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

Abstract

The application 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 application provides application of titanium ions in improving and / or promoting accumulation of tanshinone IIA and / or salvianolic acid components of Salvia miltiorrhiza. 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 results of examples that adding 2-10 μmol / L 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 Salvia miltiorrhiza hairy roots, and thus is beneficial to improving quality components 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 quality of Salvia miltiorrhiza and developing titanium fertilizer.

Description

Technical Field

[0001] This invention belongs to the field of hairy root induction technology, specifically relating to the application of titanium ions in promoting the accumulation of tanshinone IIA and / or tanshinone acid components in tanshinone. Background Technology

[0002] Titanium (Ti) is a beneficial trace element that can promote normal plant growth and improve crop yield and quality. However, titanium usually exists in the form of insoluble compounds (oxides, silicate-bound forms, or ilmenite), making it difficult to exert its effects on plants. In agriculture, titanium salts chelated with citric acid, ascorbic acid, etc., are often used as fertilizers. Wu Qi et al. found that low concentrations of titanium ions can promote oat growth and increase fresh grass yield. In flue-cured tobacco cultivation, foliar spraying and root irrigation with titanium fertilizer can promote tobacco growth and development, improve tobacco yield and quality, and improve the internal chemical composition of tobacco leaves. Soaking wheat seeds in titanium ion solutions of different concentrations increases the unit fresh weight of wheat leaves, chlorophyll content, number of grains per ear, and thousand-grain weight, thereby increasing yield. However, research on titanium fertilizers in the cultivation of traditional Chinese medicinal herbs is relatively lacking. Summary of the Invention

[0003] To address the shortcomings of existing technologies, the present invention aims to provide...

[0004] The objective of this invention is achieved through the following technical solution:

[0005] The present invention provides the use of titanium ions in at least one of the following items (1) to (4);

[0006] (1) Increase the accumulation of tanshinone IIA in tanshinone;

[0007] (2) Increase the accumulation of tanshinone compounds in tanshinone;

[0008] (3) Promotes the accumulation of tanshinone IIA in tanshinone;

[0009] (4) Promotes the accumulation of tanshinone compounds in tanshinone.

[0010] Preferably, the tanshinone acid components include any one or more of salvianolic acid B, rosmarinic acid, salvianolic acid Y, tanshinone, lithospermic acid, and salvianolic acid A.

[0011] Preferably, the molar concentration of the titanium ions is 2~10 μmol / L.

[0012] This invention provides a method for promoting the accumulation of tanshinone IIA and / or tanshinone acid components in tanshinone, comprising:

[0013] Danshen was induced in a culture medium containing titanium ions.

[0014] Preferably, the molar concentration of titanium ions in the culture medium is 2~10 μmol / L.

[0015] Preferably, the molar concentration of titanium ions in the culture medium is 5 μmol / L.

[0016] Preferably, 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.

[0017] Preferably, the Salvia miltiorrhiza includes Salvia miltiorrhiza hairy roots; before inducing the Salvia miltiorrhiza hairy roots, the Salvia miltiorrhiza hairy roots are subcultured.

[0018] Preferred methods for subculturing the hairy roots of Salvia miltiorrhiza include:

[0019] The hairy roots of Salvia miltiorrhiza were inoculated into MS medium for subculture.

[0020] Preferably, 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.

[0021] Beneficial effects:

[0022] This invention provides the application of titanium ions in enhancing and / or promoting the accumulation of tanshinone IIA and / or tanshinone acid compounds in *Salvia miltiorrhiza*. This invention investigates the effect of titanium ions on the quality components of *Salvia miltiorrhiza* by inducing hairy roots with different titanium ion concentrations. The results of the examples show that adding 2-10 μmol / L of titanium ions during the induction of hairy roots of *Salvia miltiorrhiza* is beneficial in enhancing and promoting the accumulation of the effective medicinal components tanshinone IIA and tanshinone acid compounds in the hairy roots, thereby improving the quality of *Salvia miltiorrhiza*. This invention aims to provide a theoretical basis for improving the quality of *Salvia miltiorrhiza* and developing titanium fertilizers by studying the effect of titanium ions on the quality components of *Salvia miltiorrhiza*. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 For different concentrations of TiC 12 H 12 O 14 Figure showing the effect of treatment on the fresh weight of Salvia miltiorrhiza hairy roots;

[0025] Figure 2 For different concentrations of TiC 12 H 12 O 14 Figure showing the effect of treatment on the dry weight of Salvia miltiorrhiza hairy roots;

[0026] Figure 3 For different concentrations of TiC 12 H 12 O 14 Figure showing the effect of treatment on the content of cryptotanshinone in the hairy roots of Salvia miltiorrhiza.

