Method for increasing content of dencichine in panax notoginseng cells by applying nitrogen source

Abstract

The invention relates to a method for increasing the content of dencichine in panax notoginseng cells by applying a nitrogen source, and belongs to the field of panax notoginseng cell culture.The method comprises the following steps that 1, panax notoginseng diploid cells with the good growth condition are taken and inoculated into a WPM culture medium added with nitrogen, and the WPM culture medium is placed at the temperature of 25 DEG C for dark culture for 5-15 d; (2) putting the pseudo-ginseng callus cultured in the step (1) into a mortar, adding liquid nitrogen, grinding, and extracting dencichine; and (3) making a dencichine standard curve, and determining the dencichine content through an HPLC method. The technical difficulties of pesticide residues, heavy metal pollution, long period and the like can be overcome, the content of dencichine in the panax notoginseng cells is simply and efficiently increased, and the method has important guiding significance on large-scale production of dencichine.

Description

Technical Field

[0001] This invention relates to the field of Panax notoginseng cell culture technology, and in particular to a method for increasing the content of notoginseng extract in Panax notoginseng cells by applying a nitrogen source. Background Technology

[0002] Sanqi ( Panax notoginseng Panax notoginseng is a plant belonging to the genus Panax in the family Araliaceae. Its roots and rhizomes contain a variety of bioactive components, among which dencichine is a unique component of Panax notoginseng. It has a variety of pharmacological activities, such as anti-inflammatory, antioxidant, anti-tumor, and cardiovascular protection functions, and has important application value in the pharmaceutical field.

[0003] Currently, notoginseng extract mainly comes from field-cultivated notoginseng plants. However, traditional notoginseng cultivation suffers from numerous problems, such as continuous cropping obstacles, long growth cycles, and numerous pests and diseases, which severely restrict the sustainable development of the notoginseng industry. Furthermore, field-cultivated notoginseng also faces quality and safety issues such as pesticide residues and excessive heavy metals, affecting the quality and safety of pharmaceutical products made from notoginseng.

[0004] To increase the content of notoginseng in Panax notoginseng, researchers have conducted various studies. Some studies have shown that spraying a sodium selenite solution after flowering can significantly increase the notoginseng content in Panax notoginseng leaves and roots, reaching 1.4-2.1 times and 1.2-1.7 times that of the untreated group, respectively, while also enhancing the activity of antioxidant enzyme systems in Panax notoginseng. However, this method still relies on field cultivation and cannot fundamentally solve the problems faced in Panax notoginseng cultivation.

[0005] Regarding the extraction of notoginseng, some studies have used industrial notoginseng waste as raw material, verified the presence of notoginseng in the extracted product using thin-layer chromatography, and then purified and crystallized it, thus achieving resource reuse. Although this method reduces resource waste, it still relies on the large-scale cultivation of notoginseng plants.

[0006] Further research into the pharmacological effects of notoginseng has revealed that low doses of notoginseng have a protective effect against neurogenic cell damage caused by chronic hypoxia, suggesting its protective effect against chronic neurological diseases. This further highlights the importance of notoginseng as a pharmaceutical active ingredient and increases the demand for efficient methods to obtain notoginseng.

[0007] To fully utilize Panax notoginseng resources, a process for extracting components from its stems and leaves has been developed, which can simultaneously extract three components: total saponins, total flavonoids, and notoginsenosides. This method improves the utilization rate of Panax notoginseng stems and leaves, but it still cannot solve the problems inherent in Panax notoginseng cultivation.

[0008] In the field of biotechnology, some studies have transformed Panax notoginseng cells by Agrobacterium rhizogenes to prepare Panax notoginseng hairy roots, and used the hairy roots as explants to induce callus. This method provides a new idea for Panax notoginseng cell culture, but in-depth research on improving the content of ginsenoside has not been conducted.

