Ginsenoside compound preparation for enhancing immunity of human body and preparation method of ginsenoside compound preparation

Abstract

The invention discloses a ginsenoside compound preparation for enhancing human immunity and a preparation method thereof, and belongs to the field of pharmaceutical preparations. The ginsenoside compound preparation comprises the following components in parts by weight: piperonol, NH2-PEG-OH, ginsenoside, poly (lactic-co-glycolic acid) and a surfactant. A coating carrier of ginsenoside is improved, ginsenoside is coated in nano-particles of polylactic acid-glycolic acid, and the outer layers of the nano-particles are coated with piperl modified polyethylene glycol, so that the integrity of ginsenoside in gastric juice can be improved, and an introduced piperl group can effectively inhibit P-gp; the absorbability of the intestinal tract to the ginsenoside is improved, so that the bioavailability of the ginsenoside is improved, and the performance of the ginsenoside to a human body is enhanced.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and specifically relates to a ginsenoside compound preparation for enhancing human immunity and a preparation method thereof. Background Art

[0002] Ginsenosides are a class of active ingredients extracted from ginseng and are widely believed to possess a variety of biological activities, including immunomodulatory, antioxidant, anti-inflammatory, anti-fatigue, and anti-tumor properties. However, their low bioavailability in the body limits their clinical application. The low bioavailability of ginsenosides is primarily attributed to the specificity of their molecular structure, which makes them difficult to effectively absorb in the digestive system. Even after ingestion, they are often broken down by gastric acid and intestinal enzymes, resulting in a reduction in the active ingredients, which in turn affects their immunomodulatory effects on the human body.

[0003] Traditional formulations, such as oral ginsenoside tablets or capsules, are unable to effectively overcome these bioavailability issues. To enhance the bioavailability of ginsenosides, researchers have proposed a variety of innovative preparation methods, including modification and optimization of ginsenosides through nanotechnology, liposome delivery systems, and compound formulations. The development of compound formulations, in particular, can enhance immune function and improve drug absorption efficiency by combining them with other auxiliary ingredients.

[0004] The low bioavailability of ginsenosides in the human body is mainly caused by the following factors: Poor water solubility: Most ginsenosides belong to the saponin class of compounds, which are highly hydrophilic, while the human body's digestion and absorption mechanism is mainly targeted at fat-soluble substances or substances that can be dissolved in water, resulting in difficulty in their absorption. Gastrointestinal decomposition: Ginsenosides are often hydrolyzed by enzymes in the gastrointestinal tract. Although the resulting hydrolysis products may have certain biological activity, their immunomodulatory effects are greatly weakened compared to the original saponin molecules. Limited intestinal absorption: Although ginsenosides have certain antioxidant and immune-enhancing functions, due to their large molecular structure and high polarity, they cannot effectively penetrate the cell membrane of the intestine, so their absorption rate is low and their bioavailability is limited.

[0005] To address these challenges, various approaches have been proposed to enhance the bioavailability of ginsenosides, primarily including the following strategies: Nanotechnology applications: By encapsulating ginsenosides in nanoparticles or nanocapsules, their solubility and stability can be significantly improved. These nanocarriers enhance drug solubility and intestinal absorption, thereby increasing bioavailability. Nanocarriers also protect ginsenosides from degradation by gastrointestinal enzymes, ensuring optimal release of their active ingredients. Liposome delivery systems: Liposomes are nanoscale vesicles composed of a bilayer of phospholipid molecules that effectively encapsulate and deliver lipid-soluble drugs. Liposome encapsulation of ginsenosides not only improves their stability but also facilitates intestinal absorption, enhancing bioavailability. Liposome systems facilitate the sustained release of ginsenosides in the body, prolonging their duration of action and enhancing their immunomodulatory effects. Complex formulations: Complex formulations combine ginsenosides with other auxiliary ingredients or drugs to achieve synergistic effects. Common excipients include vitamin C, selenium, and zinc, all of which have immunopotentiating properties. The compound preparation not only improves the absorption efficiency of ginsenosides but also enhances the body's immunity through multiple mechanisms of action. For example, the combination of vitamin C and zinc can promote the production of white blood cells and enhance the activity of the immune system. Summary of the Invention

[0006] In view of the above situation, in order to overcome the shortcomings of the prior art, the present invention provides a ginsenoside compound preparation for enhancing human immunity and a preparation method thereof. By improving the coating carrier of ginsenoside, ginsenoside is coated in polylactic acid glycolic acid nanoparticles, and the outer layer of the nanoparticles is coated with polyethylene glycol modified with piperonyl alcohol. This can improve the integrity of ginsenoside in gastric juice. The introduced piperonyl alcohol group can effectively inhibit P-glycoprotein (P-gp) and improve the intestinal absorption of ginsenosides, thereby increasing the bioavailability of ginsenosides and enhancing the performance of ginsenosides in the human body. Ginsenoside is an active ingredient present in ginseng and has multiple biological active functions. It plays a significant role in regulating and enhancing human immunity through multiple mechanisms such as regulating the activity of immune cells, enhancing anti-tumor ability, anti-inflammatory effects, antioxidant effects, and regulating intestinal flora.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows: the present invention provides a ginsenoside compound preparation for enhancing human immunity, wherein the raw materials for preparing the ginsenoside compound preparation include the following components in parts by weight: 26-35 parts of piperonyl alcohol, 80-100 parts of NH2-PEG-OH, 10-30 parts of ginsenosides, 30-50 parts of polylactic acid glycolic acid, and 30-50 parts of a surfactant;