[0027] Figure 4 For different concentrations of TiC 12 H 12 O 14 Figure showing the effect of treatment on the tanshinone content in the hairy roots of Salvia miltiorrhiza.

[0028] Figure 5 For different concentrations of TiC 12 H 12 O 14 Figure showing the effect of treatment on the content of dihydrotanshinone I in the hairy roots of Salvia miltiorrhiza.

[0029] Figure 6 For different concentrations of TiC 12 H 12 O 14 Figure showing the effect of treatment on the tanshinone I content in the hairy roots of Salvia miltiorrhiza.

[0030] Figure 7 For different concentrations of TiC 12 H 12 O 14 Figure showing the effect of treatment on the content of tanshinone IIA in the hairy roots of Salvia miltiorrhiza.

[0031] Figure 8 For different concentrations of TiC 12 H 12 O 14 Figure showing the effect of treatment on the total tanshinone content in the hairy roots of Salvia miltiorrhiza.

[0032] Figure 9 For different concentrations of TiC 12 H 12 O 14 Figure showing the effect of treatment on the content of salvianolic acid B in the hairy roots of Salvia miltiorrhiza.

[0033] Figure 10 For different concentrations of TiC 12 H 12 O 14 Figure showing the effect of treatment on the rosmarinic acid content in the hairy roots of Salvia miltiorrhiza.

[0034] Figure 11 For different concentrations of TiC 12 H 12 O 14 Figure showing the effect of treatment on the content of salvianolic acid Y in the hairy roots of Salvia miltiorrhiza.

[0035] Figure 12 For different concentrations of TiC 12 H 12 O 14 Figure showing the effect of treatment on tanshinone content in the hairy roots of Salvia miltiorrhiza.

[0036] Figure 13 For different concentrations of TiC 12 H 12 O 14 Figure showing the effect of treatment on the content of shikonin in the hairy roots of Salvia miltiorrhiza.

[0037] Figure 14 For different concentrations of TiC 12 H 12 O 14 Figure showing the effect of treatment on the content of salvianolic acid A in the hairy roots of Salvia miltiorrhiza.

[0038] Figure 15 For different concentrations of TiC 12 H 12 O 14 The effect of treatment on the total salvianolic acid content in the hairy roots of Salvia miltiorrhiza is shown in the figure. Detailed Implementation

[0039] This invention provides the application of titanium ions in enhancing and / or promoting the accumulation of tanshinone IIA and / or tanshinone acid components in tanshinone.

[0040] As an optional embodiment of the present invention, the molar concentration of 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 material includes TiC. 12 H 12 O 14 (Titanium citrate (IV), CAS: 01211-17-1, specification number P35098-25g, purchased from Shanghai Dingyun Reagent Business Department). This invention investigates the effect of titanium ions on the quality components of *Salvia miltiorrhiza* by inducing hairy roots with different titanium ion concentrations. The results of the examples demonstrate that adding 2-10 μmol / L of titanium ions during the induction of hairy roots of *Salvia miltiorrhiza* is beneficial in increasing and promoting the accumulation of tanshinone IIA and tanshinone acid compounds in the hairy roots, thereby improving the quality components of *Salvia miltiorrhiza*.

[0041] As an optional embodiment of the present invention, the tanshinone acid components include any one or more of salvianolic acid B, rosmarinic acid, salvianolic acid Y, tanshinone, shikonin, and salvianolic acid A. The results of the embodiments of the present invention show that adding titanium ions during the induction of hairy roots of *Salvia miltiorrhiza* is beneficial to increasing the accumulation of salvianolic acid B, rosmarinic acid, salvianolic acid Y, tanshinone, shikonin, and salvianolic acid A in the hairy roots, and increasing the content of salvianolic acid B, rosmarinic acid, salvianolic acid Y, tanshinone, shikonin, and salvianolic acid A in the hairy roots.

[0042] The present invention provides a method for promoting the accumulation of tanshinone IIA and / or tanshinone acid components in tanshinone, comprising: inducing tanshinone in a culture medium containing titanium ions.

