative component, zafniline chloride, is a key component for quality control as specified in the Pharmacopoeia of the People's Republic of China [1]. Due to the sharp decline in wild resources, current production relies on artificial cultivation. However, the content of zafniline chloride in medicinal materials of different cultivation types and origins can vary by up to 6 times (0.70~4.33mg / g) [2]. Yang et al. tested Zanthoxylum nitidum medicinal materials from 8 origins and found that the content of zafniline chloride was 0.50%~0.91% [3]. Wu Luxiang et al. tested Zanthoxylum nitidum medicinal materials from 10 origins and found that the content of zafniline chloride was 0.14%~0.19% [4]. It can be seen that the content of zafniline chloride in Zanthoxylum nitidum medicinal materials fluctuates greatly, which poses a challenge to the quality control of Zanthoxylum nitidum medicinal materials. Furthermore, traditional cultivation has a long cycle, and the accumulation of secondary metabolites is significantly affected by the environment, making quality control difficult.

[0003] Plant tissue culture technology has enabled the efficient and rapid propagation of Zanthoxylum nitidum. By optimizing bud induction, subculture, and rooting media, the explant induction rate can reach 41.7%–96.2%, with an annual proliferation rate of 3.612, and the problem of bud browning has been solved. However, current technologies focus on optimizing rapid propagation processes, and there is insufficient research on the biosynthetic regulation of Zanthoxylum nitidate in the tissue culture system. Its synthetic pathway has not yet been activated by environmental factors (such as light quality and culture medium composition), which cannot meet the needs of stable and efficient accumulation of target components in large-scale production.

[0004] In response to the above situation, it is urgent to optimize tissue culture technology to overcome the bottlenecks of long cultivation cycles and large content fluctuations in traditional cultivation, and to establish a method for targeted regulation of the biosynthesis of chlorinated thorn alkaloids, so as to provide technical support for industrial production.

[0005] References:

[0006] [1] National Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China (Part I). Beijing: China Medical Science and Technology Press, 2020;

[0007] [2] Qin Yunrui, Jiang Zhenou, Lai Maoxiang, Huang Yunfeng, Wang Xinhong. Research on the origin of Zanthoxylum nitidum and analysis of its active ingredients. Plants of Guangxi Zhuang Autonomous Region, 2019, 39(04):531-539;

[0008] [3] Yang,Y., Li, Y., Amoroso, V., Acma, F., Guiang, MM,&Wu,H. Comparison of production of bioactive components in Zanthoxylum nitidumtaproots from different regions in southern China. Biomedical chromatography: BMC, 2023, 37(5): e5602;

[0009] [4] Wu Luxiang, Huang Rugan, Lan Xiaodong, Ma Enyao, Chen Junxi, Yang Shihui, Liu Qiyue, Zhou Shouting. Quality evaluation of Zanthoxylum nitidum from different producing areas based on principal component analysis. Central South Agricultural Science and Technology, 2023, 44(10):23-26. Summary of the Invention

[0010] One object of the present invention is to address at least the aforementioned deficiencies and to provide at least the advantages described below.

[0011] This invention provides a method for increasing the content of chlorobenzamine in Zanthoxylum bungeanum tissue culture materials. The aim is to optimize tissue culture technology to directionally regulate the biosynthesis of chlorobenzamine in Zanthoxylum bungeanum tissue culture materials, thereby increasing the content of chlorobenzamine. This solves the problems of long production cycle and unstable chlorobenzamine content required for extracting chlorobenzamine from Zanthoxylum bungeanum plants using cultivation techniques, and provides a new technology for the large-scale production of chlorobenzamine.

[0012] This invention provides a method for increasing the content of chlorobenzan alkaloid in Zanthoxylum nitidum tissue culture materials, comprising the following steps:

[0013] S1. Explant selection;

[0014] S2. Bud induction culture: After disinfection, the explants are inoculated into bud induction culture medium for culture;

[0015] S3. Cultivation of clustered shoots: The tender shoots obtained in step S2 are transferred to the culture medium for cultivation.

[0016] S4. Callus induction culture: Take tender leaves from step S2, make a cut, and inoculate them into callus induction culture medium for culture;

[0017] S5. Directional controlled culture: Transfer the shoot cluster culture from step S3 or the callus induction culture from step S4 to red light conditions for 1 to 2 months. The light intensity of the red light is 150 to 250 μmol•m⁻²•s⁻¹, and the photoperiod is 10 to 14 hours / day.

[0018] Preferably, in step S1, the explants are selected from stem segments of 1-6 month old *Zanthoxylum nitidum* seedlings; in step S2, the bud induction medium is 1 / 2 MS basal medium supplemented with 0.5-1.0 mg / L 6-BA, 0.2-0.4 mg / L IBA, and 0.1-0.5 mg / L KT; in step S3, the clustered bud medium is MS basal medium supplemented with 0.5-1.0 mg / L 6-BA, 0.2-0.4 mg / L IBA, and 0.1-0.5 mg / L KT; and in step S4, the callus induction medium is MS basal medium supplemented with 2 mg / L 6-BA, 0.1-0.2 mg / L KT, 0.2-1 mg / L 2,4-D, and 1-1.5 mg / L NAA.

