Salvia miltiorrhiza endogenous oligopeptide composition and application thereof

Abstract

The invention discloses a salvia miltiorrhiza endogenous oligopeptide composition and application thereof. The composition contains salvia miltiorrhiza endogenous oligopeptides miPEP167b-5p and miPEP171b-1, wherein the concentration ratio of the salvia miltiorrhiza endogenous oligopeptides miPEP167b-5p to the miPEP171b-1 is 0.8-1.2. The oligopeptide used in the composition has high species specificity, does not promote the growth of other plants in the field, and avoids the ecological risk of a traditional broad-spectrum growth regulator. Field experiment results show that after the composition is applied to treatment, the root system yield of salvia miltiorrhiza is increased by 56% compared with that of a control group, the number of roots is increased by 435% compared with that of the control group, the rosmarinic acid content is increased by 43% compared with that of the control group, the salvianolic acid B content is increased by 81%, the tanshinone I content is increased by 214%, the tanshinone IIA content is increased by 75%, the leap-type improvement of the salvia miltiorrhiza medicinal material yield and quality is synchronously achieved, and the application prospect is wide. And reliable technical support is provided for green, efficient and high-quality development of the salvia miltiorrhiza industry.

Description

Technical Field

[0001] This invention relates to agricultural biotechnology, and more particularly to a short peptide composition derived from Danshen (Salvia miltiorrhiza) and its application. Background Technology

[0002] Salvia miltiorrhiza ( Salvia miltiorrhiza Danshen (B.) is a traditional bulk medicinal herb, used medicinally in the form of dried roots and rhizomes. Its main active ingredients are water-soluble salvianolic acid compounds and fat-soluble tanshinone compounds. Currently, the market for Danshen mainly relies on artificial cultivation; however, two major problems have long existed in its production: firstly, the yield is low and unstable, with the yield of dried medicinal materials per mu (a Chinese unit of area, approximately 0.067 hectares) fluctuating between 180 and 300 kg, and the root system exhibiting inconsistent morphology and size, resulting in poor uniformity in appearance; secondly, the quality of the medicinal materials is uneven, and the content of active ingredients is difficult to consistently meet standards.

[0003] In pursuit of higher yields, some farmers illegally use chemical growth regulators such as root-strengthening agents and plant growth promoters. While these agents can broadly increase root biomass, their effects lack specificity. While stimulating the growth of *Salvia miltiorrhiza*, they also promote weed growth and significantly reduce the relative content of active ingredients. Furthermore, this practice seriously violates the quality and safety requirements for medicinal materials stipulated in the *Good Manufacturing Practices for Chinese Medicinal Herbs*. Most existing growth regulators are broad-spectrum, having similar growth-promoting effects on both crops and weeds, and generally causing a decrease in active ingredients, making them unsuitable for the standardized production of Chinese medicinal herbs where strict quality standards are required.

[0004] In the field of biotechnology, although the molecular mechanism of Danshen quality formation has been studied in depth and the biosynthetic pathway of active ingredients has been basically elucidated, there is a lack of safe and effective technologies that can be directly applied to field production and achieve precise quality control. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a Danshen endogenous short peptide composition that promotes the improvement of Danshen production performance through synergistic effects; the second purpose is to provide the application of this composition.

[0006] Technical solution: The tanshinone endogenous short peptide composition of the present invention contains tanshinone endogenous short peptides miPEP167b-5p and miPEP171b-1 in a concentration ratio of 0.8-1.2, wherein the amino acid sequence of miPEP167b-5p is MACPYPPPNRSSFGLHQTTNSICVLVFLV; and the amino acid sequence of miPEP171b-1 is MPFSSEKSIAYP.

[0007] Preferably, the composition further contains a surfactant; more preferably, the surfactant is a nonionic surfactant, polysorbate-20.

[0008] The application of the endogenous short peptide composition of Danshen described in this invention in improving the production performance of Danshen.

[0009] Preferably, the production performance includes the root biomass and content of active ingredients in *Salvia miltiorrhiza*; more preferably, the content of active ingredients is tanshinone B, rosmarinic acid, tanshinone I, and tanshinone II. A The content of one or more of tanshinone, caffeic acid, and p-coumaric acid.

