Method for promoting ginsenoside accumulation in ginseng cambium stem cells

Abstract

The invention belongs to the technical field of biotechnology and plant tissue culture, and particularly relates to a method for promoting ginsenoside accumulation in ginseng cambium stem cells. According to the method for promoting ginsenoside accumulation in the ginseng cambium stem cells, efficient induction and accumulation of ginsenoside in the ginseng cambium stem cells are promoted by optimizing culture conditions and induction strategies. The ginseng cambium stem cells obtained by the method have high ginsenoside content, and especially saponin components such as Ro, Re, Rg2, Rh1 and the like are remarkably improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biotechnology and plant tissue culture, and particularly relates to a method for promoting accumulation of ginsenosides in ginseng cambium stem cells. BACKGROUND

[0002] Panax ginseng C.A. Mey. is a perennial herb of the Araliaceae family, and its rhizome is a precious Chinese medicinal material known as "King of Herbs". Panax ginseng C.A. Mey. is a perennial herb that prefers cool and humid climates and grows in needle-broadleaf mixed forests or shrub forests on gentle or sloping slopes at an altitude of 500-1100 meters, and is a precious medicinal material. Research shows that Panax ginseng C.A. Mey. can tonify primordial qi, restore pulse and prevent collapse, tonify the spleen and lung, produce fluid and calm the mind, and can effectively treat clinical conditions such as physical weakness, collapse, spleen deficiency, poor appetite, palpitation, insomnia, heart failure, and is known as "King of Herbs".

[0003] According to literature reports, Panax ginseng C.A. Mey. contains mainly sugars, saponins, volatile components, and organic acids and their esters, enzymes, proteins, sterols and their glycosides, nitrogen-containing compounds, polypeptides, flavonoids, lignin, inorganic elements, and vitamins. Among them, ginsenosides are the main active components of Panax ginseng C.A. Mey., and have the effects of anti-fatigue, delaying aging, regulating the central nervous system, improving the body's immunity, improving the blood supply of heart and brain, inhibiting the growth of tumor cells, etc. So far, more than 40 saponin components have been isolated and identified from Panax ginseng C.A. Mey. The physiological activity of ginsenosides determines their edible and medicinal value.

[0004] At present, due to overexploitation, environmental destruction, etc., the resources of wild Panax ginseng C.A. Mey. have almost been exhausted, and field cultivation is the main source of Panax ginseng C.A. Mey. However, due to the slow growth of Panax ginseng C.A. Mey., long planting period, and strict requirements for environmental conditions, its quality is easily affected by climate, cultivation conditions, and pests and diseases; especially the complex cultivation technology, excessive pesticide residues, and old Panax ginseng C.A. Mey. land greatly limit the development prospects of artificially cultivated Panax ginseng C.A. Mey. At present, the supply of field-cultivated Panax ginseng C.A. Mey. has been difficult to meet the market demand.

[0005] The ginseng cambium stem cell refers to a cell line with the potential of unlimited self-renewal and differentiation into any type of cells induced from a ginseng explant (such as roots, stems, and leaves) under specific conditions in a laboratory. In a suitable culture medium, these cells can continuously divide and proliferate, and theoretically can be permanently preserved and expanded, which can provide a stable and unlimited biomass source. The culture system of the ginseng cambium stem cell greatly reduces the dependence on wild ginseng and farmland planting ginseng, and is beneficial to species protection and ecological environment. However, the ginseng saponin content of the ginseng cambium stem cell cultured by the traditional method is generally not ideal. Therefore, it is expected in the field to develop a method for effectively promoting the accumulation of ginseng saponins in ginseng cambium stem cells, especially a method for increasing the content of rare ginseng saponins, which has important scientific research value and application prospect. SUMMARY

[0006] A first object of the present application is to provide a method for promoting the accumulation of ginseng saponins in ginseng cambium stem cells by optimizing the culture conditions and induction strategy, so as to solve the problems of low ginseng saponin content, especially insufficient rare ginseng saponins in ginseng cambium stem cells; A second object of the present application is to provide a ginseng cambium stem cell and its use.