[0019] Preferably, the bud induction medium in step S2 is 1 / 2 MS basal medium supplemented with 0.6 mg / L 6-BA, 0.2 mg / L IBA and 0.1 mg / L KT; the shoot clustering medium in step S3 is MS basal medium supplemented with 0.6 mg / L 6-BA, 0.2 mg / L IBA and 0.1 mg / L KT; the callus induction medium in step S4 is MS basal medium supplemented with 2 mg / L 6-BA, 0.1 mg / L KT, 0.2 mg / L 2,4-D and 1 mg / L NAA; the pure red light intensity under red light conditions is 200 μmol•m⁻²•s⁻¹, and the photoperiod is 12 hours / day.

[0020] Preferably, the red light condition activates the expression of genes in the chlorobenzanol synthesis pathway, including NCS, CYP450, and BBE.

[0021] Preferably, the culture is carried out under red light for 1 to 2 months, including:

[0022] The first phase, red light conditions, days 1-15, with a light intensity of 150-180 μmol·m⁻²·s⁻¹ and a photoperiod of 10 hours / day;

[0023] In the second stage, from day 16 to 30 under red light conditions, the light intensity was increased to 200 to 220 μmol·m⁻²·s⁻¹ and the photoperiod was extended to 12 to 14 hours / day.

[0024] In the third stage, from day 31 of the red light condition until the end of the culture, the light intensity was maintained at 200 μmol·m⁻²·s⁻¹, the photoperiod was 12 hours / day, and it was supplemented with 1 hour of blue light at an intensity of 50 μmol·m⁻²·s⁻¹ every day, with intermittent irradiation.

[0025] The red light wavelength is 620~660nm, the blue light wavelength is 450~480nm, and the intermittent irradiation is 1 hour of blue light inserted after every 6 hours of red light.

[0026] Preferably, the shoot culture medium in step S5, which is cultured under red light, also contains 0.05~0.2 mg / L SA and 5~10 μM Trp.

[0027] Preferably, the first-stage shoot-forming medium is MS basal medium supplemented with 0.6 mg / L 6-BA, 0.2 mg / L IBA, 0.1 mg / L KT and 0.05 mg / L SA; the second-stage shoot-forming medium is MS basal medium supplemented with 0.6 mg / L 6-BA, 0.2 mg / L IBA, 0.1 mg / L KT and 0.15 mg / L SA; and the third-stage shoot-forming medium is MS basal medium supplemented with 0.6 mg / L 6-BA, 0.2 mg / L IBA, 0.1 mg / L KT, 0.1 mg / L SA and 6 μM Trp.

[0028] Preferably, the budding culture medium of the first stage is used for cultivation from day 1 to day 15 under red light conditions;

[0029] On the 16th day under red light conditions, the shoot clusters were transferred to the premixed second-stage shoot cluster medium for cultivation. The replacement of the second-stage shoot cluster medium was carried out simultaneously with the red light intensity enhancement stage, with the red light intensity increasing from 150 μmol·m⁻²·s⁻¹ to 200 μmol·m⁻²·s⁻¹.

[0030] On day 31 under red light conditions, the shoot clusters were transferred to the premixed shoot cluster culture medium of the third stage; wherein the shoot cluster culture media of the first stage, the second stage and the third stage are all pre-sterilized and packaged liquid culture media, which are directly injected into the culture container or pre-packaged in the culture container after opening.

[0031] The present invention also provides the application of the above method in the production of chlorinated thorn alkali.

[0032] The present invention has at least the following beneficial effects:

[0033] This invention optimizes tissue culture technology to directionally regulate the biosynthesis of chlorobenzamine in Zanthoxylum bungeanum tissue culture materials, resulting in a 2-10 times higher content of chlorobenzamine in Zanthoxylum bungeanum tissue culture materials cultured under red light compared to those cultured under white light.

[0034] This invention utilizes targeted regulation of tissue culture technology to activate the expression of genes involved in the synthesis pathway of thorn chloride (NCS, CYP450, BBE, etc.) under red light conditions, resulting in a higher content of the target component in tissue culture materials (clustered shoots or callus tissue) compared to white light culture. This method addresses the problems of long cultivation cycles (3-5 years), large content fluctuations (up to 6-fold differences between different production areas), and insufficient regulation of secondary metabolites in existing tissue culture techniques. By controlling environmental factors to directionally promote the accumulation of thorn chloride, it provides stable technical support for industrial production.

[0035] This invention employs a phased-controlled low-intensity red light-initiated gene expression strategy, gradually enhancing red light to promote metabolic accumulation, and using blue light to synergistically avoid photoreceptor passivation. This strategy continuously activates the synthetic pathway while reducing light stress, achieving simultaneous optimization of the content and stability of target components. It verifies the positive regulatory effect of dynamic adjustment of light quality, light intensity, and photoperiod on biosynthesis.

[0036] This invention alleviates light stress by regulating plant hormone signaling pathways through SA, improving the survival rate of clustered shoots and enhancing the expression of synthetic genes (such as BBE); Trp, as a precursor for alkaloid synthesis, directly supplements metabolic pathway substrates, overcoming the limitation of insufficient precursors in single-light-quality regulation. These two components, along with staged light-quality regulation, synergistically form a multi-regulatory mechanism, further optimizing the biosynthetic efficiency and stability of the target components, providing a new technology for the large-scale production of chlorinated thorn alkaloids.

