A pretreatment method to increase the purification rate of rare saponin Rg3 in red ginseng

Abstract

The application belongs to the technical field of medicine, and provides a pretreatment method for increasing the purification rate of rare ginsenoside Rg3 in red ginseng, comprising: crushing ginseng, water extraction, to obtain ginseng water extract; heating the ginseng water extract to react, to obtain ginseng slurry; adding organic acid to the ginseng slurry, and reacting at 25-85 DEG C for 0.5-2.5 h; obtaining rare ginsenoside Rg3 extract; column chromatography separation, to obtain rare ginsenoside Rg3; the organic acid is at least one of hydroxyl cinnamic acid, mustard acid or 3-coffee acyl quinine acid. Compared with the traditional method, the Rg3 preparation method is simple and convenient, the time consumption is short, the yield is extremely high, the cost is low, and it is suitable for large-scale industrial production of rare ginsenoside Rg3 preparation and purification.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and specifically relates to a pretreatment method for increasing the purification rate of rare saponin Rg3. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Red ginseng, as an important processed product, is widely used in clinical practice both domestically and internationally. Red ginseng contains various medicinal components, including ginseng polysaccharides, maltol, arginine disaccharides, various volatile oils, and ginsenosides. Ginsenosides are present in high amounts in red ginseng and are the main component responsible for its medicinal value. Currently, many types of ginsenosides have been reported, but the most common are Rh1, Rg2, Rb2, Ro, and Rc. The rare saponin Rg3, as one of the most widely reported members, possesses various pharmacological effects, such as anti-cancer properties, reducing the toxic side effects of radiotherapy, enhancing immunity, regulating hormone levels, dilating blood vessels, protecting brain nerve cells against dementia, and inhibiting platelet aggregation. Furthermore, numerous studies have found that Rg3 has low toxicity and is recognized by scholars worldwide as a superior product combining medicinal and tonic properties. Therefore, the development and application prospects of Rg3 are extremely broad.

[0004] It has been reported that the rare saponin Rg3 has not been found in either wild or cultivated ginseng, but has only been detected in red ginseng at a concentration of 0.03%, which poses a significant challenge to its purification. Currently, Rg3 preparation processes include physical heating, acid-base treatment, and biotransformation. For example, the paper "The Effect of Citric Acid Treatment on the Chemical Composition and Bioactivity of Red Ginseng" discloses a method for acid hydrolysis of ginsenosides, and the paper "Research Progress on In Vitro Conversion and Analysis Methods of Ginsenosides" discloses various ginsenoside conversion methods. However, these preparation methods are complex, costly, have low yields, and exhibit significant instability, hindering the large-scale purification of Rg3 and making mass production difficult.

[0005] Furthermore, existing Rg3 preparation processes focus on increasing yield, neglecting the impact of fine-tuning preparation conditions on Rg3 production. This leads to the decomposition or transformation of high-content Rg3, resulting in extremely low yields. Therefore, there is an urgent need to develop and seek a mild method that specifically enhances the yield of these rare saponins under certain conditions. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a pretreatment method for increasing the purification rate of rare saponin Rg3 in red ginseng. This invention uses at least one of hydroxycinnamic acid, sinapic acid, or 3-caffeoylquinic acid as an acid catalyst, which can specifically increase the yield of rare saponin Rg3 under mild conditions. This is beneficial for the large-scale purification of Rg3, and the method is simple, yields high output, and exhibits mild reaction conditions and high stability.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention provides a pretreatment method for increasing the purification rate of rare saponin Rg3 in red ginseng, comprising:

[0009] Ginseng is crushed and extracted with water to obtain ginseng aqueous extract;

[0010] The ginseng water extract was heated to react and ginseng slurry was obtained.

[0011] Organic acid was added to the ginseng slurry, and the reaction was carried out at 25℃~85℃ for 0.5h~2.5h to obtain rare saponin Rg3 extract; column chromatography was used to separate the rare saponin Rg3.

[0012] The organic acid is at least one of hydroxycinnamic acid, sinapic acid, or 3-caffeoylquinic acid.

