A method for improving oral bioavailability of ginsenosides in ginseng

Abstract

The application discloses a method for improving oral bioavailability of ginsenosides in ginseng, which comprises the following steps: preparing a ginsenoside-natural eutectic solution by using total ginsenosides and a natural eutectic solvent, and applying the ginsenoside-natural eutectic solution as an intermediate to oral preparations, so that the degradation of ginsenosides in ginseng in a gastric acid environment can be effectively resisted, the effective medicinal material concentration of ginsenosides in ginseng entering the intestinal tract for absorption is increased, and ginsenosides can be more effectively penetrated through an artificial membrane, and finally, the intermediate can effectively improve the oral absorption effect of animals and further improve the bioavailability; and the application is suitable for further development and application of ginsenosides in ginseng.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of Chinese herbal medicine development, and particularly relates to a method for improving oral bioavailability of ginsenosides in ginseng. BACKGROUND

[0002] Ginseng is one of the most valuable Chinese herbal medicines in China, which is warm in nature and slightly sweet in taste, and has the effects of tonifying primordial qi, restoring pulse and preventing collapse, tonifying the spleen and lung, tonifying qi and generating fluid, and tranquilizing the mind and nourishing blood. The main medicinal active ingredient in ginseng is ginsenosides. It has been reported that ginsenoside Rg1 can resist aging, improve memory and learning ability, and ginsenoside Re can play a role in anti-ischemia and protecting the heart.

[0003] However, the oral bioavailability of ginsenosides has been very low, which leads to poor absorption of the medicinal active ingredients, and indirectly causes the waste of ginseng medicinal resources. SUMMARY

[0004] The present application provides a method for improving oral bioavailability of ginsenosides in ginseng, which effectively improves the oral bioavailability of animals.

[0005] The technical scheme of the present application is as follows:

[0006] The method for improving oral bioavailability of ginsenosides in ginseng uses ginsenosides and natural eutectic solvent to prepare a ginsenoside-natural eutectic solution, which is used as an intermediate for oral preparations. Water or other functional materials can also be added during application.

[0007] The natural eutectic solvent composition includes a hydrogen bond donor and a hydrogen bond acceptor. The hydrogen bond donor includes urea, glycerol, etc. The hydrogen bond acceptor includes choline chloride, betaine, etc.

[0008] The molar ratio of the hydrogen bond donor to the hydrogen bond acceptor in the natural eutectic solvent is 1-3:1-3.

[0009] The preparation method of the natural eutectic solvent is as follows:

[0010] After mixing the hydrogen bond donor and the hydrogen bond acceptor, place them in an 85-95℃ oil bath pot for heating and stirring at a stirring speed of 300-400rpm until a transparent and uniform liquid is formed, which is the natural eutectic solvent.

[0011] The preparation method of the ginsenoside-natural eutectic solution is as follows:

[0012] (1) 10-30mg of ginsenoside powder is added to 1mL of natural eutectic solvent, mixed and sealed, and ultrasonically treated for 30min;

[0013] (2) The solution of step (1) is sequentially filtered through a 0.45 μm microporous filter and a 0.22 μm sterilization filter in an ultra-clean workbench, and the obtained liquid is stored in a clean and sterile sample bottle, to obtain a sterile Panax notoginseng saponins-natural deep eutectic solution.

[0014] Compared with the prior art, the present application has the following advantages:

[0015] (1) The natural deep eutectic solvent has a low vapor pressure and is not easy to volatilize, and can stably control the concentration of Panax notoginseng saponins in the solvent.

[0016] (2) The natural deep eutectic solvent can offset the partial degradation of Panax notoginseng saponins in gastric juice.

[0017] (3) The natural deep eutectic solvent can assist Panax notoginseng saponins in the transmembrane process.

