Application of titanium ions in promoting accumulation of tanshinone IIA and / or salvianolic acid components of salvia miltiorrhiza

Abstract

The invention provides application of titanium ions in promoting accumulation of tanshinone IIA and / or salvianolic acid components of salvia miltiorrhiza, and belongs to the technical field of hairy root induction. The invention provides application of titanium ions in improving and / or promoting accumulation of salvia miltiorrhiza tanshinone IIA and / or salvianolic acid components. According to the method, hairy roots of salvia miltiorrhiza are induced by using different titanium ion concentrations, and the influence of titanium ions on quality components of salvia miltiorrhiza is researched. The result of the embodiment shows that 2-10 [mu] mol / L of titanium ions are added in the salvia miltiorrhiza hairy root induction process, accumulation of tanshinone IIA and salvianolic acid components in salvia miltiorrhiza hairy roots can be improved and promoted, and then the quality components of salvia miltiorrhiza can be improved. Through the influence of titanium ions on the quality components of the salvia miltiorrhiza, a theoretical basis is provided for improving the quality of the salvia miltiorrhiza and developing a titanium fertilizer.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of hairy root induction, and particularly relates to application of titanium ions in promoting accumulation of tanshinone IIA and / or salvianolic acid components in Salvia miltiorrhiza. BACKGROUND

[0002] Titanium (Ti) is a beneficial trace element that can promote normal growth of plants and improve crop yield and quality. However, titanium is usually in the form of insoluble compounds (oxides, silicate-bound state or ilmenite), which is difficult to play a role in plants. In agriculture, titanium salts chelated with citric acid, ascorbic acid and the like are often used as fertilizers. Wu et al. found that low-concentration titanium ions can promote growth of oats and increase fresh grass yield. In the cultivation of flue-cured tobacco, foliar spraying and root irrigation of titanium fertilizer can promote growth and development of flue-cured tobacco, improve yield and quality of flue-cured tobacco, and improve internal chemical components of tobacco leaves. Wheat seeds are soaked in titanium ion solutions with different concentrations, and the unit fresh weight of wheat leaves and the chlorophyll content are increased, the number of grains per spike and the thousand-grain weight are increased, and thus the yield is improved. However, the research on titanium fertilizer in the cultivation of traditional Chinese medicinal materials is relatively blank. SUMMARY

[0003] In view of the defects in the prior art, the purpose of the present application is to provide The purpose of the present application is achieved by the following technical solutions. The present application provides application of titanium ions in at least one of the following (1)~(4): (1) increasing accumulation of tanshinone IIA in Salvia miltiorrhiza; (2) increasing accumulation of salvianolic acid components in Salvia miltiorrhiza; (3) promoting accumulation of tanshinone IIA in Salvia miltiorrhiza; (4) promoting accumulation of salvianolic acid components in Salvia miltiorrhiza.

[0004] Preferably, the salvianolic acid components include any one or two or more of salvianolic acid B, rosmarinic acid, salvianolic acid Y, danshensu, shikonin and salvianolic acid A.

[0005] Preferably, the molar concentration of the titanium ions is 2~10 μmol / L.

[0006] The present application provides a method for promoting accumulation of tanshinone IIA and / or salvianolic acid components in Salvia miltiorrhiza, comprising: inducing Salvia miltiorrhiza in a culture medium containing titanium ions.

[0007] Preferably, the molar concentration of the titanium ions in the culture medium is 2~10 μmol / L.

[0008] Preferably, the molar concentration of titanium ions in the culture medium is 5 μmol / L.

[0009] Preferably, the temperature of the induction is 22-28 ℃; the time of the induction is 7-10 days; and the induction is performed in a shaker with a rotation speed of 90-120 rpm.

[0010] Preferably, the Salvia miltiorrhiza includes Salvia miltiorrhiza hairy roots; and the Salvia miltiorrhiza hairy roots are subcultured before being induced.

[0011] Preferably, the method for subculturing the Salvia miltiorrhiza hairy roots includes: The Salvia miltiorrhiza hairy roots are inoculated into MS medium for subculture.

