A method for producing the diterpenoid compound jolkinol A using cell suspension culture.

Abstract

This invention discloses a method for producing the diterpenoid compound jolkinol A using cell suspension culture, belonging to the field of plant cell culture technology. The method includes: using *Euphorbia hirta* stem segments or young leaves as explants, inoculating them onto MS medium containing 6-BA and NAA to induce callus formation; suspending the callus in liquid MS medium containing 6-BA and NAA; adding salicylic acid to the culture system for induction for 10-14 days; collecting the cells, and obtaining jolkinol A through methanol extraction and chromatographic separation. After induction with 550 μmol / L salicylic acid for 10 days, this invention achieved a jolkinol A yield of 47.00 mg / L, 5.4 times higher than the control group, providing technical support for the large-scale production of jolkinol A.

Description

Technical Field

[0001] This invention relates to the field of plant cell culture technology, and specifically to a method for producing the diterpenoid compound jolkinol A using cell suspension culture. Background Technology

[0002] Jolkinol A is a diterpenoid compound of the genus *Euphorbia* (CAS No. 62820-11-5), primarily derived from the roots and stems of *Euphorbia lathyris* L., a plant in the Euphorbiaceae family. Studies have shown that diterpenoid compounds of this type possess a wide range of pharmacological activities, including anti-cholestasis, anti-tumor, anti-inflammatory, and multidrug resistance reversal activities. Furthermore, they have been reported to exhibit fungicidal activity against plant pathogens. Therefore, diterpenoid compounds of this type have attracted considerable attention. However, because these compounds are found only in plants of the genus *Euphorbia* within the Euphorbiaceae family, and in very limited quantities, their source is limited by the instability of plant cultivation. Moreover, their chemical structures are relatively complex, making total chemical synthesis impractical.

[0003] Plant cell culture technology is particularly suitable for producing natural products that are difficult to synthesize chemically, have low content in plants, or are limited by resources. It can significantly increase the yield of target products synthesized by cells by culturing plant cells in sterile, controlled bioreactors and by optimizing culture conditions and adding inducers, among other strategies. Moreover, the production cycle is short and it is easy to scale up, thus showing great potential.

[0004] Currently, there is no established cell culture technology suitable for the production of alkyl diterpenes from *Euphorbia humifusa*, nor have there been reports of induction conditions that can significantly improve jolkinol A. This invention uses *Euphorbia humifusa*, a plant in the Euphorbiaceae family, as material. By adjusting the type and concentration of hormones in the culture medium and adding suitable induction conditions, the yield of jolkinol A in *Euphorbia humifusa* callus cells was significantly increased, thus providing important technical support for the application and large-scale production of the bioactive diterpenoid compound jolkinol A. Summary of the Invention

[0005] To address the aforementioned problems in the prior art, this invention aims to provide a method for producing the diterpenoid compound jolkinol A using cell suspension culture.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A method for producing the diterpenoid compound jolkinol A using cell suspension culture includes the following steps:

[0008] (1) The upper stem segments of the *Euphorbia hirta* plant were disinfected as explants and then inoculated onto callus induction medium for callus induction culture to obtain callus tissue. The composition of the callus induction medium was: MS medium + 1.0~2.0 mg / L 6-BA + 0.3~0.5 mg / L NAA + 30 g / L sucrose + 2.5~3.0 g / L plant gel, pH 5.8; the callus induction culture conditions were: 24~26℃, light intensity of 1800~2000 1x, and photoperiod of 16 h / d.

[0009] (2) The callus tissue obtained in step (1) was inoculated into liquid suspension culture medium for suspension culture to establish a callus suspension cell culture system; the liquid suspension culture medium consisted of MS medium + 1.0~2.0 mg / L 6-BA + 0.3~0.5 mg / L NAA, pH 5.8; the suspension culture conditions were: 24~26℃, light intensity of 1800~2000 1x, light duration of 16 h / d, shaking culture; subculture once every 2-3 weeks;

[0010] (3) Add elicitor salicylic acid to the suspension culture system obtained in step (2) to induce the synthesis and accumulation of jolkinol A in the jolkinol suspension cells;

[0011] (4) Collect the sutra suspension cells obtained in step (3) and extract and separate jolkinol A from them.

[0012] In some embodiments, the explants described in step (1) may also include young leaves on the upper part of the scion plant.

[0013] In some embodiments, the callus induction culture in step (1) is performed every 3 weeks for callus subculture.