[0008] Preferably, the surfactant comprises at least one of octylphenol polyoxyethylene ether, sodium dodecylbenzene sulfonate, polyvinyl pyrrolidone, polysorbate 80 and poloxamer;

[0009] Preferably, the raw materials for preparing the NH2-PEG-OH include the following components in parts by weight: 10-15 parts of polyethylene glycol, 8.5-13 parts of triphenylphosphine, 5.5-8.2 parts of phthalimide, and 6-9 parts of diethyl azodicarboxylate;

[0010] Preferably, the preparation method of NH2-PEG-OH specifically comprises the following steps:

[0011] L1. Dissolve polyethylene glycol in dry THF, add triphenylphosphine, mix well, add phthalimide, transfer to an ice-water bath, continue stirring, and slowly add diethyl azodicarboxylate dropwise. After the addition is complete, react at 30-40°C for 18-24 hours, concentrate under reduced pressure, and purify to obtain an intermediate product;

[0012] Preferably, in step L1, the mass concentration of the polyethylene glycol in THF is 75-100 mg / mL;

[0013] L2, dissolving the intermediate product prepared in step L1 in anhydrous ethanol, adding hydrazine hydrate solution dropwise, raising the reaction temperature to carry out reflux reaction, filtering, collecting the filtrate, concentrating under reduced pressure, adding dichloromethane, standing, filtering, collecting the filtrate, and concentrating under reduced pressure to obtain NH2-PEG-OH;

[0014] Preferably, in step L2, the mass concentration of the intermediate product in anhydrous ethanol is 50-80 mg / mL;

[0015] Preferably, in step L2, the mass volume ratio between the intermediate product and the hydrazine hydrate solution is 2-4 g / mL;

[0016] Preferably, in step L2, the reflux reaction temperature is 80-90°C and the reaction time is 2-3h;

[0017] The present invention also provides a method for preparing a ginsenoside compound preparation for enhancing human immunity, which specifically comprises the following steps:

[0018] S1. dissolving polylactic acid glycolic acid in an oil phase solution, adding total ginsenosides, and mixing well to obtain an oil phase;

[0019] Preferably, in step S1, the volume ratio of anhydrous ethanol to acetone in the oil phase solution is 2-3:1-2;

[0020] Preferably, in step S1, the mass concentration of the polylactic acid-co-glycolic acid in the oil phase solution is 10-20 mg / mL;

[0021] S2. Dissolve the surfactant in deionized water to obtain an aqueous phase, add the oil phase prepared in step S1 dropwise to the aqueous phase, and stir to react thoroughly. After the reaction is completed, perform vacuum distillation, wash, and dry to obtain ginsenoside nanoparticles;

[0022] Preferably, in step S2, the volume ratio between the water phase and the oil phase is 2-4:1;

[0023] Preferably, in step S2, the stirring reaction time is 3-5h;

[0024] S3, dissolving NH2-PEG-OH in a THF / DCM mixed solvent, adding piperonyl alcohol and triphenylphosphine, mixing well, transferring to an ice-water bath, adding diisopropyl azodicarboxylate dropwise, raising the reaction temperature after completion of the addition, performing a reflux reaction, concentrating under reduced pressure, washing with deionized water, and purifying to obtain piperonyl alcohol-modified PEG-NH2;

[0025] Preferably, in step S3, the mass concentration of the NH2-PEG-OH in the THF / DCM mixed solvent is 4-5 g / L;

[0026] Preferably, in step S3, the volume ratio between THF and DCM in the THF / DCM mixed solvent is 1:1-3;

[0027] Preferably, in step S3, the mass ratio between triphenylphosphine and piperonyl alcohol is 1:1.8-1.9;

[0028] Preferably, in step S3, the mass ratio between the piperonyl alcohol and diisopropyl azodicarboxylate is 1:1.35-1.5;

[0029] Preferably, in step S3, the reaction temperature of the reflux reaction is 50-60° C., and the reaction time is 18-24 h;

[0030] S4, dissolving the ginsenoside nanoparticles prepared in step S2 in DMSO, adding EDC and NHS for activation, adding the piperonyl alcohol-modified PEG-NH2 prepared in step S3 to the reaction system, and continuously stirring the reaction at room temperature for 4-5 hours. After the reaction is completed, neutralizing the reaction system, dialyzing to remove unreacted reactants, and freeze-drying to obtain a ginsenoside composite preparation;

[0031] Preferably, in step S4, the mass ratio between EDC and NHS is 1:0.8-0.9;

[0032] Preferably, in step S4, the mass concentration of the EDC in DMSO is 1-2 mg / mL.