[0012] Preferably, the temperature of the subculture is 22-28 ℃; the time of the subculture is 15-21 days; and the subculture is performed in a shaker with a rotation speed of 90-120 rpm.

[0013] Beneficial effects: The application provides application of titanium ions in improving and / or promoting accumulation of tanshinone IIA and / or salvianolic acid components in Salvia miltiorrhiza. The application studies the influence of titanium ions on quality components of Salvia miltiorrhiza by using different concentrations of titanium ions to induce Salvia miltiorrhiza hairy roots. The application shows by the results of the examples that adding 2-10 μmol / L of titanium ions in the induction process of Salvia miltiorrhiza hairy roots is beneficial to improving and promoting accumulation of the effective components tanshinone IIA and salvianolic acid components in Salvia miltiorrhiza hairy roots, and thus is beneficial to improving the quality of Salvia miltiorrhiza. The application studies the influence of titanium ions on quality components of Salvia miltiorrhiza, so as to provide a theoretical basis for improving the quality of Salvia miltiorrhiza and developing titanium fertilizer. BRIEF DESCRIPTION OF DRAWINGS

[0014] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0015] Figure 1 For different concentrations of TiC 12 H 12 O 14 The result graph of the influence of the treatment on the fresh weight of Salvia miltiorrhiza hairy roots; Figure 2 For different concentrations of TiC 12 H 12 O 14 The result graph of the influence of the treatment on the dry weight of Salvia miltiorrhiza hairy roots; Figure 3 TiC for different concentrations 12 H 12 O 14 The effect of treatment on the content of cryptotanshinone in Salvia miltiorrhiza hairy roots results chart; Figure 4 TiC for different concentrations 12 H 12 O 14 The effect of treatment on the content of tanshiny new ketone in Salvia miltiorrhiza hairy roots results chart; Figure 5 TiC for different concentrations 12 H 12 O 14 The effect of treatment on the content of dihydro tanshinone I in Salvia miltiorrhiza hairy roots results chart; Figure 6 TiC for different concentrations 12 H 12 O 14 The effect of treatment on the content of tanshinone I in Salvia miltiorrhiza hairy roots results chart; Figure 7 TiC for different concentrations 12 H 12 O 14 The effect of treatment on the content of tanshinone IIA in Salvia miltiorrhiza hairy roots results chart; Figure 8 TiC for different concentrations 12 H 12 O 14 The effect of treatment on the content of total tanshinone in Salvia miltiorrhiza hairy roots results chart; Figure 9 TiC for different concentrations 12 H 12 O 14 The effect of treatment on the content of salvianolic acid B in Salvia miltiorrhiza hairy roots results chart; Figure 10 TiC for different concentrations 12 H 12 O 14 The effect of treatment on the content of rosmarinic acid in Salvia miltiorrhiza hairy roots results chart; Figure 11 TiC for different concentrations 12 H 12 O 14 The effect of treatment on the content of salvianolic acid Y in Salvia miltiorrhiza hairy roots results chart; Figure 12 TiC for different concentrations 12 H 12 O 14 The effect of treatment on the content of danshensu in Salvia miltiorrhiza hairy roots results chart; Figure 13 TiC for different concentrations12 H 12 O 14 The result graph of the influence of the treatment on the content of the lithospermic acid in the salvia miltiorrhiza hairy root; Figure 14 TiC 12 H 12 O 14 The result graph of the influence of the treatment on the content of the salvianolic acid A in the salvia miltiorrhiza hairy root; Figure 15 TiC 12 H 12 O 14 The result graph of the influence of the treatment on the content of the total salvianolic acid in the salvia miltiorrhiza hairy root. DETAILED DESCRIPTION

[0016] The application provides application of titanium ions in improving and / or promoting accumulation of tanshinone IIA and / or salvianolic acid components in salvia miltiorrhiza.