[0010] Preferably, the application steps include: spraying the tanshinone endogenous short peptide composition onto tanshinone plants during the critical growth period.

[0011] Preferably, the critical growth period is any one of the following: mid-to-late March, mid-April (greening period), mid-May (flowering period), or early September.

[0012] Preferably, the spraying method is foliar spraying, spraying 1-2 times per week for 2-4 consecutive weeks.

[0013] Preferably, the concentrations of miPEP167b-5p and miPEP171b-1 in the tanshinone endogenous short peptide composition are both 0.5-1.5 μM.

[0014] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: 1. The miPEP167b-5p and miPEP171b-1 used in this composition are endogenous short peptides of Salvia miltiorrhiza, which have high species specificity and only target and regulate the growth, development, and metabolism of Salvia miltiorrhiza itself, without promoting the growth of other plants in the field, thus avoiding the ecological risks brought by traditional broad-spectrum growth regulators; 2. This composition does not involve genetically modified organisms or chemically synthesized pesticides, has no chemical pesticide residues, and meets the requirements of green agriculture and the "Good Manufacturing Practice for Chinese Medicinal Herbs"; 3. The application of this composition can achieve a synergistic improvement in yield and quality. Field trial results show that the application of this composition... After treatment with the compound, the root yield of Salvia miltiorrhiza increased by 56% and the number of roots increased by 435% compared with the control. It not only significantly increased the biomass of the root system of individual plants, but also significantly promoted the development of lateral roots and improved the root morphology, directly and effectively solving the problem of low yield of Salvia miltiorrhiza. At the same time, the content of rosmarinic acid in Salvia miltiorrhiza increased by 43%, the content of salvianolic acid B increased by 81%, the content of tanshinone I increased by 214%, and the content of tanshinone IIA increased by 75% compared with the control, comprehensively and significantly improving the quality of Salvia miltiorrhiza. In the same technical solution, a leapfrog improvement in the yield and quality of Salvia miltiorrhiza was achieved simultaneously, providing reliable technical support for the green, efficient and high-quality development of the Salvia miltiorrhiza industry. Attached Figure Description

[0015] Figure 1 Statistical chart showing the results of determining the content of active ingredients in Salvia miltiorrhiza hairy roots after treatment with different concentrations of miPEP167b-5p.

[0016] Figure 2 Statistical chart showing the results of determining the content of active ingredients in Salvia miltiorrhiza hairy roots after treatment with different concentrations of miPEP171b-1;

[0017] Figure 3 Appearance (A) and morphological index statistics (BG) of potted Salvia miltiorrhiza plants after treatment with endogenous short peptides.

[0018] Figure 4 Statistical chart showing the results of the determination of the content of medicinal components in potted Salvia miltiorrhiza plants after treatment with endogenous short peptides.

[0019] Figure 5 Image of the root system of *Salvia miltiorrhiza* after field spraying with endogenous short peptide composition;

[0020] Figure 6 Statistical chart of morphological indicators of Salvia miltiorrhiza root system after field spraying of Salvia miltiorrhiza endogenous short peptide composition;

[0021] Figure 7 A statistical chart showing the results of the determination of the content of medicinal components in the roots of *Salvia miltiorrhiza* after field spraying of the endogenous short peptide composition. Detailed Implementation

[0022] The technical solution of the present invention will be further described below.

[0023] Example 1: Preparation of endogenous short peptides from Tanshinone

[0024] Based on the amino acid sequence of miPEP167b-5p: MACPYPPPNRSSFGLHQTTNSICVLVFLV; and the amino acid sequence of miPEP171b-1: MPFSSEKSIAYP, Nanjing Genscript Biotech Co., Ltd. was commissioned to synthesize the above miPEPs artificially, obtaining the Danshen endogenous short peptides miPEP167b-5p and miPEP171b-1 powder with a purity >95%, which were used for subsequent experiments.

[0025] Example 2: Verification of the hairy root level of how endogenous short peptides from Tanshinone promote the synthesis of active ingredients in Tanshinone.