[0013] In some embodiments, the ginseng is red ginseng.

[0014] In some embodiments, the water extraction conditions are a solid-liquid ratio of 1:18-20, soaking for 2-3 hours, and extraction at 90-95°C for 24-32 hours.

[0015] In some implementations, the aqueous extract is passed through a 20-mesh sieve.

[0016] In some embodiments, the specific conditions for heating the reaction are 60°C to 65°C for 12 to 16 hours.

[0017] In some embodiments, the amount of organic acid added is 10% to 50%;

[0018] In some more effective implementations, the amount added is 20±1%.

[0019] In some embodiments, the acid catalysis reaction time is 60±1 min at 40±1 °C.

[0020] In a second aspect, the present invention provides rare saponin Rg3 extracted by the above-described method.

[0021] A third aspect of the invention provides the use of organic acids in improving the catalytic efficiency of rare saponins Rg3 acids, wherein the organic acid is at least one of hydroxycinnamic acid, sinapic acid, or 3-caffeoylquinic acid.

[0022] Beneficial effects of the present invention

[0023] (1) The present invention uses at least one of hydroxycinnamic acid, sinapic acid or 3-caffeoylquinic acid as an acid catalyst, which can specifically increase the yield of rare saponin Rg3 under mild conditions, which is beneficial to the large-scale purification of Rg3 product. The method is simple, the yield is large, the reaction conditions are mild and the stability is high.

[0024] (2) This invention provides an organic acid (hydroxycinnamic acid, sinapic acid or 3-caffeoylquinic acid) catalytic condition that can significantly increase the yield of rare saponin Rg3. The yield of Rg3 is highest in this temperature range. This condition is suitable for the large-scale purification of Rg3 and is a key condition for increasing the yield of Rg3.

[0025] (3) Compared with traditional methods, the Rg3 preparation method of the present invention is simple and convenient, takes less time, has a very high yield, and is low in cost, making it suitable for the preparation and purification of rare saponin Rg3 in large-scale industrial production. Detailed Implementation

[0026] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0027] The present invention will be further described in detail below with reference to specific embodiments. It should be noted that the specific embodiments are explanations of the present invention and not limitations thereof.

[0028] Example 1

[0029] S1: Crush the red ginseng rootlets, soak them in 18 times the amount of water for 2 hours, extract them at 90℃ for 24 hours, pass them through a 20-mesh sieve to obtain red ginseng water extract, treat the red ginseng water extract at 60℃ for 12 hours to obtain red ginseng paste, sterilize it at 105℃ for 20 minutes, and cool it to room temperature for later use.

[0030] S2: Design an orthogonal table with hydroxycinnamic acid addition of 10%, 20%, 30%, 40% and 50%, reaction temperature of 25℃, 40℃, 55℃, 70℃ and 85℃, and reaction time of 0.5h, 1h, 1.5h, 2h and 2.5h, and calculate the yield of Rg3 after each treatment.

[0031] S3: Using the optimal addition of 20% hydroxycinnamic acid and reaction at 40℃ for 1 h as the baseline, the addition of hydroxycinnamic acid (20±1%), reaction temperature (40±1℃), and reaction time (60±1 min) were finely adjusted to obtain the minimum range of conditions that yielded the highest Rg3. The purity of Rg3 was calculated, and Rg3 was obtained by separation by column chromatography.

[0032] Example 2

[0033] S1: Crush the red ginseng rootlets, soak them in 18 times the amount of water for 2 hours, extract them at 90℃ for 24 hours, pass them through a 20-mesh sieve to obtain red ginseng water extract, treat the red ginseng water extract at 60℃ for 12 hours to obtain red ginseng paste, sterilize it at 105℃ for 20 minutes, and cool it to room temperature for later use.

[0034] S2: Using 20% ​​sinapic acid and a reaction time of 1 h at 40°C as the baseline, the amount of sinapic acid added (20±1%), the reaction temperature (40±1°C), and the reaction time (60±1 min) were finely adjusted to obtain Rg3 extract. The purity of Rg3 was calculated, and Rg3 was obtained by separation by chromatography column.