[0014] In some embodiments, the amount of callus inoculated in step (2) is 5g of fresh callus per 50mL of liquid suspension culture medium.

[0015] In some embodiments, the concentration of 6-BA in the liquid suspension culture medium in step (2) is 1.0 mg / L and the concentration of NAA is 0.3 mg / L.

[0016] In some embodiments, the rotation speed of the oscillation culture in step (2) is 100~140 rpm.

[0017] In some embodiments, the concentration of salicylic acid added in step (3) is 400~650 μmol / L.

[0018] In some embodiments, salicylic acid in step (3) is added on days 2 to 6 after the suspension cells are subcultured, and the cells are induced and cultured for another 10 to 14 days after the addition.

[0019] In some embodiments, salicylic acid in step (3) is added on the 3rd day after the suspension cells are subcultured, and the cells are induced and cultured for another 10 days after the addition, with a final concentration of 550 μmol / L.

[0020] In some embodiments, the extraction and separation in step (4) includes the following steps: soaking the suspended cells of *Euphorbia lathyris* in methanol and filtering them; grinding the resulting suspended cells and extracting them by ultrasonication in methanol; combining the methanol extract and the methanol ultrasonic extract and concentrating them under reduced pressure to obtain a brown crude extract; suspending the crude extract in distilled water and extracting it sequentially with petroleum ether, dichloromethane and ethyl acetate; separating the obtained dichloromethane extract by MCI column chromatography with gradient elution using methanol-water as the mobile phase and collecting the components; separating the components by semi-preparative high-performance liquid chromatography to obtain the compound jolkinol A.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] 1) This invention targets the diterpenoid compound jolkinol A with specific pharmacological activity. Through screening, an optimal combination of 6-BA and NAA as plant growth regulators was determined, and a high-yield jolkinol A callus induction system and suspension cell culture system were successfully established, providing a feasible technical path for the sustainable production of jolkinol A.

[0023] 2) This invention is the first to apply salicylic acid as an inducer in the culture of *Jollyrissinus* suspension cells. By optimizing its concentration (550 μmol / L) and treatment time (added on day 3 after subculture, followed by 10 days of induction culture), the yield of jolkinol A in suspension cells reached 47.00 mg / L, which is 5.4 times higher than the control group without added salicylic acid. This technical effect far exceeds that of conventional culture methods, demonstrating the significant advantages of this invention in increasing the accumulation of the target product, jolkinol A.

[0024] 3) The method provided by this invention covers the entire process from explant induction, cell suspension culture, inducer treatment to extraction and separation of target compounds. The conditions of each step are clear and easy to control. It is not only suitable for laboratory-scale product preparation, but also lays a solid foundation for the industrial continuous production of jolkinol A. It has important supporting value for in-depth pharmacological research and application development of this compound. Attached Figure Description

[0025] Figure 1 To obtain callus tissue of *Euphorbia hirta* in culture media with different hormone formulations;

[0026] Figure 2 Suspension culture of *Jolkinol A* as a high-yield diterpenoid compound;

[0027] Figure 3 The content of the diterpenoid compound jolkinol A in callus cells obtained from culture media with different hormone formulations;

[0028] Figure 4 To investigate the promoting effect of different concentrations of salicylic acid treatment on the production of jolkinol A in suspended cells. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, or are performed according to the kit and product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0030] Example 1:

