Application of choline chloride in improving medicinal quality of rhizoma alismatis

Abstract

The invention discloses application of choline chloride in improving the medicinal quality of rhizoma alismatis. The treatment concentration of choline chloride is in the range of 40-5000 mg / L, the choline chloride and the rhizoma alismatis tubers have the effect of remarkably increasing the content of the effective components 23-acetyl alisol B and 23-acetyl alisol C of the rhizoma alismatis tubers, and the effect of increasing the content of the effective components of the rhizoma alismatis tubers by 1000 mg / L of choline chloride is most remarkable. Compared with a control group, the content of 23-acetyl alisol B in rhizoma alismatis tubers is increased by 41.46%, the content of 23-acetyl alisol C is increased by 54.05%, and the total content of effective components of rhizoma alismatis is increased by 42.31% by spraying 1000 mg / L choline chloride on leaf surfaces, so that the medicinal quality of rhizoma alismatis is remarkably improved.

Description

Technical Field

[0001] This invention belongs to the field of plant growth regulator technology, specifically relating to a plant growth regulator for improving the medicinal quality of Alisma plantago-aquatica. Background Technology

[0002] Alisma plantago-aquatica is a herbaceous plant belonging to the genus Alisma in the family Alismataceae. Its dried tubers are used medicinally, possessing diuretic, heat-clearing, turbidity-resolving, and lipid-lowering effects. It is commonly used to treat diseases such as urinary difficulty, edema, phlegm retention and dizziness, and hyperlipidemia. With increasing market demand, Alisma plantago-aquatica is being cultivated on a continuously expanding scale due to its short growth cycle, simple planting and management, and good economic benefits.

[0003] However, the current standardized cultivation technology system for Alisma plantago-aquatica is still imperfect, resulting in inconsistent medicinal quality and seriously hindering the healthy development and quality improvement of the Alisma plantago-aquatica industry. The medicinal quality of Alisma plantago-aquatica mainly depends on the content of active ingredients in its tubers. The Chinese Pharmacopoeia (2025 edition) clearly stipulates that the total content of 23-acetylalisenoside B and 23-acetylalisenoside C should be used as the quality control standard, requiring it to be no less than 0.10%. Therefore, optimizing cultivation measures to stabilize and increase the content of the main active ingredients in Alisma plantago-aquatica is crucial for ensuring the quality of medicinal materials and promoting industrial upgrading.

[0004] Among various cultivation optimization measures, the application of plant growth regulators is one of the effective means to regulate the synthesis and accumulation of secondary metabolites (i.e., active ingredients) in medicinal plants. Choline chloride is a common plant growth regulator, known to promote growth and enhance stress resistance in various crops. However, a search of domestic and international literature and patents has not yielded any research or technical reports on increasing the content of 23-acetylalisenoside B and 23-acetylalisenoside C in Alisma plantago-aquatica tubers through foliar spraying of choline chloride. Summary of the Invention

[0005] This invention provides the application of choline chloride in improving the medicinal quality of Alisma plantago-aquatica. Foliar spraying of choline chloride can increase the content of effective components (23-acetylalisenoside B and 23-acetylalisenoside C) in Alisma plantago-aquatica tubers, thereby improving the medicinal quality of Alisma plantago-aquatica.

[0006] Choline chloride was used to improve the medicinal quality of Alisma plantago-aquatica: Compared with the control, treatment with 40 mg / L choline chloride increased the content of active ingredients (23-acetylalisenoside B and 23-acetylalisenoside C) in Alisma plantago-aquatica tubers. The effect of choline chloride on increasing the content of active ingredients in Alisma plantago-aquatica tubers gradually increased with increasing concentrations. Choline chloride treatment at concentrations ranging from 200 to 1000 mg / L significantly increased the content of active ingredients in Alisma plantago-aquatica tubers, with 1000 mg / L choline chloride showing the most significant effect. Compared with the control, foliar spraying with 1000 mg / L choline chloride increased the content of 23-acetylalisenoside B in Alisma plantago-aquatica tubers by 41.46%, the content of 23-acetylalisenoside C by 54.05%, and the total content of active ingredients (23-acetylalisenoside B and 23-acetylalisenoside C) by 42.31%.