[0017] As an optional embodiment of the application, the molar concentration of the titanium ions can be 2-10 μmol / L, or 2, 3, 4, 5, 6, 7, 8, 9 or 10 μmol / L; the titanium ion-containing substance includes TiC 12 H 12 O 14 (Titanium (IV) citrate, CAS: 01211-17-1, specification number P35098-25g, purchased from Shanghai Dingyun Reagent Operating Department). The application studies the influence of titanium ions on quality components of salvia miltiorrhiza by using different titanium ion concentrations to induce salvia miltiorrhiza hairy roots. The application shows through the embodiment results that adding 2-10 μmol / L of titanium ions in the process of inducing salvia miltiorrhiza hairy roots is beneficial to improving and promoting accumulation of tanshinone IIA and salvianolic acid components in the salvia miltiorrhiza hairy roots, and thus is beneficial to improving quality components of salvia miltiorrhiza.

[0018] As an optional embodiment of the application, the salvianolic acid components include any one or two or more of salvianolic acid B, rosmarinic acid, salvianolic acid Y, danshensu, lithospermic acid and salvianolic acid A. The application shows through the embodiment results that adding titanium ions in the process of inducing salvia miltiorrhiza hairy roots is beneficial to improving accumulation of salvianolic acid B, rosmarinic acid, salvianolic acid Y, danshensu, lithospermic acid and salvianolic acid A in the hairy roots, and improving the content of salvianolic acid B, rosmarinic acid, salvianolic acid Y, danshensu, lithospermic acid and salvianolic acid A in the hairy roots.

[0019] The application provides a method for promoting accumulation of tanshinone IIA and / or salvianolic acid components in salvia miltiorrhiza, which comprises: inducing salvia miltiorrhiza in a culture medium containing titanium ions.

[0020] As an optional embodiment of the present application, the Salvia miltiorrhiza can be selected to verify the corresponding effect of the hairy roots of Salvia miltiorrhiza; and the culture medium can be a culture medium. As an optional embodiment of the present application, the hairy roots of Salvia miltiorrhiza are subcultured before being induced. The present application does not have a special limitation on the source of the hairy roots of Salvia miltiorrhiza, and the hairy roots obtained by using the conventional method in the art can be used. As an optional embodiment of the present application, the hairy roots of Salvia miltiorrhiza can be the hairy roots obtained by infecting Salvia miltiorrhiza seedlings with Agrobacterium rhizogenes Accc10060. As an optional embodiment of the present application, the hairy roots of Salvia miltiorrhiza can be the tender part of the hairy roots of Salvia miltiorrhiza. After obtaining the hairy roots of Salvia miltiorrhiza, the hairy roots of Salvia miltiorrhiza are subcultured. The present application does not have a special limitation on the method of subculture, and the conventional subculture method in the art can be used. As an optional embodiment of the present application, the method of subculture can be that the hairy roots of Salvia miltiorrhiza are inoculated into a MS culture medium for subculture. As an optional embodiment of the present application, the hairy roots of Salvia miltiorrhiza for inoculation can be 0.2-0.8 g, or 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8 g. The present application does not have a special limitation on the method of inoculation, and the conventional inoculation method in the art can be used. As an optional embodiment of the present application, the temperature of subculture can be 22-28℃, or 25℃; the time of subculture can be 15-21 d, or 15 d; the process of subculture is carried out in a shaker, and the rotation speed of the shaker can be 90-120 rpm, or 90 rpm.

[0021] After the subculture is completed, the obtained hairy roots of Salvia miltiorrhiza are preferably induced in a culture medium containing titanium ions. As an optional embodiment of the present application, the culture medium can be a MS culture medium; and the molar concentration of titanium ions in the culture medium can be 2-10 μmol / L, or 2, 3, 4, 5, 6, 7, 8, 9 or 10 μmol / L. As an optional embodiment of the present application, the titanium ions can be added to the culture medium in the form of TiCl4 to prepare the culture medium with the corresponding concentration of titanium ions. As an optional embodiment of the present application, the temperature of induction can be 22-28℃, or 25℃; the time of induction can be 7-10 d, or 8 d; the process of induction is carried out in a shaker, and the rotation speed of the shaker can be 90-120 rpm, or 90 rpm. 12 H 12 O 14 The process of induction is carried out in a shaker, and the rotation speed of the shaker can be 90-120 rpm, or 90 rpm.