[0018] (4) The natural deep eutectic solvent-Panax notoginseng saponins complex can effectively improve the oral bioavailability of animals. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Stability of Panax notoginseng saponins in artificial gastrointestinal fluid under different solvent conditions;

[0020] Figure 2 Parallel artificial membrane apparent permeability of Panax notoginseng saponins in different solvents;

[0021] Figure 3 PAMPA artificial membrane permeation amount of Panax notoginseng saponins, wherein A-D are the content changes of Panax notoginseng saponins aqueous solution in gastrointestinal juice incubation; E-H are the content changes of Panax notoginseng saponins-natural deep eutectic solution in gastrointestinal juice incubation;

[0022] Figure 4 Blood drug time curve of Panax notoginseng saponins, wherein A-C are three Panax notoginseng saponin monomers, and D is the sum of the three Panax notoginseng saponins. DETAILED DESCRIPTION

[0023] The present application will be described in detail below in conjunction with the embodiments and the accompanying drawings, which will help those skilled in the art to further understand the present application, but the scope of protection of the present application is not limited to the content described. The methods in the examples are performed according to the conventional operation if not otherwise specified. The reagents used are all conventional commercially available reagents if not otherwise specified, and the percentages in the examples are volume percentages.

[0024] Example 1

[0025] The natural deep eutectic solvent is prepared according to the following specific preparation method:

[0026] Take a clean round-bottom flask, accurately weigh the components of choline chloride and urea in a molar ratio of 1:2 into the round-bottom flask, and close the inlet. Place the round-bottom flask in a preheated 90°C oil bath, and stir at a speed of 300-400 rpm. Heat until a transparent and uniform liquid is formed, which is the natural eutectic solvent. Remove the liquid and cool it in a room temperature environment. Store for later use.

[0027] Adjust the raw materials, mix betaine-glycerol (1:2) and choline chloride-glycerol (1:2) to prepare different natural eutectic solvents.

[0028] Example 2

[0029] Preparation of natural eutectic solvents with different water contents:

[0030] Mix the three natural eutectic solvents of Example 1 with water at water contents of 15%, 30%, and 45% (v / v), i.e., add ultrapure water to the above three natural eutectic solvents in proportion and shake well to make them uniform. Nine natural eutectic solvents are obtained, including choline chloride-urea solvents with water contents of 15%, 30%, and 45% (15CU, 30CU, 45CU), choline chloride-glycerol solvents with water contents of 15%, 30%, and 45% (15CG, 30CG, 45CG), and betaine-glycerol solvents with water contents of 30% and 45% (30BG, 45BG). The specific steps are shown in Table 1 below:

[0031] Table 1

[0032]

[0033] The physicochemical properties of some of the synthesized natural eutectic solvents at 298K are shown in Table 2:

[0034] Table 2

[0035]

[0036] Mix the hydrogen bond donor and hydrogen bond acceptor and place them in an 85-95°C oil bath for heating and stirring. Stir at a speed of 300-400 rpm. A transparent and uniform liquid can still be prepared, which is the natural eutectic solvent.

[0037] Example 3

[0038] Preparation method of ginsenosides-natural eutectic solution. Prepare 15CU, 30CU, 45CU, 15CG, 30CG, 45CG, 30BG, and 45BG according to the method of Example 2 and perform experiments. The specific steps are as follows:

[0039] (1) Take 1 mL of each of the eight different natural eutectic solvents or ultrapure water into a clean centrifuge tube, and accurately weigh 30 mg of ginsenoside powder and add it to the tube. Seal the centrifuge tube and place it in an ultrasonic device for ultrasonic treatment for 30 min. Filter the above solutions using sterile 0.45 μm microporous membranes and 0.22 μm microporous membranes to obtain ginsenoside-natural eutectic solution and ginsenoside aqueous solution.

[0040] Example 4

[0041] Parallel artificial membrane permeation experiments of total ginsenosides under different solvent conditions:

[0042] (1) Preparation of phosphate buffer solution:

[0043] Weigh out 0.20g KCl, 8.00g NaCl, 0.27g KH2PO4, and 1.42g Na2HPO4, add pure water to make up to 1L in a volumetric flask, and adjust the pH to 7.4;

[0044] (2) Experiments were conducted using the Franz diffusion cell:

[0045] Before the experiment, the diffusion cell conditions were set up and the equipment was preheated at 37°C. The artificial membrane was placed between the supply chamber and the receiving chamber and fixed with clamps. Then, phosphate buffer solution was added to the supply chamber and the receiving chamber, and air bubbles were removed. The presence of leakage was observed. If there was no leakage, the phosphate buffer solution in the supply chamber was carefully aspirated, and 2 mL of different ginseng total saponin solutions prepared in Example 3 were added. The solutions were placed in the diffusion cell for incubation. Three parallel experiments were set up for each group. After incubation for 2, 4, 6, 8, 12, and 24 hours, the liquid was collected from the receiving chamber and the content was detected by high performance liquid chromatography (HPLC) according to the ginseng saponin detection method in the Chinese Pharmacopoeia.