[0009] In summary, the existing technology mainly relies on Panax notoginseng plants cultivated in the field to obtain ginsenoside, which has problems such as long growth cycle, continuous cropping obstacles, many pests and diseases, pesticide residues, and excessive heavy metals, which restricts the rapid scale production of medical products using Panax notoginseng as raw material. Although some studies have used chemicals or waste parts of Panax notoginseng to improve the efficiency of ginsenoside, these methods still cannot fundamentally solve the problems faced by Panax notoginseng cultivation. Therefore, developing a new method that does not rely on field cultivation and can efficiently improve the content of ginsenoside is of great significance for overcoming the limitations of the existing technology and promoting the development of the ginsenoside industry. SUMMARY

[0010] In order to overcome the problems in the background art, the present application provides a method for improving the content of ginsenoside in Panax notoginseng cells by applying nitrogen source, which can overcome the technical difficulties of pesticide residues, heavy metal pollution and long cycle, and simply and efficiently improve the content of ginsenoside in Panax notoginseng cells, which has important guiding significance for large-scale production of ginsenoside.

[0011] To achieve the above-mentioned purpose, the present application is realized by the following technical solutions: The present application provides a method for improving the content of ginsenoside in Panax notoginseng cells by applying nitrogen source, which comprises the following steps: (1) Taking Panax notoginseng diploid cells in good growth condition, inoculating them in WPM medium added with nitrogen, and placing them in dark culture at 25℃ for 5-15d; (2) Taking the Panax notoginseng callus cultured in step (1) and placing it in a mortar, adding liquid nitrogen for grinding, and extracting ginsenoside; (3) Preparing a standard curve of ginsenoside, and determining the content of ginsenoside by HPLC method.

[0012] In the above technical solution, the nitrogen source in step (1) includes calcium nitrate tetrahydrate and ammonium nitrate.

[0013] In the above technical solution, the concentration of calcium nitrate tetrahydrate is 55.6g / L, and the concentration of ammonium nitrate is 40g / L-120g / L.

[0014] In the above technical solution, the WPM medium is composed of 30g / L sucrose, 2mg / L 2,4-D, 0.5g / L activated carbon and 3g / L plant gel, and the pH is 5.8.

[0015] In the technical scheme, the extracting panaxin in step (2) comprises the following steps: weighing the ground panax notoginseng material powder, putting the powder in a centrifugal tube, and adding ultrapure water according to a material-liquid ratio of 1:20; ultrasonic extraction is performed for 30 min in an ultrasonic instrument with an ultrasonic power of 90 W and a frequency of 45 kHz, 1 time of extraction is performed, 10 min of centrifugation is performed at 5000 rpm, the supernatant is filtered through a 0.22 mu m microporous filter membrane, and a panaxin sample extraction solution is obtained.

[0016] Compared with the prior art, the beneficial effects of the present application are that: 1. The present application provides a simple and efficient new method for improving the content of panaxin, which can significantly affect the accumulation of panaxin in panax notoginseng cells by regulating the concentration of nitrogen source. The experimental results show that culturing panax notoginseng embryonic cells in a WPM medium with an appropriate amount of nitrogen source can effectively improve the yield of panaxin.

[0017] 2. The present application overcomes the technical difficulties of pesticide residues, heavy metal pollution and long growth cycle in traditional field cultivation of panax notoginseng. The cell culture technology is used under sterile conditions, which avoids the problems of pesticide use and heavy metal pollution, and the culture period is short, so that rapid production can be realized.

[0018] 3. The present application provides a new direction and important guiding significance for large-scale production of panaxin. Through the combination of cell culture technology and nitrogen source regulation, large-scale and standardized production of panaxin can be realized, which provides a stable raw material source for the pharmaceutical industry.

[0019] 4. Compared with traditional field cultivation, the method of the present application is not limited by season, soil and climate conditions, and can be produced continuously throughout the year, with high production efficiency and controllable cost. DETAILED DESCRIPTION

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the drawings shown.