[0035] Example 3

[0036] S1: Crush the red ginseng rootlets, soak them in 18 times the amount of water for 2 hours, extract them at 90℃ for 24 hours, pass them through a 20-mesh sieve to obtain red ginseng water extract, treat the red ginseng water extract at 60℃ for 12 hours to obtain red ginseng paste, sterilize it at 105℃ for 20 minutes, and cool it to room temperature for later use.

[0037] S2: Using 20% ​​3-caffeoylquinic acid as the baseline and reacting at 40℃ for 1 h, the amount of 3-caffeoylquinic acid added (20±1%), reaction temperature (40±1℃), and reaction time (60±1 min) were finely adjusted to obtain Rg3 extract. The purity of Rg3 was calculated, and Rg3 was obtained by separation by chromatography column.

[0038] Example 4

[0039] S1: Crush the red ginseng rootlets, soak them in 18 times the amount of water for 2 hours, extract them at 90℃ for 24 hours, pass them through a 20-mesh sieve to obtain red ginseng water extract, treat the red ginseng water extract at 60℃ for 12 hours to obtain red ginseng paste, sterilize it at 105℃ for 20 minutes, and cool it to room temperature for later use.

[0040] S2: Using 20% ​​hydroxycinnamic acid and sinapic acid (each 10%) as the baseline and 1 hour of reaction at 40°C as the reaction time, the addition of hydroxycinnamic acid and sinapic acid (20±1%), the reaction temperature (40±1°C), and the reaction time (60±1 min) were finely adjusted to obtain Rg3 extract. The purity of Rg3 was calculated, and Rg3 was obtained by separation by column chromatography.

[0041] Example 5

[0042] S1: Crush the red ginseng rootlets, soak them in 18 times the amount of water for 2 hours, extract them at 90℃ for 24 hours, pass them through a 20-mesh sieve to obtain red ginseng water extract, treat the red ginseng water extract at 60℃ for 12 hours to obtain red ginseng paste, sterilize it at 105℃ for 20 minutes, and cool it to room temperature for later use.

[0043] S2: Using 20% ​​hydroxycinnamic acid and 10% 3-caffeoylquinic acid as the baseline and reacting at 40℃ for 1 h, the addition of hydroxycinnamic acid and 3-caffeoylquinic acid was finely adjusted (20±1%), the reaction temperature (40±1℃), and the reaction time (60±1 min) to obtain Rg3 extract. The purity of Rg3 was calculated, and Rg3 was obtained by separation by column chromatography.

[0044] Example 6

[0045] S1: Crush the red ginseng rootlets, soak them in 18 times the amount of water for 2 hours, extract them at 90℃ for 24 hours, pass them through a 20-mesh sieve to obtain red ginseng water extract, treat the red ginseng water extract at 60℃ for 12 hours to obtain red ginseng paste, sterilize it at 105℃ for 20 minutes, and cool it to room temperature for later use.

[0046] S2: Using 20% ​​sinapic acid and 10% 3-caffeoylquinic acid as the baseline and reacting at 40℃ for 1 h, the addition of sinapic acid and 3-caffeoylquinic acid was finely adjusted (20±1%), the reaction temperature (40±1℃) and the reaction time (60±1 min) to obtain Rg3 extract. The purity of Rg3 was calculated, and Rg3 was obtained by separation by column chromatography.

[0047] Example 7

[0048] S1: Crush the red ginseng rootlets, soak them in 18 times the amount of water for 2 hours, extract them at 90℃ for 24 hours, pass them through a 20-mesh sieve to obtain red ginseng water extract, treat the red ginseng water extract at 60℃ for 12 hours to obtain red ginseng paste, sterilize it at 105℃ for 20 minutes, and cool it to room temperature for later use.

