A one-pot extraction method of gynura divaricata compound composition and application thereof in reducing uric acid

Abstract

The application discloses a one-pot extraction method of a Gynura divaricata compound composition and application of the Gynura divaricata compound composition in reducing uric acid, and the Gynura divaricata, sophora flower and galangal are crushed, and are weighed and uniformly mixed according to proportions; the mixture is fully extracted by using an ultrasonic-assisted ethanol extraction method, and a filtrate is obtained by filtration; the filtrate is concentrated by a rotary evaporator under reduced pressure until no alcohol taste is left, an appropriate amount of distilled water is added, and a concentrated solution is obtained; the concentrated solution is freeze-dried, and is stored in a desiccator to obtain the Gynura divaricata compound composition. The Gynura divaricata compound composition prepared by the application has the advantages of simple and rapid preparation process, low cost, high yield, good inhibition activity on xanthine oxidase, and higher inhibition rate on xanthine oxidase activity than a separately extracted and compounded composition, and is expected to be applied to preparation of a xanthine oxidase inhibiting uric acid reducing drug.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine compound application technology, and in particular to a method for one-pot extraction of Panax notoginseng compound composition and its application in lowering uric acid. Background Technology

[0002] In recent years, the incidence of gout and hyperuricemia has been rising annually, showing a trend towards affecting younger people. Studies have shown that extracts from natural plants that are both food and medicine have good effects in treating gout and lowering uric acid. Panax notoginseng, as a new resource food, contains various active ingredients such as flavonoids, phenols, nitrogenous compounds, terpenes, fatty acids, and steroids, making it highly valuable for both food and medicinal purposes. Research has found that Panax notoginseng extract can lower uric acid levels and improve gout symptoms; extracts from Sophora japonica and Alpinia galanga have excellent inhibitory activity against xanthine oxidase, a key enzyme in uric acid production; the combination of these three can exert a synergistic effect in lowering uric acid. However, extracting the three plants separately and then combining them is cumbersome, costly, and difficult to industrialize. Summary of the Invention

[0003] The purpose of this invention is to provide a one-pot extraction method for a compound composition of Panax notoginseng and its application in lowering uric acid. The compound composition of Panax notoginseng provided by this invention has good uric acid-lowering activity, and the preparation method is simple, fast and low cost.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] This invention provides a one-pot extraction method for a compound composition of Panax notoginseng, comprising the following steps:

[0006] The dried Panax notoginseng, Sophora japonica flowers, and Alpinia galanga were pulverized, weighed and mixed according to the mass ratio of Panax notoginseng, Sophora japonica flowers, and Alpinia galanga powder, which was 0.5-2:1:1. The mixture was fully extracted using ultrasound-assisted ethanol extraction, and the filtrate was obtained by filtration. The filtrate was concentrated under reduced pressure using a rotary evaporator until no alcohol odor was detected, and an appropriate amount of distilled water was added to obtain a concentrated solution. The concentrated solution was freeze-dried and stored in a desiccator to obtain the Panax notoginseng compound composition.

[0007] Preferably, the vacuum concentration temperature is 40–60°C, the rotation speed is 40–60 rpm, and the vacuum degree is 40–100 bar.

[0008] Preferably, the particle size of the three raw material powders is less than 100 mesh; the Panax notoginseng is the leaf of Panax notoginseng.

[0009] Preferably, the volume fraction of ethanol in the ethanol extraction is 80-95%, and the ratio of the raw material mixture to the ethanol solution is 1g:(20-30)mL.

[0010] Preferably, the ultrasonic extraction conditions are: ultrasonic temperature 40-60℃, ultrasonic power 80-100%, and ultrasonic time 2-4h.

[0011] The present invention provides a compound composition of Panax notoginseng prepared by the above method.

[0012] This invention provides the application of the aforementioned Panax notoginseng compound composition in the preparation of drugs or additives that inhibit xanthine oxidase and lower uric acid.

