Application of strontium ion in improving and / or promoting accumulation of tanshinone components and / or salvianolic acid components

Abstract

The application provides application of strontium ions in improving and / or promoting accumulation of tanshinone components and / or salvianolic acid components, and belongs to the technical field of hairy root induction. The application provides application of strontium ions in improving and / or promoting accumulation of tanshinone components and / or salvianolic acid components. The application induces tanshinone hairy roots through different strontium ion concentrations, studies the influence of strontium ions on tanshinone components, so as to provide a theoretical basis for improving tanshinone quality and developing strontium fertilizer. The application shows through the results of examples that, in the process of inducing tanshinone hairy roots, adding 10-100 mu mol / L of strontium ions is beneficial to improving and promoting accumulation of tanshinone components and salvianolic acid components in tanshinone hairy roots, and further beneficial to improving tanshinone quality components.

Description

Technical Field

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

[0002] Strontium is a trace element in most biological systems. Due to its chemical similarity to calcium, it is easily absorbed by plants. Plants absorb strontium from soil and hydroponic media through their roots and from atmospheric deposition through their leaves and fruits. Studies by Marčiulionienieni et al. found that low concentrations of strontium ions promoted the growth of *Gnaphalium affine*. Similarly, studies by Yao Kai and Sowa et al. found that low concentrations of strontium ions also promoted the growth of spinach and soybeans. Furthermore, studies by Wójciak-Kosior et al. found that 2 mmol / L strontium ions could promote the synthesis of secondary metabolites such as daidzein, coumarin, genistein, and gentianin in soybeans.

[0003] Currently, the application of strontium ions is still relatively limited, with limited use in traditional Chinese medicine. Summary of the Invention

[0004] In view of the deficiencies in the prior art, the purpose of this invention is to provide the application of strontium ions in increasing and / or promoting the accumulation of tanshinone and / or tanshinone acid components. It can promote the accumulation of tanshinone and tanshinone acid components in tanshinone by supplementing strontium ions in tanshinone culture, thereby increasing the content of tanshinone and tanshinone acid components in tanshinone.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] This invention provides the application of strontium ions in enhancing and / or promoting the accumulation of tanshinone and / or tanshinone acid components.

[0007] Preferably, the molar concentration of the strontium ions is 10~100 μmol / L.

[0008] Preferably, the tanshinone components include any one or more of tanshinone I, tanshinone IIA, tanshinone neotanshinone, dihydrotanshinone I, and cryptotanshinone; 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.

[0009] This invention provides a method for promoting the accumulation of tanshinone and / or tanshinone acid components in tanshinone, comprising:

[0010] Salvia miltiorrhiza was cultured in a medium containing strontium ions.

[0011] Preferably, the culture temperature is 22~28℃; the culture time is 7~10 days; the culture process is carried out in a shaker with a rotation speed of 90~120 rpm.

[0012] Preferably, the Salvia miltiorrhiza includes Salvia miltiorrhiza hairy roots; before inducing the Salvia miltiorrhiza hairy roots, the Salvia miltiorrhiza hairy roots are subcultured.

[0013] Preferred methods for subculturing the hairy roots of Salvia miltiorrhiza include:

[0014] The hairy roots of Salvia miltiorrhiza were inoculated into MS medium for subculture.

[0015] 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.

[0016] Preferably, the molar concentration of strontium ions in the culture medium is 10~100 μmol / L.

[0017] Preferably, the molar concentration of strontium ions in the culture medium is 10~50 μmol / L.

[0018] The beneficial effects of this invention are:

[0019] This invention provides the application of strontium ions in enhancing and / or promoting the accumulation of tanshinone and / or tanshinone acid components. This invention studies the effect of strontium ions on the quality components of *Salvia miltiorrhiza* by inducing hairy roots with different strontium ion concentrations, aiming to provide a theoretical basis for improving the quality of *Salvia miltiorrhiza* and developing strontium fertilizers. The results of the embodiments of this invention show that adding 10-100 μmol / L of strontium ions during the induction of hairy roots of *Salvia miltiorrhiza* is beneficial for enhancing and promoting the accumulation of tanshinone and tanshinone acid components, the effective components of traditional Chinese medicine, in the hairy roots of *Salvia miltiorrhiza*, thereby improving the quality of *Salvia miltiorrhiza*. Attached Figure Description

[0020] 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.