[0026] 0.5 g of *Salvia miltiorrhiza* hairy roots were subcultured in a 150 mL Erlenmeyer flask containing 75 mL of MS liquid medium and cultured in a constant temperature shaker at 25℃ and 110 rpm for 18 days before being used in experiments. miPEP167b-5p and miPEP171b-1 powders were dissolved in sterile water to prepare 2 mM stock solutions, which were further diluted to 0, 0.1, 0.2, 0.5, 1.0, and 2.0 μM, for a total of six concentration gradients, to treat *Salvia miltiorrhiza* hairy roots. Samples were collected after 8 days of treatment.

[0027] The fresh weight of the hairy roots was weighed, dried, and pulverized. The content of active ingredients was determined by high performance liquid chromatography (HPLC) according to the method described in "Zhang Shuncang et al., Chinese Journal of Traditional Chinese Medicine, 2011, 36 (10):1269-1274". The active ingredients included: rosmarinica acid, salvianolic acid B, tanshinol, caffeic acid, p-coumaric acid, and tanshinone II. A (Tanshinone II A ).

[0028] The results are as follows Figure 1 and Figure 2 As shown, compared with the control group, 0.5 μM and 1.0 μM miPEP171b-1 significantly increased the content of various phenolic acids and tanshinone compounds in hairy roots. miPEP167b-5p also showed a positive effect in promoting component accumulation. This example demonstrates the bioactivity of the selected miPEPs at the in vitro level.

[0029] Example 3: Pot-scale verification of the regulation of the production performance of tanshinone plants by endogenous short peptides in tanshinone.

[0030] Salvia miltiorrhiza seedlings aged 60 days were treated with 0.5 μM and 1.0 μM miPEP167b-5p and miPEP171b-1, respectively (plant culture conditions: Salvia miltiorrhiza seeds germinated on moist filter paper, and after 15 days of germination, they were transferred to nutrient pots (16 cm inner diameter, 19 cm height) for culture at 25℃, with a light condition of 16 h light / 8 h darkness). Treatments were applied by foliar spraying, with 0.01% polysorbate-20 (Tween 20) added as an adjuvant to all treatment groups. During spraying, the leaves were ensured to be evenly moistened on both sides until just enough to drip. Water (with 0.01% Tween 20 added) served as the control group (CK), and each treatment was replicated three times. Sprayed once a week for 3 weeks, followed by 30 days of continued growth. Samples were collected to analyze plant height, leaf number, fresh weight of above-ground portion, fresh weight of root, number of primary lateral roots, and root length. High-performance liquid chromatography (HPLC) was used to determine the levels of rosmarinica acid, salvianolic acid B, tanshinone I, and tanshinone II.A (TanshinoneII A The content of ).

[0031] The results are as follows Figure 3 and Figure 4 As shown, miPEP167b-5p significantly promoted the growth and root development of *Salvia miltiorrhiza*. The fresh root weight and lateral root number in the 1.0 μM miPEP167b-5p treatment group were 1.42 times and 1.39 times higher, respectively. miPEP171b-1, besides significantly increasing plant height, had no effect on other morphological parameters. 1.0 μM miPEP167b-5p simultaneously promoted the accumulation of phenolic acids and tanshinone compounds. The contents of rosmarinic acid, salvianolic acid B, tanshinone I, and tanshinone IIA in the treatment group were 1.52 times, 1.25 times, 1.65 times, and 1.44 times higher than in the control group, respectively. Compared with 1.0 μM miPEP167b-5p, 1.0 μM miPEP171b-1 showed a stronger induction effect on component accumulation. The contents of the four components in the treatment group were 2.38 times, 1.60 times, 2.05 times, and 1.84 times that in the control group, respectively. These results indicate that miPEP167b-5p can increase the content of effective components while promoting the root development of Salvia miltiorrhiza, while miPEP171b-1 mainly plays a role in regulating the biosynthesis of effective components.