[0021] Figure 1 is a panaxin standard chromatogram, peak 1: panaxin; Figure 2 is a panaxin sample chromatogram, peak 1: panaxin; Figure 3 is a panaxin standard curve; Figure 4 is a panaxin content change graph at different nitrogen levels according to the present application. DETAILED DESCRIPTION

[0022] In order to make the objects, technical solutions and advantages of the present application clearer, the following further describes the embodiments of the present application with reference to the accompanying drawings.

[0023] The following description of the embodiments refers to the accompanying drawings. The same numbers in different drawings identify the same or similar elements. The following description of the exemplary embodiments is not representative of all possible embodiments consistent with the present application. Rather, they are merely examples of devices and methods consistent with some aspects of the present application as detailed in the appended claims.

[0024] In the description of the present application, it should be understood that the terms "first", "second" and the like are used only for descriptive purposes, and cannot be construed as indicating or implying relative importance. For those of ordinary skill in the art, the specific meanings of the above terms in the present application can be understood in specific cases. In addition, in the description of the present application, unless otherwise specified, "a plurality of" means two or more. The "and / or" describes the relationship between the associated objects, which means that there can be three relationships, for example, A and / or B, which means that there are three cases of A alone, A and B together, and B alone. The character " / " generally represents an "or" relationship between the associated objects before and after it.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terms used in the specification of the present application are only for the purpose of describing the specific embodiments and are not intended to limit the present application. The term "and / or" used herein includes any and all combinations of one or more associated listed items.

[0026] Embodiment 1 The present application provides a method for improving the content of ginsenoside in Panax notoginseng cells by applying nitrogen source, which comprises the following steps: (1) Take Panax notoginseng diploid cells in good growth condition, inoculate them in WPM medium added with nitrogen, and place them in dark culture at 25℃ for 5d; Specifically, Panax notoginseng diploid cells in good growth condition, without contamination, and with high cell viability are selected as experimental materials. In a clean bench, using sterile operation techniques, Panax notoginseng diploid cells are inoculated into WPM medium added with nitrogen which has been prepared. When inoculating, about 0.5g of fresh and heavy Panax notoginseng diploid cells are selected and evenly distributed on the surface of the culture medium. After inoculation, the culture dish is sealed and placed in a 25℃ constant temperature incubator for dark culture. The dark culture condition refers to a culture environment completely avoiding light, which is conducive to the growth of Panax notoginseng cells and the accumulation of ginsenoside. The culture time is 5d, during which Panax notoginseng cells absorb nitrogen and other nutrients in the culture medium, promoting cell growth and synthesis of secondary metabolites.

[0027] In this embodiment, the nitrogen source includes calcium nitrate tetrahydrate and ammonium nitrate. The concentration of calcium nitrate tetrahydrate is 55.6 g / L, and the concentration of ammonium nitrate is 40 g / L. Specifically, when preparing the WPM medium, calcium nitrate tetrahydrate and ammonium nitrate are added as the nitrogen source according to the above concentrations. Calcium nitrate tetrahydrate provides calcium ions and nitrate ions, and ammonium nitrate provides ammonium ions and nitrate ions, which play an important role in the growth of Panax notoginseng cells and the synthesis of ginsenosides.

[0028] The WPM medium is composed of 30 g / L sucrose, 2 mg / L 2,4-D, 0.5 g / L activated carbon, and 3 g / L plant gel, and the pH is 5.8. Specifically, when preparing the WPM medium, first weigh each component, dissolve it in a suitable amount of deionized water, then adjust the pH to 5.8, and finally dilute to the desired volume. After preparation, the medium is divided into culture dishes and sterilized at 121°C for 20 minutes before use.