[0043] As an optional embodiment of the present invention, the *Salvia miltiorrhiza* hairy roots can be selected for corresponding effect verification; the culture medium can be a culture medium. As an optional embodiment of the present invention, the *Salvia miltiorrhiza* hairy roots are subcultured before inducing them. The present invention does not have a special limitation on the source of the *Salvia miltiorrhiza* hairy roots; hairy roots induced by conventional methods in the art are acceptable. As an optional embodiment of the present invention, the *Salvia miltiorrhiza* hairy roots can be hairy roots obtained by infecting *Salvia miltiorrhiza* seedlings with *Agrobacterium rhizogenes* Accc10060. As an optional embodiment of the present invention, the *Salvia miltiorrhiza* hairy roots can be the young parts of *Salvia miltiorrhiza* hairy roots. After obtaining the *Salvia miltiorrhiza* hairy roots, the present invention subcultures the *Salvia miltiorrhiza* hairy roots. The present invention does not have a special limitation on the method of subculture; conventional subculture methods in the art are acceptable. As an optional embodiment of the present invention, the subculture method can be: inoculating the *Salvia miltiorrhiza* hairy roots in MS medium for subculture. As an optional embodiment of the present invention, the amount of *Salvia miltiorrhiza* hairy roots used for inoculation can be 0.2~0.8g, or 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, or 0.8g. The present invention does not specifically limit the inoculation method; any conventional inoculation method in the art can be used. As an optional embodiment of the present invention, the subculture temperature can be 22~28℃, or 25℃; the subculture time can be 15~21 days, or 15 days; the subculture process is carried out in a shaker, and the shaker speed can be 90~120 rpm, or 90 rpm.

[0044] After subculturing, the obtained *Salvia miltiorrhiza* hairy roots are preferably induced in a culture medium containing titanium ions. As an optional embodiment of the invention, the culture medium can be MS medium; 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 invention, the titanium ions can be in the form of TiC. 12 H12 O 14 The titanium ion concentration is prepared by adding it to the culture medium in the form of titanium ions. As an optional embodiment of the present invention, the induction temperature can be 22~28℃ or 25℃; the induction time can be 7~10 days or 8 days; the induction process is carried out in a shaker, and the shaker speed can be 90~120 rpm or 90 rpm.

[0045] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0046] Example 1

[0047] 1. Culture of hairy roots of Salvia miltiorrhiza

[0048] The hairy roots of Salvia miltiorrhiza were obtained by infecting Salvia miltiorrhiza seedlings with Agrobacterium rhizogenes Accc10060. For details of the method, please refer to the reference [Zhang Xiaonan, Cui Guanghong, Jiang Xihong, et al. Establishment and analysis of in vitro culture system of transgenic hairy roots of Salvia miltiorrhiza [J]. Chinese Journal of Traditional Chinese Medicine, 2012, 37(15):2257-2261.].

[0049] 2. Subculture of hairy roots of Salvia miltiorrhiza

[0050] Under aseptic conditions, 0.2 g of the tender hairy rootlets of *Salvia miltiorrhiza* were inoculated into 50 mL of MS medium. The medium was placed on a shaker and cultured at 25°C, 90 rpm, in the dark for 15 days as the experimental material.

[0051] 3. Preparation of inducers

[0052] Weigh 0.347 g of AgNO3 powder and add it to 100 mL of distilled water to prepare a 20 mmol / L AgNO3 stock solution. Dissolve 1.265 g of Na2S2O3 in 100 mL of water to prepare an 80 mmol / L Na2S2O3 stock solution. Weigh 0.856 g of TiC... 12 H 12 O 14 The powder was added to 250 mL of distilled water to prepare an 8 mmol / L TiC solution. 12 H 12 O 14 Mother liquor. Prepare AgNO3 to Na2S2O3 solution by mixing 500 μL of AgNO3 mother liquor and 500 μL of Na2S2O3 mother liquor, with a molar ratio of AgNO3 to Na2S2O3 of 1:4. + Mother liquor. All mother liquors were filtered through a 0.22 μm filter membrane.

[0053] References [Wang Xueyong, Cui Guanghong, Huang Luqi, et al. Effects of inducers on the accumulation of tanshinone components in the hairy roots of Salvia miltiorrhiza [J]. Chinese Journal of Traditional Chinese Medicine, 2007, 32(10):976-978.] Methods: Preparation of 250 mg / mL YE (yeast extract) stock solution: Dissolve 25 g of yeast extract (purchased from Beijing Solarbio Science & Technology Co., Ltd.) in 125 mL of distilled water, add 100 mL of anhydrous ethanol, place in a refrigerator at 4℃ for 4 days, discard the supernatant, dissolve the gelatinous precipitate in 125 mL of distilled water, add anhydrous ethanol until the ethanol content reaches 80%. After precipitation, centrifuge, dissolve the precipitate in 100 mL of distilled water, and sterilize.

