Efficient in vitro accumulation method of camptothecin

Abstract

The present application relates to the field of plant tissue culture, and particularly discloses a high-efficiency in-vitro accumulation method of camptothecin. The present application uses young stem segments of camptotheca acuminata as explants, and induces accumulation of camptothecin in camptotheca acuminata whole plants through the steps of bud induction, adventitious bud induction, camptothecin in-vitro enrichment phase I culture, camptothecin in-vitro enrichment phase II culture, camptothecin in-vitro enrichment phase III culture, and adventitious root camptothecin in-vitro enrichment, thereby establishing a high-efficiency in-vitro accumulation method of camptothecin. The method of the present application can produce camptothecin under in-vitro conditions, and meets the requirements of season-unrestricted production of camptothecin, land saving, and natural camptothecin without toxic and side residues.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture, specifically relating to a method for efficient in vitro accumulation of camptothecin. Background Technology

[0002] Camptotheca acuminata, a plant belonging to the genus Camptotheca in the family Nephrolepisaceae, is a tall, deciduous tree endemic to my country and is a Class II protected wild plant in China. In 1966, Monroe E. Wall of the United States isolated camptothecin (CPT) from the bark of Camptotheca acuminata. Tumor studies have demonstrated that this tryptophan-terpene alkaloid possesses anticancer activity. [1] This has attracted widespread attention. Since the 1990s, countries such as the United States, Japan, Canada, and the United Kingdom have actively invested significant human and material resources in the research of *Camptotheca acuminata* and the development of camptothecin, making *Camptotheca acuminata* the second most important woody anticancer medicinal plant after the yew and a globally popular research topic. Because camptothecin itself is toxic, it has the potential to be developed into a plant-derived pesticide for controlling field pests. [2] .

[0003] Besides its medicinal value, Camptotheca acuminata also has considerable economic value. As a fast-growing species, it can mature into a forest in about 3 years. Its high yield means that large-scale planting of Camptotheca acuminata can effectively alleviate the pressure of timber shortage in my country and meet the country's timber demand. [3] However, current research on the production process of camptothecin and the artificial cultivation of Camptotheca acuminata is limited both domestically and internationally, mainly focusing on its artificial synthesis and drug development. The chemical synthesis of camptothecin is complex and the system is relatively imperfect. Producing camptothecin through suspension cell and callus culture methods is simpler and faster. In recent decades, researchers have established a regeneration system for Camptotheca acuminata using explants such as shoot tips, stem segments, embryos, and leaves, exploring the effects of explants, plant hormones, basic culture media, and additives on Camptotheca acuminata regeneration and camptothecin synthesis. However, the camptothecin content in Camptotheca acuminata tissue culture products is currently not ideal. [4] .

[0004] Due to the important pharmacological effects of camptothecin in anticancer activity, *Camptotheca acuminata* has become an important woody medicinal plant for anticancer treatment and a hot research area globally. Based on current research and trends both domestically and internationally, CPT and its analogues are primarily obtained through isolation from *Camptotheca acuminata*. [5] The development of the anticancer effects of camptothecin should be based on sufficient camptothecin resources. However, wild camptothecin resources are very limited, making it impossible to achieve the industrialization and large-scale comprehensive utilization of camptothecin resources. Camptothecin seeds are prone to dormancy and have a low germination rate, which is a bottleneck for the artificial cultivation of camptothecin. In addition, the problems of diseases and pests of camptothecin have become increasingly prominent in recent years. [6,7] .

[0005] To rationally utilize, develop, and protect Camptotheca acuminata resources, tissue culture technology can be used to rapidly propagate Camptotheca acuminata seedlings, thus addressing the shortage of these resources. Simultaneously, by establishing an in vitro regeneration system with high camptothecin accumulation, using the entire Camptotheca acuminata plant as a carrier for camptothecin accumulation, the production of camptotheca acuminata seedlings rich in camptothecin can be quantified. This ensures vigorous growth of medicinal organs, a short growth cycle, and that raw material harvesting is not limited by season, providing a stable supply of high-camptothecin-rich pharmaceutical materials to the market with consistent characteristics and quality, thereby solving the problem of camptothecin raw material sourcing.

[0006] References:

[0007] [1] "The isolation and structure of camptothecin, a novel alkaloidalleukemia and tumor inhibitor from camptotheca acuminata1, 2", Wall, MonroeE., et al. Plant antitumor agents. I. Journal of the American Chemical Society, Volume 88, Issue 16, Pages 3888-3890, 1966.