[0029] (2) Take the Panax notoginseng callus cultured in step (1) and grind it in a mortar with liquid nitrogen to extract ginsenosides; Specifically, after the culture is completed, the Panax notoginseng callus in the culture dish is taken out, the residual medium on the surface is washed with sterile water, and then the surface moisture is absorbed with filter paper. Weigh 0.5 g of material and place it in a mortar, then add an appropriate amount of liquid nitrogen to freeze the material quickly. The low temperature of liquid nitrogen can effectively inhibit the activity of enzymes and prevent the degradation of ginsenosides. After the liquid nitrogen evaporates, immediately grind the frozen tissue into fine powder with a pestle. During the grinding process, liquid nitrogen can be added multiple times as needed to ensure that the sample is always in a low-temperature state until the tissue is completely ground into fine powder.

[0030] Weigh 0.3 g of ground Panax notoginseng material powder and place it in a centrifuge tube. Add 6 mL of ultrapure water at a solid-liquid ratio of 1:20 (g / mL). Ultrasonic treatment is performed in an ultrasonic instrument with a power of 90 W and a frequency of 45 kHz for 30 minutes. Extract once, centrifuge at 5000 rpm for 10 minutes, filter the supernatant through a 0.22 μm microporous filter membrane, and obtain the ginsenoside sample extract.

[0031] (3) Prepare a ginsenoside standard curve and determine the ginsenoside content by HPLC method.

[0032] First, prepare an appropriate amount of ginsenoside standard powder. Accurately weigh a certain amount of ginsenoside standard and add ultrapure water to prepare a 0.1 mg / mL standard solution, which is stored at 4°C. Set the sequence injection volume to 2, 4, 6, 8, 10, and 12 μL. Determine the peak area value of the ginsenoside standard under the chromatographic conditions for ginsenoside determination. Plot the standard curve with sample amount (x) as the horizontal coordinate and peak area value (y) as the vertical coordinate.

[0033] The standard working solution was analyzed using a high performance liquid chromatograph (HPLC), and the peak areas of different concentrations of standard samples were recorded. The chromatographic conditions were as follows: the chromatographic column type was Hypersil GOLD (250 mm x 4.6 mm, 5 μm), the mobile phase was methanol (A)-0.03% trifluoroacetic acid (B) (85:15), isocratic elution for 15 min, the flow rate was 0.4 mL / min, the detection wavelength was 220 nm, the column temperature was 25°C, and the injection volume was 5 μL. According to the standard sample concentration and the corresponding peak area, a standard curve was drawn, and linear regression analysis was performed to obtain the regression equation and the correlation coefficient.

[0034] Then, the panaxin sample extract obtained in step (2) was subjected to HPLC analysis under the same chromatographic conditions as the standard sample, and the peak area of panaxin in the sample was recorded. According to the standard curve regression equation, the concentration of panaxin in the sample was calculated, and combined with the extraction volume and sample weight, the content of panaxin in the panax ginseng cells (mg / g) was calculated. Through the above method, the content of panaxin in panax ginseng cells can be accurately determined, and the effect of different nitrogen source treatments on the synthesis of panaxin can be evaluated.

[0035] Example 2 The present application provides a method for increasing the content of panaxin in panax ginseng cells by applying nitrogen source, which comprises the following steps: (1) Take well-grown panax ginseng diploid cells, inoculate them in WPM medium added with nitrogen, and place them in a 25°C dark culture for 10 days; Specifically, well-grown, uncontaminated, and high-viability panax ginseng diploid cells were selected as experimental materials. In a clean bench, sterile operation techniques were used to inoculate the panax ginseng diploid cells into the WPM medium prepared and added with nitrogen. The WPM medium was composed of 30 g / L sucrose, 2 mg / L 2,4-D, 0.5 g / L activated carbon, and 3 g / L plant gel, and the pH was 5.8. When preparing the WPM medium, first, weigh each component, dissolve it in a suitable amount of deionized water, then adjust the pH to 5.8, and finally make up to the required volume. After preparation, the medium was divided into culture dishes, sterilized by high-pressure sterilization at 121°C for 20 minutes, and then used. The nitrogen source included calcium nitrate tetrahydrate and ammonium nitrate, the concentration of calcium nitrate tetrahydrate was 55.6 g / L, and the concentration of ammonium nitrate was 80 g / L. When inoculating, about 0.5 g of fresh and heavy panax ginseng diploid cells were selected and evenly distributed on the surface of the medium. After inoculation, the culture dishes were sealed and placed in a 25°C constant temperature incubator for dark culture. The dark culture condition refers to a completely light-free culture environment, which is conducive to the growth of panax ginseng cells and the accumulation of panaxin. The culture time was 10 days, during which the panax ginseng cells absorbed the nitrogen and other nutrients in the medium, promoting cell growth and the synthesis of secondary metabolites.