[0013] This invention uses Panax notoginseng, Sophora japonica flowers, and Alpinia galanga as raw materials. A one-pot method is employed, involving ultrasonic-assisted ethanol-water extraction, filtration, vacuum concentration, and freeze-drying to prepare a Panax notoginseng compound composition. High-performance liquid chromatography (HPLC) was used to determine the index components of the obtained Panax notoginseng compound composition. The contents of chlorogenic acid, rutin, and galangin were similar to those of the separately extracted and compounded compositions. Xanthine oxidase activity assays were used to evaluate the inhibitory activity of the obtained Panax notoginseng compound composition against xanthine oxidase. The inhibition rate of xanthine oxidase activity was higher than that of the separately extracted and compounded compositions, suggesting its potential application in the preparation of drugs that inhibit xanthine oxidase and lower uric acid.

[0014] The preparation process of the compound composition of Panax notoginseng obtained by this invention is simple and rapid, with low cost, high yield, and good inhibitory activity against xanthine oxidase. Attached Figure Description

[0015] Figure 1 The HPLC data chromatograms of chlorogenic acid, rutin and galangin in Example 1 of the present invention are shown.

[0016] Figure 2 This is the HPLC data chromatogram of the separately extracted and then compounded composition in Example 1 of the present invention;

[0017] Figure 3 The HPLC data chromatogram of the compound composition of Panax notoginseng extracted by one-pot method in Example 1 of this invention;

[0018] Figure 4 The HPLC data chromatogram of the compound composition of Panax notoginseng extracted by one-pot method in Example 2 of the present invention;

[0019] Figure 5 The HPLC data chromatogram of the Panax notoginseng compound composition extracted by one-pot method in Example 3 of the present invention;

[0020] Figure 6 A graph showing the inhibition rate of xanthine oxidase (XOD) by alcohol extracts of Panax notoginseng, Sophora japonica, and Alpinia galanga;

[0021] Figure 7 Data graphs showing the XOD inhibition rate of the compounded combination of extracts of Panax notoginseng, Sophora japonica, and Alpinia galanga;

[0022] Figure 8 The data graph shows the inhibition rate of xanthine oxidase (XOD) on the compound composition of Panax notoginseng extracted by one-pot method in the embodiments of the present invention; Detailed Implementation

[0023] This invention provides a one-pot method for preparing a compound composition of Panax notoginseng, comprising the following steps:

[0024] The dried Panax notoginseng, Sophora japonica flowers, and Alpinia galanga were pulverized, weighed and mixed in proportion; the mixture was fully extracted by ultrasonic-assisted ethanol extraction and filtered to obtain filtrate; the filtrate was concentrated under reduced pressure using a rotary evaporator until no alcohol odor was detected, and an appropriate amount of distilled water was added to obtain a concentrated solution; the concentrated solution was freeze-dried and stored in a desiccator to obtain the Panax notoginseng compound composition.

[0025] Unless otherwise specified, all raw materials used in this invention are commercially available products well known in the art.

[0026] In this invention, a mixture of Panax notoginseng, Sophora japonica flowers, and Alpinia galanga powders is soaked in an ethanol aqueous solution for ultrasonic extraction, followed by filtration to obtain the extract.

[0027] In this invention, the particle size of the powders of Panax notoginseng, Sophora japonica, and Alpinia galanga is preferably less than 100 mesh; the Panax notoginseng refers to the leaves of Panax notoginseng.

[0028] In this invention, the volume fraction of ethanol in the aqueous ethanol solution is preferably 80-95%, more preferably 85-90%, and specifically 95% in the embodiments of this invention. In this invention, the preferred ratio of the mixture of Panax notoginseng, Sophora japonica, and Alpinia galanga powders to the aqueous ethanol solution is 1g:(20-30)mL, more preferably 1g:(24-27)mL. Specifically, in the embodiments of this invention, it is 1g:20mL.

[0029] After obtaining the extract, the present invention concentrates the extract under reduced pressure to dryness, disperses the obtained concentrate with an appropriate amount of distilled water, freeze-dries it, and obtains the Panax notoginseng compound composition.

[0030] The present invention preferably performs the vacuum concentration in a rotary evaporator. In the present invention, the temperature of the vacuum concentration is preferably 40-60°C, more preferably 45-55°C, and even more preferably 50°C; the rotation speed is preferably 40-60 rpm, more preferably 45-55 rpm, and even more preferably 50 rpm; the vacuum level is preferably 40-100 bar, more preferably 50-80 bar, and even more preferably 60-70 bar.