[0007] In order to solve the above technical problems, the present application provides a method for promoting the accumulation of ginseng saponins in ginseng cambium stem cells, comprising the following steps: (1) inoculating a ginseng explant into a first induction culture medium, and obtaining ginseng primary callus by first induction culture; (2) inoculating the ginseng primary callus into a second induction culture medium, and obtaining ginseng subculture callus by second induction culture; (3) inoculating the ginseng subculture callus into a third induction culture medium for third induction culture, thereby obtaining the desired ginseng cambium stem cell; wherein, The third induction culture medium comprises 50-200 μM jasmonic acid and / or methyl jasmonate and 50-100 μM aesculin.

[0008] Specifically, in the method for promoting the accumulation of ginseng saponins in ginseng cambium stem cells, the first induction culture medium in step (1) comprises MS basic medium and is added with 0.5-2.0 mg / L 6-benzylaminopurine and 0.1-1.0 mg / L naphthaleneacetic acid.

[0009] Specifically, in the method for promoting the accumulation of ginseng saponins in ginseng cambium stem cells, the first induction culture step in step (1) is dark culture, the culture temperature is 22-28°C, and the culture time is 25-30 days.

[0010] Specifically, in the method for promoting accumulation of ginsenosides in ginseng cambium stem cells, the second induction medium in step (2) comprises a MS basic medium.

[0011] Specifically, in the method for promoting accumulation of ginsenosides in ginseng cambium stem cells, the second induction culture step in step (2) is dark culture, the culture temperature is 20-25 DEG C, and the culture time is 40-45 days.

[0012] Specifically, in the method for promoting accumulation of ginsenosides in ginseng cambium stem cells, the third induction medium in step (3) comprises: 1 / 2 MS basic medium 1.5-2 g / L, WPM basic medium 0.5-1 g / L, methyl jasmonate 50-200 μM, and aesculin 50-100 μM.

[0013] Specifically, in the method for promoting accumulation of ginsenosides in ginseng cambium stem cells, the third induction culture step in step (3) is dark culture, the culture temperature is 20-25 DEG C, the ventilation amount is 0.05-0.15 vvm, and the culture time is 28-32 days.

[0014] Specifically, in the method for promoting accumulation of ginsenosides in ginseng cambium stem cells, the third induction culture step in step (3) is carried out in a bubble-type bioreactor.

[0015] Specifically, in the method for promoting accumulation of ginsenosides in ginseng cambium stem cells, the methyl jasmonate and aesculin are added in the third induction culture for 20-25 days.

[0016] The application further provides a ginseng cambium stem cell cultured by the method.

[0017] The application further provides a use of the ginseng cambium stem cell for preparing an anti-emetic toxin drug.

[0018] The method for promoting accumulation of ginsenosides in ginseng cambium stem cells promotes high-efficiency induction and accumulation of ginsenosides in ginseng cambium stem cells by optimizing culture conditions and induction strategies, promotes generation of primary callus in ginseng explants by optimizing plant hormone ratio and using a first induction medium containing 6-benzylaminopurine and naphthaleneacetic acid, obtains subculture callus by long-term dark culture of the second induction medium without hormones, and further obtains cambium stem cells by high-efficiency induction of the third induction medium. The ginseng cambium stem cells obtained by the method have high ginsenoside content, and especially, the contents of saponin components such as Ro, Re, Rg2 and Rh1 are significantly increased.

[0019] The method for promoting accumulation of ginsenosides in ginseng cambium stem cells in the application can effectively stimulate the expression of key enzyme genes of ginsenoside synthesis pathway and improve the yield of ginsenosides by adding jasmonic acid / jasmonic acid methyl ester and aesculin as inducers in the active ingredient induction culture and by adding the inducers at a proper stage of culture.

[0020] The method for promoting accumulation of ginsenosides in ginseng cambium stem cells in the application uses a bubbling bioreactor based on the optimization of a bioreactor culture system, the aeration amount is 0.05-0.15 vvm, and a plurality of air inlet devices are arranged at the bottom of the tank body to form uniform airflow stirring and promote uniform distribution of nutrients and cell growth.

[0021] The method for promoting accumulation of ginsenosides in ginseng cambium stem cells in the application can also be based on enzyme conversion of rare saponins, i.e., after the culture is completed, β-glycosidase is used to enzymatically hydrolyze the obtained ginsenosides to convert common saponins into rare ginsenosides such as Rh2, Rg3 and CK.