[0022] In order to further illustrate the present application, the technical solutions provided by the present application are described in detail below in combination with the drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0023] Example 1 1. Culture of Salvia miltiorrhiza hairy roots Salvia miltiorrhiza hairy roots were obtained by infection of Salvia miltiorrhiza seedlings with Agrobacterium rhizogenes Accc10060. The detailed method is described in the reference [Zhang X N, Cui G H, Jiang X H, et al. Establishment and analysis of transgenic hairy root culture system of Salvia miltiorrhiza[J]. China Journal of Chinese Materia Medica, 2012, 37(15): 2257-2261.].

[0024] 2. Subculture of Salvia miltiorrhiza hairy roots Under sterile conditions, 0.2 g of the tender part of Salvia miltiorrhiza hairy roots was inoculated into 50 mL of MS medium. It was placed on a shaker at 25°C, 90 rpm, and cultured in the dark for 15 days as test material.

[0025] 3. Preparation of elicitors 0.347 g of AgNO3 powder was added to 100 mL of distilled water to prepare a 20 mmol / L AgNO3 stock solution. 1.265 g of Na2S2O3 was added to 100 mL of water to prepare an 80 mmol / L Na2S2O3 stock solution. 0.856 g of TiCl4 powder was added to 250 mL of distilled water to prepare an 8 mmol / L TiCl4 stock solution. 500 μL of the AgNO3 stock solution and 500 μL of the Na2S2O3 stock solution were mixed to prepare an AgNO3 / Na2S2O3 stock solution with a molar ratio of AgNO3 to Na2S2O3 of 1:4. All stock solutions were filtered through a 0.22 μm filter membrane. 12 H 12 O 14 12 H 12 O 14 +

[0026] The 250 mg / mL YE (yeast extract) stock solution was prepared according to the method described in the reference [Wang X Y, Cui G H, Huang L Q, et al. Effects of elicitors on the accumulation of tanshinones in Salvia miltiorrhiza hairy roots [J]. China Journal of Chinese Materia Medica, 2007, 32(10): 976-978.]: 25 g of yeast extract (purchased from Beijing Solabio Technology Co., Ltd.) was dissolved in 125 mL of distilled water, 100 mL of absolute ethanol was added, and it was placed in a 4°C refrigerator for 4 days. The supernatant was discarded, and the gelatinous precipitate was dissolved in 125 mL of distilled water. Absolute ethanol was added to a concentration of 80%. After centrifugation, the precipitate was dissolved in 100 mL of distilled water and sterilized.

[0027] 4. Induction of Salvia miltiorrhiza hairy roots 4.1 YE+Ag + Induction of Salvia miltiorrhiza hairy roots by elicitors ​​​Take 500 μL YE stock solution and 250 μL AgNO3 stock solution, add to the conical flask containing 50 mL MS medium, get the final concentration of 2500 mg / L YE + 100 μmol / L Ag induction medium, set 4 parallel tests, the control group (CK) does not add inducer. The hairy roots of Salvia miltiorrhiza cultured for 15 days were moved into the medium, placed in a shaker, cultured in the dark environment at 25°C, 90 rpm for 8 days, then sampled. This treatment group is recorded as "YE+Ag + " group.

[0028] 4.2 Ag + induction on hairy roots of Salvia miltiorrhiza Take 75 μL Ag + stock solution, add to the conical flask containing 50 mL MS medium, get the final concentration of 15 μmol / L Ag + induction medium, set 4 parallel tests, the control group (CK) does not add inducer. The hairy roots of Salvia miltiorrhiza cultured for 15 days were moved into the medium, placed in a shaker, cultured in the dark environment at 25°C, 90 rpm for 8 days, then sampled. This treatment group is recorded as "Ag + " group.