[0031] The present invention provides a compound composition of Panax notoginseng prepared by the preparation method described above.

[0032] This invention uses Panax notoginseng, Sophora japonica flowers, and Alpinia galanga as raw materials. A one-pot method is employed, involving ultrasound-assisted ethanol-water extraction, filtration, vacuum concentration, and freeze-drying, to prepare a Panax notoginseng compound composition. High-performance liquid chromatography (HPLC) was used to determine the index components of the obtained Panax notoginseng compound composition. The contents of chlorogenic acid, rutin, and galangin were similar to those of the separately extracted and compounded compositions. Xanthine oxidase activity was measured to evaluate the inhibitory activity of the obtained Panax notoginseng compound composition against xanthine oxidase. The inhibition rate of xanthine oxidase activity was higher than that of the separately extracted and compounded compositions, suggesting its potential application in the preparation of drugs that inhibit xanthine oxidase and lower uric acid.

[0033] This invention provides the application of the above-described compound composition of Panax notoginseng in the preparation of drugs or additives that inhibit xanthine oxidase and lower uric acid.

[0034] The following detailed description of the compound composition of Panax notoginseng, its preparation method and application provided by the present invention, with reference to the embodiments, should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1

[0036] Dried Panax notoginseng, Sophora japonica flowers, and Alpinia galanga were pulverized and passed through a 100-mesh sieve. A certain weight of the powder was weighed according to a 1:2:2 ratio, and 95% ethanol aqueous solution was added at a ratio of 1g:20mL. The mixture was ultrasonically extracted at 60℃ for 3 hours. The filtrate was filtered using a Buchner funnel and concentrated under reduced pressure using a rotary evaporator until no alcohol odor remained. The concentration was set at 50℃, 50 rpm, and 40 bar. An appropriate amount of distilled water was added, and the mixture was ultrasonically dispersed to obtain a concentrated solution. The concentrated solution was freeze-dried and stored to obtain the Panax notoginseng compound composition, with a yield of 18.0%.

[0037] The contents of the index components, chlorogenic acid, rutin and galangin of the obtained Panax notoginseng compound composition were determined by high performance liquid chromatography.

[0038] Specifically, chlorogenic acid, rutin, and galangin standards were prepared at 2 mg / mL using methanol, and a 100 μg / mL mixed standard was prepared for testing at a ratio of 1:1:1. Stock solutions of Panax notoginseng extract, Sophora japonica extract, and galangal extract at 10 mg / mL were prepared using methanol, and a 10 mg / mL mixed sample was prepared at a ratio of 2:5:3. This mixed sample was then diluted with methanol to 1 mg / mL for testing. A stock solution of Panax notoginseng compound was prepared using methanol and diluted to 1 mg / mL for testing. Gradient elution was performed using a C18 column with methanol and formic acid-water as the mobile phase. The detection temperature was 35℃, and the wavelength was 327 nm to analyze the index components of the Panax notoginseng compound.

[0039] The results showed that the compound composition of Panax notoginseng prepared by the one-pot method contained indicator components such as chlorogenic acid, rutin, and galangin. Figure 3 The content of these substances, from highest to lowest, is rutin, galangin, and chlorogenic acid, similar to the composition of extracts extracted individually and then compounded.

[0040] The xanthine oxidase inhibitory activity of the compound composition of Panax notoginseng was further evaluated using the xanthine oxidase activity assay.

[0041] Specifically, accurately weigh the sample mass, add DMSO to prepare a 100 mg / mL stock solution, and dissolve by sonication. Dilute the stock solution 10 times with DMSO to obtain a 10 mg / mL sample solution for later use. Prepare the base solution according to the instructions: take one vial of component A, add 1 mL of component B, and store at 4℃ for later use. Prepare the working solution: take 990 μL of reaction solution, add 10 μL of base solution, and incubate at 37℃ for 20 min before use. The xanthine oxidase solution is prepared by taking 380 μL of phosphate buffer, adding 20 μL of xanthine oxidase stock solution (i.e., diluted 20 times), and mixing thoroughly. The experiment includes a blank group, an enzyme reaction group, and an experimental group, with three replicates for each group. The reaction was carried out in a 96-well UV plate. The blank well contained 20 μL PBS + 180 μL working solution; the enzyme reaction well contained 10 μL PBS + 10 μL enzyme solution + 180 μL working solution; and the experimental well contained 8 μL PBS + 10 μL enzyme solution + 2 μL sample + 180 μL working solution. After adding the working solution, the plate was immediately placed in a microplate reader and shaken for 10 seconds. The absorbance at 295 nm was measured (A0). After waiting for 1 minute, the absorbance at 295 nm was measured again (A1), and the xanthine oxidase activity inhibition rate was calculated.