[0037] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0038] Figure 1 It exhibits a clustered budding morphology characteristic of Zanthoxylum bungeanum;

[0039] Figure 2 The content of alkaloid chloride in Zanthoxylum bungeanum buds cultured under different light qualities;

[0040] Figure 3 Clustering heatmap of genes related to alkaloid synthesis pathways in young shoots of Zanthoxylum bungeanum cultured under different light qualities;

[0041] Figure 4 The morphology of callus tissue of Zanthoxylum nitidum;

[0042] Figure 5 The content of alkaloid chloride in Zanthoxylum nitidum callus cultured under different light qualities;

[0043] Figure 6 Clustering heatmap of genes related to alkaloid synthesis pathways in Zanthoxylum nitidum callus cultured under different light qualities;

[0044] Among them are berberine bridge enzyme (BBE); codeine 3-O-demethylase (DIOX); thebaine synthase (MLP31); caffeic acid 3-O-methyltransferase (COMT1); cytochrome P450 enzymes (cytochrome P450 CYP82D47, CYP82D6); cytochrome P450 enzymes (cytochrome P450 81E8, CYP81Q32); and 7-O-methyltransferase ((R,S)-reticuline 7-O-methyltransferase, 7OMT). Detailed Implementation

[0045] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0046] It should be noted that, unless otherwise specified, the experimental methods described in the following embodiments are conventional methods, and the reagents and materials mentioned are commercially available. In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "setting" should be interpreted broadly. For example, they can refer to fixed connection or setting, detachable connection or setting, or integral connection or setting. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances. The terms "lateral," "longitudinal," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing this invention and simplifying the description. They do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this invention.

[0047] This invention provides a method for increasing the content of chlorobenzan alkaloid in Zanthoxylum nitidum tissue culture materials, comprising the following steps:

[0048] S1 Select explants, using stem segments of seedlings aged 1 to 6 months as explants;

[0049] S2 bud induction culture: After sterilization with 0.1% mercuric chloride, explants were inoculated onto bud induction medium (1 / 2 MS + 0.5-1.0 mg / L 6-BA + 0.2-0.4 mg / L 6-BA + 0.1-0.5 mg / L KT). After one month of culture, the budding rate was over 90%, with 2-5 buds per explant.

[0050] S3. Sprout culture: The shoots with a length of 2-3 cm obtained in step S2 were transferred to the sprout culture medium (MS + 0.5-1.0 mg / L 6-BA + 0.2-0.4 mg / L 6-BA + 0.1-0.5 mg / L KT). After 2 months of culture, each shoot differentiated into 3-10 shoots.

[0051] S4 Callus induction culture: The tender leaves obtained in step S2 were slightly scratched and transferred to callus induction medium (MS + 2 mg / L 6-BA + 0.1~0.2 mg / L KT + 0.2~1 mg / L 2,4-D + 1~1.5 mg / L NAA). After 2 months of culture, the callus induction rate was over 90%, and each tender leaf grew 0.2~0.1 g of callus.

[0052] S5 directed regulation of nephrite chloride biosynthesis culture involves transferring clustered shoots or callus tissue to red light for 1–2 months, with a light intensity of 150–250 μmol•m⁻²•s⁻¹ and a photoperiod of 10–14 hours / day. This activates the expression of genes related to the nephrite chloride biosynthesis pathway (NCS, CYP450, BBE, etc.) in vivo, resulting in a 2–10 times higher nephrite chloride content compared to materials cultured under white light.

[0053] The material is in the form of clustered buds or callus tissue.

[0054] Example 1

[0055] The method for increasing the content of chlorobenzan alkaloid in Zanthoxylum nitidum tissue culture materials includes the following steps:

[0056] On September 20, 2023, 500g of Zanthoxylum nitidum seeds were harvested and directly sown into the germination bed.

[0057] On October 20, 2023, the germinated seeds were selected and transferred to seedling trays, and nutrient solution was watered regularly to promote the growth of seedlings.

[0058] On December 23, 2023, the main stems of seedlings were used as explants for tissue culture experiments. Each stem segment was cut into 2-3 cm long pieces, rinsed thoroughly with running water, and sterilized in 0.1% mercuric chloride solution for 5 minutes on a sterile operating table. Then, it was rinsed 3 times with sterile water and inoculated into bud induction medium (1 / 2 MS + 0.6 mg / L 6-BA + 0.2 mg / L IBA + 0.1 mg / L KT). A total of 150 bottles were inoculated, with one material inoculated in each bottle.

[0059] On January 8, 2024, 15 bottles were recorded as contaminated, and of the remaining 135 bottles, 96 bottles had sprouted new shoots.

[0060] On January 23, 2024, 1 bottle was contaminated. Of the remaining 134 bottles, 125 bottles sprouted new shoots (i.e., after deducting contamination, the total germination rate was 125 / 134=93.3%). The number of new shoots on 10 bottles was randomly selected, and the average number of sprouts was 2.4 per plant. The minimum number of sprouts was 1, and the maximum number of sprouts was 4.

[0061] On March 5, 2024, tender shoots that had grown to 2-3 cm in length were cut off from the shoot induction medium on a sterile operating table and transferred to the shoot cluster medium (MS + 0.6 mg / L 6-BA + 0.2 mg / L LIBA + 0.1 mg / L KT). A total of 50 bottles were inoculated, with 3 shoots inoculated in each bottle.

[0062] On May 6, 2024, the growth status of the clustered buds was recorded. Three bottles were randomly selected, totaling nine materials. The average number of buds per material was calculated to be 5.6 (with a minimum of 3 buds and a maximum of 18 buds). The average bud length was 2.74 cm (the shortest bud was 0.32 cm and the longest bud was 5.62 cm). Figure 1 (The morphology of clustered buds of Zanthoxylum nitidum).

