Preparation method of rare ginsenosides Rk1 and Rg5

Abstract

The invention belongs to the field of compound preparation, and particularly relates to a preparation method of rare ginsenosides Rk1 and Rg5. The invention particularly relates to a method for preparing a mixture of ginsenosides Rk1 and Rg5 by directly converting panaxadiol saponins through high-pressure steam heating. The preparation method comprises the following steps: dissolving panaxadiol saponins by using a theanine aqueous solution, and then preparing a mixture of ginsenosides Rk1 and Rg5 by utilizing a high-pressure steam sterilization pot reaction. The technology is simple to operate and suitable for industrial production. The theanine used in the invention is easy to obtain and safe to use, and can also be used in the fields of food, medicine, health care and the like.

Description

Technical Field

[0001] The invention belongs to the field of compound preparation, and particularly relates to a method for preparing rare ginsenosides Rk1 and Rg5. Background Art

[0002] Panax ginseng Panax ginseng The dried roots and rhizomes of CA Meyer are precious medicinal materials that have the functions of replenishing vital energy, restoring pulse and strengthening deficiency, and tonifying the spleen and lungs. The low-polarity and rare ginsenosides contained in ginseng are important active ingredients in ginsenosides. Modern research shows that rare ginsenosides have strong pharmacological activities such as anti-tumor, lowering blood pressure, improving immunity, and anti-inflammatory. (See: Lv Qing. Study on the mechanism of apoptosis induced by ginsenosides Rk1 and Rg5 in human liver cancer MHCC-97H cells [D]. Changchun: Jilin University, 2019; Zhang Jing et al. Effects of ginsenosides Rg3(R), Rg3(S), Rg5 / Rk1 on the improvement of ethanol-induced memory impairment in mice [J]. Journal of Jilin Agricultural University, 2006, 28: 283;)

[0003] At present, the preparation methods of rare ginseng saponins mainly include enzymatic hydrolysis, acid hydrolysis and alkaline hydrolysis. Enzymatic hydrolysis has the advantages of high efficiency and non-toxicity, but it also has the disadvantages of slow reaction and high price, so it cannot be widely used in industrial production. Strong acid and strong alkali hydrolysis has the advantages of low price, fast reaction speed and simple operation, and is also the most commonly used saponin hydrolysis method, but they have the disadvantages of strong corrosiveness, high toxicity, more by-products and environmental pollution.

[0004] For example: CN 201610344506.3 discloses a method for mass production of ginsenoside Rk1 using protopanaxadiol saponins as raw materials, the method comprising adding protopanaxadiol saponins and water into a fermentation tank, and passing N 2 After online sterilization, organic acid and catalytic amount of Keggin structure heteropoly acid H-xYW-(12)O-(40) were added. nH2O catalyst, wherein Y is selected from P, Si, Fe or Zn, x is 3 or 4, and n is a positive integer of 0-30, reacting at 80-105°C for 24-48 hours, and finally collecting the reaction product for purification to obtain high-purity ginsenoside Rk1.

[0005] CN 201710865039.3 discloses a method for extracting and applying ginsenoside Rg5 and Rk1. The invention refluxes the steamed raw materials of Panax plants with a solvent, and the extract is subjected to macroporous adsorption resin column chromatography to obtain total saponins containing ginsenoside Rg5 and Rk1. The total saponins, steamed total ginsenosides of Panax, total saponins of Notoginseng, or steamed ginsenoside Rb1 are subjected to macroporous adsorption resin column chromatography to obtain grouped saponins with a total content of ginsenoside Rg5 and Rk1 greater than 97%. The grouped saponins are then repeatedly subjected to macroporous adsorption resin column chromatography to obtain monomeric ginsenoside Rg5 with a purity greater than 90% and monomeric ginsenoside Rk1.