[0054] 4. Induction of hairy roots of Salvia miltiorrhiza

[0055] 4.1 YE+Ag + Induction of Salvia miltiorrhiza hairy roots by inducers

[0056] 500 μL of YE stock solution and 250 μL of AgNO3 stock solution were added to an Erlenmeyer flask containing 50 mL of MS medium to obtain an induction medium with a final concentration of 2500 mg / L YE + 100 μmol / L Ag. Four parallel experiments were set up, with the control group (CK) receiving no inducing agent. After 15 days of culture, the hairy roots of *Salvia miltiorrhiza* were transferred to the medium and placed in a shaker. After 8 days of culture in the dark at 25°C and 90 rpm, samples were taken. This treatment group was designated "YE+Ag". + "Group.

[0057] 4.2 Ag + Induction of Salvia miltiorrhiza hairy roots by inducers

[0058] Take 75 μL Ag + The stock solution was added to an Erlenmeyer flask containing 50 mL of MS medium to obtain a final concentration of 15 μmol / L Ag. + The induction medium was prepared, and four parallel experiments were set up, with the control group (CK) receiving no inducing agent. After 15 days of culture, the hairy roots of *Salvia miltiorrhiza* were transferred to the medium and placed in a shaker. They were cultured for 8 days in the dark at 25°C and 90 rpm, and samples were taken afterward. This treatment group was designated "Ag". + "Group.

[0059] 4.3 TiC 12 H 12 O 14 Induction of hairy roots of Salvia miltiorrhiza

[0060] 12.5 μL, 31.2 μL, and 62.5 μL of TiC were respectively pipetted. 12 H 12 O 14The stock solution was added to an Erlenmeyer flask containing 50 mL of MS medium to obtain induction medium with final concentrations of 2 μmol / L, 5 μmol / L, and 10 μmol / L, with four parallel experiments for each concentration. The control group (CK) received no inducing agent. After 15 days of culture, the hairy roots of *Salvia miltiorrhiza* were transferred to the medium and placed in a shaker. After 8 days of culture in the dark at 25°C and 90 rpm, samples were taken. These treatment groups were designated as 2Ti or 2μM Ti, respectively. 4+ Group, 5Ti or 5μM Ti 4+ Group and 10Ti or 10μM Ti 4+ Group.

[0061] 5. Growth Measurement

[0062] After rinsing the hairy roots twice with distilled water, carefully blot the moisture off the hairy roots with absorbent paper and determine the fresh weight. Place them in a 40℃ oven and dry for 2 days until constant weight. Remove them, weigh them, and record the results.

[0063] 6. Determination of chemical composition content

[0064] 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.

[0065] 7. Data Processing

[0066] 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.

[0067] 8. Results

[0068] 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.

[0069] Table 1 TiC 12 H 12 O 14 Effects on the biomass of hairy roots of Salvia miltiorrhiza (unit: g, mean ± standard deviation)

[0070]

[0071] 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.

[0072] 2) TiC 12 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.

[0073] Table 2 TiC 12 H 12 O 14 Effect of tanshinone content on hairy roots of Salvia miltiorrhiza (unit: mg / g, mean ± standard deviation)

[0074]

[0075] 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.

[0076] 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.

[0077] 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)

[0078]

[0079] 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 12 H 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.

[0080] 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; When the titanium ion concentration is 5 μmol / L, it increases or promotes the accumulation of tanshinone IIA in tanshinone. When the concentration of titanium ions is 2~10 μmol / L, it increases or promotes the accumulation of tanshinone acid components; the tanshinone acid components are any one or more of rosmarinic acid, salvianolic acid Y, tanshinone, lithospermic acid and salvianolic acid A. The titanium ions are provided in the form of titanium citrate (IV).

2. A method for promoting the accumulation of tanshinone IIA and / or tanshinone acid components in Salvia miltiorrhiza, characterized in that, include: Salvia miltiorrhiza was induced in a culture medium containing titanium ions; When the titanium ion concentration is 5 μmol / L, it increases or promotes the accumulation of tanshinone IIA in tanshinone. When the concentration of titanium ions is 2~10 μmol / L, it increases or promotes the accumulation of tanshinone acid components; the tanshinone acid components are any one or more of rosmarinic acid, salvianolic acid Y, tanshinone, lithospermic acid and salvianolic acid A. The titanium ions are provided in the form of titanium citrate (IV).

3. The method according to claim 2, 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.

4. The method according to claim 2, 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.

5. The method according to claim 4, 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.

6. The method according to claim 5, 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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