[0033] The beneficial effects achieved by the present invention are as follows:

[0034] The present invention provides a ginsenoside compound preparation for enhancing human immunity and a preparation method thereof. By improving the coating carrier of ginsenoside, ginsenoside is coated in polylactic acid glycolic acid nanoparticles, and the outer layer of the nanoparticles is coated with polyethylene glycol modified with piperonyl alcohol. This can improve the integrity of ginsenoside in gastric juice. The introduced piperonyl alcohol group can effectively inhibit P-gp, improve the intestinal absorbability of ginsenosides, thereby improving the bioavailability of ginsenosides and enhancing the performance of ginsenosides on the human body. Ginsenoside is an active ingredient present in ginseng and has multiple biologically active functions. It plays a significant role in regulating and enhancing human immunity through multiple mechanisms such as regulating the activity of immune cells, enhancing anti-tumor ability, anti-inflammatory effects, antioxidant effects, and regulating intestinal flora. The present invention forms ginsenoside nanoparticles by preparing polylactic acid glycolic acid to coat ginsenosides. P-gp is a transmembrane transport protein present in intestinal epithelial cells. It can actively transport exogenous substances (including drugs) from the cell to the outside of the cell, thereby limiting the absorption of drugs. Piperine can enhance the intestinal absorption of some drugs by inhibiting P-glycoprotein (P-gp). The main group playing a role is the aromatic 3,4-methylenedioxybenzene region. The 3,4-methylenedioxyphenyl group on the aromatic ring can interact with the drug binding site of P-gp, affecting its activity. The present invention reacts piperonyl alcohol with polyethylene glycol to form a 3,4-methylenedioxyphenyl group. , a 3,4-methylenedioxyphenyl group with P-gp inhibitory activity is grafted onto one end of the polyethylene glycol, and the other end of the polyethylene glycol is connected to the nanoparticle through a -CO-NH- bond. When the ginsenoside nanoparticles enter the intestine, the relevant protease can hydrolyze the -CO-NH- bond, and the 3,4-methylenedioxyphenyl-modified polyethylene glycol is more likely to interact with the cell membrane of the intestinal epithelial cells, promoting the endocytosis of the nanoparticles into the intestinal cells through the cell membrane, which not only improves the absorption capacity of the nanoparticles, but also may make the release of ginsenosides in the intestine more efficient. Modifying the surface of the nanoparticles with 3,4-methylenedioxybenzene can improve the absorption capacity of the coated ginsenosides in the intestine. This is achieved through multiple mechanisms, mainly including inhibiting the elimination effect of P-gp, enhancing the cellular absorption of nanoparticles, improving stability and bioavailability, and improving the intestinal absorption rate of drugs through targeted delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The infrared spectroscopic images of the ginsenoside nanoparticles and ginsenoside composite preparation prepared in Example 1;

[0036] Figure 2 The graph shows the in vitro release performance of the ginsenoside composite preparations prepared in Examples 1-3 and Comparative Examples 1-2 of the present invention;

[0037] Figure 3 Graph showing the transport and absorption performance of the ginsenoside composite preparations prepared in Examples 1-3 and Comparative Examples 1-2 of the present invention;

[0038] Figure 4 This is a graph showing the effects of the ginsenoside compound preparations prepared in Examples 1-3 of the present invention and Comparative Examples 1-2 on mouse immune factors.

[0039] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention. DETAILED DESCRIPTION

[0040] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only and are not intended to limit the scope of this application.

[0042] The experimental methods in the following examples, unless otherwise specified, are conventional methods; the test materials and test strains used in the following examples, unless otherwise specified, are purchased from commercial channels.

[0043] Polyethylene glycol, derived from PEG-600, brand: Aladdin, item number: P103727, product specification: averageMn600;

[0044] Ginsenosides, derived from total ginsenosides, brand: Yuanye, item number: S25997, product specification: UV ≥ 80% (root extract);

[0045] Polylactic acid-co-glycolic acid, derived from the product polylactic acid-co-glycolic acid copolymer, brand: Yuanye, item number: S24436, product specifications: molecular weight: 10,000-20,000 PLGA 50:50, carboxyl terminated, carboxyl terminated.

[0046] Example 1

[0047] This embodiment provides a ginsenoside compound preparation for enhancing human immunity. The raw materials for preparing the ginsenoside compound preparation include the following components in parts by weight: 30 parts of piperonyl alcohol, 90 parts of NH2-PEG-OH, 30 parts of ginsenoside, 50 parts of polylactic acid glycolic acid, and 50 parts of poloxamer;

[0048] The raw materials for preparing NH2-PEG-OH include the following components in parts by weight: 12 parts of polyethylene glycol, 10.5 parts of triphenylphosphine, 6.5 parts of phthalimide, and 7.0 parts of diethyl azodicarboxylate;

[0049] The preparation method of NH2-PEG-OH specifically comprises the following steps:

[0050] L1. Accurately weigh 12 g of polyethylene glycol (PEG-600) and place it in a flask. Add 200 mL of anhydrous THF to completely dissolve the polyethylene glycol. Add 10.5 g of triphenylphosphine to the reaction system and stir at 300 rpm. After mixing evenly, add 6.5 g of phthalimide and transfer the reaction system to an ice-water bath and continue stirring the reaction. Dissolve 7.0 g of diethyl azodicarboxylate in 50 mL of anhydrous THF and add it dropwise to the reaction system at a rate of 1 mL / min. After the addition is complete, place the mixture at 30 ° C for stirring and react. After reacting for 24 hours, concentrate under reduced pressure to remove excess reaction cosolvent, purify the reactant by column chromatography, and dry it to obtain an intermediate product.