[0008] [2] "Biotechnological Applications in the Study of Camptotheca acuminata and Camptotheca alkaloid", Liu Zhanmei, Cui Yingde, Bin Shuying, Guangdong Chemical Industry, No. 6, pp. 52, 67-70, 2006.

[0009] [3] "Progress in the development and utilization of Camptotheca acuminata and Camptotheca alkaloid", Feng Jiancan, Zhang Yujie, Tan Yunde, Forestry Science, No. 5, pp. 100-108, 2000.

[0010] [4] "Research progress on tissue culture and rapid propagation technology of Camptotheca acuminata", Zhang Yanbo, Zhang Xiaojie, Xiao Lei, et al., Hebei Agricultural Sciences, Vol.19, No.4, pp.67-70, 2015.

[0011] [5] "Greening and medicinal functions of Camptotheca acuminata", Feng Yuyuan, Yunnan Forestry, Vol. 29, No. 6, p. 37, 2008.

[0012] [6] "Research progress on the cultivation techniques of Camptotheca acuminata and the accumulation of camptothecin", Zeng Ming, Yu Fa, Wang Luyu, Anhui Agricultural Sciences, Vol. 37, No. 34, pp. 16849-16850, 2009.

[0013] [7] "Study on the infection cycle of *Camptotheca acuminata* in *Pseudomonas pulcherrima*", Cai Yinfeng, Yunnan Agricultural University, 2023. Summary of the Invention

[0014] To address the aforementioned problems, this invention uses young stem segments of *Camptotheca acuminata* as explants and employs a series of steps including bud induction, adventitious bud induction, in vitro enrichment culture of camptothecin (stage I), in vitro enrichment culture of camptothecin (stage II), in vitro enrichment culture of camptothecin (stage III), and in vitro enrichment of camptothecin from adventitious roots. This process induces the accumulation of camptothecin in the entire *Camptotheca acuminata* plant, establishing a method for efficient in vitro accumulation of camptothecin. This method allows for the production of camptothecin under in vitro conditions, eliminating seasonal limitations on camptothecin production, saving land, and producing naturally occurring camptothecin without any toxic residues.

[0015] The technical solution of the present invention is as follows:

[0016] A highly efficient in vitro accumulation method for camptothecin includes the following steps:

[0017] (1) Take young, sterile stem segments of Camptotheca acuminata; wherein the young, sterile stem segments of Camptotheca acuminata refer to stem segments cut from semi-lignified branches of Camptotheca acuminata;

[0018] (2) Bud induction: The stem segments from step (1) were inoculated into MS medium containing 1-5 mg / L 6-BA and 0.1-0.5 mg / L NAA and cultured for 2-4 weeks until the buds sprouted.

[0019] (3) Adventitious bud induction: The fixed buds from step (2) are inoculated into an adventitious bud induction medium to induce adventitious buds of Camptotheca acuminata. The buds are cultured for 4-8 weeks to obtain adventitious bud clusters. The adventitious bud induction medium is MS medium containing 0.1-2 mg / L 6-BA, 0-0.5 mg / L NAA, and 0-1 mg / L KT.

[0020] (4) Camptothecin in vitro enrichment stage I culture: The adventitious bud clusters from step (3) were transferred to the camptothecin in vitro enrichment stage I culture medium to induce synchronous regeneration of Camptothecin adventitious buds. After culturing for 3-5 weeks, synchronous growth and development of adventitious bud clusters were obtained. The camptothecin in vitro enrichment stage I culture medium was MS medium containing 0.05-1.2 mg / L 6-BA, 0.005-0.12 mg / L NAA, 0.1-1.0 mg / L GA3, and 0-0.01 mg / L tryptophan.

[0021] (5) Camptothecin in vitro enrichment phase II culture: The adventitious bud clusters from step (4) were inoculated into the camptothecin phase II in vitro enrichment medium to induce the elongation of the camptothecin adventitious buds. After culturing for 4-6 weeks, the elongated camptothecin seedlings were obtained; the camptothecin phase II in vitro enrichment medium was MS medium containing 0.4-0.6 mg / L 6-BA, 0.04-0.06 mg / L NAA, 0-10 mg / L phloroglucinol and 0-50 mg / L CaCl2.

