Vietnamese sophora root decoction process based on component dynamic change and activity correlation and application

Abstract

The invention discloses a vietnamese sophora root decoction process based on component dynamic change and activity correlation and application, and belongs to the technical field of traditional Chinese medicine processing process optimization. The process disclosed by the invention comprises the following steps: S1, decocting subprostrate sophora at different times to obtain samples at different decoction times; s2, detecting the components of the sample at different decoction times; s3, detecting the anti-inflammatory activity of the sample at different decoction times; and S4, analyzing the correlation between the sample component content and the sample anti-inflammatory activity at different decoction times, determining the appropriate decoction time, and obtaining the decoction according to the decoction time. The invention finds that the contents of seven index components in the vietnamese sophora root change along with the decoction time and have a certain correlation with the anti-inflammatory activity, and by taking the index components as a reference, the toxicity and the anti-inflammatory activity can be balanced, and a scientific basis is provided for clinical reasonable application of'toxicity reduction and effect storage 'of the vietnamese sophora root.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine processing technology, specifically relating to a decoction process and application of Sophora tonkinensis root based on the dynamic changes of components and their activity correlation. Background Technology

[0002] Sophora tonkinensis Gagnep. is the dried root and rhizome of Sophora tonkinensis, a plant in the Fabaceae family. It is a commonly used traditional Chinese medicine for clearing heat and detoxifying, and has the effects of relieving sore throat and swelling, anti-inflammation, and immune regulation.

[0003] Pharmacological studies have shown that Sophora tonkinensis root possesses multiple pharmacological activities, including anti-inflammatory, anti-tumor, immunomodulatory, hepatoprotective, cardiovascular protective, and hypoglycemic effects. However, literature indicates that Sophora tonkinensis root exhibits a "drug-toxicity coexistence" characteristic; excessive use or improper decoction can easily induce neurotoxicity (such as dizziness and respiratory depression). Therefore, the decoction time may have a significant impact on the content and activity of the effective components in Sophora tonkinensis root.

[0004] Currently, only the daily dosage of Sophora tonkinensis root is specified as 3-6g, but the decoction process parameters (such as decoction time) are not clearly defined. Existing research mainly focuses on the extraction of Sophora tonkinensis root components and its pharmacological effects, lacking research on the "dynamic changes of components and their correlation with activity" during the decoction process. This results in a lack of scientific guidance for clinical decoction processes, leading to insufficient efficacy or toxicity risks. Therefore, as a typical Chinese medicinal herb with both medicinal and toxic properties, the efficacy and toxicity of Sophora tonkinensis root are closely related to its alkaloids and flavonoids. This patent's research on a decoction time control process and its application based on the dynamic changes of components and their correlation with activity is of great significance for the rational clinical application of Sophora tonkinensis root. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a decoction process and application of Sophora tonkinensis root based on the dynamic changes of components and their correlation with activity. This invention found that the content of seven indicator components in Sophora tonkinensis root (oxymatrine, oxysophoraine, matrine, sophoraine, pterostilbene, sophoridine, and cytisine) changes with the decoction time. At the same time, there is a certain correlation between the above indicator components and the anti-inflammatory activity of the Sophora tonkinensis root decoction. Using the above indicator components as a reference, toxicity and anti-inflammatory activity can be balanced. Finally, it was found that the decoction time of 0.5-2.5 hours has good anti-inflammatory activity and low toxicity.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides a decoction process for Sophora tonkinensis root based on the correlation between dynamic changes in components and activity, comprising the following steps:

[0008] S1. Sophora tonkinensis was decocted for different times to obtain samples with different decoction times;

[0009] S2. Detect the components of samples under different decoction times;

[0010] S3. The anti-inflammatory activity of samples under different decoction times was detected;

[0011] S4. Conduct correlation analysis between the content of sample components and the anti-inflammatory activity of the sample at different decoction times, determine the appropriate decoction time, and obtain the decoction based on the decoction time.

[0012] Preferably, the different simmering times in S2 are 0.5-5h.

[0013] Preferably, the sample components in S2 include cytisine, oxymatrine, oxysodium, matrine, sophoridine, trifolin, and sophoridine.