[0029] 4.3 TiC 12 H 12 O 14 induction on hairy roots of Salvia miltiorrhiza Take 12.5 μL, 31.2 μL, 62.5 μL TiC 12 H 12 O 14 stock solution, add to the conical flask containing 50 mL MS medium, get the final concentration of 2 μmol / L, 5 μmol / L, 10 μmol / L induction medium, set 4 parallel tests for each concentration, the control group (CK) does not add inducer. The hairy roots of Salvia miltiorrhiza cultured for 15 days were moved into the medium, placed in a shaker, cultured in the dark environment at 25°C, 90 rpm for 8 days, then sampled. This treatment group is recorded as 2Ti or 2 μM Ti 4+ group, 5Ti or 5 μM Ti 4+ group and 10Ti or 10 μM Ti 4+ group.

[0030] 5. Growth determination After rinsing the hairy roots with distilled water twice, carefully absorb the water on the hairy roots with absorbent paper, determine the fresh weight. Put into a 40°C oven, dry for 2 days to constant weight, then take out, weigh the dry weight, and record the results.

[0031] 6. Determination of chemical composition content The chemical components were determined using the method described in the literature [Wang Qiao, Yu Yongjie, Fu Haiyan, et al. Analysis of quality differences in Salvia miltiorrhiza from different producing areas based on multi-index content determination combined with chemometrics [J]. Journal of Analytical Testing, 2023, 42(04):389-401.]. The contents of 11 chemical components in the hairy roots of Salvia miltiorrhiza, including tanshinone I, tanshinone IIA, tanshinone neoketone, dihydrotanshinone I, cryptotanshinone, salvianolic acid B, rosmarinic acid, salvianolic acid Y, tanshinone, shikonin, and salvianolic acid A, were determined using UPLC and UPLC-QQQ-MS. The total tanshinone content was defined as the sum of the contents of tanshinone I, tanshinone IIA, tanshinone neoketone, dihydrotanshinone I, and cryptotanshinone. The total salvianolic acid content was defined as the sum of the contents of salvianolic acid B, rosmarinic acid, salvianolic acid Y, tanshinone, shikonin, and salvianolic acid A.

[0032] 7. Data Processing Experimental data were processed and calculated using Excel 365, and analyzed using R 4.2.0. The Dunnett test was used to analyze the differences in fresh weight and content of *Salvia miltiorrhiza* hairy roots under different treatments.

[0033] 8. Results 1) TiC 12 H 12 O 14 The effects on the biomass of hairy roots of Salvia miltiorrhiza are shown in Table 1 and Figures 1-2 As shown in the figure. In the figure, ns indicates that the difference is not significant. , or The symbol indicates a significant difference, and the same applies below.

[0034] Table 1 TiC 12 H 12 O 14 Effects on the biomass of hairy roots of Salvia miltiorrhiza (unit: g, mean ± standard deviation)

[0035] From Table 1 and Figures 1-2 The control experiment showed that the hairy roots of Salvia miltiorrhiza in YE + Ag + Under the induction of Ag, the fresh weight decreased significantly; + Under the induction of [the specific chemical], the dry weight increased significantly. However, at concentrations of 2 μmol / L, 5 μmol / L, and 10 μmol / L TiC [the dry weight], the dry weight increased significantly. 12 H 12 O 14 Under the induction of [the drug], the fresh weight and dry weight of the hairy roots of Salvia miltiorrhiza were not significantly different from those of the control group.

[0036] 2) TiC12 H 12 O 14 The effects of tanshinone content on the hairy roots of Salvia miltiorrhiza are shown in Table 2 and Figures 3-8 As shown.

