Method for increasing solubility of portulaca oleracea flavonoid compounds

Abstract

The invention discloses a method for increasing solubility of portulaca oleracea flavonoid compounds, which comprises the following steps: dissolving the portulaca oleracea flavonoid compounds in methanol or ethanol, and carrying out ultrasonic dissolution to obtain a clear, transparent and uniform phase A; mixing cationic amino acid, a dissolution promoter, polyol and water to form a uniform phase B; slowly dropwise adding the phase A into the phase B under an ultrasonic condition, and reducing pressure to remove methanol after dropwise adding is completed, so as to prepare a mixed solution; placing the mixed solution in liquid nitrogen, rapidly cooling and freezing, and freeze-drying to obtain high-content purslane flavonoid compound freeze-dried powder; the surfactant, the arginine and the polyhydric alcohol system are synergistically compounded through the hydrophobic effect, the cation-pi and the solvation effect to interfere the pi-pi stacking degree between flavonoid molecules, and finally the excellent dissolution promoting and solubilizing effect on the purslane flavonoid compound is achieved, and then the bioavailability of the purslane flavonoid compound is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of preparation of bioactive substances, and in particular relates to a method for increasing the solubility of purslane flavonoid compounds. Background Art

[0002] Purslane is a herb widely distributed in the world. Its main active ingredient is flavonoids, which account for 7.67% of the total weight of Purslane. It has outstanding antioxidant, anti-inflammatory, anti-diabetic, antibacterial, and anti-cancer medicinal values.

[0003] Flavonoids are one of the largest plant secondary metabolites. They are compounds with a common C6-C3-C6 skeleton, in which two aromatic rings are connected by a three-carbon bridge, usually forming a benzoic acid arrangement. This structure makes it easy for π-π stacking to occur between the molecules of these active substances, resulting in their extremely poor solubility in aqueous solutions, which in turn limits their widespread application in the fields of biomedicine and daily chemicals.

[0004] Researchers have applied various technologies to improve the solubility of poorly water-soluble drugs, such as active molecule complexation, micelle and microemulsion solubilization, cosolvents, polymer micelles, prodrug technology, drug nanocrystals, solid dispersion methods, crystal engineering technology and nanomorphology technology. In 2018, Rita Caparica et al. prepared two choline-based ILs as ionic liquids to improve the solubility of poorly soluble ferulic acid and rutin. In 2021, Xuan Jingjing et al. used low-toxic choline bicarbonate and choline hydroxide as cations and geranic acid, malic acid, malonic acid, phenylalanine, sorbic acid, and succinic acid as anions to prepare ionic liquids that showed significant solubilization efficiency for active substances such as quercetin and retinoic acid. In 2023, Guo Youjia et al. solubilized the poorly soluble drugs rifampicin and famotidine based on the molecular recognition ability of open-ring cucurbituril, and found that their solubilization properties in water were improved by 147 and 39 times, respectively; this shows that improving the water solubility of candidate drugs by salification, co-crystal formation, and utilizing metastable crystal forms or introducing polar functional groups into the molecular structure is a common main strategy and method to improve the water-soluble drugs.

[0005] However, the use of green and mild solvents to effectively solubilize purslane flavonoids is still a problem in the field to improve drug bioavailability. Summary of the invention

[0006] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0007] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0008] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for increasing the solubility of purslane flavonoid compounds.

[0009] In order to solve the above technical problems, the present invention provides the following technical solution: a method for increasing the solubility of purslane flavonoid compounds, comprising:

[0010] The purslane flavonoids are dissolved in methanol or ethanol, and ultrasonically dissolved to obtain a clear, transparent and uniform phase A;

[0011] Mixing cationic amino acid, dissolution agent, polyol and water into a uniform phase B;

[0012] Phase A is slowly added dropwise to phase B under ultrasonic conditions, and after the addition is complete, methanol is removed under reduced pressure to obtain a mixed solution;

[0013] The mixed solution is placed in liquid nitrogen for rapid cooling and freezing, and freeze-dried to obtain a high-content purslane flavonoids complex freeze-dried powder;

[0014] Wherein, the cationic amino acid is one or more of arginine, polyarginine, lysine and histidine;

[0015] The dissolution promoter is one or more than two of sodium lauroyl glutamate, potassium lauroyl sarcosinate, sodium subtilisin, sodium cocoyl glycinate, Tween 80, polyether-26, cyclodextrin, lecithin, and hydroxypropyl cyclodextrin.