[0046] Calculate the apparent permeability coefficient using the following formula:

[0047]

[0048] Where ΔQ / Δt is the permeation rate (μg·s⁻¹) -1 A represents the effective area of ​​the artificial membrane (0.85 cm²). 2 C0 is the initial drug concentration (μg·mL); -1 ).

[0049] The results are as follows Figure 1 As shown, after 4 hours of incubation, the apparent permeability coefficient of total ginsenosides in a natural eutectic solvent was: 15CU: 0.95 × 10⁻⁶. -6 cm·s -1 30CU: 0.92×10 -6 cm·s -1; 45CU: 0.9 x 10 -6 cm s -1 ; 15CG: 0.37 x 10 -6 cm s -1 ; 30CG: 0.39 x 10 -6 cm s -1 ; 45CG: 0.46 x 10 -6 cm s -1 ; 30BG: 0.80 x 10 -6 cm s -1 ; 45BG: 1.03 x 10 -6 cm s -1 , and only 0.43 x 10 -6 cm s -1 ; it is shown that natural eutectic solution (except CG) can better assist ginsenosides to penetrate the artificial membrane than water, and further, from Figure 2 and Table 3, it can be seen that natural eutectic solution can increase the bioavailability by increasing the intake of ginsenosides, especially ginsenoside Rd, through the membrane.

[0050] Table 3

[0051]

[0052] Example 5

[0053] Stability experiment of ginsenosides in different solvents in artificial gastrointestinal fluid environment, ginsenosides-30CU solution prepared by the method of Example 3 and ginsenosides aqueous solution were used for experiment, and the specific steps were as follows:

[0054] (1) Preparation of artificial gastrointestinal fluid:

[0055] According to the Chinese Pharmacopoeia, artificial gastrointestinal fluid and artificial blank gastrointestinal fluid were prepared, and the specific method was as follows:

[0056] Preparation of artificial gastric juice: take dilute hydrochloric acid 16.4 mL, then add water 800 mL and pepsin 10 g, fully dissolve, adjust pH to 1.3 with 3.65 g / L hydrochloric acid solution, dilute with water and constant volume to 1000 mL, and the artificial gastric juice is obtained.

[0057] Preparation of artificial intestinal juice: take potassium dihydrogen phosphate 6.8 g, add water 500 mL to dissolve, adjust pH to 6.8 with 4 g / L sodium hydroxide solution; weigh trypsin 10 g, add appropriate amount of water to fully dissolve; mix the above two solutions, then dilute with water and constant volume to 1000 mL, and the artificial intestinal juice is obtained.

[0058] Blank artificial gastrointestinal fluid does not contain pepsin and trypsin, and the rest is the same as the preparation method of artificial gastrointestinal fluid;

[0059] (2) Artificial gastrointestinal fluid incubation experiment:

[0060] Take 100 μL of prepared ginsenoside-30CU solution and ginsenoside aqueous solution respectively in 1 mL of preheated artificial gastrointestinal fluid and blank artificial gastrointestinal fluid at 37°C, and mix thoroughly, set up three parallel experiments;

[0061] Gastric juice and blank gastric juice group experimental method:

[0062] Incubate in a 37°C water bath, take 300 μL of sample at 0 min, 40 min, 80 min, 120 min, 160 min, and 200 min, add 700 μL of ice acetonitrile (previously stored in a -20°C refrigerator for 2 h), vortex to terminate the reaction, centrifuge at 12000 r / min at 4°C for 10 min, pass the supernatant through a 0.22 μm filter membrane, and perform HPLC analysis to determine the content.

[0063] Intestinal juice and blank intestinal juice group experimental method:

[0064] Incubate in a 37°C water bath, take 300 μL of sample at 0 min, 80 min, 160 min, 240 min, 320 min, and 400 min, add 700 μL of ice acetonitrile (previously stored in a -20°C refrigerator for 2 h), vortex to terminate the reaction, centrifuge at 12000 r / min at 4°C for 10 min, pass the supernatant through a 0.22 μm filter membrane, and perform HPLC analysis to determine the content.