[0042]

[0043] The results showed that the compound composition of Panax notoginseng prepared by the one-pot method had a higher inhibition rate on xanthine oxidase activity than the compound composition prepared by extraction alone. Figures 7-8 ),in, Figure 7The results show the inhibition rate of xanthine oxidase activity of the single-extraction and compounded compositions obtained using the uniform design principle. The extraction process parameters for the single extraction were the same as in Example 1. The compounding ratios were by mass: Combination 1 was *Gynostemma pentaphyllum* extract: *Sophora japonica* flower extract: *Alpinia galanga* extract = 7:1:5; Combination 2 was *Gynostemma pentaphyllum* extract: *Sophora japonica* flower extract: *Alpinia galanga* extract = 3:2:10; Combination 3 was *Gynostemma pentaphyllum* extract: *Sophora japonica* flower extract: *Alpinia galanga* extract = 10:3:4; Combination 4 was *Gynostemma pentaphyllum* extract: *Sophora japonica* flower extract: *Alpinia galanga* extract = 6:4:9; Combination 5 was *Gynostemma pentaphyllum* extract: *Sophora japonica* flower extract: *Alpinia galanga* extract = 2:5:3; Combination 6 was *Gynostemma pentaphyllum* extract: *Sophora japonica* flower extract: *Alpinia galanga* extract = 2:5:3. Flower extract: Alpinia galanga extract = 9:6:8, combination 7 is Panax notoginseng extract: Sophora japonica flower extract: Alpinia galanga extract = 5:7:2, combination 8 is Panax notoginseng extract: Sophora japonica flower extract: Alpinia galanga extract = 1:8:7, combination 9 is Panax notoginseng extract: Sophora japonica flower extract: Alpinia galanga extract = 8:9:1, combination 10 is Panax notoginseng extract: Sophora japonica flower extract: Alpinia galanga extract = 4:10:6. Among the 10 combinations, combinations 2, 5, and 8 showed better inhibition rates on xanthine oxidase activity, at 78.04%, 78.12%, and 79.0%, respectively. Figure 8 The results show the inhibition rates of the compound composition of Panax notoginseng obtained by one-pot extraction and combination 5 on xanthine oxidase activity. The compound composition of Panax notoginseng obtained by one-pot extraction 1 (Example 1) had an inhibition rate of 84.21% on xanthine oxidase activity, which is significantly higher than that of combination 5. It is expected to be used in the preparation of drugs that inhibit xanthine oxidase and lower uric acid.

[0044] Example 2

[0045] Dried Panax notoginseng, Sophora japonica flowers, and Alpinia galanga were pulverized and passed through a 100-mesh sieve. A certain weight of the powder was weighed according to a 1:1:1 ratio, and 95% ethanol aqueous solution was added at a ratio of 1g:20mL. The mixture was ultrasonically extracted at 60℃ for 3 hours. The filtrate was filtered using a Buchner funnel and concentrated under reduced pressure using a rotary evaporator until no alcohol odor remained. The concentration was set at 50℃, 50 rpm, and 40 bar. An appropriate amount of distilled water was added, and the mixture was ultrasonically dispersed to obtain a concentrated solution. The concentrated solution was freeze-dried and stored to obtain the Panax notoginseng compound composition, with a yield of 14.5%.

[0046] The contents of the index components, chlorogenic acid, rutin and galangin of the obtained Panax notoginseng compound composition were determined by high performance liquid chromatography.