[0031] (1) Induction of callus tissue from *Euphorbia hirta*: The young leaves of the upper part of the *Euphorbia hirta* plant were used as explants. After rinsing with running water, the explants were sterilized in a clean bench. They were first soaked in 75% ethanol for 45 seconds, rinsed twice with sterile water, disinfected with 2% sodium hypochlorite solution for 8 minutes, and rinsed 4 times with sterile distilled water. After sterilization, the explants were placed on sterile filter paper to absorb the water, cut into small pieces, and inoculated into callus induction medium. Eight different hormone types and concentrations of medium were prepared, namely: (1) MS medium + 1.0 mg / L 2,4-D + 0.3 mg / L 6-BA; (2) MS medium + 1.5 mg / L 2,4-D + 0.2 mg / L 6-BA; (3) MS medium + 2.0 mg / L 2,4-D + 0.5 mg / L 6-BA; (4) MS medium + 1.0 mg / L 6-BA + 0.3 mg / L (5) MS medium + 2.0 mg / L 6-BA + 0.5 mg / L NAA; (6) MS medium + 1.0 mg / L NAA; (7) MS medium + 0.5 mg / L TDZ + 0.1 mg / L NAA; (8) MS medium + 1.0 mg / L TDZ + 0.2 mg / L NAA, with 30 g / L sucrose and 2.8 g / L plant gel added to the medium, pH 5.8, and sterilized under high temperature and high pressure. After inoculation, the explants were cultured at a temperature of 24-26℃, a light intensity of 1800-2000 1x, and a photoperiod of 16h / d. Yellow-green callus was visible after 20 days of induction culture. Subculture of callus tissue was performed every 3 weeks thereafter, with the medium and culture conditions unchanged. The callus state obtained from different mediums after two months of culture was as follows. Figure 1 As shown: Callus obtained from MS medium supplemented with 2,4-D as the main auxin and cultured in combination with 6-BA was pale yellow, soft, and brittle; callus obtained from medium supplemented with NAA only was green and firmer; callus obtained from medium supplemented with a combination of 6-BA and NAA grew vigorously, was pale green, and had a loose granular texture; callus obtained from medium supplemented with a combination of TDZ and NAA was pale green and had a firmer granular texture. Overall, callus could be induced using all eight different hormone formulations. Further analysis of the jolkinol A content in callus cells obtained from different media is needed to select the optimal hormone formulation.

[0032] (2) Establishment of a callus cell suspension culture system for high production of diterpenoid compound jolkinol A: 5g (fresh weight) of callus tissue was inoculated into an Erlenmeyer flask containing 50 mL of liquid MS medium. The liquid medium with 8 different hormone formulations was prepared in the same way as in step (1) above, except that it did not contain plant gel. The cell suspension culture was placed at 25 ℃, light intensity of 1800 1x, and light duration of 16 h / d on a shaker at 120 rpm. The suspension cells were subcultured every two weeks. Figure 2 Suspension culture cells were collected on day 14 after subculture, and the content of the diterpenoid compound jolkinol A in callus cells obtained from different culture media formulations was detected. Specifically, the collected suspension cells were washed with distilled water to remove residual culture medium, followed by freeze-drying and mechanical pulverization. The freeze-dried powder sample (100 mg) was extracted with 2 mL of methanol by sonication for 30 minutes, centrifuged at 12,000 rpm for 10 minutes, and the supernatant was collected. The jolkinol A content was determined by HPLC-QTOF-MS / MS. Detection instrument: Agilent HPLC-Q-TOF-MS / MS (1260 HPLC + 6530 Q-TOF); Column: Agilent Poroshell C18 (4.6 mm × 100 mm, 2.7 μm); Column temperature: 30℃; Mobile phase: Water (0.1% formic acid, phase A), methanol (0.1% formic acid, phase B); Detection program: 0–5 min, 40–75% B; 5–15 min, 75% B; 15–30 min, 75–100% B; 30–40 min, 100% B; 40.01–50 min, 40% B; Flow rate: 0.5 mL·min -1Injection volume: 5 μL; Detection wavelength: 280 nm. Mass spectrometry ion source: Electrospray ionization (ESI), positive ion mode; Gas flow rate: 10.0 L / min; Ion source temperature: 350℃; Capillary voltage: 3500 V; OCT RFV: 750 V; Fragmentation voltage: 130 V; Sprayer: 35 psig; Acquisition range: m / z 100~1300; Secondary collision voltage: 30 eV. Quantitative analysis was performed using the standard curve of jolkinol A. The method for constructing the jolkinol A standard curve was as follows: Accurately weigh 1 mg of jolkinol A, place it in a 1 mL volumetric flask, add chromatographic grade methanol, sonicate to dissolve, and dilute to 1 mL to prepare a 1 mg / mL standard stock solution. The stock solution of jolkinol A reference standard was diluted with chromatographic grade methanol to five concentration gradients from high to low: 250 μg / mL, 100 μg / mL, 50 μg / mL, 10 μg / mL, and 1 μg / mL. The standard was detected using the LC-MS method described above, and a standard curve was plotted. The results showed that the linear regression equation for the standard curve of jolkinol A was: y = 29.306x - 5.2384, R0 2 = 0.9999, with a linear range of 1–250 μg / mL. The content of the diterpenoid compound jolkinol A in callus cells obtained from culture media with different hormone formulations is shown in [reference needed]. Figure 3 The results showed that the highest content of jolkinol A was found in suspension cells cultured in MS liquid medium supplemented with 1.0 mg / L 6-BA + 0.3 mg / L NAA, reaching 410.85 ± 16.60 μg / g (dry weight).