[0006] CN202010007323.9 discloses a method for preparing ginsenoside Rk1 and Rg5, which includes the following steps: preparing ginsenoside with a macroporous resin column, subjecting the aqueous solution of ginsenoside acid to ultra-high pressure treatment, low-temperature treatment, centrifugation, heating the supernatant at a high temperature after precipitation filtration, centrifuging and filtering again to obtain a precipitate, and drying the precipitate to obtain high-purity ginsenoside Rk1 and Rg5.

[0007] From the above hydrolysis saponin process, it can be seen that currently, the separation and purification of ginsenoside Rk1 and Rg5 usually combine acid conversion and macroporous resin purification methods. This method has a long separation time and is prone to waste of resources and components. Moreover, the reaction method is complex and unsafe.

[0008] L-Theanine is a unique free amino acid in tea, which is γ-ethylamide of glutamic acid and has a sweet taste. It was first isolated from green tea, accounting for 2% of the dry weight of tea and more than 50% of the total free amino acids in tea. At the same time, L-Theanine is a substance with high safety and low toxicity.

[0009] The L-Theanine used in the present invention can make up for the deficiencies of the existing conversion technology of rare ginsenosides. The hydrolysis of ginsenosides by L-Theanine has the advantages of rapidity, high efficiency, fewer by-products, low price, and environmental friendliness. Summary of the Invention

[0010] The present invention can fully make up for the deficiencies of the existing ginsenoside hydrolysis process. By directly hydrolyzing the ginsenoside of the protopanaxadiol group with economically safe L-Theanine, ginsenosides can be hydrolyzed efficiently, greenly, and environmentally friendly to prepare rare ginsenosides, solving the disadvantages of the existing preparation process such as poor hydrolysis specificity, low yield, strong corrosiveness, environmental pollution, and more by-products caused by the use of a large amount of strong acids and bases.

[0011] The present invention uses L-Theanine to hydrolyze the ginsenoside of the protopanaxadiol group. Compared with inorganic acids and organic acids, L-Theanine is safe to use, has a mild reaction, a small sour taste, and has medicinal values such as health care, antioxidant, and nerve protection.

[0012] To remedy the existing process for preparing rare ginsenosides by hydrolysis, the present invention provides a method for efficiently hydrolyzing ginsenoside of the dammarenediol type with L-theanine to obtain a mixture of ginsenoside Rk1 and Rg5. The specific implementation is as follows:

[0013] The method for preparing a mixture of rare ginsenoside Rk1 and Rg5 from ginsenoside of the dammarenediol type according to the present invention comprises the following steps: Weigh ginsenoside of the dammarenediol type, and select an aqueous solution of L-theanine with a mass fraction of 30-40% as the reaction solution. Dissolve L-theanine with hot water at 40-60°C, and then add ginsenoside of the dammarenediol type in a mass ratio of 1:15-25 while stirring. Then, react in a high-pressure steam sterilizer at 100-120°C for 1-2 h. After the reaction is completed, adjust the pH of the solution to neutral with sodium hydroxide solution, extract twice with ethyl acetate, combine the ethyl acetate layers, and recover the solvent under reduced pressure to obtain the L-theanine hydrolysis product.

[0014] In the above method, through a large number of research experiments, it is found that: when the mass fraction of L-theanine is 30-40%, and the reaction is carried out in a high-pressure steam sterilizer at 110-120°C for 1.5-2 h, the hydrolysis effect is the best, and the yield of the mixture of ginsenoside Rk1 and Rg5 is the highest.

[0015] Through the study of the optimal reaction concentration, it is found that under the above reaction conditions, when using L-theanine with a mass fraction of 30-40% and a mass ratio of ginsenoside of the dammarenediol type to the aqueous solution of L-theanine of 1:15-20, the hydrolysis effect is the best, and the yield of the mixture of ginsenoside Rk1 and Rg5 is the highest. At the same time, it is found that when the concentration of L-theanine is lower than the above mass fraction, or the addition ratio is less than 1:15-20, the reaction rate significantly slows down and the yield is low; in addition, when the concentration of L-theanine is higher than the above mass fraction, or the addition ratio is greater than 1:15-20, a large amount of other impurities are generated, which has a great impact on the yield of the target mixture.