[0022] (6) Phase III culture of camptothecin in vitro enrichment: The seedlings from step (5) were transferred to MS medium supplemented with 1 / 2-4 / 3 times the amount of macroelements for Phase III in vitro enrichment of camptothecin. The culture was carried out for 3-5 weeks to obtain seedlings with increased biomass. The medium contained 0.4-0.6 mg / L 6-BA, 0.04-0.06 mg / L NAA, 4-6 mg / L phloroglucinol and 15-25 mg / L CaCl2.

[0023] (7) In vitro enrichment of camptothecin in adventitious roots: The seedlings in step (6) were transferred to 1 / 2 MS medium for in vitro enrichment culture of camptothecin in adventitious roots. The culture was carried out for 4-8 weeks to induce the rooting of adventitious buds of Camptotheca acuminata. The medium contained 0.5-2 mg / L IBA, 0.1-1 mg / L NAA, 4-6 mg / L phloroglucinol, and 1-3 g / L AC; or contained 0.5-2 mg / L IBA, 0.1-1 mg / L NAA, and 4-6 mg / L phloroglucinol.

[0024] Preferably, in step (1), the sterile stem segments of young Camptotheca acuminata are taken from healthy young Camptotheca acuminata stem segments and disinfected. The disinfection process is as follows: after rinsing with running water, the segments are placed in a clean bench, then soaked in ethanol-treated plant tissue culture antibacterial protectant, and then rinsed with sterile water. Preferably, the process involves rinsing with running water for 1 hour, placing the segments in a clean bench, treating with 75% ethanol for 30 seconds, soaking in 50% plant tissue culture antibacterial protectant PPM for 10-15 minutes, and rinsing with sterile water 3-5 times. More preferably, soaking in PPM for 12 minutes provides the best disinfection effect, with a cleanliness rate of 98.50% and a survival rate of 100%.

[0025] As a preferred option, in step (2), 1.5-3 mg / L 6-BA and 0.15-0.3 mg / L NAA are added to the MS medium; preferably, after culturing for 2-3 weeks, the 6-BA concentration of 2.5 mg / L and the NAA concentration of 0.25 mg / L have the best effect on inducing bud germination, with a bud germination rate of 95.70%.

[0026] As a preferred option, in step (3), 0.5-1.5 mg / L 6-BA, 0-0.2 mg / L NAA, and 0-0.5 mg / L KT are added to the culture medium; preferably, culturing with 1.0 mg / L 6-BA and 0.1 mg / L NAA for 3-5 weeks has the best effect on inducing adventitious buds, with an adventitious bud induction rate of 92.75% and an average number of adventitious buds of 22.64.

[0027] Preferably, in step (4), the MS medium contains 0.1-0.8 mg / L 6-BA, 0.01-0.08 mg / L NAA, 0.3-0.8 mg / L GA3, and 0-0.008 mg / L tryptophan Trp; more preferably, the medium contains 0.5 mg / L 6-BA, 0.05 mg / L NAA, and 0.5 mg / L GA3. 3、 The optimal high-frequency synchronization effect of adventitious shoots was achieved by culturing with 0.005 mg / L tryptophan Trp for 3-5 weeks, with an induction rate of up to 83.45%.

[0028] As a preferred option, in step (5), the MS medium containing 0.5 mg / L 6-BA, 0.05 mg / L NAA, 5 mg / L phloroglucinol and 20 mg / L CaCl2 showed the best seedling elongation and growth, with an adventitious bud elongation rate of 76.83% and an average seedling length of 6.57 cm.

[0029] As a preferred option, in step (6), adding 2 / 3 times the amount of MS medium containing 0.5 mg / L 6-BA, 0.05 mg / L NAA, 5 mg / L phloroglucinol and 20 mg / L CaCl2 has the best effect on inducing the biomass expansion of Camptotheca acuminata seedlings, with the biomass reaching up to 5.02 g.

[0030] As a preferred option, in step (7), 1.0 mg / L IBA, 0.5 mg / L NAA, 5 mg / L phloroglucinol and 2 g / L AC are added to the culture medium, the rooting rate is 100%, the number of adventitious roots reaches 10.2, and the content of camptothecin in adventitious roots reaches 125 µg / g.