[0063] On June 5, 2024, vigorous shoots from the bud induction medium of Zanthoxylum nitidum were taken, and single shoots longer than 2 cm were cut off and transferred to the bud clustering medium (MS + 0.6 mg / L 6-BA + 0.2 mg / L LIBA + 0.1 mg / L KT). A total of 100 bottles were inoculated, with 3 shoots inoculated in each bottle. They were cultured under pure red light (620~660 nm), pure blue light (450~480 nm), pure green light (500~570 nm), and white light (400~760 nm), respectively. The light intensity was 200 μmol•m⁻²•s⁻¹, and the photoperiod was 12 hours / day. 25 bottles were cultured under each light quality.

[0064] On August 6, 2024, *Zanthoxylum nitidum* shoots subcultured under different light qualities were harvested, and the content of chlorophyll alkaloids was determined using UPLC-MS-MS, along with transcriptome sequencing. Analysis revealed significant differences in chlorophyll alkaloid content under the four light quality conditions, with the highest relative expression level observed under red light, which was 13.7 times higher than under white light. Figure 2 The relative content of zephyranthes chloride in zephyranthes shoots cultured under different light qualities. Based on transcriptome data, the expression of related genes (BBE, STOX, TNMT) in the zephyranthes chloride biosynthesis pathway was significantly upregulated under red light. Figure 3 Clustering heatmap of genes related to alkaloid synthesis pathway in young shoots of Zanthoxylum bungeanum cultured under different light qualities.

[0065] The nutrient solution regularly applied to the seedling trays is a modified Hoagland nutrient solution, with the specific formula as follows:

[0066] Macroelements (mg / L): Calcium nitrate (Ca(NO3)2・4H2O) 1000, Potassium nitrate (KNO3) 810, Potassium dihydrogen phosphate (KH2PO4) 136, Magnesium sulfate (MgSO4・7H2O) 493, Ammonium sulfate ((NH4)2SO4) 132;

[0067] Trace elements (μmol / L): Sodium iron ethylenediaminetetraacetate (NaFe-EDTA) 100, boric acid (H3BO3) 20, manganese sulfate (MnSO4·H2O) 1, zinc sulfate (ZnSO4·7H2O) 0.2, copper sulfate (CuSO4·5H2O) 0.01, sodium molybdate (Na2MoO4·2H2O) 0.01;

[0068] Preparation method: Dissolve the macro-elements and micro-elements separately in distilled water, mix them, adjust the pH to 5.8~6.2, and water once every 7~10 days.

[0069] I. UPLC-MS-MS detection method:

[0070] 1. Sample pretreatment

[0071] Take 0.1g of fresh tissue culture material (bud or callus), add 1mL of 80% methanol, grind with liquid nitrogen and then extract by ultrasonication for 30min (power 200W, frequency 40kHz), centrifuge at 12,000rpm for 15min, take the supernatant and filter it through a 0.22μm microporous membrane, and use the filtrate for UPLC-MS-MS detection.

[0072] 2. High-performance liquid chromatography (UPLC) conditions

[0073] Column: Agilent SB-C18 (1.8μm, 2.1mm × 100mm)

[0074] Mobile phases: Phase A is ultrapure water containing 0.1% formic acid, and Phase B is acetonitrile containing 0.1% formic acid.

[0075] Elution gradient:

[0076] 0.00min: Phase B 5%

[0077] 0.01–9.00 min: Phase B linearly increases to 95% and is maintained for 1 min.

[0078] 10.00–11.10 min: Phase B decreases to 5%, equilibrate to 14 min.

[0079] Flow rate: 0.35 mL / min

[0080] Column temperature: 40℃

[0081] Injection volume: 2 μL

[0082] 3. Mass spectrometry (MS-MS) conditions

[0083] Ion source: Electrospray ionization (ESI+, positive ion mode)

[0084] Ion source parameters:

[0085] Temperature: 500℃

[0086] Spray voltage: 5500V

[0087] Curtain gas (CUR): 25psi

[0088] Nebulizer gas (GS1): 50psi

[0089] Auxiliary gas (GS2): 60 psi

[0090] Scanning mode: Multiple Response Monitoring (MRM)

[0091] Target ion pair:

[0092] Mother ion m / z 340.2, daughter ion m / z 192.1 (quantitative ion)

[0093] Collision energy (CE): 35eV

[0094] De-clustering voltage (DP): 80V

[0095] 4. Content Calculation

[0096] Relative content: Peak area of ​​target group / Peak area of ​​white light group (white light group is set to 1)

[0097] Absolute content conversion: A standard curve was plotted using alkali chloride standard (purity ≥98%), and the absolute content of alkali chloride in the sample (mg / g fresh sample) was calculated.

[0098] II. Transcriptome sequencing methods:

[0099] 1. RNA extraction and library construction

[0100] Total RNA extraction: Using the PlantRNA Purification Reagent kit (Invitrogen), 50 mg of fresh tissue culture material was ground in liquid nitrogen and RNA was extracted according to the instructions. Purity was detected by NanoDrop 2000 (OD260 / 280=1.8~2.0) and integrity was detected by Agilent 2100 Bioanalyzer (RIN≥8.0).

[0101] cDNA library construction:

[0102] Magnetic beads enrich poly(A)+ mRNA, and fragmentation buffer is added to break it into 100~300bp fragments;

[0103] The first-strand cDNA was synthesized using a six-base random primer, followed by the synthesis of the second-strand cDNA.

[0104] End repair, A-tailing, ligation of sequencing adapters (including index), and screening of 200-400bp fragments with magnetic beads;

[0105] PCR amplification was performed for 12 cycles to construct a cDNA library.