[0051] L2, take 8.0 g of the intermediate product prepared in step L1 and place it in a dry flask. After adding 150 mL of anhydrous ethanol to fully dissolve the intermediate product, take 4 mL of hydrazine hydrate solution and add it dropwise to the reaction system at 0.5 mL / min. After the addition is complete, raise the reaction temperature to 80 ° C. and reflux reaction. After reacting for 3 hours, filter, collect the filtrate and concentrate under reduced pressure. Add dichloromethane and the concentrated substance and mix evenly. After standing at 0 ° C. for 4 hours, filter, collect the filtrate and concentrate under reduced pressure to obtain NH2-PEG-OH;

[0052] This embodiment also provides a method for preparing a ginsenoside compound preparation for enhancing human immunity, which specifically comprises the following steps:

[0053] S1. Prepare an oil phase solution by mixing anhydrous ethanol and acetone in a volume ratio of 2:1. Accurately weigh 50 mg of poly(lactic-co-glycolic acid) and dissolve it in 5 mL of the oil phase solution. Stir at 500 rpm to completely dissolve the poly(lactic-co-glycolic acid). Add 30 mg of total ginsenosides and continue stirring until the total ginsenosides are completely dissolved. After the reaction system is evenly mixed, obtain an oil phase.

[0054] S2. Dissolve 50 mg of poloxamer in 10 mL of deionized water and mix uniformly at 500 rpm to obtain an aqueous phase. Add the oil phase prepared in step S1 dropwise to the aqueous phase at 2 mL / min while increasing the stirring speed to 1000 rpm for stirring and mixing. After the addition is complete, continue stirring and react for 4 hours. Remove excess reaction solvent by distillation under reduced pressure, repeatedly wash with deionized water and saturated NaCl solution, and freeze-dry to obtain ginsenoside nanoparticles.

[0055] S3. Accurately weigh 90 mg of NH2-PEG-OH and place it in a flask. Mix THF and DCM in a volume ratio of 1:1 to obtain a THF / DCM mixed solvent. Take 5 mL of the THF / DCM mixed solvent and add it to the flask. Add 30 mg of piperonyl alcohol and 57 mg of triphenylphosphine. After mixing evenly, transfer the reaction system to an ice-water bath. Accurately weigh 0.45 g of diisopropyl azodicarboxylate and dissolve it in 5 mL of anhydrous THF. Take 0.5 mL and add it dropwise to the reaction system at 0.1 mL / min. After the addition is complete, increase the reaction temperature to 60°C, reflux for 18 hours, concentrate under reduced pressure, wash repeatedly with deionized water to remove excess reaction solvent, and purify by column chromatography to obtain piperonyl alcohol-modified PEG-NH2.

[0056] S4. The ginsenoside nanoparticles prepared in step S2 were dissolved in 10 mL of DMSO, and 10 mg of EDC and 8 mg of NHS were added for activation. The piperonyl alcohol-modified-PEG-NH2 prepared in step S3 was added to the reaction system, and the mixture was stirred continuously at room temperature for 4 h. After the reaction was completed, the reaction system was neutralized, and the unreacted reactants were removed by dialysis. After freeze-drying, a ginsenoside composite preparation was obtained.

[0057] Example 2

[0058] This embodiment provides a ginsenoside compound preparation for enhancing human immunity. The raw materials for preparing the ginsenoside compound preparation include the following components in parts by weight: 35 parts of piperonyl alcohol, 100 parts of NH2-PEG-OH, 10 parts of ginsenoside, 30 parts of polylactic acid glycolic acid, and 30 parts of poloxamer;

[0059] The raw materials for preparing NH2-PEG-OH include the following components in parts by weight: 10 parts of polyethylene glycol, 8.75 parts of triphenylphosphine, 5.5 parts of phthalimide, and 6.0 parts of diethyl azodicarboxylate;

[0060] The preparation method of NH2-PEG-OH specifically comprises the following steps:

[0061] L1. Accurately weigh 10 g of polyethylene glycol (PEG-600) and place it in a flask. Add 200 mL of anhydrous THF to completely dissolve the polyethylene glycol. Add 8.75 g of triphenylphosphine to the reaction system and stir at 300 rpm. After mixing evenly, add 5.5 g of phthalimide and transfer the reaction system to an ice-water bath and continue stirring the reaction. Dissolve 6.0 g of diethyl azodicarboxylate in 50 mL of anhydrous THF and add it dropwise to the reaction system at a rate of 1 mL / min. After the addition is complete, place the mixture at 40° C. and stir to react. After reacting for 18 hours, concentrate under reduced pressure to remove excess reaction cosolvent, purify the reactant by column chromatography, and dry to obtain an intermediate product.

[0062] L2, take 10.0 g of the intermediate product prepared in step L1 and place it in a dry flask. After adding 150 mL of anhydrous ethanol to fully dissolve the intermediate product, take 2.5 mL of hydrazine hydrate solution and add it dropwise to the reaction system at 0.5 mL / min. After the addition is complete, raise the reaction temperature to 90 ° C. and reflux reaction. After reacting for 2 hours, filter, collect the filtrate and concentrate under reduced pressure. Add dichloromethane and the concentrated substance and mix evenly. After standing at 0 ° C. for 4 hours, filter, collect the filtrate and concentrate under reduced pressure to obtain NH2-PEG-OH;

[0063] This embodiment also provides a method for preparing a ginsenoside compound preparation for enhancing human immunity, which specifically comprises the following steps:

[0064] S1. Anhydrous ethanol and acetone were prepared in a volume ratio of 3:2 to form an oil phase solution. 30 mg of poly(lactic-co-glycolic acid) was accurately weighed and dissolved in 2 mL of the oil phase solution. The mixture was stirred at 500 rpm to completely dissolve the poly(lactic-co-glycolic acid). 10 mg of total ginsenosides was added and the mixture was stirred and mixed until the total ginsenosides were completely dissolved. After the reaction system was evenly mixed, an oil phase was obtained.