[0031] Preferably, in steps (2)-(7), 20-40 g / L of sucrose and 4-6 g / L of agar powder are added to the MS medium to adjust the pH of the medium to 5.5-6; preferably, 30 g / L of sucrose and 5 g / L of agar powder are added to adjust the pH of the medium to 5.8.

[0032] As a preferred option, in steps (2)-(7), the culture conditions are 25±2 ℃, light intensity 2000-3000 lx, and light exposure 14-18 h / d; preferably, the light intensity is 2500 lx and the light exposure is 16 h / d.

[0033] Using the above-mentioned efficient in vitro accumulation method for camptothecin, camptothecin was prepared from in vitro culture materials of Camptothecin and camptothecin was extracted from them.

[0034] The beneficial effects of this invention are as follows:

[0035] This invention uses young stem segments of *Camptotheca acuminata* as explants and, through steps including bud induction, adventitious bud induction, adventitious bud elongation, in vitro accumulation of camptothecin (stages I, II, and III), and in vitro accumulation of camptothecin in adventitious roots, induces the accumulation of camptothecin in the entire *Camptotheca acuminata* plant, establishing a method for efficient in vitro accumulation of camptothecin. This method allows for the production of camptothecin under in vitro conditions, satisfying the requirements of seasonal production, saving land, and producing naturally occurring camptothecin without toxic residues. The high-camptothecin in vitro culture method of this invention significantly increases the camptothecin content. Experimental results show that the camptothecin content in cultured seedlings can reach up to 795 µg / g, which is five times the content of natural materials. Attached Figure Description

[0036] Figure 1 Young branches of the Camptotheca acuminata.

[0037] Figure 2 The buds of the Camptotheca acuminata sprout.

[0038] Figure 3 Camptotheca acuminata adventitious buds.

[0039] Figure 4 Phase I enrichment of camptothecin in vitro.

[0040] Figure 5 Phase II enrichment of camptothecin in vitro.

[0041] Figure 6 Phase III enrichment of camptothecin in vitro.

[0042] Figure 7 Camptothecin is enriched in vitro in adventitious roots. Detailed Implementation

[0043] To more clearly illustrate the purpose, technical solution, and advantages of this invention, a detailed description will be provided below in conjunction with the accompanying drawings and specific embodiments. The drawings illustrate only exemplary embodiments of the invention and are not intended to limit its implementation. This invention can be implemented in various forms, and its design concept and core technology are not limited to the embodiments shown in the drawings. These embodiments are provided to facilitate understanding of the principles, structure, and function of this invention by those skilled in the art, thereby enabling them to better master and apply its technical solutions. The terminology used in this specification is only for describing specific embodiments and does not constitute a limitation of the invention.

[0044] Example 1:

[0045] (1) Preparation of explant materials

[0046] Take healthy, semi-lignified branches of trees that are resistant to lignification. Figure 1Stem segments with apical buds were taken, rinsed with running water for 1 hour, and placed in a clean bench. They were treated with 75% ethanol for 30 seconds, then soaked in 50% plant tissue culture antimicrobial protectant (Plant Preservative Mixture, PPM) for 10-20 minutes (Table 1). The treated material was then inoculated into MS medium supplemented with 1 mg / L 6-BA and 0.1 mg / L NAA. After 2 weeks of culture, the contamination and growth status of each treatment were recorded. The results showed that the cleanliness rate gradually increased with prolonged PPM treatment, reaching 100% with 20 minutes of PPM treatment. However, the survival rate gradually decreased from 100% to 83.54%. Excessive PPM treatment led to the inactivation or growth stunting of some explants. The optimal disinfection treatment was 12 minutes of PPM, achieving a cleanliness rate of 98.5% and a survival rate of 100%.

[0047] Table 1. Effect of disinfection methods on the sterilization effect of starting materials

[0048]

[0049] (2) Bud induction

[0050] The explants were sterilized using the treatment conditions described in example 1.2 of step (1). After sterilization, the explants were inoculated into MS medium containing 1.5–3 mg / L 6-BA and 0.15–0.3 mg / L NAA and cultured for 2–5 weeks. The germination time and germination rate of the primary shoots were recorded. The results are shown in Table 2. With increasing concentrations of 6-BA and NAA, both the germination time and germination rate initially increased and then decreased. The best germination induction effect was observed when the concentration of 6-BA was 2.5 mg / L and the concentration of NAA was 0.25 mg / L, with the shortest germination time being 17 days and a germination rate of 95.7%. Figure 2 Demonstrates the sprouting of buds from a Camptotheca acuminata tree.