[0106] 2. Sequencing and Data Analysis

[0107] Sequencing platform: Illumina NovaSeq 6000, PE150 mode, single sample sequencing data volume ≥6Gb.

[0108] Data processing:

[0109] The data from the machine is filtered by Fastpv0.23.2 (removing connectors and low-quality reads, Q≥30) to obtain CleanData;

[0110] Reference genome: Zanthoxylum nitidum genome;

[0111] Comparison tool: Hisat2v2.2.1, Mapped rate ≥85%;

[0112] Differential gene analysis: DESeq2v1.34.0, with selection criteria of |log2(FoldChange)|≥1 and FDR<0.05, and KEGG pathway enrichment analysis (p<0.05).

[0113] III. Absolute content data of chlorinated thorn alkali:

[0114] 1. Example 1 (Bud Material)

[0115] Absolute content of white light group: 0.52mg / g fresh buds (the content of dried product can be converted according to the moisture content of fresh sample of 60%).

[0116] The absolute content of the red light group was 6.85 mg / g of fresh buds (13.7 times higher than that of the white light group, and 1.58 times that of the highest value of 4.33 mg / g of dried product in traditional cultivation).

[0117] 2. Example 2 (Callus Tissue)

[0118] Absolute content of white light group: 0.31 mg / g fresh callus tissue;

[0119] Absolute content of red light group: 0.65mg / g fresh callus tissue (relative content 2.1 times, higher than that of white light group and some medicinal materials from different producing areas);

[0120] Explanation of the rationale for testing fresh samples:

[0121] Characteristics of tissue culture materials: During the tissue culture process, the samples need to be kept fresh to maintain metabolic activity. Drying may lead to the degradation of secondary metabolites. Therefore, using fresh samples for testing can better reflect the real-time synthesis capability.

[0122] Conversion to dried product: The moisture content of fresh samples is about 60%~70%. Based on this conversion, the moisture content of dried buds from the Hongguang group is 17.13~22.83 mg / g (significantly higher than the pharmacopoeia standard of 0.13% and the highest value in traditional cultivation), which meets the requirements of industrial extraction.

[0123] Compared with existing technologies:

[0124] In the red light-treated clustered shoots of this invention, the absolute content of chlorinated thorn alkaloids was 6.85 mg / g of fresh sample. Based on the fact that the water content of fresh thorn buds is approximately 60%~70%, the content in dried product can reach 17.13~22.83 mg / g (calculated on a dried basis).

[0125] Compared with the content of dried Zanthoxylum nitidum (0.70~4.33 mg / g) of different cultivation types reported by Qin Yunrui et al. (2019), the conversion value of fresh clustered buds in this invention significantly exceeds that of existing medicinal materials.

[0126] Compared with the 0.14%~0.19%, i.e. 1.4~1.9mg / g, detected by Wu Luxiang et al. (2023) in 10 medicinal materials from different origins, the content of this invention is significantly increased and significantly higher than the minimum standard stipulated in the Chinese Pharmacopoeia (0.13%, i.e. 1.3mg / g).

[0127] More importantly, this invention reduces the range of content fluctuations by controlling the tissue culture environment, whereas the content difference in existing cultivation techniques can reach 6 times (Qin Yunrui et al., 2019), thus solving the core problem of "unstable content" in traditional production.

[0128] It is evident that this invention can increase the alkaloid content of sphagnum moschata in clustered buds, improve quality stability (in the analysis of 20 samples from each treatment group, the alkaloid content of fresh buds in the red light treatment group was the highest at 6.89 mg / g and the lowest at 5.91 mg / g; the alkaloid content of fresh buds in the white light group was the highest at 1.79 mg / g and the lowest at 0.27 mg / g), and it only takes 3-4 months from explant inoculation to the end of red light treatment, while traditional cultivation requires 3-5 years to harvest, demonstrating a significant cycle advantage of this invention.

[0129] Example 2

[0130] The method for increasing the content of chlorobenzan alkaloid in Zanthoxylum nitidum tissue culture materials includes the following steps:

[0131] On May 6, 2024, leaves from vigorous buds of Zanthoxylum nitidum bud induction medium were taken, slightly scratched with a scalpel, and transferred to callus induction medium (MS + 2 mg / L 6-BA + 0.1 mg / L KT + 0.2 mg / L 2,4-D + 1 mg / L NAA) on a sterile operating table. A total of 50 bottles were inoculated, with 3 leaves inoculated in each bottle.

[0132] On July 6, 2024, the growth of callus tissue was recorded. Ten bottles, totaling 30 materials, were randomly selected, and the callus induction rate was calculated to be 100%. Two more bottles were randomly selected, and the average weight of the materials was measured to be 0.74g. Figure 4 Morphology of Zanthoxylum nitidum callus.

[0133] On July 6, 2024, the light green callus tissue was transferred to a new callus induction medium (MS + 2 mg / L 6-BA + 0.1 mg / L KT + 0.2 mg / L 2,4-D + 1 mg / L NAA), totaling 80 bottles, with 3 materials per bottle. They were cultured under pure red light (620~660nm), pure blue light (450~480nm), pure green light (500~570nm), and white light (400~760nm), respectively, with a light intensity of 200 μmol•m⁻²•s⁻¹ and a photoperiod of 12 hours / day. 20 bottles were cultured under each light quality.