[0016] As a preferred embodiment of the method of the present invention, the purslane flavonoid compound is any one of quercetin, myricetin, kaempferol, apigenin and luteolin.

[0017] As a preferred embodiment of the method of the present invention, the polyol is one or more of butanediol, propylene glycol, pentanediol and hexanediol.

[0018] As a preferred embodiment of the method of the present invention, wherein: in terms of the mass percentage of each raw material,

[0019]

[0020] As a preferred embodiment of the method of the present invention, the pH of the homogeneous phase B is 7.5.

[0021] As a preferred embodiment of the method of the present invention, the pH of the solution is adjusted by adding 5 mol / L NaOH.

[0022] As a preferred embodiment of the method of the present invention, the rapid cooling and freezing in liquid nitrogen takes place for 30 minutes.

[0023] As a preferred embodiment of the method of the present invention, the step of slowly dropping phase A into phase B under ultrasonic conditions further comprises ultrasonic treatment, wherein the ultrasonic temperature is 60° C. and the ultrasonic treatment time is 30 min.

[0024] As a preferred embodiment of the method of the present invention, the reduced pressure removal of methanol comprises slowly removing the methanol under reduced pressure at room temperature using a rotary evaporator.

[0025] Beneficial effects of the present invention:

[0026] The present invention provides a method for increasing the solubility of purslane flavonoid compounds, wherein a surfactant, arginine and a polyol system are synergistically compounded to interfere with the π-π stacking degree between flavonoid molecules through hydrophobic interaction, cation-π and solvation, thereby ultimately achieving an excellent solubilization-enhancing effect on the purslane flavonoid compounds, thereby improving their bioavailability. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0028] Figure 1 : are the XRD spectra of the products in the examples of the present invention, wherein a is the XRD spectrum of quercetin and Que-Sur-Arg system freeze-dried powder, and b is the XRD spectrum of myricetin and Myr-sur-Arg system freeze-dried powder.

[0029] Figure 2 : is the dissolution standard curve of the freeze-dried powder in the embodiment of the present invention, wherein a is the dissolution standard curve of quercetin, b is the dissolution standard curve of luteolin, c is the dissolution standard curve of apigenin, d is the dissolution standard curve of kaempferol, and e is the dissolution standard curve of myricetin. DETAILED DESCRIPTION

[0030] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0031] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0032] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0033] In the embodiments of the present invention: the purity of quercetin, kaempferol, apigenin, luteolin and myricetin is ≥ 97%, Shanghai Myrrel Biochemical Technology Co., Ltd.;

[0034] Methanol, ethanol, 1,2-propylene glycol, 1,2-pentanediol, 1,3-butanediol, 1,2-hexanediol, L-arginine, L-histidine, L-phenylalanine, sodium hydroxide, lecithin, Sinopharm Chemical Reagent Co., Ltd.;

[0035] Ethylhexylglycerin, Shanghai Green Butterfly Industrial Co., Ltd.; Sodium Bacillus subtilisin, Japan KANEKA CORPORATION; Deionized water, homemade in the laboratory.

[0036] Example 1

[0037] 25 mg of quercetin was added to 10 mL of methanol solution to obtain a uniform quercetin solution A.

[0038] Then, 25 mg / mL, 10 mL of polyarginine solution was prepared, and 250 mg of sodium subtilisin was added to the solution. After mixing evenly, 5 mol / L NaOH was used to adjust the pH of the mixed solution to 7.5 to obtain flavonoid medium interfering agent B.