[0033] (3) Adding the elicitor salicylic acid to promote jolkinol A synthesis: Salicylic acid (SA) was prepared into a stock solution of 100 mmol / L using 70% ethanol and filtered through a 0.22 μm filter membrane for sterilization. On the 3rd day after subculturing the suspension cells, different volumes of SA stock solution were added to the suspension culture system. Five different final concentrations of SA were set up for treatment: 50 µmol / L, 200 µmol / L, 400 µmol / L, 550 µmol / L and 650 µmol / L. The control group suspension cells were treated with solvent (70% ethanol). Subsequently, these suspension cultures were placed in the optimal liquid medium determined in step (2) above and cultured on a shaker under the same culture conditions. The induction treatment experiments with different SA concentrations were repeated three times. Suspension cells were collected on days 0, 3, 6, 10 and 14 after induction, and the content of jolkinol A in suspension cells treated with different concentrations of SA was detected by the same method as described above. The promoting effects of different concentrations of SA treatment on jolkinol A production in suspended cells are shown in [reference needed]. Figure 4 The results showed that, compared with the control group and the low-concentration SA treatment group, high concentrations of SA significantly induced the synthesis and accumulation of jolkinol A in *Euphorbia lathyris* suspension cells. After 10 days of treatment with 550 μmol / L SA, the yield of jolkinol A reached its maximum value of 47.00 mg / L, which was 5.4 times that of the control group.

[0034] (4) Extraction and separation of jolkinol A from suspended cells: 5 kg of *Euphorbia lathyris* suspended cells were soaked in methanol for one week, filtered, and the resulting suspended cells were ground and ultrasonically extracted in methanol for 1 hour. The resulting methanol extract and ultrasonic extract were concentrated under reduced pressure to obtain a brown crude extract, which was then suspended in distilled water. This suspension was extracted sequentially with petroleum ether, dichloromethane, and ethyl acetate. The dichloromethane extract was concentrated under reduced pressure to obtain a dichloromethane fraction (3.18 g). The dichloromethane fraction was separated by MCI column chromatography with methanol-water (20:80 to 100:0, v / v) as the mobile phase to obtain five fractions (Fr. A1 to Fr. A5). Subsequently, the Fr. A2 fraction (0.91 g) was separated by semi-preparative high-performance liquid chromatography to finally obtain 29.8 mg of compound jolkinol A.

[0035] Example 2:

[0036] (1) Induction of callus from *Euphorbia hirta*: The upper stem segments of *Euphorbia hirta* plants were used as explants. After rinsing with running water, the explants were sterilized in a clean bench. They were first soaked in 75% ethanol for 45 seconds, rinsed twice with sterile water, disinfected with 2% sodium hypochlorite solution for 8 minutes, and rinsed 4 times with sterile distilled water. The sterilized explants were placed on sterile filter paper to absorb the moisture, cut into small segments of 0.5-0.8 cm in length, and inoculated into callus induction medium: MS medium + 2.0 mg / L 6-BA + 0.5 mg / L NAA + 30 g / L sucrose + 2.8 g / L plant gel (pH 5.8). The culture temperature was 24-26℃, the light intensity was 1800-2000 1x, and the photoperiod was 16 h / d. After 20 days of induction culture, yellow-green callus was visible. Subculture of callus was performed every 3 weeks thereafter, with the culture medium and culture conditions remaining unchanged.

[0037] (2) Establishment of a callus suspension culture system for high production of diterpenoid compound jolkinol A: 5 g (fresh weight) of callus tissue was inoculated into an Erlenmeyer flask containing 50 mL of liquid MS medium. 1.0 mg / L 6-BA, 0.3 mg / L NAA and 30 g / L sucrose were added to the liquid medium, and the pH value was 5.8. The cell suspension culture was placed at 25 ℃, with a light intensity of 1800 1x and a photoperiod of 16 h / d, and cultured on a shaker at 120 rpm. The suspension cells were subcultured every two weeks.

[0038] (3) Adding the elicitor salicylic acid to promote the synthesis of jolkinol A: The salicylic acid (SA) stock solution was prepared with 70% ethanol and filtered with a 0.22 μm filter membrane for sterilization. On the 3rd day after the suspension cells were subcultured, the SA stock solution was added to the suspension culture system to make the final concentration 550 μmol / L. After adding, the cells were cultured for 10 days. The suspension cells were collected, rinsed with distilled water to remove residual culture medium, and then soaked in methanol.