[0051] Table 2. Effects of different plant hormone combinations on bud germination

[0052]

[0053] (3) Adventitious bud induction

[0054] Adventitious shoot induction was performed using the treatment conditions described in example 2.3 of step (2). The induced adventitious shoots were inoculated into adventitious shoot induction medium. The basic medium was MS medium, supplemented with 0.5-1.5 mg / L 6-BA, 0-0.2 mg / L NAA, and 0-0.5 mg / L KT. The medium was cultured for 3-5 weeks, and the adventitious shoot induction rate and average number of adventitious shoots were recorded. The experimental results are shown in Table 3. The combination of 6-BA and NAA was more effective than the combination of 6-BA and KT in inducing adventitious shoots. Among them, the ratio of 1 mg / L 6-BA + 0.1 mg / L NAA showed the best effect in inducing adventitious shoot regeneration, with an adventitious shoot induction rate of 92.75% and an average number of adventitious shoots of 22.64. Figure 3 Showing the adventitious buds of the Camptotheca acuminata.

[0055] Table 3. Effects of different plant hormone combinations on adventitious shoot induction

[0056]

[0057] (4) Phase I culture for in vitro enrichment of camptothecin

[0058] Adventitious shoot induction was performed using the treatment conditions described in example 3.2 of step (3). The adventitious shoot clusters were transferred to MS medium containing 0.1-0.8 mg / L 6-BA + 0.01-0.08 mg / L NAA + 0.1-0.8 mg / L GA3 + 0-0.008 mg / L Trp for in vitro enrichment of camptothecin in phase I culture for 3-5 weeks to obtain synchronously growing adventitious shoot clusters. The number of effective shoots with a height of 1 cm or more, the high-frequency synchronization rate, and the uniformity were counted. The results are shown in Table 4. Among them, MS medium containing 0.5 mg / L 6-BA + 0.05 mg / L NAA + 0.5 mg / L GA3 + 0.005 mg / L Trp showed the best high-frequency synchronization induction effect and the highest induction rate, reaching 83.45%. Figure 4 This shows the in vitro enrichment of camptothecin in stage I.

[0059] Table 4. Effects of different plant hormone combinations on high-frequency synchronous adventitious shoot induction

[0060]

[0061] (5) Phase II culture for in vitro enrichment of camptothecin

[0062] The camptothecin in vitro enrichment stage I culture was carried out using the treatment conditions of example number 4.3 in step (4). The induced adventitious buds were inoculated into the camptothecin in vitro enrichment stage II medium. The basic medium was MS medium, supplemented with 0.5 mg / L 6-BA, 0.05 mg / L NAA, 1-10 mg / L phloroglucinol and 0-40 mg / L CaCl2. The culture was carried out for 4-6 weeks, and the height and elongation rate of the adventitious buds were recorded. The results showed that phloroglucinol and CaCl2 could promote the elongation of the adventitious buds of Camptotheca acuminata, but the elongation effect varied greatly due to different treatment concentrations (Table 5). With the increase of phloroglucinol and CaCl2 concentrations, the elongation rate of adventitious buds and the average seedling height showed a trend of first increasing and then decreasing. The seedling elongation effect and growth were best in the treatment of 5 mg / L phloroglucinol + 20 mg / L CaCl2, with an adventitious bud elongation rate of 76.83% and an average seedling length of 6.57 cm, indicating good growth. Figure 5 This image shows a photograph of the second stage of in vitro enrichment of camptothecin.

[0063] Table 5. Effects of different hormone combinations on the elongation of adventitious buds in Camptotheca acuminata.

[0064]

[0065] (6) Phase III culture for in vitro enrichment of camptothecin

[0066] The in vitro enrichment phase II culture of camptothecin was carried out using the treatment conditions of example number 5.3 in step (5). Adventitious shoots were transferred to MS medium supplemented with 1 / 3 to 4 / 3 times the amount of the medium. Each medium was supplemented with 0.5 mg / L 6-BA, 0.05 mg / L NAA, 5 mg / L phloroglucinol, and 20 mg / L CaCl2. The culture was carried out for 3-5 weeks, and the biomass was counted. The experimental results are shown in Table 6. The 2 / 3 times MS medium supplemented with the medium had the best effect on the biomass amplification of Camptothecin adventitious shoots, with a maximum biomass of 5.02 g. The plants grew vigorously and the leaves were bright green. Figure 6 Photographs showing the third stage of in vitro enrichment of camptothecin.