[0016] In addition, the present invention first discovers and confirms that dissolving L-theanine with hot water at 40-60°C in the above method is crucial for the reaction and is more conducive to the complete conversion of ginsenoside of the dammarenediol type.

[0017] As a preferred embodiment of the present invention, an aqueous solution of L-theanine with a mass fraction of 40% is selected as the reaction solution.

[0018] As a preferred embodiment of the present invention, the ratio of ginsenoside of the dammarenediol type to the reaction solution is 1:20.

[0019] As a preferred embodiment of the present invention, place it in a high-pressure steam sterilizer at 120°C and heat it for 1.5 h.

[0020] The beneficial effects of the present invention compared with the prior art include:

[0021] The present invention can fully make up for the deficiencies of the existing hydrolysis process of ginsenosides. Using ginsenoside Rd as the raw material, through the hydrolysis of L-theanine, strictly controlling factors such as the concentration of L-theanine, conversion temperature, and conversion time, it fully ensures the efficient hydrolysis of ginsenoside Rd and maximizes the yield of ginsenoside Rk1 and Rg5. This method is economical, safe, does not pollute the environment, is suitable for industrial production, and has strong practical significance.

[0022] It is found that under the same conditions, the use of L-theanine (γ-ethylamide of glutamic acid) is superior to L-glutamine in terms of conversion effect. Description of the Drawings

[0024] Figure 1 It is a schematic diagram of the structural transformation of ginsenosides in the preparation process of the present invention (taking diol-type ginsenosides as an example).

[0025] Figure 2 It is a comparison between the chromatogram of the L-theanine hydrolysis product and the chromatogram of ginsenoside Rd under the optimal conditions of the present invention. Detailed Embodiments

[0027] The present invention can be further illustrated by the following specific embodiments, but the present invention is not limited thereto.

[0028] Example 1:

[0029] Weigh 2 g of ginsenoside Rd (purity ≥ 95%), select an aqueous solution of L-theanine (CAS No.: 3081-61-6) with a mass fraction of 40% as the reaction solution, dissolve L-theanine in hot water at 40-60 °C, then add ginsenoside Rd in a ratio of 1:20, stir while adding, and then place it in a high-pressure steam sterilizer at 120 °C for high-temperature heating reaction for 1.5 h. After the reaction is completed, adjust the pH of the solution to neutral with sodium hydroxide solution, extract twice with ethyl acetate, combine the ethyl acetate layers, and recover the solvent under reduced pressure to obtain the L-theanine hydrolysis product, that is, a mixture of ginsenoside Rk1 and Rg5.

[0030] Among them, the process of converting ginsenoside Rd into Rk1 and Rg5 is as Figure 1 shown.

[0031] Example 2:

[0032] Weigh 2 g of ginsenoside group A (purity ≥ 95%), select an aqueous solution of glutamine (CAS No.: 56 - 85 - 9) with a mass fraction of 40% as the reaction solution. Then, add ginsenoside group A at a ratio of 1:20, stirring while adding. Then, place it in a high-pressure steam sterilizer at 120 °C for high-temperature heating reaction for 1.5 h. After the reaction is completed, adjust the pH of the solution to neutral with sodium hydroxide solution, extract it twice with ethyl acetate, combine the ethyl acetate layers, and recover the solvent under reduced pressure to obtain the glutamine hydrolysis product, that is, a mixture of ginsenoside Rk1 and Rg5.