[0014] Preferably, the correlation analysis in S4 uses Pearson correlation analysis to evaluate IC. 50 The interdependence of values ​​on the components of Sophora tonkinensis samples with different decoction times.

[0015] This invention provides the application of the above-mentioned Sophora tonkinensis decoction process in the preparation of Sophora tonkinensis extract with low toxicity and high anti-inflammatory activity.

[0016] As a preferred method, the specific method for preparing a low-toxicity, high-anti-inflammatory Sophora tonkinensis root extract is as follows:

[0017] Mix Sophora tonkinensis powder with purified water, stir well and decoct. Cool to room temperature, centrifuge and collect the supernatant to obtain Sophora tonkinensis decoction. Take Sophora tonkinensis decoction, add an equal volume of dichloromethane-ammonia solution and shake well. Extract and separate the layers, collect the dichloromethane layer, and evaporate it to dryness by rotary evaporation. Add an appropriate amount of methanol to dissolve the residue to obtain the test solution.

[0018] Preferably, the Sophora tonkinensis root powder has a mesh size of 30-60 mesh.

[0019] Preferably, the mass ratio of the Sophora tonkinensis root powder to purified water is 1:(30-50).

[0020] Preferably, the decocting time is 0.5-2.5 hours.

[0021] Preferably, the volume ratio of dichloromethane to ammonia is 100:1.

[0022] It contains at least the following beneficial technical effects:

[0023] This invention provides a decoction process for Sophora tonkinensis root based on the correlation between "decoction time-components-activity". Using Sophora tonkinensis root powder as raw material, it is mixed with water at a specific material-liquid ratio and decocted for different durations to obtain Sophora tonkinensis root extracts with varying decoction times. The results show that the content of seven indicator components (oxymatrine, oxysophoraine, matrine, sophoraine, pterostilbene, sophoridine, and cytisine) in Sophora tonkinensis root changes with decoction time. Furthermore, these indicator components are correlated with the anti-inflammatory activity of the Sophora tonkinensis root decoction. Using these indicator components as a reference, toxicity and anti-inflammatory activity can be balanced. Ultimately, it was found that decoction times of 0.5-2.5 hours exhibit better anti-inflammatory activity and lower toxicity. This process is simple, easy to operate, and low in cost, improving the safety and efficacy of Sophora tonkinensis root in clinical use and providing a scientific basis for the rational clinical application of Sophora tonkinensis root with "reduced toxicity and preserved efficacy". Attached Figure Description

[0024] Figure 1 HPLC overlay chromatograms of Sophora tonkinensis samples and mixed reference standards at different decoction times.

[0025] Figure 2 The graph shows the trend of the content of 7 indicator components with the decoction time.

[0026] Figure 3 Correlation analysis of the main components of Sophora tonkinensis and its anti-inflammatory effects at different decoction times. Detailed Implementation

[0027] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. The invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the invention in any way.

[0028] Example 1

[0029] Take Sophora tonkinensis root, pulverize it and pass it through a 60-mesh sieve. Mix the Sophora tonkinensis root powder with purified water at a ratio of 1:30 (g / mL), stir well, and decoct for different times (0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 h). After cooling to room temperature, centrifuge and collect the supernatant to obtain Sophora tonkinensis root decoction. Take the Sophora tonkinensis root decoction, add an equal volume of dichloromethane-ammonia water (100:1, v / v) solution and shake well. Extract and separate the layers, collect the dichloromethane layer, and evaporate it to dryness by rotary evaporation. Add an appropriate amount of methanol to dissolve the residue to obtain the test solution.

[0030] Experimental Example 1

[0031] (1) Plotting the standard curve

[0032] Accurately weigh appropriate amounts of oxymatrine, matrine, cytisine, sophoridine, oxysophoridine, pterostilbene, and sophoridine reference standards, dissolve them in methanol, and prepare mixed reference solutions containing 1.00 mg oxymatrine, 1.20 mg matrine, 1.00 mg cytisine, 0.10 mg sophoridine, 0.20 mg oxysophoridine, 0.50 mg pterostilbene, and 0.25 mg sophoridine per 1 mL.