[0067] Table 6. Effects of basic culture medium on the biomass expansion of Camptotheca acuminata adventitious buds

[0068]

[0069] (7) In vitro enrichment culture of camptothecin in adventitious roots

[0070] The in vitro enrichment of camptothecin was carried out under the treatment conditions of example number 6.3 in step (6). Adventitious shoots were transferred to a medium containing 1 / 2 MS + 0.5-1.5 mg / L IBA + 0.1-1 mg / L NAA + 5 mg / L phloroglucinol + 0-2 g / L AC and cultured for 4-8 weeks to accumulate camptothecin in the whole plant. The rooting rate and number of roots of each treatment were counted. The results are shown in Table 7. All treatments can promote the rooting of aseptic seedlings of Camptothecin. Among them, the culture formula of 1 / 2 MS + 1 mg / L IBA + 0.5 mg / L NAA + 5 mg / L phloroglucinol + 2 g / L AC has the best effect on inducing adventitious roots, with a rooting rate of 100% and an average number of 10.2 roots, and the adventitious root system is well developed. Figure 7 Photographs showing the in vitro enrichment stage of camptothecin in adventitious roots.

[0071] Table 7. Effects of different culture formulas on rooting of Camptotheca acuminata seedlings

[0072]

[0073] (8) Determination of camptothecin content

[0074] The camptothecin content was determined by taking the aerial parts of seedlings cultured in 2MS medium with 0.5 mg / L 6-BA + 0.05 mg / L NAA + 5 mg / L phloroglucinol + 20 mg / L CaCl2 (sample 1, i.e. example number 6.3), the aerial parts of adventitious roots cultured in 1 / 2MS medium with 1 mg / L IBA + 0.5 mg / L NAA + 5 mg / L phloroglucinol + 2 g / L AC (sample 2, i.e. example number 7.5), the root system of seedlings cultured in 1 / 2MS medium with 1 mg / L IBA + 0.5 mg / L NAA + 5 mg / L phloroglucinol + 2 g / L AC (sample 3, i.e. example number 7.5), and young leaf material from mature trees (sample 4).

[0075] In (1)-(8), the MS medium was: 30 g / L sucrose, 5 g / L agar powder, and the pH of the medium was adjusted to 5.8; the culture conditions were 25±2 ℃, light intensity 2500 lx, and light exposure 16 h / d.

[0076] The content of camptothecin was analyzed using liquid chromatography-mass spectrometry (LC-MS). The specific experimental steps are as follows:

[0077] ① Sample pretreatment

[0078] Fresh tissue samples were freeze-dried to obtain dry weight samples. An appropriate amount of plant sample was weighed, and MeOH was added to prepare a sample of suitable concentration. The sample was sonicated for 30 min, centrifuged (15000 rpm) for 10 min, and the supernatant was taken. The supernatant was diluted with MeOH / H2O = 1:1 and then used for LC-MS analysis.

[0079] ② Preparation of standard solutions

[0080] Accurately weigh the camptothecin standard, dissolve it in DMSO to obtain a 1 mg / mL reference solution, and dilute it with MeOH / H2O at a ratio of 1:1 to obtain an external standard working solution with a concentration of 1 μg / mL (L6). Take 500 μL of L6 and add it to 500 μL of MeOH / H2O at a ratio of 1:1 to obtain L5; take 200 μL of L5 and add it to 800 μL of MeOH / H2O at a ratio of 1:1 to obtain L4; take 500 μL of L4 and add it to 500 μL of MeOH / H2O at a ratio of 1:1 to obtain L3; take 200 μL of L3 and add it to 800 μL of MeOH / H2O at a ratio of 1:1 to obtain L2; take 500 μL of L2 and add it to 500 μL of MeOH / H2O at a ratio of 1:1 to obtain L1. These standard solutions are used to determine the camptothecin content in plant samples.

[0081] ③ Liquid chromatography and mass spectrometry conditions

[0082] Chromatographic conditions: The column was an ACQUITY UPLC BEH C18 (50 mm × 2.1 mm, 1.7 μm); the mobile phase was aqueous phase (A) – 0.1% formic acid (FA) in H2O, and organic phase (B) – 0.1% FA in acetonitrile (ACN); the column temperature was 45℃; the flow rate was 0.5 mL / min; the injection volume was 0.5 μL; and the liquid phase gradient is shown in the table below.