[0065] From the results, it can be seen that Figure 3 ), ginsenoside aqueous solution is not stable in artificial gastric juice (acidic) environment, the content is inversely proportional to the incubation time, after 200 min of artificial gastric juice incubation, the content of ginsenoside components is greatly reduced, among which ginsenoside Rd is relatively stable, while ginsenoside Rg1 and ginsenoside Re decrease significantly, with an average reduction of about 50%, the content of ginsenoside in ginsenoside-30CU solution incubated with gastric acid does not change significantly, indicating that natural eutectic solution can effectively resist the degradation of ginsenoside components by gastric acid, further indicating that choline chloride-urea solution can make ginsenoside components more effectively enter the small intestine, which is more conducive to absorption. At the same time, it can be known from the experimental results that ginsenoside components are relatively stable in intestinal juice environment.

[0066] Example 6

[0067] Comparison of animal oral bioavailability of ginsenoside components in different solvents:

[0068] (1) Sample preparation:

[0069] A, Panax ginseng total saponins (natural deep eutectic solvent 30CU) (10 mg / mL): accurately weigh 100 mg of Panax ginseng total saponins in a 10 mL volumetric flask, add an appropriate amount of 30CU natural deep eutectic solvent, ultrasonic dissolution for 30 min, constant volume, pass the solution through 0.45 μm microporous filter and 0.22 μm sterilization filter in turn on the clean bench, store the obtained liquid in a clean and sterile sample bottle, and obtain a sterile Panax ginseng total saponins-natural deep eutectic solution;

[0070] B, Panax ginseng total saponins (aqueous solution) (10 mg / mL): accurately weigh 100 mg of Panax ginseng total saponins in a 10 mL volumetric flask, add an appropriate amount of ultrapure water, ultrasonic dissolution, constant volume, pass the solution through 0.45 μm microporous filter and 0.22 μm sterilization filter in turn on the clean bench, store the obtained liquid in a clean and sterile sample bottle, and obtain a sterile Panax ginseng total saponins-aqueous solution;

[0071] (2) Drug administration:

[0072] After 4-5 days of adaptive feeding of 60 SPF male Kunming mice, the mice were randomly divided into 2 groups, 30 in each group, namely the oral Panax ginseng total saponins (natural deep eutectic solvent 30CU) group and the oral Panax ginseng total saponins (aqueous solution) group. The mice were fasted but not water deprived the night before administration and continued to be fasted after administration. Among them: the oral Panax ginseng total saponins (natural deep eutectic solvent 30CU) group, the administration dose was 50 mg / kg, the concentration was 10 mg / mL, and the administration volume was 5 mL / kg; the oral Panax ginseng total saponins (aqueous solution) group, the administration dose was 50 mg / kg, the concentration was 10 mg / mL, and the administration volume was 5 mL / kg;

[0073] (3) Blood collection:

[0074] After gavage administration of mice, the mice were slightly anesthetized with ether at 15 min, 30 min, 45 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h, and blood was collected about 0.6 mL by damaging the intraocular venous plexus and placed in a centrifuge tube containing sodium citrate (9:1) anticoagulant. Immediately place in ice bath, then centrifuge at 3000 rpm for 10 min, take the upper plasma and place in a-80℃ refrigerator for standby;

[0075] (4) Blood sample processing:

[0076] The blood plasma of each blood sampling point was accurately taken 100 μL, 500 μL of methanol / acetonitrile (4 / 1) solution was added, vortexed for 3 min, then centrifuged at 4 ℃, 12000 rpm for 10 min, 500 μL of supernatant was taken in a clean centrifuge tube, dried by nitrogen at 40 ℃, 100 μL of methanol was added for redissolution, vortexed for 3 min, then placed in a 1.5 mL sharp bottom polypropylene centrifuge tube, centrifuged at 4 ℃, 12000 rpm for 15 min, the supernatant was taken in a liquid phase vial, and analyzed by ultra-high performance liquid chromatography-mass spectrometry (LC-MS);

[0077] The chromatographic conditions were as follows: chromatographic column Agilent C18 (4.6 mm x 50 mm, 1.8 μm); flow rate: 0.30 mL / min; column temperature: 35 ℃; injection volume: 5 μL; mobile phase: acetonitrile (B) / water (A); gradient elution: (0-5 min, 80% A, 5-6 min, 80%-25% A, 6-12 min, 25% A);

[0078] Standard curve establishment:

[0079] The standard solutions of ginsenoside Rg1, ginsenoside Re and ginsenoside Rd with concentrations of 100000, 50000, 10000, 5000, 1000, 500, 100, 50, 10, 5, 1 ng / mL were prepared, and LC-MS analysis was performed to establish the drug regression equation, i.e. the standard curve.