[0065] S2. Dissolve 30 mg of sodium dodecylbenzenesulfonate in 6 mL of deionized water and mix uniformly at 500 rpm to obtain an aqueous phase. Add the oil phase prepared in step S1 dropwise to the aqueous phase at 2 mL / min while increasing the stirring speed to 1000 rpm for stirring and mixing. After the addition is complete, continue stirring and react for 3 h. Remove excess reaction solvent by distillation under reduced pressure, repeatedly wash with deionized water and saturated NaCl solution, and freeze-dry to obtain ginsenoside nanoparticles.

[0066] S3. Accurately weigh 100 mg of NH2-PEG-OH and place it in a flask. Mix THF and DCM in a volume ratio of 1:2 to obtain a THF / DCM mixed solvent. Take 5 mL of the THF / DCM mixed solvent and add it to the flask. Add 35 mg of piperonyl alcohol and 65 mg of triphenylphosphine. After mixing evenly, transfer the reaction system to an ice-water bath. Accurately weigh 0.5 g of diisopropyl azodicarboxylate and dissolve it in 5 mL of anhydrous THF. Take 0.5 mL and add it dropwise to the reaction system at 0.1 mL / min. After the addition is complete, increase the reaction temperature to 55°C, reflux for 21 hours, concentrate under reduced pressure, wash repeatedly with deionized water to remove excess reaction solvent, and purify by column chromatography to obtain piperonyl alcohol-modified PEG-NH2.

[0067] S4. Dissolve the ginsenoside nanoparticles prepared in step S2 in 10 mL of DMSO, add 20 mg of EDC and 18 mg of NHS for activation, add the piperonyl alcohol-modified-PEG-NH2 prepared in step S3 to the reaction system, and continue stirring to react at room temperature for 5 hours. After the reaction is completed, neutralize the reaction system, dialyze to remove unreacted reactants, and freeze-dry to obtain a ginsenoside composite preparation.

[0068] Example 3

[0069] This embodiment provides a ginsenoside compound preparation for enhancing human immunity. The raw materials for preparing the ginsenoside compound preparation include the following components in parts by weight: 26 parts of piperonyl alcohol, 80 parts of NH2-PEG-OH, 14 parts of ginsenoside, 40 parts of polylactic acid glycolic acid, and 40 parts of poloxamer;

[0070] The raw materials for preparing NH2-PEG-OH include the following components in parts by weight: 15 parts of polyethylene glycol, 13 parts of triphenylphosphine, 8.2 parts of phthalimide, and 8.8 parts of diethyl azodicarboxylate;

[0071] The preparation method of NH2-PEG-OH specifically comprises the following steps:

[0072] L1. Accurately weigh 15 g of polyethylene glycol (PEG-600) and place it in a flask. Add 200 mL of anhydrous THF to completely dissolve the polyethylene glycol. Add 13 g of triphenylphosphine to the reaction system and stir at 300 rpm. After mixing evenly, add 8.2 g of phthalimide and transfer the reaction system to an ice-water bath and continue stirring the reaction. Dissolve 8.8 g of diethyl azodicarboxylate in 50 mL of anhydrous THF and add it dropwise to the reaction system at a rate of 1 mL / min. After completion of the addition, place the mixture at 35 ° C. and stir to react. After reacting for 21 hours, concentrate under reduced pressure to remove excess reaction cosolvent, purify the reactant by column chromatography, and dry to obtain an intermediate product.

[0073] L2, take 12.0g of the intermediate product prepared in step L1 and place it in a dry flask. After adding 150mL of anhydrous ethanol to fully dissolve the intermediate product, take 4mL of hydrazine hydrate solution and add it dropwise to the reaction system at 0.5mL / min. After the addition is complete, raise the reaction temperature to 85°C and reflux reaction. After reacting for 2.5h, filter, collect the filtrate and concentrate under reduced pressure. Add dichloromethane and mix the concentrated material evenly, place it at 0°C and let it stand for 4h, filter, collect the filtrate and concentrate under reduced pressure to obtain NH2-PEG-OH;

[0074] This embodiment also provides a method for preparing a ginsenoside compound preparation for enhancing human immunity, which specifically comprises the following steps:

[0075] S1. Anhydrous ethanol and acetone were prepared in a volume ratio of 1:1 to form an oil phase solution. 40 mg of polylactic-co-glycolic acid was accurately weighed and dissolved in 2 mL of the oil phase solution. The mixture was stirred at 500 rpm to completely dissolve the polylactic-co-glycolic acid. 14 mg of total ginsenosides was added and the mixture was stirred and mixed until the total ginsenosides were completely dissolved. After the reaction system was evenly mixed, an oil phase was obtained.

[0076] S2. Dissolve 40 mg of octylphenol polyoxyethylene ether in 8 mL of deionized water and mix uniformly at 500 rpm to obtain an aqueous phase. Add the oil phase prepared in step S1 dropwise to the aqueous phase at 2 mL / min while increasing the stirring speed to 1000 rpm for stirring and mixing. After the addition is complete, continue stirring and react for 5 h. Remove excess reaction solvent by distillation under reduced pressure, repeatedly wash with deionized water and saturated NaCl solution, and freeze-dry to obtain ginsenoside nanoparticles.