[0036] (2) Take the three-leaf ginseng callus after step (1) and place it in a mortar, add liquid nitrogen and grind, extract panaxin; Specifically, after the culture is completed, the three-leaf ginseng callus in the culture dish is taken out, the residual medium on the surface is washed with sterile water, and then the surface moisture is absorbed with filter paper. 0.5 g of material is weighed and placed in a mortar, and an appropriate amount of liquid nitrogen is added to freeze the material quickly. The low temperature of liquid nitrogen can effectively inhibit the activity of enzymes and prevent panaxin from being degraded. After the liquid nitrogen evaporates, the frozen tissue is immediately ground into fine powder with a pestle. During the grinding process, liquid nitrogen can be added multiple times as needed to ensure that the sample is always in a low temperature state until the tissue is completely ground into fine powder.

[0037] Weigh 0.3 g of ground three-leaf ginseng material powder and place it in a centrifuge tube. Add ultrapure water at a material to liquid ratio of 1:20 (g / mL) and 6 mL of ultrapure water. Ultrasonic at a power of 90 W and a frequency of 45 kHz for 30 min, extract 1 time, centrifuge at 5000 rpm for 10 min, take the supernatant and filter through a 0.22 μm microporous filter membrane to obtain the panaxin sample extract.

[0038] (3) Prepare a standard curve of panaxin and determine the content of panaxin by HPLC method.

[0039] First, prepare an appropriate amount of panaxin standard powder, accurately weigh a certain amount of panaxin standard, add ultrapure water, and prepare a 0.1 mg / mL standard solution, which is stored at 4°C. Set the sequence injection volume to 2, 4, 6, 8, 10, and 12 μL, and determine the peak area value of the panaxin standard according to the chromatographic conditions for determining panaxin. Draw a standard curve with sample amount (x) as the abscissa and peak area value (y) as the ordinate.

[0040] Use a high-performance liquid chromatograph (HPLC) to analyze the standard working solution and record the peak areas of the standard samples of different concentrations. The chromatographic conditions are as follows: the chromatographic column type is Hypersil GOLD (250 mm x 4.6 mm, 5 μm), the mobile phase is methanol (A) - 0.03% trifluoroacetic acid (B) (85:15), isocratic elution for 15 min, the flow rate is 0.4 mL / min, the detection wavelength is 220 nm, the column temperature is 25°C, and the injection volume is 5 μL. According to the standard concentration and the corresponding peak area, a standard curve is drawn and linear regression analysis is performed to obtain the regression equation and the correlation coefficient.

[0041] Then, the sample extract of panaxin obtained in step (2) is subjected to HPLC analysis under the same chromatographic conditions as the standard sample, and the peak area of panaxin in the sample is recorded. According to the standard curve regression equation, the concentration of panaxin in the sample is calculated, and combined with the extraction volume and sample weight, the content of panaxin in the panax ginseng cells (mg / g) is calculated. Through the above method, the content of panaxin in panax ginseng cells can be accurately determined, and the effect of different nitrogen source treatments on the synthesis of panaxin can be evaluated.