[0134] On August 6, 2024, callus tissues of *Zanthoxylum nitidum* cultured under different light qualities were harvested, and the content of alkaloid chloride was determined by UPLC-MS-MS. Simultaneously, transcriptome sequencing was performed (the measurement method was the same as in Example 1). Analysis showed significant differences in the alkaloid chloride content under the four light quality conditions, with the highest relative expression level observed under red light, which was 2.1 times that under white light. Figure 5 The relative content of zafyne chloride in Zanthoxylum nitidum callus cultured under different light qualities. Based on transcriptome data, the expression of related genes (NCS, CYP450, BBE, etc.) in the zafyne chloride biosynthesis pathway was significantly upregulated under red light. Figure 6 Clustering heatmap of genes related to alkaloid biosynthesis pathway in Zanthoxylum nitidum callus cultured under different light qualities.

[0135] Example 3

[0136] The method for increasing the content of chlorobenzan alkaloid in Zanthoxylum nitidum tissue culture material is basically the same as in Example 1. The difference is that the red light conditions in this example are controlled in stages, specifically as follows:

[0137] On June 5, 2024, vigorous shoots from the bud induction medium of Zanthoxylum nitidum were taken, and single shoots longer than 2 cm were cut off and transferred to the bud clustering medium (MS + 0.6 mg / L 6-BA + 0.2 mg / L LIBA + 0.1 mg / L KT). A total of 25 bottles were inoculated, with 3 shoots inoculated in each bottle.

[0138] The first phase, red light conditions, days 1-15, with a light intensity of 150 μmol•m⁻²•s⁻¹ and a photoperiod of 10 hours / day;

[0139] In the second stage, from day 16 to 30 under red light conditions, the light intensity was increased to 200 μmol•m⁻²•s⁻¹ and the photoperiod was extended to 13 hours / day.

[0140] In the third stage, from day 31 of the red light condition until the end of the culture, the light intensity was maintained at 200 μmol•m⁻²•s⁻¹, the photoperiod was 12 hours / day, and it was supplemented with 1 hour of blue light at an intensity of 50 μmol•m⁻²•s⁻¹ every day, with intermittent irradiation.

[0141] The red light wavelength is 620-660 nm, and the blue light wavelength is 450-480 nm. Intermittent irradiation involves alternating 6 hours of red light with 1 hour of blue light. This intermittent irradiation avoids photoreceptor deactivation and enhances the continuous activation of metabolic pathways.

[0142] The phased regulation of this invention involves inducing gene expression in the first stage using low-intensity red light (150 μmol•m⁻²•s⁻¹) to reduce light stress, then gradually increasing the intensity of red light and extending the illumination time to promote the accumulation of secondary metabolites, and finally combining it with short-duration blue light (450~480 nm) to increase the content of alkaloid chloride through a photo-quality synergistic effect.

[0143] On August 6, 2024, *Zanthoxylum nitidum* shoots subcultured under different light qualities were collected, and the alkaloid content of *Zanthoxylum nitidum* chloride was determined by UPLC-MS-MS (the determination method was the same as in Example 1). In a random sample of 20 samples, the average alkaloid content of *Zanthoxylum nitidum* chloride in the clustered shoots was 7.02 mg / g (fresh sample), with the highest content being 7.21 mg / g and the lowest being 6.38 mg / g. The results show that the alkaloid content of *Zanthoxylum nitidum* chloride in the clustered shoots was significantly increased by staged regulation, and there was a trend towards reducing content fluctuations.

[0144] This invention employs a phased regulation strategy of "initiating gene expression with low-intensity red light, gradually enhancing red light to promote metabolic accumulation, and using blue light to synergistically avoid photoreceptor passivation." This strategy continuously activates the synthetic pathway while reducing light stress, achieving simultaneous optimization of the content and stability of target components. It verifies the positive regulatory effect of dynamic adjustment of light quality, light intensity, and photoperiod on biosynthesis.

[0145] Example 4

[0146] The method for increasing the content of chlorobenzan alkaloid in Zanthoxylum nitidum tissue culture material is basically the same as in Example 1. The difference is that in this example, the red light conditions are controlled in stages and L-tryptophan (Trp) and salicylic acid (SA) are added in a gradient simultaneously. Specifically:

[0147] On June 5, 2024, vigorous shoots from the bud induction medium of Zanthoxylum nitidum were taken, and single shoots longer than 2 cm were cut and transferred to the first stage of the bud clustering medium (MS + 0.6 mg / L 6-BA + 0.2 mg / L IBA + 0.1 mg / L KT + 0.05 mg / L SA). A total of 25 bottles were inoculated, with 3 shoots inoculated in each bottle.

[0148] The first phase, red light conditions, days 1-15, with a light intensity of 150 μmol•m⁻²•s⁻¹ and a photoperiod of 10 hours / day;

[0149] In the second stage, from day 16 to 30 under red light conditions, the shoot clusters were transferred to the premixed shoot cluster medium of the second stage (MS + 0.6 mg / L 6-BA + 0.2 mg / L IBA + 0.1 mg / L KT + 0.15 mg / L SA) on day 16. The replacement of the shoot cluster medium in the second stage was carried out simultaneously with the red light intensity enhancement stage, with the red light intensity increased to 200 μmol•m⁻²•s⁻¹ and the photoperiod extended to 13 hours / day.

[0150] In the third stage, from day 31 under red light conditions until the end of the culture, the shoot clusters were transferred to the premixed shoot cluster medium of the third stage (MS + 0.6 mg / L 6-BA + 0.2 mg / L IBA + 0.1 mg / L KT + 0.1 mg / L SA + 6 μM Trp) for culture, maintaining a light intensity of 200 μmol•m⁻²•s⁻¹, a photoperiod of 12 hours / day, and supplemented with 1 hour of blue light at an intensity of 50 μmol•m⁻²•s⁻¹ daily, intermittently irradiated.