[0077] S3. Accurately weigh 80 mg of NH2-PEG-OH and place it in a flask. Mix THF and DCM in a volume ratio of 1:3 to obtain a THF / DCM mixed solvent. Take 5 mL of the THF / DCM mixed solvent and add it to the flask. Add 26 mg of piperonyl alcohol and 42 mg of triphenylphosphine. After mixing evenly, transfer the reaction system to an ice-water bath. Accurately weigh 0.36 g of diisopropyl azodicarboxylate and dissolve it in 5 mL of anhydrous THF. Take 0.5 mL and add it dropwise to the reaction system at 0.1 mL / min. After the addition is complete, increase the reaction temperature to 50°C, reflux for 24 hours, concentrate under reduced pressure, wash repeatedly with deionized water to remove excess reaction solvent, and purify by column chromatography to obtain piperonyl alcohol-modified PEG-NH2.

[0078] S4. The ginsenoside nanoparticles prepared in step S2 were dissolved in 10 mL of DMSO, 15 mg of EDC and 12 mg of NHS were added for activation, and the piperonyl alcohol-modified-PEG-NH2 prepared in step S3 was added to the reaction system. The mixture was stirred continuously at room temperature for 4.5 h. After the reaction was completed, the reaction system was neutralized, dialyzed to remove unreacted reactants, and freeze-dried to obtain a ginsenoside composite preparation.

[0079] Comparative Example 1

[0080] This comparative example provides a ginsenoside compound preparation and a preparation method thereof, which differs from Example 1 only in that all components do not contain piperonyl alcohol, and the remaining components and component contents are the same as those in Example 1.

[0081] Comparative Example 2

[0082] This comparative example provides a ginsenoside compound preparation and a preparation method thereof, which differs from Example 1 only in that all components do not contain NH2-PEG-OH, and the remaining components and component contents are the same as those in Example 1.

[0083] Experimental Example 1

[0084] In this experimental example, the chemical structure of the ginsenoside composite preparation prepared in Example 1 was analyzed by infrared spectroscopy. According to the preparation process of potassium bromide tableting method, the ginsenoside nanoparticles and ginsenoside composite preparation prepared in Example 1 were dried, mixed with potassium bromide powder, ground and tableted, and the samples were scanned using a Nicolet 710 Fourier transform infrared spectrometer. Figure 1 The infrared spectra of the ginsenoside nanoparticles and ginsenoside composite preparation prepared in Example 1 are shown in Figure 1, where A is the ginsenoside nanoparticles and B is the ginsenoside composite preparation. As shown in Figure 1, in A, since ginsenosides contain phenolic and organic acid active groups and are coated with polylactic acid glycolic acid, the absorption peak at 3440-3410 cm-1 indicates that the active groups in ginsenosides still exist, indicating that ginsenosides have not been completely destroyed or changed. At the same time, the absorption peak at 1640-1610 cm-1 indicates that the active groups in ginsenosides still exist. -1 The absorption peak at 1635-1615 cm is the C=O carbonyl stretching vibration in PLGA, indicating that the PLGA polymer successfully exists in the final prepared nanoparticles. In the preparation of ginsenoside nanoparticles, a nanoprecipitation method is used, in which ginsenoside and PLGA are mixed by dissolving in an organic phase, and then precipitated by adding an aqueous phase to form nanoparticles. According to the infrared spectrum, the absorption peak of ginsenoside did not show a drastic shift change, indicating that ginsenoside was not completely changed or exposed to the external environment, indicating that it was encapsulated inside the nanoparticles. In B, at 1635-1615 cm -1 and 1515-1495cm-1 The characteristic absorption vibration peak of the benzene ring appears at 1225-1200cm -1 、945-925cm -1 The stretching vibration of the epoxy ring skeleton is at 1125-1100cm -1 The characteristic absorption peak of COC appears at , indicating that the characteristic groups of piperonyl alcohol exist on the surface of the ginsenoside composite preparation prepared in Example 1 of the present invention.

[0085] Experimental Example 2

[0086] In this experimental example, the in vitro release performance of the ginsenoside compound preparations prepared in Examples 1-3 and Comparative Examples 1-2 was tested. A phosphate buffer solution containing 2 wt% tween 80 was used as the release medium, and the pH of the release medium was adjusted to 6.5. The in vitro release ability of the ginsenoside compound preparation was determined by dialysis. The ginsenoside compound preparations prepared in Examples 1-3 and Comparative Examples 1-2 were dissolved in 5 wt% glucose injection at a concentration of 1 mg / mL to prepare a ginsenoside compound preparation solution, which was transferred to an activated dialysis bag with an analytical cutoff of 30 kDa. The bag was sealed after air bubbles were removed and placed in the release medium. Under sealed conditions, it was placed at 37°C and shaken at a speed of 100 rpm. Samples were taken at the sampling points, the concentration of ginsenosides in the release medium was determined, and the cumulative release rate of ginsenosides was calculated.