[0022] The method for promoting accumulation of ginsenosides in ginseng cambium stem cells in the application significantly improves the total ginsenoside content of the ginseng cambium stem cells obtained by culture, and the content of rare saponins such as Ro, Re, Rg2 and Rh1 is also significantly improved.

[0023] The method for promoting accumulation of ginsenosides in ginseng cambium stem cells in the application is stable and has good repeatability, is suitable for industrial production, provides reliable technical support for the industrial production of ginsenosides, and solves the problems of lack of wild ginseng resources and unstable saponin content. DETAILED DESCRIPTION

[0024] Example 1 Healthy ginseng root segments are used as explants, and after conventional disinfection treatment, they are inoculated into MS basic medium containing 1.0 mg / L 6-benzylaminopurine and 0.5 mg / L naphthaleneacetic acid, and are subjected to conventional culture at 25℃ in the dark for 4 weeks to induce the generation of ginseng primary callus.

[0025] From the obtained primary callus, cell masses with embryonic characteristics (light yellow, loose and granular) are selected. The selected primary callus is transferred to MS basic medium without plant hormones and is continuously subjected to dark culture at 23℃ for 6 weeks to obtain ginseng subculture callus.

[0026] The obtained subculture callus was inoculated into liquid medium containing 1.8 g / L 1 / 2MS basal medium and 0.8 g / L WPM basal medium, and cultured in a bioreactor with aeration amount of 0.1 vvm, and dark culture was continuously carried out at 23°C. When cultured for 25 days, 100 μM methyl jasmonate and 80 μM aesculin were added to the liquid medium to a final concentration, and culture was continuously carried out for 5 days under the above conditions, and ginseng meristem stem cells were harvested.

[0027] Example 2 Healthy ginseng root segments were used as explants, and after routine disinfection treatment, they were inoculated into MS basal medium containing 0.5 mg / L 6-benzylaminopurine and 1.0 mg / L naphthalene acetic acid, and routine culture was carried out at 22°C in dark culture conditions for 4 weeks to induce ginseng primary callus.

[0028] From the obtained primary callus, cell masses with embryonic characteristics (light yellow, loose, granular) were selected. The selected primary callus was transferred to MS basal medium without plant hormones, and dark culture was continuously carried out at 20°C for 6 weeks to obtain ginseng subculture callus.

[0029] The obtained subculture callus was inoculated into liquid medium containing 1.5 g / L 1 / 2MS basal medium and 1 g / L WPM basal medium, and cultured in a bioreactor with aeration amount of 0.05 vvm, and dark culture was continuously carried out at 20°C. When cultured for 25 days, 50 μM methyl jasmonate and 100 μM aesculin were added to the liquid medium to a final concentration, and culture was continuously carried out for 5 days under the above conditions, and ginseng meristem stem cells were harvested.

[0030] Example 3 Healthy ginseng root segments were used as explants, and after routine disinfection treatment, they were inoculated into MS basal medium containing 2.0 mg / L 6-benzylaminopurine and 0.1 mg / L naphthalene acetic acid, and routine culture was carried out at 28°C in dark culture conditions for 4 weeks to induce ginseng primary callus.

[0031] From the obtained primary callus, cell masses with embryonic characteristics (light yellow, loose, granular) were selected. The selected primary callus was transferred to MS basal medium without plant hormones, and dark culture was continuously carried out at 25°C for 6 weeks to obtain ginseng subculture callus.

[0032] The obtained subculture callus was inoculated into liquid medium containing 2.0 g / L 1 / 2MS basal medium and 0.5 g / L WPM basal medium, and cultured in a bioreactor with aeration amount of 0.15 vvm at 25°C in the dark. When cultured to the 25th day, 200 μM methyl jasmonate and 50 μM aesculin were added to the liquid medium to a final concentration, and the culture was continued under the above conditions for 5 days, and the ginseng meristem stem cells were harvested.

[0033] Comparative Example 1 The method for promoting the accumulation of ginsenosides in ginseng meristem stem cells in the present comparative example is the same as that in Example 1, except that the methyl jasmonate and aesculin are not added to the third medium.