[0021] Figure 1 Figure 1 shows the effect of different concentrations of SrCl2 treatment on the fresh weight of Salvia miltiorrhiza hairy roots.

[0022] Figure 2 Figure 1 shows the effect of different concentrations of SrCl2 treatment on the dry weight of hairy roots of Salvia miltiorrhiza.

[0023] Figure 3 Figure 1 shows the effect of different concentrations of SrCl2 treatment on the content of cryptotanshinone in the hairy roots of Salvia miltiorrhiza.

[0024] Figure 4 The figure shows the effect of different concentrations of SrCl2 treatment on the tanshinone content in the hairy roots of Salvia miltiorrhiza.

[0025] Figure 5 Figure 1 shows the effect of different concentrations of SrCl2 treatment on the content of dihydrotanshinone I in the hairy roots of Salvia miltiorrhiza.

[0026] Figure 6 The effect of different concentrations of SrCl2 treatment on the tanshinone I content in the hairy roots of Salvia miltiorrhiza is shown in the figure.

[0027] Figure 7 Figure 1 shows the effect of different concentrations of SrCl2 treatment on the content of tanshinone IIA in the hairy roots of Salvia miltiorrhiza.

[0028] Figure 8 Figure 1 shows the effect of different concentrations of SrCl2 treatment on the total tanshinone content in the hairy roots of Salvia miltiorrhiza.

[0029] Figure 9 The effect of different concentrations of SrCl2 treatment on the content of salvianolic acid B in the hairy roots of Salvia miltiorrhiza is shown in the figure.

[0030] Figure 10 Figure 1 shows the effect of different concentrations of SrCl2 treatment on the rosmarinic acid content in the hairy roots of Salvia miltiorrhiza.

[0031] Figure 11 The effect of different concentrations of SrCl2 treatment on the content of salvianolic acid Y in the hairy roots of Salvia miltiorrhiza is shown in the figure.

[0032] Figure 12 The figure shows the effect of different concentrations of SrCl2 treatment on the tanshinone content in the hairy roots of Salvia miltiorrhiza.

[0033] Figure 13 The effect of different concentrations of SrCl2 treatment on the content of shikonin in the hairy roots of Salvia miltiorrhiza is shown in the figure.

[0034] Figure 14 The effect of different concentrations of SrCl2 treatment on the content of salvianolic acid A in the hairy roots of Salvia miltiorrhiza is shown in the figure.

[0035] Figure 15 The figure shows the effect of different concentrations of SrCl2 treatment on the total salvianolic acid content in the hairy roots of Salvia miltiorrhiza. Detailed Implementation

[0036] This invention provides the application of strontium ions in enhancing and / or promoting the accumulation of tanshinone and / or tanshinone acid components.

[0037] As an optional embodiment of the present invention, the molar concentration of strontium ions can be 10~100 μmol / L, or 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 μmol / L; the strontium-containing substance includes SrCl2. The present invention studies the effect of strontium ions on the quality components of *Salvia miltiorrhiza* by culturing hairy roots with different strontium ion concentrations. The results of the embodiments of the present invention show that adding 10~100 μmol / L of strontium ions during the induction of hairy roots of *Salvia miltiorrhiza* is beneficial to increasing and promoting the accumulation of tanshinone and tanshinone acid components in the hairy roots of *Salvia miltiorrhiza*, thereby improving the quality components of *Salvia miltiorrhiza*.

[0038] As an optional embodiment of the present invention, the tanshinone components include any one or more of tanshinone I, tanshinone IIA, tanshinone neoformate, dihydrotanshinone I, and cryptotanshinone; 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 strontium ions during the induction of hairy roots of *Salvia miltiorrhiza* is beneficial to increasing the content of tanshinone I, tanshinone IIA, tanshinone neoformate, dihydrotanshinone I, and cryptotanshinone in the hairy roots. Adding strontium 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.

[0039] The present invention provides a method for promoting the accumulation of tanshinone and / or tanshinone acid components in tanshinone, comprising: culturing tanshinone in a culture medium containing strontium ions.