[0037] Table 2 TiC 12 H 12 O 14 Effect of tanshinone content on hairy roots of Salvia miltiorrhiza (unit: mg / g, mean ± standard deviation)

[0038] From Table 2 and Figures 3-8 It can be seen that the hairy roots of Salvia miltiorrhiza in 5 μmol / L TiC 12 H 12 O 14 Under the induction of [the substance], tanshinone IIA significantly increased. Other tanshinone components showed [a decrease] at 2 μmol / L, 5 μmol / L, and 10 μmol / L TiC [concentrations]. 12 H 12 O 14 No significant changes were observed in the hairy roots of *Salvia miltiorrhiza* under induction with YE + Ag. + Under the induction of [a specific substance], cryptotanshinone, tanshinone, dihydrotanshinone I, and total tanshinone significantly increased. Tanshinone I and tanshinone IIA significantly decreased. [The text abruptly ends here, likely due to an incomplete sentence or missing information.] + Under the induction of tanshinone, cryptotanshinone, tanshinone I, tanshinone IIA, and total tanshinone in the hairy roots of Salvia miltiorrhiza were significantly increased.

[0039] 3) TiC 12 H 12 O 14 The effects of these effects on the content of phenolic acids in the hairy roots of *Salvia miltiorrhiza* are shown in Table 3 and... Figures 9-15 As shown.

[0040] Table 3 TiC 12 H 12 O 14 Effect of phenolic acid content on hairy roots of Salvia miltiorrhiza (unit: mg / g, mean ± standard deviation)

[0041] From Table 3 and Figures 9-15 It can be seen that in 2 ~ 10 μmol / L TiC 12 H 12 O 14 Under certain conditions, the contents of rosmarinic acid, salvianolic acid Y, lithospermic acid, salvianolic acid A, and total salvianolic acid in the hairy roots of Salvia miltiorrhiza increased with TiC. 12 H 12 O 14 The concentration increases with increasing concentration. At 5 μmol / L TiC 12H 12 O 14 The highest concentration of tanshinone was observed at TiC, with a significant difference compared to the control group. Tanshinone content increased with increasing TiC content. 12 H 12 O 14 The concentration increases and then decreases, at 2 μmol / L and 5 μmol / L TiC 12 H 12 O 14 Under induction, the content differed significantly from the control group. In the positive control group, the content of rosmarinic acid and tanshinone was significantly different in YE+Ag. + and Ag + Under the induction of [the substance], all [the levels] significantly increased.

[0042] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of titanium ions in at least one of the following items (1) to (4); (1) Increase the accumulation of tanshinone IIA in tanshinone; (2) Increase the accumulation of tanshinone compounds in tanshinone; (3) Promotes the accumulation of tanshinone IIA in tanshinone; (4) Promotes the accumulation of tanshinone compounds in tanshinone.

2. The application according to claim 1, characterized in that, The tanshinone acid components include any one or more of the following: tanshinone B, rosmarinic acid, tanshinone Y, tanshinone, lithospermic acid, and tanshinone A.

3. The application according to claim 1, characterized in that, The molar concentration of titanium ions is 2~10 μmol / L.

4. A method for promoting the accumulation of tanshinone IIA and / or tanshinone acid components in tanshinone, characterized in that, include: Salvia miltiorrhiza was induced in a culture medium containing titanium ions.

5. The method according to claim 4, characterized in that, The molar concentration of titanium ions in the culture medium is 2~10 μmol / L.

6. The method according to claim 4 or 5, characterized in that, The molar concentration of titanium ions in the culture medium is 5 μmol / L.

7. The method according to claim 4, characterized in that, The induction temperature is 22~28℃; the induction time is 7~10 days; the induction process is carried out in a shaker with a rotation speed of 90~120 rpm.

8. The method according to claim 4, characterized in that, The *Salvia miltiorrhiza* includes *Salvia miltiorrhiza* hairy roots; before inducing the *Salvia miltiorrhiza* hairy roots, the *Salvia miltiorrhiza* hairy roots are subcultured.

9. The method according to claim 8, characterized in that, Methods for subculturing the hairy roots of Salvia miltiorrhiza include: The hairy roots of Salvia miltiorrhiza were inoculated into MS medium for subculture.

10. The method according to claim 9, characterized in that, The subculture temperature is 22~28℃; the subculture time is 15~21 days; the subculture process is carried out in a shaker with a rotation speed of 90~120 rpm.

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