[0033] The mixed reference solution was determined using high-performance liquid chromatography (HPLC) under the following chromatographic conditions: Agilent ZORBAX Bonus-RP C 18 (4.6×250mm, 5μm) chromatographic column; mobile phase: acetonitrile (A)-0.1% phosphoric acid, 0.1% triethylamine (adjusted to pH=7 with triethylamine) (B), gradient elution (0–21 min, 5%–22% A; 21–26 min, 22%–26% A; 26–39 min, 26%–41% A; 39–45 min, 41%–46% A; 45–55 min, 46%–58% A; 55–60 min, 58%–80% A); flow rate: 0.8 mL / min; detection wavelength: 220 nm; column temperature: 35℃; injection volume: 10 μL.

[0034] Accurately pipette 2, 5, 10, 15, 20, and 25 μL of the mixed reference solution and inject them into the HPLC system for analysis. Record the peak areas of the seven components. Plot a standard curve with the injection volume (μg) of each compound as the x-axis and the peak area as the y-axis. Perform linear regression to obtain the regression equation. The results are shown in Table 1. The correlation coefficients of the seven components were all greater than 0.9960, indicating a good linear relationship.

[0035] Table 1. Linear regression equations and linear ranges for the seven components.

[0036]

[0037] (2) The test solution from Example 1 was analyzed by HPLC in step (1), and the results are as follows: Figure 1 As shown; substituting the peak area of ​​the measured results into the standard curve equation of step (1), the contents of the seven index components (cytisine, oxymatrine, oxysophoridine, matrine, sophoridine, trifolin stigmacin, and sophoridine) were calculated, and the results are shown in Table 2 and Figure 2 As shown.

[0038] Table 2. Content of 7 components in Sophora tonkinensis at different decoction times

[0039]

[0040]

[0041] Note: Different lowercase letters after the data in the same column indicate significant differences (P<0.05).

[0042] From Table 2 and Figure 2 It was found that the content of cytisine did not fluctuate significantly with increasing decoction time; the contents of oxymatrine and oxysodium increased slightly during decoction times of 0.5–2.5 h and 0.5–1.0 h, respectively, and then decreased with increasing decoction time; the contents of matrine, sosodium, and pterostilbene increased with increasing decoction time; the content of sophoridine increased continuously during decoction times of 0.5–2.5 h, reaching a maximum at 2.5 h, and then decreased continuously with continued decoction. The method provided by this invention found that, except for cytisine, the contents of the other six components changed significantly with different decoction times, which may be related to the degradation, transformation, or decreased stability of these components during the decoction process.

[0043] Experimental Example 2

[0044] (1) Samples of Sophora tonkinensis with different decoction times were prepared according to the method in Example 1. The obtained extracts were dried in an oven at 60°C and stored in a desiccant for later use. An appropriate amount of Sophora tonkinensis extract decocted for 0.5–2.0 h was dissolved in DMSO aqueous solution (20% DMSO, v / v) to prepare a stock solution of 18.96 mg / mL, which was then diluted sequentially to prepare test solutions of 14.22, 10.67, 8.00, 6.00, 4.50, and 3.38 mg / mL and stored at 4°C for later use. An appropriate amount of Sophora tonkinensis extract decocted for 2.5–5.0 h was dissolved in the buffer solution in the COX-2 kit to prepare a stock solution of 7.11 mg / mL, which was then diluted sequentially to prepare test solutions of 5.33, 4.00, 3.00, 2.25, and 1.69 mg / mL.

[0045] (2) Determination of in vitro anti-inflammatory activity of Sophora tonkinensis samples with different decoction times

[0046] The COX-2 inhibitor screening kit was operated according to the instructions. Under light-protected conditions, the fluorescence intensity of the samples was measured using a microplate reader with an excitation wavelength of 560 nm and an emission wavelength of 590 nm. The average fluorescence value (RFU) of each sample well and blank control well was recorded. The inhibition rate of each sample at different concentrations was calculated using formula (1), and a regression curve was fitted to calculate the IC50. 50 The results are shown in Table 3.