[0080] (5) Data analysis:

[0081] DAS2.0 non-compartment model fitting was used for pharmacokinetic parameter analysis, and the pharmacokinetic parameters t 1 / 2 , T max , C max , AUC 0-t of the three representative ginsenosides (ginsenoside Re, ginsenoside Rg1, ginsenoside Rd) in the oral ginsenoside (natural eutectic solvent 30CU) group and the oral ginsenoside aqueous solution group were calculated. The pharmacokinetic parameters C max and T max are measured values. When the test component exists in the liver and intestine circulation, etc. in the body, it will cause the measured blood concentration-time curve to exist double peak phenomenon. At this time, the first peak is the absorption peak of the drug itself released in the body without liver and intestine circulation and directly absorbed into the blood. Therefore, when there are double peaks or triple peaks, T max and C max are still recorded as the peak time and peak area of the first peak.

[0082] Relative bioavailability (Fr) calculation:

[0083] Fr=AUCt AUC r x 100%

[0084] In the formula, the footnote t represents the test solution, and r represents the reference solution.

[0085] In the experiment, the total ginsenoside aqueous solution is used as the reference solution, and the relative bioavailability of each component in the total ginsenoside (natural deep eutectic solvent 30CU) is calculated.

[0086] Figure 4 The pharmacokinetic curve of the total ginsenoside in the animal body after gavage is shown in Table 4, and the AUC and relative bioavailability of each component after oral administration of the total ginsenoside (natural deep eutectic solvent 30CU) and the total ginsenoside aqueous solution are known.

[0087] Table 4

[0088]

[0089] In summary, the ginsenoside components in the natural deep eutectic solvent (choline chloride-urea) have more advantages in terms of artificial membrane penetration and oral absorption bioavailability. The natural deep eutectic solvent (choline chloride-urea) developed by the application can more effectively assist the ginsenoside components to penetrate the artificial membrane and resist the degradation of the acidic environment in the stomach, so that they can more effectively reach the intestine for absorption. At the same time, according to the animal oral bioavailability table, the oral absorption effect of the three ginsenoside components in the natural deep eutectic solvent 30CU is higher than that in the aqueous solution, which further proves its positive effect on improving the bioavailability.

Claims

1. A method for improving oral bioavailability of ginsenosides in ginseng, characterized in that, The total ginsenoside-natural deep eutectic solvent solution is prepared by using total ginsenoside and natural deep eutectic solvent as an intermediate for oral preparations. The preparation method of the total ginsenoside-natural deep eutectic solvent solution is as follows: (1) 10-30 mg of total ginsenoside powder is added into 1 mL of natural deep eutectic solvent, mixed, sealed, and ultrasonically treated for 30 min; (2) The solution of step (1) is sequentially filtered through 0.45 μm microporous filter and 0.22 μm sterilization filter in an ultraclean bench, and the obtained liquid is stored in a clean and sterile sample bottle to obtain a sterile total ginsenoside-natural deep eutectic solvent solution.

2. The method for improving oral bioavailability of ginsenosides in ginseng according to claim 1, wherein, The natural deep eutectic solvent composition comprises a hydrogen bond donor and a hydrogen bond acceptor, wherein the hydrogen bond donor comprises urea and glycerol, and the hydrogen bond acceptor comprises choline chloride and betaine.

3. The method for improving oral bioavailability of ginsenosides in ginseng according to claim 2, wherein, The molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 1-3:1-3.

4. The method for improving oral bioavailability of ginsenosides in ginseng according to claim 2, wherein, The preparation method of the natural deep eutectic solvent is as follows: The hydrogen bond donor and the hydrogen bond acceptor are mixed and heated and stirred at 85-95 ℃ at a stirring speed of 300-400 rpm until a transparent and uniform liquid is obtained, which is the natural deep eutectic solvent.

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