[0087] Figure 2 The in vitro release performance results of the ginsenoside composite preparations prepared in Examples 1-3 and Comparative Examples 1-2 of the present invention are shown. As shown in the figure, the release rate of the ginsenoside composite preparations prepared in Examples 1-3 is significantly lower than that of the ginsenoside composite preparations prepared in Comparative Examples 1 and 2. In Comparative Example 1, due to the lack of modification with piperonyl alcohol, the surface hydrophilicity of the ginsenoside composite preparation is relatively strong, and its release efficiency is relatively fast in a water-based release medium. In Comparative Example 2, due to the lack of NH2-PEG-OH coating, the surface structure of the nanoparticles is loose, resulting in a low release efficiency. The binding of piperonyl alcohol to the ginsenoside nanoparticles is poor, and hydrophobic surface modification cannot be achieved, resulting in a high hydrophilicity of the ginsenoside nanoparticles, which is easily decomposed in a water-based environment and thus has a high release rate.

[0088] Example 3

[0089] In this example, the ginsenoside compound preparations prepared in Examples 1-3 and Comparative Examples 1-2 were subjected to transport and absorption experiments. A Caco-2 cell monolayer model was established. Caco-2 cells were cultured to 80% to 90% confluence, digested with trypsin, and resuspended. The cells were then inoculated into Transwell culture plates at a density of 1.5×10 cells per well.5 The upper microporous membrane of the Transwell culture plate (pore size of 3 μm) simulates the barrier of intestinal epithelial cells, allowing cells to grow and form a closed cell monolayer. 2 mL of complete cell culture medium was added to the lower layer per well and cultured until the resistance value (TEER) was greater than 400 Ω·cm 2 When , it indicates that the cell monolayer is well formed and the Caco-2 cell monolayer model is successfully established.

[0090] The Caco-2 cell monolayer model was rinsed twice in preheated HBSS buffer. After removing the HBSS, the transport of danshensu from the cell villus side to the basal surface in the Caco-2 cell model was tested to simulate the process of the drug entering the blood through the intestinal epithelial cells. The cell villus side was used as the supply pool, and the ginsenoside compound preparations prepared in Examples 1-3 and Comparative Examples 1-2 were added. The basal surface side was used as the receiving pool, and HBSS buffer solution was added as the receiving pool. After 180 minutes, samples were taken from the receiving pools to detect the concentration of ginsenosides. The effective permeability coefficient was used to evaluate the permeation rate of ginsenosides on the cell monolayer (cm·s -1 ), the specific formula is as follows:

[0091]

[0092] in, is the rate of change of drug concentration in the receiving pool; A is the area of ​​the cell membrane, cm 2 ; C0 is the initial concentration of the drug in the supply pool, mol·mL -1 .

[0093] Figure 3 The transport and absorption performance results of the ginsenoside composite preparations prepared in Examples 1-3 and Comparative Examples 1-2 of the present invention are shown in the figure. As shown in the figure, the effective permeability of ginsenosides in the receiving pool in Examples 1-3 was significantly higher than that in Comparative Examples 1 and 2. In Comparative Example 2, piperonyl alcohol has the effect of inhibiting P-gp protein and can also increase the effective permeability of its ginsenosides. However, since piperonyl alcohol is free in the supply pool, its instantaneous inhibitory effect on P-gp is more obvious, but the ginsenosides are not initially released from the carrier. Therefore, the effective permeability of Comparative Example 2 is lower than that of the treatment group in Examples 1-3.

[0094] Experimental Example 4

[0095] In this experimental example, the functionality of the ginsenoside compound preparations prepared in Examples 1-3 and Comparative Examples 1-2 was tested. SPF grade 5-8 week old ICR male mice weighing 20±2 g were taken. After one week of adaptation, the mice were randomly divided into 8 groups, namely, Example 1, 2, 3 groups, Comparative Example 1, 2 groups, normal group, disease group and positive group, with 5 mice in each group. On the 1st to 3rd day of the experiment, except for the normal group, the mice in the other groups were intraperitoneally injected with cyclophosphamide at a dose of 80 mg / (kg BW) to establish the model. Drug intervention was performed on the 4th to 28th day of the experiment, among which the positive group The mice were gavaged with levamisole hydrochloride solution at a dose of 40 mg / (kg BW). The Example 1, 2, and 3 groups and the Comparative Example 1 and 2 groups were treated with 8.14 mg / kg ginsenoside compound preparation. The normal group and the disease group were gavaged with an equal amount of 0.5% CMC-Na solution daily. After the culture, the levels of immunoglobulin A (IgA), immunoglobulin G (IgG), interleukin 2 (IL-2), interleukin 6 (IL-6), and interferon gamma (IFN-γ) in the serum of each group of mice were determined strictly according to the instructions of the ELISA kit.

[0096] Figure 4 This is a graph showing the effects of the ginsenoside compound preparations prepared in Examples 1-3 of the present invention and Comparative Examples 1-2 on mouse immune factors. As shown in the figure, in the Example 1-3 treatment groups, the levels of various immune factors are relatively high, indicating that the ginsenoside compound preparations prepared in Examples 1-3 of the present invention have a certain ability to enhance immunity. The modified compound preparations improve the absorption capacity of ginsenosides in the mouse intestine, further enhancing the positive effects of ginsenosides.

[0097] While the embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that various changes, modifications, substitutions, and alterations can be made to the embodiments without departing from the principles and spirit of the invention.