[0033] Example 3:

[0034] Weigh 2 g of ginsenoside group A (purity ≥ 95%), select an aqueous solution of L-theanine with a mass fraction of 20% as the reaction solution, dissolve L-theanine with hot water at 40 - 60 °C. Then, add ginsenoside group A at a ratio of 1:20, stirring while adding. Then, place it in a high-pressure steam sterilizer at 120 °C for high-temperature heating reaction for 1.5 h. After the reaction is completed, adjust the pH of the solution to neutral with sodium hydroxide solution, extract it twice with ethyl acetate, combine the ethyl acetate layers, and recover the solvent under reduced pressure to obtain the L-theanine hydrolysis product, that is, a mixture of ginsenoside Rk1 and Rg5.

[0035] Example 4:

[0036] Weigh 2 g of ginsenoside group A (purity ≥ 95%), select an aqueous solution of L-theanine with a mass fraction of 30% as the reaction solution, dissolve L-theanine with hot water at 40 - 60 °C. Then, add ginsenoside group A at a ratio of 1:20, stirring while adding. Then, place it in a high-pressure steam sterilizer at 120 °C for high-temperature heating reaction for 1.5 h. After the reaction is completed, adjust the pH of the solution to neutral with sodium hydroxide solution, extract it twice with ethyl acetate, combine the ethyl acetate layers, and recover the solvent under reduced pressure to obtain the L-theanine hydrolysis product, that is, a mixture of ginsenoside Rk1 and Rg5.

[0037] Example 5:

[0038] Weigh 2 g of ginsenoside group A (purity ≥ 95%), select an aqueous solution of L-theanine with a mass fraction of 50% as the reaction solution, dissolve L-theanine with hot water at 40 - 60 °C. Then, add ginsenoside group A at a ratio of 1:20, stirring while adding. Then, place it in a high-pressure steam sterilizer at 120 °C for high-temperature heating reaction for 1.5 h. After the reaction is completed, adjust the pH of the solution to neutral with sodium hydroxide solution, extract it twice with ethyl acetate, combine the ethyl acetate layers, and recover the solvent under reduced pressure to obtain the L-theanine hydrolysis product, that is, a mixture of ginsenoside Rk1 and Rg5.

[0039] Example 6:

[0040] Weigh 2 g of ginsenoside group A (purity ≥ 95%), and select an aqueous solution of L-theanine with a mass fraction of 40% as the reaction solution. Dissolve L-theanine in hot water at 40 - 60 °C, and then add ginsenoside group A at a ratio of 1:10 while stirring. Then, place it in a high-pressure steam sterilizer at 120 °C for high-temperature heating reaction for 1.5 h. After the reaction is completed, adjust the pH of the solution to neutral with sodium hydroxide solution, extract it twice with ethyl acetate, combine the ethyl acetate layers, and recover the solvent under reduced pressure to obtain the L-theanine hydrolysis product, that is, a mixture of ginsenoside Rk1 and Rg5.

[0041] Example 7:

[0042] Weigh 2 g of ginsenoside group A (purity ≥ 95%), and select an aqueous solution of L-theanine with a mass fraction of 40% as the reaction solution. Dissolve L-theanine in hot water at 40 - 60 °C, and then add ginsenoside group A at a ratio of 1:15 while stirring. Then, place it in a high-pressure steam sterilizer at 120 °C for high-temperature heating reaction for 1.5 h. After the reaction is completed, adjust the pH of the solution to neutral with sodium hydroxide solution, extract it twice with ethyl acetate, combine the ethyl acetate layers, and recover the solvent under reduced pressure to obtain the L-theanine hydrolysis product, that is, a mixture of ginsenoside Rk1 and Rg5.

[0043] Example 8:

[0044] Weigh 2 g of ginsenoside group A (purity ≥ 95%), and select an aqueous solution of L-theanine with a mass fraction of 40% as the reaction solution. Dissolve L-theanine in hot water at 40 - 60 °C, and then add ginsenoside group A at a ratio of 1:25 while stirring. Then, place it in a high-pressure steam sterilizer at 120 °C for high-temperature heating reaction for 1.5 h. After the reaction is completed, adjust the pH of the solution to neutral with sodium hydroxide solution, extract it twice with ethyl acetate, combine the ethyl acetate layers, and recover the solvent under reduced pressure to obtain the L-theanine hydrolysis product, that is, a mixture of ginsenoside Rk1 and Rg5.