[0034] Comparative Example 2 The method for promoting the accumulation of ginsenosides in ginseng meristem stem cells in the present comparative example is the same as that in Example 1, except that the methyl jasmonate is not added to the third medium, and only aesculin is added to a final concentration of 180 μM.

[0035] Comparative Example 3 The method for promoting the accumulation of ginsenosides in ginseng meristem stem cells in the present comparative example is the same as that in Example 1, except that the aesculin is not added to the third medium, and only methyl jasmonate is added to a final concentration of 180 μM.

[0036] Experimental Example 1. Ginsenoside content In the present experimental example, the content of ginsenosides in the ginseng meristem stem cells obtained by the methods in Example 1 and Comparative Examples 1-3 was detected according to the method described in Chinese Patent CN118360233A (see paragraphs 【0065-0072】), including the contents of 9 common ginsenosides Rb1 (Ginsenoside Rb1), Rb2 (Ginsenoside Rb2), Rc (Ginsenoside Rc), Rd (Ginsenoside Rd), Re (Ginsenoside Re), Rf (Ginsenoside Rf), Rg1 (Ginsenoside Rg1), Rg2 (Ginsenoside Rg2), and Ro (Ginsenoside Ro), and the contents of 4 rare ginsenosides Rg3 (Ginsenoside Rg3), Rh1 (Ginsenoside Rh1), F1 (Ginsenoside F1), and F2 (Ginsenoside F2), and the results are shown in Table 1 below.

[0037]

[0038] It can be seen that, in the method for promoting accumulation of ginsenosides in ginseng cambium stem cells, methyl jasmonate and aesculin are added as inducers in the active ingredient induction culture, and the key enzyme gene expression of the ginsenoside synthesis pathway can be effectively stimulated by adding the inducers at a proper stage of the culture, and the yield of various ginsenosides is improved.

[0039] It can be seen from the data in Comparative Example 2-3 that methyl jasmonate itself has the effect of promoting accumulation of common ginsenosides in the ginseng cambium stem cells, and aesculin itself has no obvious effect of promoting common ginsenosides, but has a certain effect of promoting accumulation of rare ginsenosides, and can assist methyl jasmonate to play the active induction effect, and further increase the content of ginsenosides.

[0040] 2, Enzymatic conversion of rare ginsenosides In this experimental example, the ginseng cambium stem cells obtained by the method in the foregoing Example 1 are extracted by a conventional method to obtain total ginsenoside extract.

[0041] In this experimental example, the ginseng cambium stem cells are subjected to enzymatic hydrolysis by using immobilized β-glycosidase BglPm and Bgp1, the reaction pH is 5.0, and the temperature is 45°C.

[0042] After the enzymolysis is completed, the content of rare ginsenosides is determined. The results show that various rare ginsenosides such as Rh2 and Rg3 can be obtained.

[0043] 3, Application of ginseng cambium stem cells Vomitoxin is one of the mycotoxins with the highest incidence and the strongest toxicity in the world. Vomitoxin has strong toxicity to both humans and animals, and can cause vomiting, diarrhea, skin irritation, refusal to eat, nervous disorders, miscarriage, stillbirth, and the like in humans and animals. Vomitoxin is an immunosuppressant, which can inhibit the synthesis of protein and DNA, RNA and the function of mitochondria, and prevent cell division and membrane function. Vomitoxin in feed will affect the palatability of the feed, and also cause oral ulcer, intestinal flora disorder and reduction of feed conversion rate of livestock and poultry, which seriously affects the economic benefits of the breeding industry.

[0044] In this experimental example, based on the ginseng cambium stem cells prepared in the foregoing Example 1, the effect and result of inducing cells to resist vomitoxin are verified.

[0045] In this example, the Hela cells are pretreated with different concentrations of ginseng cambium stem cells for 24 h, and then treated with different concentrations of vomitoxin, and the IC50 of the cells in the blank control DMSO group and the ginseng extract pretreatment group is compared to determine the cytotoxic effect of the active substance on resisting vomitoxin.