[0098] The present invention and its embodiments are described above. Such description is not restrictive. The drawings show only one embodiment of the present invention, and actual applications are not limited thereto. In short, if a person skilled in the art is inspired by the above, and does not deviate from the purpose of the present invention, any method and embodiment similar to the technical solution without creative design shall fall within the scope of protection of the present invention.

Claims

1. A ginsenoside compound preparation for enhancing human immunity, characterized by: The raw materials for preparing the ginsenoside compound preparation include the following components in parts by weight: 26-35 parts of piperonyl alcohol, 80-100 parts of NH2-PEG-OH, 10-30 parts of ginsenoside, 30-50 parts of polylactic acid glycolic acid, and 30-50 parts of surfactant; The raw materials for preparing the NH2-PEG-OH include the following components in parts by weight: 10-15 parts of polyethylene glycol, 8.5-13 parts of triphenylphosphine, 5.5-8.2 parts of phthalimide, and 6-9 parts of diethyl azodicarboxylate.

2. The ginsenoside compound preparation for enhancing human immunity according to claim 1, characterized in that: The surfactant includes at least one of octylphenol polyoxyethylene ether, sodium dodecylbenzene sulfonate, polyvinyl pyrrolidone, polysorbate 80 and poloxamer.

3. The ginsenoside compound preparation for enhancing human immunity according to claim 2, characterized in that: The preparation method of the NH2-PEG-OH specifically comprises the following steps: L1. Dissolve polyethylene glycol in dry THF, add triphenylphosphine, mix well, add phthalimide, transfer to an ice-water bath, continue stirring, and slowly add diethyl azodicarboxylate dropwise. After the addition is complete, react at 30-40°C for 18-24 hours, concentrate under reduced pressure, and purify to obtain an intermediate product; L2. Dissolve the intermediate product prepared in step L1 in anhydrous ethanol, add hydrazine hydrate solution dropwise, increase the reaction temperature to perform reflux reaction, filter after the reaction, collect the filtrate, concentrate under reduced pressure, add dichloromethane, let stand, filter, collect the filtrate, and concentrate under reduced pressure to obtain NH2-PEG-OH.

4. The ginsenoside compound preparation for enhancing human immunity according to claim 3, characterized in that: In step L1, the mass concentration of the polyethylene glycol in THF is 75-100 mg / mL.

5. The ginsenoside compound preparation for enhancing human immunity according to claim 4, characterized in that: In step L2, the mass concentration of the intermediate product in anhydrous ethanol is 50-80 mg / mL; the mass-to-volume ratio of the intermediate product to the hydrazine hydrate solution is 2-4 g / mL.

6. The ginsenoside compound preparation for enhancing human immunity according to claim 5, characterized in that: In step L2, the reflux reaction temperature is 80-90° C., and the reaction time is 2-3 h.

7. A method for preparing the ginsenoside compound preparation for enhancing human immunity according to claim 6, characterized in that: The specific steps include: S1. dissolving polylactic acid glycolic acid in an oil phase solution, adding total ginsenosides, and mixing well to obtain an oil phase; S2. Dissolve the surfactant in deionized water to obtain an aqueous phase, add the oil phase prepared in step S1 dropwise to the aqueous phase, and stir to react for 3-5 hours. After the reaction is completed, distill under reduced pressure, wash, and dry to obtain ginsenoside nanoparticles; S3, dissolving NH2-PEG-OH in a THF / DCM mixed solvent, adding piperonyl alcohol and triphenylphosphine, mixing well, transferring to an ice-water bath, adding diisopropyl azodicarboxylate dropwise, raising the reaction temperature after completion of the addition, performing a reflux reaction, concentrating under reduced pressure, washing with deionized water, and purifying to obtain piperonyl alcohol-modified PEG-NH2; S4. Dissolve the ginsenoside nanoparticles prepared in step S2 in DMSO, add EDC and NHS for activation, add the piperonyl alcohol-modified PEG-NH2 prepared in step S3 to the reaction system, and continue stirring to react at room temperature for 4-5 hours. After the reaction is completed, neutralize the reaction system, dialyze to remove unreacted reactants, and freeze-dry to obtain a ginsenoside composite preparation.

8. The method for preparing a ginsenoside compound preparation for enhancing human immunity according to claim 7, characterized in that: In step S1, the volume ratio of anhydrous ethanol to acetone in the oil phase solution is 2-3:1-2; the mass concentration of polylactic acid glycolic acid in the oil phase solution is 10-20 mg / mL; in step S2, the volume ratio of the aqueous phase to the oil phase is 2-4:

1.

9. The method for preparing a ginsenoside compound preparation for enhancing human immunity according to claim 8, characterized in that: In step S3, the mass concentration of the NH2-PEG-OH in the THF / DCM mixed solvent is 4-5 g / L; the volume ratio of THF and DCM in the THF / DCM mixed solvent is 1:1-3; the mass ratio of triphenylphosphine to piperonyl alcohol is 1:1.8-1.9; the mass ratio of piperonyl alcohol to diisopropyl azodicarboxylate is 1:1.35-1.5; the reaction temperature of the reflux reaction is 50-60°C, and the reaction time is 18-24 hours.

10. The method for preparing a ginsenoside compound preparation for enhancing human immunity according to claim 9, characterized in that: In step S4, the mass ratio of the EDC to NHS is 1:0.8-0.9; and the mass concentration of the EDC in DMSO is 1-2 mg / mL.

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