[0045] Example 9:

[0046] Weigh 2 g of ginsenoside group A (purity ≥ 95%), and select an aqueous solution of L-theanine with a mass fraction of 40% as the reaction solution. Dissolve L-theanine in hot water at 40 - 60 °C, and then add ginsenoside group A at a ratio of 1:20 while stirring. Then, place it in a high-pressure steam sterilizer at 100 °C for high-temperature heating reaction for 1.5 h. After the reaction is completed, adjust the pH of the solution to neutral with sodium hydroxide solution, extract it twice with ethyl acetate, combine the ethyl acetate layers, and recover the solvent under reduced pressure to obtain the L-theanine hydrolysis product, that is, a mixture of ginsenoside Rk1 and Rg5.

[0047] Example 10:

[0048] Weigh 2 g of ginsenoside group A (purity ≥ 95%), select an aqueous solution of L-theanine with a mass fraction of 40% as the reaction solution, dissolve L-theanine with hot water at 40 - 60 °C, then add ginsenoside group A in a ratio of 1:20, stirring while adding, and then place it in a high-pressure steam sterilizer at 110 °C for high-temperature heating reaction for 1.5 h. After the reaction is completed, adjust the pH of the solution to neutral with sodium hydroxide solution, extract with ethyl acetate twice, combine the ethyl acetate layers, recover the solvent under reduced pressure to obtain the L-theanine hydrolysis product, that is, a mixture of ginsenoside Rk1 and Rg5.

[0049] Example 11:

[0050] Weigh 2 g of ginsenoside group A (purity ≥ 95%), select an aqueous solution of L-theanine with a mass fraction of 40% as the reaction solution, dissolve L-theanine with hot water at 40 - 60 °C, then add ginsenoside group A in a ratio of 1:20, stirring while adding, and then place it in a high-pressure steam sterilizer at 130 °C for high-temperature heating reaction for 1.5 h. After the reaction is completed, adjust the pH of the solution to neutral with sodium hydroxide solution, extract with ethyl acetate twice, combine the ethyl acetate layers, recover the solvent under reduced pressure to obtain the L-theanine hydrolysis product, that is, a mixture of ginsenoside Rk1 and Rg5.

[0051] Example 12:

[0052] Weigh 2 g of ginsenoside group A (purity ≥ 95%), select an aqueous solution of L-theanine with a mass fraction of 40% as the reaction solution, dissolve L-theanine with hot water at 40 - 60 °C, then add ginsenoside group A in a ratio of 1:20, stirring while adding, and then place it in a high-pressure steam sterilizer at 120 °C for high-temperature heating reaction for 0.5 h. After the reaction is completed, adjust the pH of the solution to neutral with sodium hydroxide solution, extract with ethyl acetate twice, combine the ethyl acetate layers, recover the solvent under reduced pressure to obtain the L-theanine hydrolysis product, that is, a mixture of ginsenoside Rk1 and Rg5.

[0053] Example 13:

[0054] Weigh 2 g of ginsenoside group A (purity ≥ 95%), select an aqueous solution of L-theanine with a mass fraction of 40% as the reaction solution, dissolve L-theanine with hot water at 40 - 60 °C, then add ginsenoside group A in a ratio of 1:20, stirring while adding, and then place it in a high-pressure steam sterilizer at 120 °C for high-temperature heating reaction for 1 h. After the reaction is completed, adjust the pH of the solution to neutral with sodium hydroxide solution, extract with ethyl acetate twice, combine the ethyl acetate layers, recover the solvent under reduced pressure to obtain the L-theanine hydrolysis product, that is, a mixture of ginsenoside Rk1 and Rg5.