[0046] CCK-8 method for detecting cell viability: Hela cells were pretreated with ginseng formation layer stem cells at concentrations of 0, 25, 50, 100 nM for 24 h. Subcultured into 96-well plates, and after the cells adhered, 50, 25, of emetic toxin were added to the serum-free DMEM medium, 3 replicate wells were set for each concentration (n=3). After 24 h of emetic toxin treatment, 10 μL of CCK-8 reagent was added to each well, and the reaction was carried out at 37℃ for 2 h. The absorbance was measured at 450 nm wavelength using an enzyme-labeled instrument.

[0047] According to the formula: cell survival rate=[1-(experimental hole-control hole) / (control hole-blank hole)]x100%, the cell survival rate of each concentration experimental hole was calculated, and the results are shown in Table 2 below.

[0048]

[0049] It can be seen that the ginseng formation layer stem cells obtained by the present application have anti-emetic toxin effect, although the anti-emetic toxin effect is slightly different from that of conventional chemical drugs, but subsequent optimization can be considered by increasing the concentration or further purifying the active ingredients. It can be seen that the ginseng formation layer stem cells can be used for the development of anti-emetic toxin drugs, and have good specificity to emetic toxin, and can be used for the development and research of anti-emetic toxin drugs, and have wide application prospect.

[0050] The embodiments of the present application are described in detail above, and the specific examples are applied to the principles and implementation modes of the present application. The above embodiment description is only used to help understand the method of the present application and its core idea; at the same time, for those skilled in the art, according to the idea of the present application, the specific implementation mode and application range will be changed, and the above description should not be understood as a limitation of the present application.

Claims

1. A method for promoting the accumulation of ginsenosides in ginseng cambium stem cells, characterized in that, Includes the following steps: (1) Take ginseng explants and inoculate them into the first induction culture medium. After the first induction culture, ginseng primary callus tissue is obtained. (2) The ginseng primary callus was inoculated into the second induction culture medium and subjected to the second induction culture to obtain ginseng subcultured callus; (3) The ginseng subcultured callus is inoculated into a third induction culture medium for third induction culture to obtain the desired ginseng cambium stem cells; wherein, The third induction medium comprises: 50-200 μM jasmonic acid and / or methyl jasmonic acid and 50-100 μM fraxin.

2. The method for promoting the accumulation of ginsenosides in ginseng cambium stem cells according to claim 1, characterized in that, In step (1), the first induction medium includes MS basal medium and is supplemented with 0.5-2.0 mg / L 6-benzylaminopurine and 0.1-1.0 mg / L naphthaleneacetic acid.

3. The method for promoting the accumulation of ginsenosides in ginseng cambium stem cells according to claim 2, characterized in that, In step (1), the first induction culture step is dark culture, the culture temperature is 22-28℃, and the culture time is 25-30 days.

4. The method for promoting the accumulation of ginsenosides in ginseng cambium stem cells according to claim 1, characterized in that, In step (2), the second induction medium includes MS basal medium.

5. The method for promoting the accumulation of ginsenosides in ginseng cambium stem cells according to claim 4, characterized in that, In step (2), the second induction culture step is dark culture, the culture temperature is 20-25℃, and the culture time is 40-45 days.

6. The method for promoting the accumulation of ginsenosides in ginseng cambium stem cells according to any one of claims 1-5, characterized in that, In step (3), the third induction medium includes: 1 / 2 MS basal medium 1.5-2 g / L, WPM basal medium 0.5-1 g / L, methyl jasmonate 50-200 μM, and fraxin 50-100 μM.

7. The method for promoting the accumulation of ginsenosides in ginseng cambium stem cells according to claim 6, characterized in that, In step (3), the third induction culture step is dark culture, the culture temperature is 20-25℃, the aeration rate is 0.05-0.15vvm, and the culture time is 28-32 days.

8. The method for promoting the accumulation of ginsenosides in ginseng cambium stem cells according to claim 7, characterized in that, In step (3), the third induction culture step is carried out in a bubble-type bioreactor.

9. The method for promoting the accumulation of ginsenosides in ginseng cambium stem cells according to claim 8, characterized in that, In step (3), methyl jasmonate and fraxin are added after the third induction culture has been carried out for 20-25 days.

10. A ginseng cambium stem cell cultured by any one of claims 1-9.

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