A biological inducer for efficiently biosynthesizing chlorogenic acid substances using Panax notoginseng cells, and its preparation method and application

Abstract

The present invention relates to the technical field of plant cell culture, and discloses a method for efficiently producing chlorogenic acid substances by combining plant cell culture technology with a new inducer. The main plant cells used are: Panax notoginseng suspension cells. The steps include: preparation of a new inducer, addition of a new inducer, efficient synthesis of chlorogenic acid substances, extraction and identification of products. The present invention uses Ensifer adhaerens inactivated by adding a new inducer, and further utilizes plant cell liquid fermentation to improve the synthesis of chlorogenic acid substances in Panax notoginseng suspension cells. The method is simple to operate, efficient in raw material preparation, less post-processing pollution, and the chlorogenic acid extract is close to the plant content level. The chlorogenic acid extract provided by the present invention has excellent antioxidant and anti-inflammatory functions, and can provide raw materials for the preparation of medicinal or skin care product additives.

Description

Technical Field

[0001] The present invention relates to the technical field of plant cell culture, and in particular to a biological inducer for efficiently biosynthesizing chlorogenic acid substances using Panax notoginseng cells, and a preparation method and application thereof. Background Art

[0002] Chlorogenic acid (CA) is a type of phenolic acid compound generated by caffeic acid and quinic acid (1-hydroxyhexahydrogallic acid). Chlorogenic acid substances are commonly found in plants, and are abundant in plants such as honeysuckle, eucommia, chrysanthemum, coffee, and gardenia. Currently, chlorogenic acid is mainly extracted from wild plants. Although plants are a renewable resource, they grow slowly, are difficult to cultivate, have low content of active ingredients, and are easily affected by many factors such as geography, seasons, and environmental changes, which limits the extraction of active ingredients from wild plant resources. Therefore, an alternative production method is needed.

[0003] Plant cell suspension culture refers to the process of inoculating plant tissues or small cell aggregates into liquid culture medium and oscillating the culture. It usually selects fast-growing, loose, and well-conditioned callus tissues for suspension culture, and obtains a large number of plant cells in good growth status in a short period of time through suspension culture methods similar to microbial fermentation. Subsequently, inducers are added to obtain a considerable yield of effective secondary metabolites, such as chlorogenic acids. Due to its many advantages such as short cycle, strong controllability, little influence by natural conditions, convenient operation, good repeatability, and easy industrial production, it has been ideally applied to the small-scale production of some important biologically active substances and natural products.

[0004] Elicitors are a special type of triggering factor that can activate the activity of enzymes in the metabolic process, thereby increasing the content of secondary metabolites and sometimes even inducing new compounds. Generally, they can be divided into biological elicitors and abiotic elicitors. Biological elicitors include elicitors from fungi, bacteria or herbivores, plant cell wall fragments, chemicals released when plants are invaded by pathogens or herbivores, etc. Their functions are coupled with receptors and work by activating or inhibiting certain enzymes or ion channels. Abiotic elicitors are not natural components in plant cells themselves, but can cause plants to produce defense responses through chemical or physical pathways, thereby triggering the synthesis of plant secondary active substances. Their types mainly include plant growth regulators, salicylic acid, methyl jasmonate, heavy metal salts, rare earth elements, etc.

[0005] The present invention combines a novel inducer, Ensifer adhaerens, with a plant cell suspension culture technique to find a method for efficiently biosynthesizing chlorogenic acid substances. Summary of the invention

[0006] The purpose of the present invention is to develop a new type of inducer and a method for producing chlorogenic acid substances using plant cell culture technology to solve the problems of long growth cycle of traditional plants, shortage of raw material resources, great environmental impact, and long-term reliance on methyl jasmonate (Methyl Jasmonate, MeJA), salicylic acid (Salicylic acid, SA), yeast extract (Yeastextract, YE) and the like to induce secondary metabolites.

[0007] The technical solution of the present invention is as follows:

[0008] A biological inducer for efficiently biosynthesizing chlorogenic acid substances using Panax notoginseng cells, which is inactivated Ensifer adhaerens.

[0009] The preparation method thereof is:

[0010] (1) Activation of bacterial strains: Ensifer adhaerens stored at -80°C was inoculated onto a sterile solid culture medium slant and cultured at 28°C for 3 days for bacterial activation; the composition of the solid culture medium was: 10 g / L soluble starch, 1 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate, 1 g / L sodium chloride, 2 g / L ammonium sulfate, 2 g / L calcium carbonate, 0.001 g / L ferrous sulfate, 0.001 g / L zinc sulfate heptahydrate, 0.5 g / L yeast extract, 1 g / L peptone, 20 g / L agar, the pH was controlled at 7.2 by NaOH, and the culture was sterilized at 115°C for 25 min in advance; Ensifer adhaerens was preserved by the State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology.