[0042] Example 3 The embodiment of the present application proposes a method for increasing the content of panaxin in panax ginseng cells by applying nitrogen source, which comprises the following steps: (1) Take well-grown panax ginseng diploid cells, inoculate them in WPM medium added with nitrogen, and place them in a 25°C dark culture for 15 days; Specifically, well-grown, uncontaminated, and high-viability panax ginseng diploid cells are selected as experimental materials. In a clean bench, using sterile operation techniques, the panax ginseng diploid cells are inoculated into the WPM medium added with nitrogen which has been prepared. The WPM medium is composed of 30 g / L sucrose, 2 mg / L 2,4-D, 0.5 g / L activated carbon, and 3 g / L plant gel, and the pH is 5.8. When preparing the WPM medium, first weigh each component, dissolve it in a suitable amount of deionized water, then adjust the pH to 5.8, and finally make up to the required volume. After preparation, the medium is divided into culture dishes, sterilized by 121°C high-pressure sterilization for 20 minutes, and then used. The nitrogen includes calcium nitrate tetrahydrate and ammonium nitrate, the concentration of calcium nitrate tetrahydrate is 55.6 g / L, and the concentration of ammonium nitrate is 120 g / L. When inoculating, about 0.5 g of fresh and heavy panax ginseng diploid cells are selected and evenly distributed on the surface of the medium. After inoculation, the culture dish is sealed and placed in a 25°C constant temperature incubator for dark culture. The dark culture condition refers to a completely light-free culture environment, which is conducive to the growth of panax ginseng cells and the accumulation of panaxin. The culture time is 15 days, during which the panax ginseng cells absorb the nitrogen and other nutrients in the medium, promoting cell growth and the synthesis of secondary metabolites.

[0043] (2) Take the panax ginseng callus cultured in step (1) and place it in a mortar, add liquid nitrogen to grind, and extract panaxin; Specifically, after the culture is completed, the panax ginseng callus in the culture dish is taken out, the residual medium on the surface is washed with sterile water, and then the surface moisture is absorbed with filter paper. 0.5 g of material is weighed and placed in a mortar, and an appropriate amount of liquid nitrogen is added to freeze the material quickly. The low temperature of liquid nitrogen can effectively inhibit the activity of enzymes and prevent the degradation of panaxin. After the liquid nitrogen evaporates, the frozen tissue is immediately ground into fine powder with a pestle. During the grinding process, liquid nitrogen can be added multiple times as needed to ensure that the sample is always in a low-temperature state until the tissue is completely ground into fine powder.

[0044] Weigh 0.3 g of ground Panax notoginseng material powder into a centrifuge tube, add ultrapure water at a material to liquid ratio of 1:20 (g / mL), and add 6 mL of ultrapure water. Ultrasonic extraction is performed in an ultrasonic instrument with an ultrasonic power of 90 W and a frequency of 45 kHz for 30 min, 1 time, 5000 rpm centrifugation for 10 min, and the supernatant is filtered through a 0.22 μm microporous filter to obtain a Panax notoginseng saponin sample extract.

[0045] (3) Prepare a Panax notoginseng saponin standard curve and determine the Panax notoginseng saponin content by HPLC.

[0046] First, prepare an appropriate amount of Panax notoginseng saponin standard powder, accurately weigh a certain amount of Panax notoginseng saponin standard, add ultrapure water, and prepare a 0.1 mg / mL standard solution, which is stored at 4°C. Set the sequence injection volume to 2, 4, 6, 8, 10, and 12 μL, and determine the peak area value of the Panax notoginseng saponin standard under the chromatographic conditions for Panax notoginseng saponin determination. Draw a standard curve with the sample amount (x) as the abscissa and the peak area value (y) as the ordinate.