[0040] 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, before culturing the *Salvia miltiorrhiza* hairy roots in a culture medium containing strontium ions, the *Salvia miltiorrhiza* hairy roots are subcultured. 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 22, 23, 24, 25, 26, 27, or 28℃; the subculture time can be 15~21 days, or 15, 16, 17, 18, 19, 20, or 21 days; the subculture process is carried out in a shaker, and the shaker speed can be 90~120 rpm, or 90, 100, 110, or 120 rpm.

[0041] After subculturing, the obtained *Salvia miltiorrhiza* hairy roots are preferably cultured in a medium containing strontium ions. As an optional embodiment of the invention, the medium can be MS medium; the molar concentration of strontium ions in the medium can be 10-100 μmol / L, or 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μmol / L. As an optional embodiment of the invention, the strontium ions can be added to the medium in the form of SrCl2 to prepare a medium with the corresponding strontium ion concentration. As an optional embodiment of the invention, the culture temperature can be 22-28℃, or 22, 23, 24, 25, 26, 27, or 28℃; the culture time can be 7-10 days, or 7, 8, 9, or 10 days; the culture process is carried out in a shaker, and the shaker speed can be 90-120 rpm, or 90, 100, 110, or 120 rpm.

[0042] 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.

[0043] Example 1

[0044] 1. Culture of hairy roots of Salvia miltiorrhiza

[0045] 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.].

[0046] 2. Subculture of hairy roots of Salvia miltiorrhiza

[0047] Under aseptic conditions, 0.2 g of the tender hairy rootlets of *Salvia miltiorrhiza* were weighed and inoculated into 50 mL of MS medium. The medium was placed on a shaker and cultured at 25°C, 90 rpm, and in the dark for 15 days to obtain the experimental material.

[0048] 3. Preparation of inducers

[0049] 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. Weigh 1.265 g of Na2S2O3 and add it to 100 mL of water to prepare an 80 mmol / L Na2S2O3 stock solution. Weigh 0.318 g of SrCl2 powder and add it to 100 mL of distilled water to prepare a 20 mmol / L SrCl2 stock solution. Prepare an AgNO3 to Na2S2O3 solution with a molar ratio of 1:4 using 500 μL of the AgNO3 stock solution and 500 μL of the Na2S2O3 stock solution. + Mother liquor. All mother liquors were filtered through a 0.22 μm filter membrane.

[0050] 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 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 4℃ refrigerator and stand for 4 days, discard the supernatant, dissolve the gelatinous precipitate in 125 mL of distilled water, add anhydrous ethanol until the ethanol volume fraction reaches 80%. Precipitate, centrifuge, dissolve the precipitate in 100 mL of distilled water, and sterilize.

[0051] 4. Induction of hairy roots of Salvia miltiorrhiza

[0052] 4.1 YE+Ag + Induction of Salvia miltiorrhiza hairy roots by inducers

[0053] Pipette 500 μL of YE stock solution and 250 μL of AgNO3 stock solution into an Erlenmeyer flask containing 50 mL of MS medium to obtain a final concentration of 2500 mg / L YE + 100 μmol / L Ag + Five parallel experiments were conducted using the induction medium, with the control group (CK) receiving no inducing agent. After 15 days of culture, all the hairy roots of *Salvia miltiorrhiza* were transferred to the medium and placed in a shaker. The mixture was cultured for 8 days in the dark at 25°C and 90 rpm, and samples were taken. This treatment group was designated "YE+Ag". + "Group.

[0054] 4.2 Ag + Induction of Salvia miltiorrhiza hairy roots by inducers

[0055] 75 μL of Ag+ stock solution was added to an Erlenmeyer flask containing 50 mL of MS medium to obtain an induction medium with a final concentration of 15 μmol / L Ag+. Five parallel experiments were set up, with the control group (CK) receiving no inducing agent. After 15 days of culture, all 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 "Ag". + "Group.

[0056] 4.3 Induction of Salvia miltiorrhiza hairy roots by SrCl2

[0057] 25 μL, 125 μL, and 250 μL of SrCl2 stock solution were respectively added to Erlenmeyer flasks containing 50 mL of MS medium to obtain induction medium with final concentrations of 10 μmol / L, 50 μmol / L, and 100 μmol / L. Five parallel experiments were set up for each concentration. The control group (CK) received no inducing agent. After 15 days of culture, all 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℃ and 90 rpm, samples were taken. These treatment groups were designated as 10Sr or 10μMSr, respectively. 2+ Group, 50Sr or 50μM Sr 2+ Group and 100Sr or 100μM Sr 2+ Group.