[0047]

[0048] Table 3. IC50 of Sophora tonkinensis samples against COX-2 at different decoction times. 50

[0049]

[0050]

[0051] Table 3 shows the IC50 values ​​of the Sophora tonkinensis samples. 50 The IC50 value decreased with increasing decoction time, with the IC50 value of the Sophora tonkinensis sample decocted for 5.0 h being the highest. 50 The lowest IC50 value was found in samples of Sophora tonkinensis root decocted for 0.5 hours. 50 Highest.

[0052] Experimental Example 3

[0053] Correlation analysis was conducted between the content of major components of Sophora tonkinensis root at different decoction times and its anti-inflammatory efficacy. Pearson correlation analysis was used to evaluate the IC50. 50 The interdependence between the value and the content of the main components of Sophora tonkinensis at different decoction times is shown in the figure. Figure 3 Analysis shows that IC 50 The levels of matrine, sophoridine, pteroside, kosmosiderin, and cytisine were all negatively correlated, indicating that the higher the levels of these components, the better the inhibitory effect. Among them, IC50 showed the highest levels. 50 The best correlation was observed with pterostilbene (r = -0.983), followed by matrine (r = -0.979), sophoridine (r = -0.969), and cytisine (r = -0.303), while the worst correlation was observed with sophoridine (r = -0.093). 50 The content of oxymatrine (r = 0.546) and oxysodium (r = 0.586) was positively correlated, indicating that the higher the content of these components, the worse the inhibitory effect.

[0054] In summary, the balance between the "content of major components and anti-inflammatory activity" of *Sophora tonkinensis* is highly dependent on the decoction time: moderately extending the decoction time can promote the conversion of oxidized alkaloids to reduced alkaloids (enhancing anti-inflammatory activity) and the dissolution of flavonoids (such as pterostilbene), but excessive decoction may increase the risk of toxicity (such as neurotoxicity) due to excessively high concentrations of reduced alkaloids, while also causing structural damage to some flavonoids (such as sophoridine). Therefore, targeted optimization of the decoction process (controlling the decoction time to ≤2.5 h) is necessary to reduce the risk of toxicity while increasing the content of effective substances and improving anti-inflammatory activity.

[0055] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A decoction process for Sophora tonkinensis root based on the correlation between dynamic changes in components and activity, characterized in that, Includes the following steps: S1. Sophora tonkinensis was decocted for different times to obtain samples with different decoction times; S2. Detect the components of samples under different decoction times; S3. The anti-inflammatory activity of samples under different decoction times was detected; S4. Conduct correlation analysis between the content of sample components and the anti-inflammatory activity of the sample at different decoction times, determine the appropriate decoction time, and obtain the decoction based on the decoction time.

2. As described in claim 1, characterized in that, The different decocting times in S2 are 0.5-5h.

3. As described in claim 1, characterized in that, The sample components in S2 include cytisine, oxymatrine, oxysodium, matrine, sophoridine, stigmataside, and sophoridine.

4. As described in claim 1, characterized in that, In S4, the correlation analysis uses Pearson correlation analysis to evaluate IC. 50 The interdependence of values ​​on the components of Sophora tonkinensis samples with different decoction times.

5. The application of the decoction process of Sophora tonkinensis root as described in claim 1 in the preparation of Sophora tonkinensis root extract with low toxicity and high anti-inflammatory activity.

6. The application according to claim 5, characterized in that, The specific method for preparing a low-toxicity, high-anti-inflammatory extract of Sophora tonkinensis root is as follows: Mix Sophora tonkinensis powder with purified water, stir well and decoct. Cool to room temperature, centrifuge and collect the supernatant to obtain Sophora tonkinensis decoction. Take Sophora tonkinensis decoction, add an equal volume of dichloromethane-ammonia solution and shake well. Extract and separate the layers, collect the dichloromethane layer, and evaporate it to dryness by rotary evaporation. Add an appropriate amount of methanol to dissolve the residue to obtain the test solution.

7. The application according to claim 6, characterized in that, The powder from Sophora tonkinensis has a mesh size of 30-60.

8. The application according to claim 6, characterized in that, The mass ratio of the Sophora tonkinensis root powder to purified water is 1:(30-50).

9. The application according to claim 6, characterized in that, The decoction time is 0.5-2.5 hours.

10. The application according to claim 6, characterized in that, The volume ratio of dichloromethane to ammonia is 100:1.

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