[0011] (2) Shake flask culture of sticky sword fungus:

[0012] Wash the cells from the fresh solid culture medium slant and inoculate into the liquid culture medium, and culture at 260rpm and 32°C for 28h; the composition of the liquid culture medium is: sucrose 40g / L, corn steep liquor 20g / L, betaine 5g / L, ammonium sulfate 1g / L, diammonium phosphate 2g / L, manganese sulfate monohydrate 0.8g / L, cobalt chloride hexahydrate 0.02g / L, magnesium oxide 0.5g / L, DMBI 0.05g / L, zinc sulfate heptahydrate 0.08g / L, calcium carbonate 1g / L, pH is controlled at 7.2 by NaOH, and sterilized at 115°C for 25min in advance;

[0013] (3) Preparation of the elicitor of Sword fungus:

[0014] The mycelium obtained in step (2) is ground, and the ground mycelium is dissolved with a phosphate buffer solution (pH=6.8). The resulting mixed solution is sterilized at 121° C. for 25 min to obtain inactivated Ensifer adhaerens, i.e., the bio-inducer of the present invention.

[0015] The obtained inactivated Xanthophyllales is used to promote Panax notoginseng cells to produce secondary metabolites - chlorogenic acid substances. The specific method is: Panax notoginseng suspension cells are cultured to the logarithmic growth phase, 50 mg / L of Xanthophyllales is added to the liquid culture medium of Panax notoginseng suspension cells, and the action time of the inducer is set to 3 days.

[0016] Technical principle of the present invention:

[0017] The present invention adds a novel inducer, i.e. inactivated Ensiferadhaerens, into the Panax notoginseng suspension cells to promote the synthesis and accumulation of chlorogenic acid substances, which are secondary metabolites in the Panax notoginseng cells.

[0018] Panax notoginseng suspension cells were cultured to the logarithmic growth phase, and different concentrations of sticky sword fungi and different concentrations of common inducers - methyl jasmonate, salicylic acid and yeast extract were added to the Panax notoginseng cell liquid culture medium. The action time of the inducer was set to 1, 2, 3, and 4 days. Next, high performance liquid chromatography and liquid chromatography-mass spectrometry were used to detect the content of chlorogenic acid substances in Panax notoginseng cells to obtain the optimal inducer and action time.

[0019] Beneficial effects of the present invention:

[0020] 1. Since the traditional whole plant material is not used, but Panax notoginseng plant cell culture is used instead to produce chlorogenic acid substances, it can save resources and is of great significance to protecting wild resources, maintaining ecological balance and promoting industrial development.

[0021] 2. The formed suspension cells have fast growth rate, strong cell vitality, easy control, short experimental cycle and simple operation, efficient raw material preparation, less post-processing pollution and good experimental repeatability.

[0022] 3. The use of Xanthoceras adheringensis as a new type of inducer has an inducing effect greater than that of methyl jasmonate (MeJA), salicylic acid (SA), and yeast extract (YE), which has not been reported in inducing secondary metabolites in plant cells.

[0023] 4. The new inducer of the sticky sword fungus added in the present invention breaks the barrier of using conventional inducers (salicylic acid, MeJA, heavy metal salts, rare earth elements) and saves experimental costs.

[0024] 5. The present invention adds a new inducer, Xanthophyllales, and the yield of chlorogenic acid substances is increased to 1.38 times, and the content is increased by 3.82% (per gram dry weight), which lays a foundation for the subsequent application of chlorogenic acid substances in the cosmetics and pharmaceutical industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : Total ion current diagram of LC-MS / MS analysis of intracellular substances of Panax notoginseng obtained in Example: qualitative analysis was performed by mass spectrometry database retrieval using NIST14 mass spectrometry database, and relative quantification was performed by chromatographic peak area normalization method; it can be seen from the figure that there are chlorogenic acid characteristic substances such as 5-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 4,5-O-dicaffeoylquinic acid glucoside derivatives, and 4,5-O-dicaffeoylquinic acid;

[0026] Figure 2 : The effects of different inducer concentrations obtained in the example on the chlorogenic acid content in Panax notoginseng cells. By comparison, the adhesion sword fungus has the greatest effect on the accumulation of chlorogenic acid in Panax notoginseng cells, with the highest content, which can reach 61.75±0.32 mg / g (per gram dry weight content);

[0027] Figure 3 : The effects of different inducer concentrations obtained in the examples on the biomass of Panax notoginseng cells. By comparison, the effect of Sword fungus on the biomass of Panax notoginseng cells was minimal;

[0028] Figure 4 : The effects of different action times of the inducers obtained in the example on the chlorogenic acid content in Panax notoginseng cells. By comparison, the adhesion sword fungus has the greatest effect on the accumulation of chlorogenic acid in Panax notoginseng cells, and the content is the highest, which can reach 61.75±0.32 mg / g (content per gram of dry weight). DETAILED DESCRIPTION

[0029] The present invention is further described below through specific embodiments in conjunction with the accompanying drawings, but the present invention is not limited thereto.