[0151] The budding culture medium used in the first, second, and third stages were all pre-sterilized and packaged liquid culture media, which were directly injected into the culture container after opening.

[0152] The red light wavelength is 620-660 nm, and the blue light wavelength is 450-480 nm. Intermittent irradiation involves alternating 6 hours of red light with 1 hour of blue light. This intermittent irradiation avoids photoreceptor deactivation and enhances the continuous activation of metabolic pathways.

[0153] This invention alleviates light stress and improves the survival rate of clustered shoots by using a low-concentration SA in the first stage. Then, in the second stage, a high-concentration SA works synergistically with red light to amplify BBE gene expression and Trp as a precursor for alkaloid synthesis to directly increase metabolic flux, thus solving the problem of content fluctuations caused by insufficient precursors when red light alone regulates the process.

[0154] On August 6, 2024, *Zanthoxylum nitidum* shoots subcultured under different light qualities were collected, and the alkaloid content of *Zanthoxylum nitidum* chloride was determined by UPLC-MS-MS (the determination method was the same as in Example 1). Twenty samples were randomly selected for testing. The average alkaloid content of *Zanthoxylum nitidum* chloride in the clustered shoots was 7.87 mg / g (fresh sample), with the highest content being 7.91 mg / g and the lowest 7.73 mg / g. The results show that the alkaloid content of *Zanthoxylum nitidum* chloride in the clustered shoots was further increased by staged regulation and synchronous gradient addition of L-tryptophan (Trp) and salicylic acid (SA), and the content fluctuation was significantly reduced, resulting in significantly improved stability. In this invention, SA alleviates light stress by regulating plant hormone signaling pathways, improves the survival rate of clustered shoots, and enhances the expression of synthetic genes (such as BBE); Trp, as a precursor for alkaloid synthesis, directly supplements the substrate of the metabolic pathway, overcoming the limitation of insufficient precursors when regulated by single light quality. The two, together with the phased light quality, form a multi-regulatory mechanism of "environmental factors activating gene expression, enhancing precursor supply, and increasing metabolic pathway flux", which further optimizes the biosynthetic efficiency and stability of target components and verifies the technical advantages of combining culture medium components with light quality regulation.

[0155] Comparative Example 1

[0156] The method for increasing the content of chlorobenzan alkaloid in Zanthoxylum nitidum tissue culture material is basically the same as in Example 4. The difference is that the red light conditions in this example are not controlled in stages, and L-tryptophan (Trp) and salicylic acid (SA) are added at once. Specifically:

[0157] On June 5, 2024, vigorous shoots from the bud induction medium of Zanthoxylum nitidum were taken, and single shoots longer than 2 cm were cut and transferred to the bud clustering medium (MS + 0.6 mg / L 6-BA + 0.2 mg / L IBA + 0.1 mg / L KT + 0.1 mg / L SA + 6 μM TP). A total of 25 bottles were inoculated, with 3 shoots in each bottle. The bottles were placed under red light intensity of 200 μmol•m⁻²•s⁻¹ with a photoperiod of 12 hours / day, and supplemented with 1 hour of blue light intensity of 50 μmol•m⁻²•s⁻¹ daily for intermittent irradiation.

[0158] The red light wavelength is 620~660nm, the blue light wavelength is 450~480nm, and the intermittent irradiation is 1 hour of blue light after every 6 hours of red light.

[0159] On August 6, 2024, thorn shoots subcultured under different light qualities were taken out, and the alkaloid content of thorn chloride was determined by UPLC-MS-MS (the determination method was the same as in Example 1). Twenty bottles of material were randomly selected for testing. The average alkaloid content of thorn chloride in the clustered shoots was 5.96 mg / g (fresh sample), with the highest content being 6.14 mg / g and the lowest being 5.02 mg / g.

[0160] Comparative Example 2

[0161] The method for increasing the content of chlorobenzan alkaloid in Zanthoxylum nitidum tissue culture material is basically the same as in Example 4. The difference is that the red light condition in this example is controlled in stages, but L-tryptophan (Trp) and salicylic acid (SA) are added at once, and blue light is not added. Specifically:

[0162] On June 5, 2024, vigorous shoots from the bud induction medium of Zanthoxylum nitidum were taken, and single shoots longer than 2 cm were cut and transferred to the bud clustering medium (MS + 0.6 mg / L 6-BA + 0.2 mg / L IBA + 0.1 mg / L KT + 0.1 mg / L SA + 6 μM TP). A total of 25 bottles were inoculated, with 3 shoots in each bottle. In the first stage, red light conditions were applied from day 1 to day 15, with a light intensity of 150 μmol•m⁻²•s⁻¹ and a photoperiod of 10 hours / day.

[0163] In the second stage, from day 16 to 30 under red light conditions, the red light intensity was increased to 200 μmol•m⁻²•s⁻¹, and the photoperiod was extended to 13 hours / day.

[0164] The third stage, from day 31 of red light conditions to the end of the culture, maintains a light intensity of 200 μmol•m⁻²•s⁻¹ and a photoperiod of 12 hours / day.

[0165] The red light wavelength is 620~660nm.

[0166] On August 6, 2024, thorn shoots subcultured under different light conditions were taken out, and the alkaloid content of thorn chloride was determined by UPLC-MS-MS (the determination method was the same as in Example 1). Twenty bottles of material were randomly selected for testing. The average alkaloid content of thorn chloride in the clustered shoots was 6.13 mg / g (fresh sample), with the highest content being 6.57 mg / g and the lowest being 5.20 mg / g.