[0007] The lack of standardized cultivation techniques for Alisma plantago-aquatica has led to inconsistent medicinal quality. Foliar application of choline chloride can increase the content of active ingredients (23-acetylalisolicol B and 23-acetylalisolicol C) in the tubers, thus improving the medicinal quality of Alisma plantago-aquatica. Foliar application of choline chloride is an ideal method for improving the medicinal quality of Alisma plantago-aquatica due to its good efficacy and simple, quick operation. Attached Figure Description

[0008] Figure 1 HPLC chromatograms of 23-acetylalisinol B in different treatment groups. Figure 1 In the chromatograms, A is the HPLC chromatogram of 23-acetylalizool B standard, B is the HPLC chromatogram of 23-acetylalizool B treated with control (H2O), C is the HPLC chromatogram of 23-acetylalizool B treated with 40 mg / L choline chloride, D is the HPLC chromatogram of 23-acetylalizool B treated with 200 mg / L choline chloride, E is the HPLC chromatogram of 23-acetylalizool B treated with 1000 mg / L choline chloride, and F is the HPLC chromatogram of 23-acetylalizool B treated with 5000 mg / L choline chloride.

[0009] Figure 2 HPLC chromatograms of 23-acetylalisinol C in different treatment groups. Figure 2In the chromatograms, A is the HPLC chromatogram of 23-acetylalizool C standard, B is the HPLC chromatogram of 23-acetylalizool C sample treated with control (H2O), C is the HPLC chromatogram of 23-acetylalizool C sample treated with 40 mg / L choline chloride, D is the HPLC chromatogram of 23-acetylalizool C sample treated with 200 mg / L choline chloride, E is the HPLC chromatogram of 23-acetylalizool C sample treated with 1000 mg / L choline chloride, and F is the HPLC chromatogram of 23-acetylalizool C sample treated with 5000 mg / L choline chloride. Detailed Implementation

[0010] Examples 1-4: The effect of choline chloride treatment on improving the medicinal quality of Alisma plantago-aquatica. A certain mass of choline chloride was dissolved in water to prepare choline chloride treatment solutions of different concentrations (40 mg / L, 200 mg / L, 1000 mg / L, 5000 mg / L), corresponding to Examples 1-4. Using *Alisma plantago-aquatica* from Honghu Lake as the experimental material, at the early stage of tuber enlargement, the choline chloride treatment solutions of different concentrations were sprayed onto the leaves using a sprayer; the control treatment was sprayed with H2O. Spraying was performed once every 8 days, for a total of 3 sprays. After each spraying, the leaves of *Alisma plantago-aquatica* were kept moist (but without obvious droplets). After the *Alisma plantago-aquatica* matured, the tubers were taken to determine the content of its effective components (23-acetylalisenoside B and 23-acetylalisenoside C). The specific detection methods and conditions are as follows: (1) Preparation of standard solutions Weigh 10 mg of 23-acetylalizool B (CAS No.: 26575-95-1) and 23-acetylalizool C (CAS No.: 26575-93-9) standards respectively, dissolve them in chromatographic grade acetonitrile, and dilute to 10 mL to obtain stock solutions of 23-acetylalizool B and 23-acetylalizool C standards with a concentration of 1000.00 μg / mL. Serially dilute the stock solutions of 23-acetylalizool B and 23-acetylalizool C standards to 500.00, 250.00, 125.00, 62.50, and 31.25 μg / mL, respectively.

[0011] (2) Detection conditions The instrument used was an Agilent 1260 high-performance liquid chromatograph; the column model was Kromasil 100-5-C18 (4.6×250 mm, Ф5 μm); the mobile phase was as shown in Table 1.

[0012] Table 1 Mobile phase conditions for high performance liquid chromatography 23-Acetylalizool B was detected at a wavelength of 208 nm; 23-Acetylalizool C was detected at a wavelength of 246 nm; flow rate: 1 mL / min; injection volume: 20 μL; column temperature: 25℃.