[0030] Used in the embodiments of the present invention:

[0031] Strain name: Xanthophyll adherans, ATCC 33212, maintained by the State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology.

[0032] Cell name: Panax notoginseng cells, preserved by the State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology.

[0033] The HPLC and UPLC-MS / MS analysis methods used in the examples of the present invention are as follows:

[0034] Mobile phase: 0.05% orthophosphoric acid aqueous solution (A) and acetonitrile (B); flow rate: 1.0 mL / min; elution conditions: 0-50 min, 5-43% A; 50-52 min, 43-80% A; 52-55 min, 80-5% A; 55-58 min, 5-5% A; chromatographic column: Waters Symmetry C18 column (5 μm, 4.6×250 mm); column temperature: 40 °C; detection wavelength: 327 nm; injection volume: 10 μL. The HPLC conditions of UPLC-MS / MS were the same as above, and the parameters of MS / MS were set as follows: scanning mode: positive and negative ions; charge-to-mass ratio m / z scanning range: 100-1000; scanning speed: 13000Da / s; drying gas: nitrogen; drying gas flow rate: 10L / min; drying gas temperature: 365℃; nebulizer pressure: 50psi; capillary voltage: +4500V; cone voltage: 40V.

[0035] The names, specifications and manufacturer information of the main reagents used in the present invention are as follows:

[0036]

[0037] The names, specifications and manufacturers of the main instruments used in the present invention are as follows:

[0038]

[0039] Statistics

[0040] Data are presented as mean ± standard error The Student's t-test (GraphPad Software Inc., San Diego, CA, USA) was used. Graphical and statistical analysis of data were performed using GraphPad Prism 8. p < 0.05 was considered statistically significant.

[0041] Example

[0042] (1) Preparation of novel inducers

[0043] A small amount of Xanthophyllales preserved in a low-temperature refrigerator was selected and inoculated onto a sterile solid slope, and cultured at 28°C for 3 days to activate the strain.

[0044] Shake flask culture of Xanthophyll adhering to the slant: wash the bacteria from the fresh slant and inoculate them into 500 mL of liquid culture medium with a liquid volume of 100 mL, and culture them at 260 rpm and 32°C for 28 h.

[0045] The solid culture medium is composed of: 10 g / L soluble starch, 1 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate, 1 g / L sodium chloride, 2 g / L ammonium sulfate, 2 g / L calcium carbonate, 0.001 g / L ferrous sulfate, 0.001 g / L zinc sulfate heptahydrate, 0.5 g / L yeast extract, 1 g / L peptone, 20 g / L agar, pH is controlled at 7.2 by NaOH, and the solid culture medium is sterilized at 115° C. for 25 min before use.

[0046] The liquid culture medium is composed of: 40 g / L sucrose, 20 g / L corn steep liquor, 5 g / L betaine, 1 g / L ammonium sulfate, 2 g / L diammonium phosphate, 0.8 g / L manganese sulfate monohydrate, 0.02 g / L cobalt chloride hexahydrate, 0.5 g / L magnesium oxide, 0.05 g / L DMBI, 0.08 g / L zinc sulfate heptahydrate, 1 g / L calcium carbonate, pH is controlled at 7.2 by NaOH, and the liquid culture medium is sterilized at 115° C. for 25 min before use.

[0047] Preparation of Sword Fungus: Take the fermentation liquid into a 10mL centrifuge tube, centrifuge at 3000rpm for 10min, wash twice with deionized water, remove the supernatant, grind the precipitate in a mortar, dissolve the ground bacteria with phosphate buffer, and sterilize the mixture at 121℃ for 25min for use.

[0048] (2) Preparation of common inducers

[0049] Preparation of salicylic acid inducer (SA): Accurately weigh 138.00 mg of salicylic acid solid powder, dissolve it with an appropriate amount of distilled water, and dilute to 10 mL to prepare a 100 mmol / L mother solution. Then, the mother solution was sterilized by filtering with a 0.22 μm microporous filter membrane and set aside;

[0050] Preparation of methyl jasmonate inducer (MeJA): Accurately pipette 235.0 μL of methyl jasmonate, dilute to 10 mL with anhydrous ethanol, and prepare a 100 mmol / L mother solution. Then, the mother solution is sterilized by filtering with a 0.22 μm microporous filter membrane and set aside.