[0047] Specifically, chlorogenic acid, rutin, and galangin standards were prepared at 2 mg / mL using methanol, and a 100 μg / mL mixed standard was prepared for testing at a ratio of 1:1:1. Stock solutions of Panax notoginseng extract, Sophora japonica extract, and galangal extract at 10 mg / mL were prepared using methanol, and a 10 mg / mL mixed sample was prepared at a ratio of 2:5:3. This mixed sample was then diluted with methanol to 1 mg / mL for testing. A stock solution of Panax notoginseng compound was prepared using methanol and diluted to 1 mg / mL for testing. Gradient elution was performed using a C18 column with methanol and formic acid-water as the mobile phase. The detection temperature was 35℃, and the wavelength was 327 nm to analyze the index components of the Panax notoginseng compound.

[0048] The results showed that the compound composition of Panax notoginseng prepared by the one-pot method contained indicator components such as chlorogenic acid, rutin, and galangin. Figure 4 The content of these compounds, from highest to lowest, is rutin, galangin, and chlorogenic acid.

[0049] The xanthine oxidase inhibitory activity of the compound composition of Panax notoginseng was further evaluated using the xanthine oxidase activity assay.

[0050] Specifically, accurately weigh the sample mass, add DMSO to prepare a 100 mg / mL stock solution, and dissolve by sonication. Dilute the stock solution 10 times with DMSO to obtain a 10 mg / mL sample solution for later use. Prepare the base solution according to the instructions: take one vial of component A, add 1 mL of component B, and store at 4℃ for later use. Prepare the working solution: take 990 μL of reaction solution, add 10 μL of base solution, and incubate at 37℃ for 20 min before use. The xanthine oxidase solution is prepared by taking 380 μL of phosphate buffer, adding 20 μL of xanthine oxidase stock solution (i.e., diluted 20 times), and mixing thoroughly. The experiment includes a blank group, an enzyme reaction group, and an experimental group, with three replicates for each group. The reaction was carried out in a 96-well UV plate. The blank well contained 20 μL PBS + 180 μL working solution; the enzyme reaction well contained 10 μL PBS + 10 μL enzyme solution + 180 μL working solution; and the experimental well contained 8 μL PBS + 10 μL enzyme solution + 2 μL sample + 180 μL working solution. After adding the working solution, the plate was immediately placed in a microplate reader and shaken for 10 seconds. The absorbance at 295 nm was measured (A0). After waiting for 1 minute, the absorbance at 295 nm was measured again (A1), and the xanthine oxidase activity inhibition rate was calculated.

[0051]

[0052] The results showed that the inhibition rate of the Panax notoginseng compound composition obtained by one-pot method 2 (Example 2) on xanthine oxidase activity was higher than that of combination 5 (Example 2). Figure 8 This technology holds promise for applications in the preparation of drugs that inhibit xanthine oxidase and lower uric acid levels.

[0053] Example 3

[0054] Dried Panax notoginseng, Sophora japonica flowers, and Alpinia galanga were pulverized and passed through a 100-mesh sieve. A certain weight of the powder was weighed according to a 2:1:1 ratio, and 95% ethanol aqueous solution was added at a ratio of 1g:20mL. The mixture was ultrasonically extracted at 60℃ for 3 hours. The filtrate was filtered using a Buchner funnel and concentrated under reduced pressure using a rotary evaporator until no alcohol odor remained. The concentration was set at 50℃, 50 rpm, and 40 bar. An appropriate amount of distilled water was added, and the mixture was ultrasonically dispersed to obtain a concentrated solution. The concentrated solution was freeze-dried and stored to obtain the Panax notoginseng compound composition, with a yield of 14.0%.

[0055] The contents of the index components, chlorogenic acid, rutin and galangin of the obtained Panax notoginseng compound composition were determined by high performance liquid chromatography.

[0056] Specifically, chlorogenic acid, rutin, and galangin standards were prepared at 2 mg / mL using methanol, and a 100 μg / mL mixed standard was prepared for testing at a ratio of 1:1:1. Stock solutions of Panax notoginseng extract, Sophora japonica extract, and galangal extract at 10 mg / mL were prepared using methanol, and a 10 mg / mL mixed sample was prepared at a ratio of 2:5:3. This mixed sample was then diluted with methanol to 1 mg / mL for testing. A stock solution of Panax notoginseng compound was prepared using methanol and diluted to 1 mg / mL for testing. Gradient elution was performed using a C18 column with methanol and formic acid-water as the mobile phase. The detection temperature was 35℃, and the wavelength was 327 nm to analyze the index components of the Panax notoginseng compound.