[0039] (4) Extraction and separation of jolkinol A from suspended cells: 5 kg of remdesivir suspension cells were soaked in methanol for one week, filtered, and the resulting suspension cells were ground and ultrasonically extracted in methanol for 1 hour. The resulting methanol extract and ultrasonic extract were concentrated under reduced pressure to obtain a brown crude extract, which was then suspended in distilled water. This suspension was extracted sequentially with petroleum ether, dichloromethane, and ethyl acetate. The dichloromethane extract was concentrated under reduced pressure to obtain a dichloromethane fraction (3.10 g). The dichloromethane fraction was separated by MCI column chromatography with methanol-water (20:80 to 100:0, v / v) as the mobile phase to obtain five fractions (Fr. A1 to Fr. A5). Subsequently, the Fr. A2 fraction (0.88 g) was separated by semi-preparative high performance liquid chromatography to finally obtain 26.2 mg of compound jolkinol A.

[0040] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. A method for producing the diterpenoid compound jolkinol A using cell suspension culture, characterized in that, Includes the following steps: (1) The upper stem segments of the *Euphorbia hirta* plant were disinfected as explants and then inoculated onto callus induction medium for callus induction culture to obtain callus tissue. The composition of the callus induction medium was: MS medium + 1.0~2.0 mg / L 6-BA + 0.3~0.5 mg / L NAA + 30 g / L sucrose + 2.5~3.0 g / L plant gel, pH 5.8; the conditions for callus induction culture were: 24~26℃, light intensity of 1800~2000 1x, and photoperiod of 16 h / d. (2) The callus tissue obtained in step (1) was inoculated into liquid suspension culture medium for suspension culture to establish a callus suspension cell culture system; the liquid suspension culture medium consisted of MS medium + 1.0~2.0 mg / L 6-BA + 0.3~0.5 mg / L NAA, pH 5.8; the suspension culture conditions were: 24~26℃, light intensity of 1800~2000 1x, light duration of 16h / d, shaking culture; subculture once every 2-3 weeks; (3) Add elicitor salicylic acid to the suspension culture system obtained in step (2) to induce the synthesis and accumulation of jolkinol A in the jolkinol suspension cells; (4) Collect the sutra suspension cells obtained in step (3) and extract and separate jolkinol A from them.

2. The method according to claim 1, characterized in that, The explants mentioned in step (1) also include the young leaves on the upper part of the succulent plant.

3. The method according to claim 1, characterized in that, In step (1), the callus induction culture is performed every 3 weeks for callus subculture.

4. The method according to claim 1, characterized in that, In step (2), the amount of callus inoculated is 5g of fresh callus per 50mL of liquid suspension culture medium.

5. The method according to claim 1, characterized in that, The concentration of 6-BA in the liquid suspension culture medium described in step (2) is 1.0 mg / L and the concentration of NAA is 0.3 mg / L.

6. The method according to claim 1, characterized in that, The rotation speed of the oscillation culture in step (2) is 100~140 rpm.

7. The method according to claim 1, characterized in that, In step (3), the concentration of salicylic acid added is 400~650 μmol / L.

8. The method according to claim 1 or 7, characterized in that, In step (3), salicylic acid is added on days 2 to 6 after the suspension cells are subcultured, and the cells are induced and cultured for another 10 to 14 days after the addition.

9. The method according to claim 8, characterized in that, In step (3), salicylic acid was added on the 3rd day after the suspension cells were subcultured, and the cells were induced and cultured for another 10 days after the addition. The final concentration of the added salicylic acid was 550 μmol / L.

10. The method according to claim 1, characterized in that, The extraction and separation described in step (4) includes the following steps: the suspended cells of *Euphorbia lathyris* are soaked in methanol and then filtered. The resulting suspended cells are ground and extracted by ultrasonication in methanol. The methanol extract and the methanol ultrasonic extract are combined and concentrated under reduced pressure to obtain a brown crude extract. The crude extract is suspended in distilled water and extracted sequentially with petroleum ether, dichloromethane and ethyl acetate. The resulting dichloromethane extract is separated by MCI column chromatography with gradient elution using methanol-water as the mobile phase, and the components are collected. The components are separated by semi-preparative high-performance liquid chromatography to obtain the compound jolkinol A.

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