[0083] Table 8. Liquid phase gradient analysis by LC-MS

[0084]

[0085] Mass spectrometry conditions: Ionization mode: ES+; Data type: SIR data; Function type: SIR of 1 channel; Chan Mass: 349.05.

[0086] LC-MS results showed that the camptothecin-rich in vitro culture method of Camptothecin obtained through the above steps significantly increased the camptothecin content: the camptothecin content of natural young leaves (sample 4) was 159 µg / g, and after the third stage of in vitro enrichment culture, the camptothecin content of the aboveground parts of Camptothecin seedlings (sample 1) was 677 µg / g; after the in vitro enrichment culture of adventitious roots, the camptothecin content of adventitious roots (sample 3) was 125 µg / g, which was not much different from the camptothecin content of natural young leaves; and the camptothecin content of the aboveground parts of Camptothecin seedlings (sample 2) was 795 µg / g, which was 5 times that of the natural material, thus achieving the accumulation of camptothecin throughout the entire plant. From the time the material is placed in the bottle to the in vitro enrichment stage of camptothecin in adventitious roots, the cultivation time is one year. In contrast, the natural tender leaves are taken from mature trees that have grown for 16 years. It can be seen that the method of the present invention can significantly shorten the time for obtaining camptothecin and produce a natural camptothecin content that is 5 times higher than that of natural materials.

[0087] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention. The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the protection scope of the present invention.

Claims

1. A high-efficiency in vitro accumulation method of camptothecin, characterized in that, Comprising the following steps: (1) Take young sterile stem segments of Coptis deltoidea; (2) Bud induction: the stem segments of step (1) are inoculated into bud induction medium, and cultured for 2-4 weeks until the buds germinate; the bud induction medium is MS+1-5 mg / L 6-BA+0.1-0.5 mg / L NAA; (3) Adventitious bud induction: the buds of step (2) are inoculated in the adventitious bud induction medium, and cultured for 4-8 weeks to obtain the adventitious bud clusters; the adventitious bud induction medium is MS+0.1-2 mg / L 6-BA+0.05-0.5 mg / L NAA, or MS+0.1-2 mg / L 6-BA+0.1-1 mg / L KT, or MS+0.1-2 mg / L 6-BA+0.05-0.5 mg / L NAA+0.1-1 mg / L KT; (4) Coptis in vitro enrichment phase I culture: the adventitious bud clusters of step (3) are transferred to the Coptis in vitro enrichment medium phase I, and cultured for 3-5 weeks to obtain the growth and development synchronous adventitious bud clusters; the Coptis in vitro enrichment medium phase I is MS+0.05-1.2 mg / L 6-BA+0.005-0.12 mg / L NAA+0.1-1.0 mg / L GA3, or MS+0.05-1.2 mg / L 6-BA+0.005-0.12 mg / L NAA+0.1-1.0 mg / L GA3+0.003-0.01 mg / L tryptophan; (5) Coptis in vitro enrichment phase II culture: the adventitious bud clusters of step (4) are inoculated in the Coptis in vitro enrichment medium phase II, and cultured for 4-6 weeks to obtain the elongated Coptis seedlings; the Coptis in vitro enrichment medium phase II is MS+0.4-0.6 mg / L 6-BA+0.04-0.06 mg / L NAA+1-10 mg / L phloroglucinol+10-50 mg / L CaCl2; (6) Coptis in vitro enrichment phase III culture: the seedlings in step (5) are transferred to the enrichment medium phase III, and cultured for 3-5 weeks to obtain the biomass amplified seedlings; the enrichment medium phase III is 1 / 3-4 / 3 times MS+0.4-0.6 mg / L 6-BA+0.04-0.06 mg / L NAA+4-6 mg / L phloroglucinol+15-25 mg / L CaCl2; The amount of sucrose added in the MS medium is 20-40 g / L, and the amount of agar powder is 4-6 g / L.