[0049] S2: Using 20% ​​hydroxycinnamic acid, sinapic acid, and 3-caffeoylquinic acid (6.667% : 6.667% : 6.667%) as the baseline and 1 h of reaction at 40℃, the amounts of hydroxycinnamic acid, sinapic acid, and 3-caffeoylquinic acid (20±1%), reaction temperature (40±1℃), and reaction time (60±1 min) were finely adjusted to obtain Rg3 extract. The purity of Rg3 was calculated, and Rg3 was obtained by column chromatography.

[0050] Comparative Example 1

[0051] S1: Crush the red ginseng rootlets, soak them in 18 times the amount of water for 2 hours, extract them at 90℃ for 24 hours, pass them through a 20-mesh sieve to obtain red ginseng water extract, treat the red ginseng water extract at 60℃ for 12 hours to obtain red ginseng paste, sterilize it at 105℃ for 20 minutes, and cool it to room temperature for later use.

[0052] S2: Using 20% ​​citric acid and 1 hour of reaction at 40°C as the baseline, the amount of citric acid added (20±1%), reaction temperature (40±1°C), and reaction time (60±1 min) were finely adjusted to obtain Rg3 extract. The purity of Rg3 was calculated, and Rg3 was obtained by separation by chromatography column.

[0053] Table 1. Yield and purity of rare saponin Rg3 extracted in each example and comparative example.

[0054]

[0055]

[0056] Wherein, Rg3 yield = Rg3 mass (g) / red ginseng pulp volume (mL).

[0057] As shown in Table 1, replacing citric acid with hydroxycinnamic acid, sinapic acid, or 3-caffeoylquinic acid as the acid catalyst can effectively increase the yield of rare saponin Rg3, indicating that the three have stronger specificity for rare saponin Rg3.

[0058] In Examples 4-7, this application attempted to combine three acid catalysts—hydroxycinnamic acid, sinapic acid, or 3-caffeoylquinic acid—in order to obtain better catalytic effects. The results showed that the combination of sinapic acid and 3-caffeoylquinic acid, as well as the combination of hydroxycinnamic acid, sinapic acid, or 3-caffeoylquinic acid, could achieve better catalytic efficiency and further increase the yield of rare saponin Rg3.

[0059] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A pretreatment method for increasing the purification rate of rare saponin Rg3 in red ginseng, characterized in that, include: Ginseng is crushed and extracted with water to obtain ginseng aqueous extract; The ginseng water extract was heated to react and ginseng slurry was obtained. Organic acid was added to the ginseng slurry and reacted at 25℃~40℃ for 0.5h~2.5h to obtain rare saponin Rg3 extract; column chromatography was used to separate the rare saponin Rg3. The organic acid is at least one of hydroxycinnamic acid, sinapic acid, or 3-caffeoylquinic acid.

2. The pretreatment method for increasing the purification rate of rare saponin Rg3 in red ginseng as described in claim 1, characterized in that, The ginseng mentioned is red ginseng.

3. The pretreatment method for increasing the purification rate of rare saponin Rg3 in red ginseng as described in claim 1, characterized in that, The water extraction conditions are a solid-liquid ratio of 1:18~20, soaking for 2h~3h, and extraction at 90℃~95℃ for 24h~32h.

4. The pretreatment method for increasing the purification rate of rare saponin Rg3 in red ginseng as described in claim 1, characterized in that, The aqueous extract was passed through a 20-mesh sieve.

5. The pretreatment method for increasing the purification rate of rare saponin Rg3 in red ginseng as described in claim 1, characterized in that, The specific conditions for the reaction by heating are 60℃~65℃ for 12h~16h.

6. The pretreatment method for increasing the purification rate of rare saponin Rg3 in red ginseng as described in claim 1, characterized in that, The amount of organic acid added is 10% to 50%.

7. The pretreatment method for increasing the purification rate of rare saponin Rg3 in red ginseng as described in claim 1, characterized in that, The amount of organic acid added is 20±1%.

8. The pretreatment method for increasing the purification rate of rare saponin Rg3 in red ginseng as described in claim 1, characterized in that, The acid-catalyzed reaction time was 60±1 min at 40±1℃.

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