[0055] Example 14:

[0056] Weigh 2 g of ginsenoside group A (purity ≥ 95%), and select an aqueous solution of L-theanine with a mass fraction of 40% as the reaction solution. Dissolve L-theanine in hot water at 40 - 60 °C, and then add ginsenoside group A in a ratio of 1:20 while stirring. Then, place it in a high-pressure steam sterilizer at 120 °C for high-temperature heating reaction for 2 h. After the reaction is completed, adjust the pH of the solution to neutral with sodium hydroxide solution, extract it twice with ethyl acetate, combine the ethyl acetate layers, and recover the solvent under reduced pressure to obtain the L-theanine hydrolysis product, that is, a mixture of ginsenoside Rk1 and Rg5.

[0057] By performing HPLC analysis on the results of the above examples, calculate the yields of ginsenoside Rk1 and Rg5 and their total yield. As shown in Table 1, Table 2, Table 3, Table 4, and Table 5, it is proved that when an aqueous solution of L-theanine with a mass fraction of 40% is used as the reaction solution, and the ratio of ginsenoside group A to the reaction solution is 1:20, and it is placed in a high-pressure steam sterilizer at 120 °C for high-temperature heating reaction for 1.5 h, the yields of ginsenoside Rk1 and Rg5 and their total yield are the highest.

[0058] Table 1 Effects of L-theanine and glutamine on hydrolysis products

[0059] Table 2 Effects of L-theanine with different mass fractions on hydrolysis products

[0060] Table 3 Effects of different material ratios on hydrolysis products

[0061] Table 4 Effects of different reaction temperatures on hydrolysis products

[0062] Table 5 Effects of different reaction times on hydrolysis products

[0063] Through the hydrolysis of ginsenoside group A by L-theanine in the examples to prepare ginsenoside Rk1 and Rg5, and comprehensively comparing with the existing ginsenoside hydrolysis processes, it can be seen that the present invention uses L-theanine to hydrolyze ginsenoside group A, effectively reducing the cost increase and resource waste caused by saponin grouping. In addition, this technology can enrich ginsenoside Rk1 and Rg5 to the greatest extent, and is economical and safe. The hydrolysis process is simple and efficient, effectively avoiding the generation of a large amount of other impurities.

[0064] The above embodiments only describe and illustrate the method of hydrolyzing ginsenoside group A with theanine to obtain a mixture of ginsenoside Rk1 and Rg5, and give a brief example. However, the present invention is not limited to the above embodiments. Modifications or substitutions can be made to the details and forms of the technical solutions of the present invention without departing from the core gist of the present invention, and the present invention can be implemented in various ways, but these modifications and substitutions should all be within the protection scope of the present invention, and will not be described one by one here.

Claims

1. A method for preparing rare ginsenosides Rk1 and Rg5, characterized in that: The method comprises the following steps: weighing ginsenosides of the ginsenoside group, selecting an aqueous solution of theanine with a mass fraction of 30-40% as a reaction solution, dissolving theanine with hot water at 40-60°C, then adding ginsenosides of the ginsenoside group to the reaction solution at a mass ratio of 1:15-25, stirring while adding, reacting in a high-pressure steam sterilizer at 100-120°C for 1-2 h, adjusting the pH value of the solution to neutral with a sodium hydroxide solution after the reaction is completed, extracting twice with ethyl acetate, combining the ethyl acetate layers, and recovering the solvent under reduced pressure to obtain a theanine hydrolyzate.

2. The method according to claim 1, characterized in that: The ginsenoside purity of the ginsenoside group is ≥95%, and mainly includes ginsenosides Rb1, Rc, Rb2, Rb3 and Rd.

3. The method according to claim 1, characterized in that: The mass fraction of the theanine aqueous solution is 40%.

4. The method according to claim 1, characterized in that: The mass ratio of the ginsenoside group saponins to the reaction solution is 1:

20.

5. The method according to claim 1, characterized in that: The reaction temperature is 120° C. and the reaction time is 1.5 h.

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