2. The method for efficient in vitro accumulation of camptothecin according to claim 1, wherein, Further comprising the following steps: (7) Adventitious root camptothecin in vitro enrichment: the bud seedling in step (6) is transferred to an adventitious root camptothecin in vitro enrichment medium, and adventitious root camptothecin in vitro enrichment culture is carried out, and the culture is carried out for 4-8 weeks to induce the camptothecin bud seedling to root; the adventitious root camptothecin in vitro enrichment medium is 1 / 2MS+0.5-2 mg / L IBA+0.1-1 mg / L NAA+4-6 mg / L phloroglucinol+1-3 g / L AC; or 1 / 2MS+0.5-2 mg / L IBA+0.1-1 mg / L NAA+4-6 mg / L phloroglucinol.

3. The method of claim 1, wherein the camptothecin is accumulated in a high yield in vitro. In step (1), the young camptotheca acuminata sterile stem segment is taken from a young camptotheca acuminata stem segment growing healthily, and is subjected to disinfection treatment; the disinfection treatment is: after being washed with running water, being placed on an ultra-clean workbench, being treated with ethanol, and being soaked with a plant tissue culture antibacterial protective agent (Plant Preservative Mixture) PPM, being washed with sterile water.

4. The method of claim 1, wherein the camptothecin is accumulated in a high yield in vitro. The disinfection treatment is: being washed with running water for 1 h, being placed on an ultra-clean workbench, being treated with 75 % ethanol for 30 s, being soaked with 50 % PPM for 10-15 min, and being washed with sterile water for 3-5 times.

5. The method of claim 4, wherein the concentration of the camptothecin is about 0.1 to 0.5 mg / mL. The disinfection treatment is to soak with PPM for 12 min.

6. The camptothecin high-yield in vitro accumulation method according to claim 1, characterized by, In step (2), the fixed bud induction medium is MS+1.5-3 mg / L 6-BA+0.15-0.3 mg / L NAA, and the culture is carried out for 2-3 weeks; in step (3), the adventitious bud induction medium is MS+0.5-1.5 mg / L 6-BA+0.05-0.2 mg / L NAA, or MS+0.5-1.5 mg / L 6-BA+0.1-0.5 mg / L KT, or MS+0.5-1.5 mg / L 6-BA+0.05-0.2 mg / L NAA+0.1-0.5 mg / L KT, and the culture is carried out for 3-5 weeks to induce adventitious buds; in step (4), the first-stage camptothecin in vitro enrichment medium is MS+0.1-0.8 mg / L 6-BA+0.01-0.08 mg / L NAA+0.3-0.8 mg / L GA3+0.003-0.008 mg / L tryptophan Trp, and the culture is carried out for 3-5 weeks.

7. The camptothecin high-yield in vitro accumulation method according to claim 6, characterized by, In step (2), the fixed bud induction medium is MS+2.5 mg / L 6-BA+0.25 mg / L NAA; in step (3), the adventitious bud induction medium is MS+1.0 mg / L 6-BA+0.1 mg / L NAA; in step (4), the first-stage camptothecin in vitro enrichment medium is MS+0.5 mg / L 6-BA+0.05 mg / L NAA+0.5 mg / L GA3+0.005 mg / L tryptophan Trp.

8. The camptothecin high-yield in vitro accumulation method according to claim 1, characterized by, In step (5), the camptothecin II phase in vitro enrichment medium is MS+0.5 mg / L 6-BA+0.05 mg / L NAA+5 mg / L phloroglucinol+20 mg / L CaCl2; in step (6), the bud seedlings in step (5) are transferred to a 2 / 3 times amount of MS medium+0.5 mg / L 6-BA+0.05 mg / L NAA+5 mg / L phloroglucinol+20 mg / L CaCl2.

9. The camptothecin high-yield in vitro accumulation method according to claim 2, characterized by, In step (7), the adventitious root camptothecin in vitro enrichment medium is 1 / 2MS+1 mg / L IBA+0.5 mg / L NAA+5 mg / L phloroglucinol+2 g / L AC.

10. The method of claim 1-9, wherein the method is a high-yield in vitro accumulation method of camptothecin, characterized in that, The pH value of the MS medium is 5.5-6; the culture condition is 25±2 ℃, the light intensity is 2000-3000 lx, and the light illumination is 14-18 h / d.

11. The camptothecin high-efficiency in vitro accumulation method according to claim 10, characterized in that, In the MS medium, the addition amount of sucrose is 30 g / L, the addition amount of inositol is 0.1 g / L, the amount of agar powder is 5 g / L, and the pH value is 5.8; the culture condition is a light intensity of 2500 lx and a light illumination of 16 h / d. ​

Images