[0032] Example 4: Field verification of the effect of the endogenous short peptide composition of Salvia miltiorrhiza on the regulation of Salvia miltiorrhiza production performance

[0033] Salvia miltiorrhiza seedlings were transplanted in mid-March, planted on raised beds with a height of 25 cm and a width of 45 cm, and a plant spacing of 25 cm × 30 cm. Treatment groups included a 1.0 μM miPEP167b-5p treatment group (167b), a 1.0 μM miPEP171b-1 treatment group (171b), and a mixture of 1.0 μM each of miPEP167b-5p and miPEP171b-1 (Mix). Water was used as the control group (CK). Each treatment was replicated three times.

[0034] To determine the optimal application window, spraying was conducted during four key growth periods: mid-to-late March, mid-April (greening stage), mid-May (flowering stage), and early September. Each period involved weekly spraying for three consecutive weeks. All treatment groups included 0.01% polysorbate-20 (Tween 20) as an adjuvant. Spraying was performed on sunny, windless days before 10:00 AM or after 4:00 PM, ensuring even wetting of both sides of the leaves until just enough to cause droplets to fall.

[0035] Salvia miltiorrhiza was harvested manually in early October. The roots were washed with clean water and drained. Root fresh weight, root length, lateral root number, and root diameter were measured. A portion of the root sample was dried in an oven at 45℃ to constant weight. The contents of rosmarinica acid, salvianolic acid B, tanshinone I, and tanshinone IIA were determined using high-performance liquid chromatography (HPLC).

[0036] The results are as follows Figure 5 , Figure 6 and Figure 7 As shown, the timing of spraying significantly affects the effect. Treatment during the greening period in mid-April has the most prominent effect on promoting root development and accumulation of effective components, while treatment in early September has the worst effect because the roots have entered a period of slow growth. Among the treatments during the greening period, the treatment group with the endogenous short peptide composition of tanshinone showed a synergistic effect with excellent results, increasing the fresh weight of roots and the number of roots to 1.56 times and 5.35 times that of the control group, respectively. Moreover, the root color of this group and the miPEP171b-1 treatment group was more reddish, which directly indicates that the accumulation of tanshinone components was richer. HPLC analysis further confirmed that the treatment group with the endogenous short peptide composition of tanshinone had the most comprehensive and significant effect on quality improvement during the greening period, with the contents of rosmarinic acid, salvianolic acid B, tanshinone I, and tanshinone IIA increasing to 1.43 times, 1.81 times, 3.14 times, and 1.75 times that of the control group, respectively.

[0037] In summary, spraying the leaves of *Salvia miltiorrhiza* with a 1.0 μM mixture of miPEP167b-5p and miPEP171b-1 once a week during the regrowth period (mid-April) for three consecutive weeks can significantly improve the production performance of *Salvia miltiorrhiza*, including the yield of the medicinal material and the content of the core effective components.

Claims

1. A composition of endogenous short peptides from Tanshinone, characterized in that, The composition contains the endogenous short peptides miPEP167b-5p and miPEP171b-1 from tanshinone, in a concentration ratio of 0.8-1.

2.

2. The tanshinone endogenous short peptide composition according to claim 1, characterized in that, The composition also contains a surfactant.

3. The Tanshinone endogenous short peptide composition according to claim 2, characterized in that, The surfactant is a nonionic surfactant.

4. The application of the endogenous short peptide composition of Danshen according to any one of claims 1-3 in improving the production performance of Danshen.

5. The application according to claim 4, characterized in that, The production performance includes the root biomass and content of active ingredients in Salvia miltiorrhiza.

6. The application according to claim 5, characterized in that, The effective ingredients are salvianolic acid B, rosmarinic acid, tanshinone I, and tanshinone II. A The content of one or more of tanshinone, caffeic acid, and p-coumaric acid.

7. The application according to claim 4, characterized in that, The application steps include: spraying the endogenous short peptide composition of Salvia miltiorrhiza onto the Salvia miltiorrhiza plants during the critical growth period.

8. The application according to claim 7, characterized in that, The critical growth period is any period in late March, mid-April (greening period), mid-May (flowering period), or early September.

9. The application according to claim 7, characterized in that, The spraying method is foliar spraying, spraying 1-2 times a week for 2-4 consecutive weeks.

10. The application according to claim 4, characterized in that, The concentrations of miPEP167b-5p and miPEP171b-1 in the tanshinone endogenous short peptide composition are both 0.5-1.5 μM.

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