[0013] (3) Preparation of standard curve Standards of 23-acetylalisolicol B and 23-acetylalisolicol C were prepared into 1 mg / mL stock solutions, serially diluted, and analyzed under their respective chromatographic conditions. Peak areas were measured. Regression analysis was performed with the concentrations of 23-acetylalisolicol B and 23-acetylalisolicol C as the abscissa (X) and peak areas as the ordinate (Y), respectively. Standard curves for the two active ingredients, 23-acetylalisolicol B and 23-acetylalisolicol C, were plotted, and the corresponding regression equations were obtained. The linear range, retention time, regression equation, and coefficient of determination for the HPLC analysis of the two active ingredients are shown in Table 2. The results in Table 2 show that the two active ingredients have good linearity within their respective ranges.

[0014] Table 2. Linear range, retention time, regression equation, and coefficient of determination of effective components of Alisma plantago-aquatica by HPLC analysis. (4) Preparation and detection of test sample solutions a) Wash the Alisma plantago-aquatica tubers with clean water, air dry naturally, then sterilize in a constant temperature drying oven at 105℃ for 30 minutes, and dry at 60℃ until constant weight. Grind the powder using a Chinese herbal medicine pulverizer and pass it through a 60-mesh sieve. Pack the resulting powder into a resealable bag and store it in a desiccator.

[0015] (b) Referring to the Chinese Pharmacopoeia (2025, Part I), accurately weigh 0.5 g of the sieved sample, add 25 ml of acetonitrile, weigh again, and sonicate (250 W, 50 kHz) for 30 min. After cooling, make up the weight with acetonitrile, shake well, and filter. Pipette 1 mL of the filtrate into a 10 mL volumetric flask, add 70% methanol to the mark, and shake well. Filter through a 0.45 μm pore size filter membrane and store in a brown liquid chromatography vial at -20°C.

[0016] c) The sample solution was tested according to the method used to prepare the standard curve. The peak areas of the two active ingredients, 23-acetylalizool B and 23-acetylalizool C, were counted respectively. Then, the peak areas of the two active ingredients were substituted into the corresponding regression equations to calculate the contents of 23-acetylalizool B and 23-acetylalizool C.

[0017] Table 3. Effects of choline chloride treatment on the content of 2,3-acetylalizool B and 2,3-acetylalizool C in Alisma plantago-aquatica tubers. The HPLC chromatograms of 2,3-acetylalisone B and 2,3-acetylalisone C are shown below. Figure 1 and Figure 2 As shown in Table 3, the specific contents of choline chloride treatment can increase the content of 2,3-acetylalipurol B and 2,3-acetylalipurol C in Alisma plantago-aquatica tubers, thereby improving the medicinal quality of Alisma plantago-aquatica. Even at a low concentration of choline chloride treatment (40 mg / L), choline chloride can still increase the content of 2,3-acetylalipurol B and 2,3-acetylalipurol C in Alisma plantago-aquatica tubers. With the increase of choline chloride treatment concentration, the effect of choline chloride on increasing the content of 2,3-acetylalipurol B and 2,3-acetylalipurol C in Alisma plantago-aquatica tubers is enhanced, but excessively high concentrations of choline chloride treatment will weaken its effect on increasing the content of 2,3-acetylalipurol B and 2,3-acetylalipurol C in Alisma plantago-aquatica tubers. Choline chloride treatment can improve the medicinal quality of Alisma plantago-aquatica. Compared with the control, choline chloride treatment increased the content of 23-acetylalipurol B in Alisma plantago-aquatica tubers by 7.32%–41.46%, and the content of 23-acetylalipurol C by 12.16%–54.05%. The total content of the effective components (23-acetylalipurol B and 23-acetylalipurol C) of Alisma plantago-aquatica increased by 7.64%–42.31%.

Claims

1. Application of choline chloride in increasing the content of 2,3-acetylalizool B and 2,3-acetylalizool C, the effective components of Alisma plantago-aquatica.

2. The application according to claim 1, characterized in that, Choline chloride is applied by foliar spraying.

3. The application according to claim 2, characterized in that, The concentration of choline chloride used is 40-5000 mg / L.

4. The application according to claim 3, characterized in that, The concentration of choline chloride used is 200-1000 mg / L.

5. A method for increasing the content of the effective components of Alisma plantago-aquatica, 2,3-acetylalisenoside B and 2,3-acetylalisenoside C, characterized in that: During the early stage of tuber enlargement of Alisma plantago-aquatica, choline chloride was sprayed on the leaves at a concentration of 40-5000 mg / L.

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