[0058] 5. Growth Measurement

[0059] 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.

[0060] 6. Determination of chemical composition content

[0061] 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.

[0062] 7. Data Processing

[0063] 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.

[0064] 8. Results

[0065] 1) The effect of SrCl2 on the biomass of hairy roots of Salvia miltiorrhiza is shown in Table 1 and Figures 1-2 As shown.

[0066] Table 1. Effects of SrCl2 on the biomass of hairy roots of Salvia miltiorrhiza (mean ± standard deviation)

[0067]

[0068] 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 100 μmol / L SrCl2, the dry weight of the hairy roots of Salvia miltiorrhiza increased significantly.

[0069] 2) The effect of SrCl2 on the tanshinone content in the hairy roots of Salvia miltiorrhiza is shown in Table 2 and... Figures 3-8 As shown.

[0070] Table 2 Effect of SrCl2 on tanshinone content in hairy roots of Salvia miltiorrhiza (unit: mg / g, mean ± standard deviation)

[0071]

[0072] From Table 2 and Figures 3-8 It was found that under the induction of 10 μmol / L SrCl2, the contents of cryptotanshinone, dihydrotanshinone I, and total tanshinone in the hairy roots of *Salvia miltiorrhiza* were significantly increased. Under the induction of 100 μmol / L SrCl2, the content of tanshinone I was significantly increased. The content of tanshinone I in the hairy roots of *Salvia miltiorrhiza* under the influence of YE + Ag + Under the induction of [a specific substance], cryptotanshinone, tanshinone, dihydrotanshinone I, and total tanshinone significantly increased, while tanshinone I and tanshinone IIA significantly decreased. [The text then abruptly shifts to a seemingly unrelated topic:] In Ag [a specific substance]... + Under the induction of tanshinone, cryptotanshinone, tanshinone I, tanshinone IIA, and total tanshinone in the hairy roots of Salvia miltiorrhiza were significantly increased.

[0073] 3) The effect of SrCl2 on the phenolic acid content of Salvia miltiorrhiza hairy roots is shown in Table 3 and Figures 9-15 As shown.

[0074] Table 3 Effect of SrCl2 on the content of phenolic acids in the hairy roots of Salvia miltiorrhiza (unit: mg / g, mean ± standard deviation)

[0075]

[0076] From Table 3 and Figures 9-15 It was found that under the induction of 10 ~ 100 μmol / L SrCl2, the content of phenolic acid components decreased with the increase of SrCl2 concentration. Under the induction of 10 μmol / L SrCl2, the contents of salvianolic acid Y, tanshinone, shikonin, and salvianolic acid A in the hairy roots of Salvia miltiorrhiza were significantly increased.

[0077] 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. Application of strontium ions in enhancing and / or promoting the accumulation of tanshinone and / or tanshinone compounds in the hairy roots of Salvia miltiorrhiza; When the strontium ion concentration is 10 μmol / L, the tanshinone components are cryptotanshinone and / or dihydrotanshinone I; When the strontium ion concentration is 100 μmol / L, the tanshinone component is tanshinone I; When the strontium ion concentration is 10 μmol / L, the tanshinone acid components are any one or more of salvianolic acid Y, tanshinone, shikonin, and salvianolic acid A.

2. A method for promoting the accumulation of tanshinone and / or tanshinone acid components in Salvia miltiorrhiza, characterized in that, include: The Salvia miltiorrhiza was cultured in a culture medium containing strontium ions; the Salvia miltiorrhiza was the hairy root of Salvia miltiorrhiza. When the strontium ion concentration is 10 μmol / L, the tanshinone components are cryptotanshinone and / or dihydrotanshinone I; When the strontium ion concentration is 100 μmol / L, the tanshinone component is tanshinone I; When the strontium ion concentration is 10 μmol / L, the tanshinone acid components are any one or more of salvianolic acid Y, tanshinone, shikonin, and salvianolic acid A.

3. The method according to claim 2, characterized in that, The culture temperature is 22~28℃; the culture time is 7~10 days; the culture 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, Before inducing the hairy roots of Salvia miltiorrhiza, the hairy roots of Salvia miltiorrhiza 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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