[0057] The results showed that the compound composition of Panax notoginseng prepared by the one-pot method contained indicator components such as chlorogenic acid, rutin, and galangin. Figure 5 The content of these compounds, from highest to lowest, is rutin, chlorogenic acid, and galangin.

[0058] The xanthine oxidase inhibitory activity of the compound composition of Panax notoginseng was further evaluated using the xanthine oxidase activity assay.

[0059] Specifically, accurately weigh the sample mass, add DMSO to prepare a 100 mg / mL stock solution, and dissolve by sonication. Dilute the stock solution 10 times with DMSO to obtain a 10 mg / mL sample solution for later use. Prepare the base solution according to the instructions: take one vial of component A, add 1 mL of component B, and store at 4℃ for later use. Prepare the working solution: take 990 μL of reaction solution, add 10 μL of base solution, and incubate at 37℃ for 20 min before use. The xanthine oxidase solution is prepared by taking 380 μL of phosphate buffer, adding 20 μL of xanthine oxidase stock solution (i.e., diluted 20 times), and mixing thoroughly. The experiment includes a blank group, an enzyme reaction group, and an experimental group, with three replicates for each group. The reaction was carried out in a 96-well UV plate. The blank well contained 20 μL PBS + 180 μL working solution; the enzyme reaction well contained 10 μL PBS + 10 μL enzyme solution + 180 μL working solution; and the experimental well contained 8 μL PBS + 10 μL enzyme solution + 2 μL sample + 180 μL working solution. After adding the working solution, the plate was immediately placed in a microplate reader and shaken for 10 seconds. The absorbance at 295 nm was measured (A0). After waiting for 1 minute, the absorbance at 295 nm was measured again (A1), and the xanthine oxidase activity inhibition rate was calculated.

[0060]

[0061] The results showed that the *Gynostemma pentaphyllum* compound composition obtained by one-pot method 3 (Example 3) inhibited xanthine oxidase activity by 80.9%, which was significantly higher than that of combination 5 (Example 3). Figure 8 This technology holds promise for applications in the preparation of drugs that inhibit xanthine oxidase and lower uric acid levels.

[0062] 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 method for one-pot extraction of a compound composition of Panax notoginseng, characterized in that, Includes the following steps: The dried Panax notoginseng, Sophora japonica flowers, and Alpinia galanga were pulverized, weighed and mixed according to the mass ratio of Panax notoginseng, Sophora japonica flowers, and Alpinia galanga powder, which was 0.5~2:1:

1. The mixture was fully extracted using an ultrasonic-assisted ethanol-water extraction method, wherein the volume fraction of ethanol in the ethanol-water solution was 80~95%. The filtrate was obtained by vacuum filtration. The filtrate was concentrated under reduced pressure using a rotary evaporator until no alcohol odor was detected. An appropriate amount of distilled water was added to obtain a concentrated solution. The concentrated solution was freeze-dried and stored in a desiccator to obtain the Panax notoginseng compound composition.

2. The method according to claim 1, characterized in that, The vacuum concentration is carried out at a temperature of 40~60℃, a rotation speed of 40~60rpm, and a vacuum degree of 40~100bar.

3. The method according to claim 1, characterized in that, The particle size of the three raw material powders is less than 100 mesh; the Panax notoginseng is the leaf of Panax notoginseng.

4. The method according to claim 1, characterized in that, The ratio of the mixture of Panax notoginseng, Sophora japonica, and Alpinia galanga powder to the ethanol aqueous solution is 1g:20~30mL.

5. The method according to claim 1, 3, or 4, characterized in that, The ultrasonic extraction conditions are as follows: ultrasonic temperature 40~60℃, ultrasonic power 80~100%, and ultrasonic time 2~4h.

6. The Panax notoginseng compound composition prepared by the method according to any one of claims 1 to 5.

7. The use of the Panax notoginseng compound composition as described in claim 6 in the preparation of a drug or drug additive that inhibits xanthine oxidase and lowers uric acid.

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