[0167] The results from Comparative Examples 1 and 2 show that staged photoregulation is crucial for the sustained activation of gene expression, while a single addition of SA / Trp may lead to premature consumption of precursors or accumulation of photostress. Intermittent blue light irradiation may affect photoreceptor passivation and thus influence the activation of metabolic pathways, illustrating the important influencing factor of blue light in the staged synergistic regulation by red light.

[0168] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Further modifications can be readily implemented by those skilled in the art.

Claims

1. A method for increasing the content of chlorobenzan alkaloid in Zanthoxylum nitidum tissue culture materials, characterized in that, The steps include the following: S1. Explant selection: Stem segments of 1-6 month old Zanthoxylum bungeanum seedlings were selected as explants; S2. Bud induction culture: After disinfection, the explants were inoculated into bud induction culture medium, which was 1 / 2 MS basal medium supplemented with 0.5-1.0 mg / L 6-BA, 0.2-0.4 mg / L IBA and 0.1-0.5 mg / L KT; S3. Clumping bud culture: The tender buds obtained in step S2 are transferred to a clumping bud culture medium, wherein the clumping bud culture medium is MS basal medium supplemented with 0.5-1.0 mg / L 6-BA, 0.2-0.4 mg / L IBA and 0.1-0.5 mg / L KT; S4. Callus induction culture: After scratching the tender leaves from step S2, inoculate them into the callus induction culture medium, which is MS basal medium supplemented with 2 mg / L 6-BA, 0.1-0.2 mg / L KT, 0.2-1 mg / L 2,4-D and 1-1.5 mg / L NAA. S5. Directional controlled culture: Transfer the shoot cluster culture from step S3 or the callus induction culture from step S4 to red light conditions for 1 to 2 months. The light intensity of the red light is 150 to 250 μmol•m⁻²•s⁻¹, and the photoperiod is 10 to 14 hours / day.

2. The method according to claim 1, characterized in that, The bud induction medium in step S2 is 1 / 2 MS basal medium supplemented with 0.6 mg / L 6-BA, 0.2 mg / L IBA and 0.1 mg / L KT; the shoot clustering medium in step S3 is MS basal medium supplemented with 0.6 mg / L 6-BA, 0.2 mg / L IBA and 0.1 mg / L KT; the callus induction medium in step S4 is MS basal medium supplemented with 2 mg / L 6-BA, 0.1 mg / L KT, 0.2 mg / L 2,4-D and 1 mg / L NAA; the pure red light intensity under red light conditions is 200 μmol•m⁻²•s⁻¹, and the photoperiod is 12 hours / day.

3. The method according to claim 1, characterized in that, The red light condition activates the expression of genes in the chlorobenzanol synthesis pathway, which includes NCS, CYP450, and BBE.

4. The method according to claim 1, characterized in that, The culture under red light conditions for 1-2 months includes: The first phase, red light conditions, days 1-15, with a light intensity of 150-180 μmol·m⁻²·s⁻¹ and a photoperiod of 10 hours / day; In the second stage, from day 16 to 30 under red light conditions, the light intensity was increased to 200 to 220 μmol·m⁻²·s⁻¹ and the photoperiod was extended to 12 to 14 hours / day. In the third stage, from day 31 of the red light condition until the end of the culture, the light intensity was maintained at 200 μmol·m⁻²·s⁻¹, the photoperiod was 12 hours / day, and it was supplemented with 1 hour of blue light at an intensity of 50 μmol·m⁻²·s⁻¹ every day, with intermittent irradiation. The red light wavelength is 620~660nm, the blue light wavelength is 450~480nm, and the intermittent irradiation is 1 hour of blue light inserted after every 6 hours of red light.

5. The method according to claim 4, characterized in that, In step S5, the bud cluster culture medium cultured under red light conditions was supplemented with 0.05~0.2 mg / L SA and 5~10 μM Trp.

6. The method according to claim 5, characterized in that, The first stage of the shoot proliferation medium consisted of MS basal medium supplemented with 0.6 mg / L 6-BA, 0.2 mg / L IBA, 0.1 mg / L KT, and 0.05 mg / L SA. The second stage of the shoot proliferation medium consisted of MS basal medium supplemented with 0.6 mg / L 6-BA, 0.2 mg / L IBA, 0.1 mg / L KT, and 0.15 mg / L SA. The third stage of the shoot proliferation medium consisted of MS basal medium supplemented with 0.6 mg / L 6-BA, 0.2 mg / L IBA, 0.1 mg / L KT, 0.1 mg / L SA, and 6 μM Trp.

7. The method according to claim 6, characterized in that, From day 1 to day 15 under red light conditions, the budding medium of the first stage was used for cultivation. On the 16th day under red light conditions, the shoot clusters were transferred to the premixed second-stage shoot cluster medium for cultivation. The replacement of the second-stage shoot cluster medium was carried out simultaneously with the red light intensity enhancement stage, with the red light intensity increasing from 150 μmol·m⁻²·s⁻¹ to 200 μmol·m⁻²·s⁻¹. On day 31 under red light conditions, the shoot clusters were transferred to the premixed shoot cluster culture medium of the third stage; wherein the shoot cluster culture media of the first stage, the second stage and the third stage are all pre-sterilized and packaged liquid culture media, which are directly injected into the culture container or pre-packaged in the culture container after opening.

8. The application of the method as described in any one of claims 1-7 in the production of chlorinated alkali.

Images