[0047] Use a high-performance liquid chromatograph (HPLC) to analyze the standard working solution and record the peak areas of the standard samples of different concentrations. The chromatographic conditions are as follows: the chromatographic column model is Hypersil GOLD (250 mm x 4.6 mm, 5 μm), the mobile phase is methanol (A) - 0.03% trifluoroacetic acid (B) (85:15), isocratic elution for 15 min, the flow rate is 0.4 mL / min, the detection wavelength is 220 nm, the column temperature is 25°C, and the injection volume is 5 μL. According to the standard sample concentration and the corresponding peak area, a standard curve is drawn, and linear regression analysis is performed to obtain the regression equation and the correlation coefficient.

[0048] Then, the Panax notoginseng saponin sample extract obtained in step (2) is subjected to HPLC analysis under the same chromatographic conditions as the standard sample, and the peak area of Panax notoginseng saponin in the sample is recorded. According to the standard curve regression equation, the concentration of Panax notoginseng saponin in the sample is calculated, and combined with the extraction volume and sample weight, the content of Panax notoginseng saponin in the Panax notoginseng cells (mg / g) is calculated. Through the above method, the content of Panax notoginseng saponin in Panax notoginseng cells can be accurately determined, and the effect of different nitrogen sources on Panax notoginseng saponin synthesis can be evaluated.

[0049] As can be seen from Figure 4 , the content of Panax notoginseng saponin in Panax notoginseng cells is significantly improved after the addition of nitrogen sources, and this method overcomes the technical difficulties of pesticide residue, heavy metal pollution, and long cycle time, providing a new direction for improving the content of Panax notoginseng saponin.

[0050] Finally, it should be noted that the above preferred embodiments are merely intended to illustrate the technical solutions of the present application, not to limit the present application. Even though the present application has been described in detail by the above preferred embodiments, those skilled in the art should understand that they can make various modifications in form and details without departing from the scope of the present application defined by the claims.

Claims

1. A method for increasing the content of ginsenoside in Panax notoginseng cells by applying a nitrogen source, characterized in that: The method for improving the content of ginsenoside in Panax notoginseng cells by applying nitrogen source comprises the following steps: ​ (1) taking Panax notoginseng diploid cells in good growth condition, inoculating them into WPM culture medium added with nitrogen, and placing them in dark culture at 25℃ for 5-15 days; (2) taking the Panax notoginseng callus after culture in step (1), grinding them in a mortar with the addition of liquid nitrogen, and extracting ginsenoside; (3) preparing a ginsenoside standard curve, and determining the content of ginsenoside by HPLC method.

2. The method for increasing the content of ginsenoside in Panax notoginseng cells by applying nitrogen source according to claim 1, characterized in that: The nitrogen in step (1) comprises calcium nitrate tetrahydrate and ammonium nitrate.

3. The method for increasing the content of ginsenoside in Panax notoginseng cells by applying nitrogen source according to claim 2, characterized in that: The concentration of the calcium nitrate tetrahydrate is 55.6 g / L, and the concentration of the ammonium nitrate is 40 g / L-120 g / L.

4. The method for increasing the content of ginsenoside in Panax notoginseng cells by applying nitrogen source according to claim 1, characterized in that: The WPM culture medium comprises 30 g / L sucrose, 2 mg / L 2,4-D, 0.5 g / L activated carbon and 3 g / L plant gel, and has a pH of 5.

8.

5. The method for increasing the content of ginsenoside in Panax notoginseng cells by applying nitrogen source according to claim 1, characterized in that: The extraction of ginsenoside in step (2) specifically comprises the following steps: weighing the ground Panax notoginseng material powder, placing it in a centrifuge tube, adding ultrapure water at a material-liquid ratio of 1:20, ultrasonically treating it in an ultrasonic instrument with an ultrasonic power of 90 W and a frequency of 45 kHz for 30 min, extracting it once, centrifuging it at 5000 rpm for 10 min, filtering the supernatant through a 0.22 μm microporous filter, and obtaining a ginsenoside sample extract.

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