[0051] (3) Addition of inducers

[0052] The Panax notoginseng suspension culture cells were cultured to the logarithmic growth phase for the inducer induction experiment. At this time, different concentrations of inducers were added and acted for 1, 2, 3, and 4 days respectively, and the effects of different concentrations of inducers on the biomass (dry weight) of Panax notoginseng suspension culture cells and the content of chlorogenic acid substances in suspension cells were determined. The final concentrations of each inducer in the culture medium are as follows: SA: 0, 50, 100, 200, 300, 400 μM; MeJA: 0, 50, 100, 200, 300, 400 μM; YE: 0, 50, 100, 200, 300, 400 mg / L; Adhesive sword fungus cell concentration: 0, 50, 100, 200, 300, 400 mg / L; Three parallel test groups were set up for each treatment group.

[0053] (4) Product extraction and identification

[0054] After 1, 2, 3, and 4 days of culture, the Panax notoginseng suspension cells were placed in a 60°C oven and dried to constant weight by hot air. 100 mg of cell powder was accurately weighed in a 10 mL centrifuge tube, and 2 mL of 70% methanol solution was added. The mixture was ultrasonicated in a water bath for 50 min. Then, the supernatant was taken and filtered through a 0.22 μm organic filter membrane to identify the components. The NIST14 mass spectrometry database was used for qualitative analysis, and the chromatographic peak area normalization method was used for relative quantification.

[0055] The key to the present invention is to add 50 mg / L of a new inducer, inactivated sticky sword fungus, on the 6th day of cultivation at the plant cell culture level. After 3 days of induction, the content of chlorogenic acid substances in Panax notoginseng cells is the highest. Compared with the content of chlorogenic acid substances in common inducers (MeJA, SA and YE), after the addition of the new inducer, the content of chlorogenic acid substances increased by 0.38 times (per gram dry weight content), and the content reached 61.75±0.32 mg / g (dry weight).

[0056] The addition of the Xanthophyllum adhensive inducer will have an inhibitory effect on cell biomass, and the higher the concentration of Xanthophyllum adhensive, the stronger the inhibitory effect on cell biomass, but compared with other inducers, Xanthophyllum adhensive has the lowest inhibitory effect. The innovation of this method is that the inactivated Xanthophyllum adhensive inducer can significantly increase the total content of chlorogenic acids in cells, and with the increase of Xanthophyllum adhensive concentration, it shows a trend of first increasing and then decreasing. On the third day of the action of 50 mg / L Xanthophyllum adhensive, the total content of chlorogenic acids in Panax notoginseng cells reached a maximum value of 61.75±0.32 mg / g (dry weight), which is 1.38 times that of the control group (content per gram of dry weight). See the results. Figure 2 , 3 、4.

[0057] The above description is only an example of the implementation mode of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of inactivated Ensifer adhaerens as a biological inducer to promote the production of chlorogenic acid by Panax notoginseng cells, It is characterized in that The preparation of the biological inducer comprises the following steps: (1) Activation of bacterial strains: Take the sticky sword bacteria stored at -80°C and inoculate it onto a sterile solid culture medium slant, and culture it at 28°C for 3 days to activate the bacterial strains; the composition of the solid culture medium is: 10 g / L soluble starch, 1 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate, 1 g / L sodium chloride, 2 g / L ammonium sulfate, 2 g / L calcium carbonate, 0.001 g / L ferrous sulfate, 0.001 g / L zinc sulfate heptahydrate, 0.5 g / L yeast extract, 1 g / L peptone, 20 g / L agar, the pH is controlled at 7.2 by NaOH, and the mixture is sterilized at 115°C for 25 min in advance; (2) Shake flask culture of sticky sword fungus: Wash the cells from the fresh solid culture medium slant and inoculate into the liquid culture medium, and culture at 260rpm and 32°C for 28h; the composition of the liquid culture medium is: sucrose 40g / L, corn steep liquor 20g / L, betaine 5g / L, ammonium sulfate 1g / L, diammonium phosphate 2g / L, manganese sulfate monohydrate 0.8g / L, cobalt chloride hexahydrate 0.02g / L, magnesium oxide 0.5g / L, DMBI 0.05g / L, zinc sulfate heptahydrate 0.08g / L, calcium carbonate 1g / L, pH is controlled at 7.2 by NaOH, and sterilized at 115°C for 25min in advance; (3) Preparation of the elicitor of Sword fungus: The bacterial cells obtained in step (2) are ground, and the ground bacterial cells are dissolved in a phosphate buffer solution with a pH of 6.

8. The obtained mixed solution is sterilized at 121° C. for 25 minutes to obtain inactivated sticky sword fungi, which are the biological inducers.

2. The use of the inactivated Ensifer adhaerens according to claim 1 as a biological inducer for promoting Panax notoginseng cells to produce chlorogenic acid, the specific method being: culturing Panax notoginseng suspension cells to the logarithmic growth phase, adding 50 mg / L inactivated Ensifer adhaerens to the Panax notoginseng suspension cell liquid culture medium, and setting the action time of the inducer to 3 days.

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