[0039] Then, A was slowly added into B and ultrasonicated at 60°C for 30 min to obtain a uniform flavonoid active ingredient mixed solution C;

[0040] Then, C was transferred to a rotary evaporator and the methanol was slowly removed under reduced pressure at room temperature;

[0041] Subsequently, the mixed solution was placed in a centrifuge tube and placed in liquid nitrogen for 30 minutes for rapid cooling and freezing, and freeze-dried to obtain a high-content flavonoid active ingredient freeze-dried powder, which can be efficiently and quickly dissolved in an aqueous solution containing a 10% 1,3-butanediol system (the polyol systems hereinafter are all 1,3-butanediol systems), and the quercetin solubility concentration can reach 1.48 mg / mL.

[0042] Example 2

[0043] 25 mg of myricetin was added to 10 mL of methanol solution to obtain a uniform myricetin solution A.

[0044] Then, 25 mg / mL, 10 mL of histidine solution was prepared, and 50 mg of sodium subtilisin and 200 mg of tween 80 were added to the solution. After mixing evenly, 5 mol / L NaOH was used to adjust the pH of the mixed solution to 7.5 to obtain flavonoid medium interfering agent B.

[0045] Then, A was slowly added into B and ultrasonicated at 60°C for 30 min to obtain a uniform flavonoid active ingredient mixed solution C;

[0046] Then, C was transferred to a rotary evaporator, and the methanol was slowly removed under reduced pressure at room temperature; subsequently, the mixed solution was placed in a centrifuge tube and placed in liquid nitrogen for 30 minutes for rapid cooling and freezing, and freeze-dried to obtain a high-content flavonoid active ingredient freeze-dried powder, which can be efficiently and quickly dissolved in an aqueous solution containing a 10% polyol system, and the solubility concentration of myricetin can reach 2.75 mg / mL.

[0047] Example 3

[0048] 25 mg of kaempferol was added to 10 mL of methanol solution to obtain a uniform kaempferol solution A.

[0049] Then, 25 mg / mL, 10 mL of histidine solution was prepared, and 250 mg of sodium cocoyl glycinate was added to the solution. After mixing evenly, 5 mol / L NaOH was used to adjust the pH of the mixed solution to 7.5 to obtain flavonoid medium interfering agent B.

[0050] Then, A was slowly added to B and ultrasonicated at 60°C for 30 minutes to obtain a uniform mixed solution C of flavonoid active substances; then, C was transferred to a rotary evaporator and the methanol was slowly removed under reduced pressure at room temperature;

[0051] Subsequently, the mixed solution was placed in a centrifuge tube and placed in liquid nitrogen for 30 minutes for rapid cooling and freezing, and freeze-dried to obtain a high-content flavonoid active ingredient freeze-dried powder. The freeze-dried powder can be efficiently and quickly dissolved in an aqueous solution containing a 10% polyol system, and the kaempferol solubility concentration can reach 1.13 mg / mL.

[0052] Example 4

[0053] 25 mg of apigenin was added to 10 mL of ethanol solution to obtain a uniform apigenin ethanol solution A.

[0054] Then, 25 mg / mL, 10 mL of histidine solution was prepared, and 250 mg of sodium subtilisin was added to the solution. After mixing evenly, 5 mol / L NaOH was used to adjust the pH of the mixed solution to 7.5 to obtain flavonoid medium interfering agent B.

[0055] Then, A was slowly added to B and ultrasonicated at 60°C for 30 minutes to obtain a uniform mixed solution C of flavonoid active substances; then, C was transferred to a rotary evaporator and the alcohol was slowly removed under reduced pressure at room temperature;

[0056] Subsequently, the mixed solution was placed in a centrifuge tube and placed in liquid nitrogen for 30 minutes for rapid cooling and freezing, and freeze-dried to obtain a high-content flavonoid active ingredient freeze-dried powder. The freeze-dried powder can be efficiently and quickly dissolved in an aqueous solution containing a 10% polyol system, and the apigenin solubility concentration can reach 1.04 mg / mL.

[0057] Example 5

[0058] 25 mg of luteolin was added to 10 mL of ethanol solution to obtain a uniform luteolin ethanol solution A.

[0059] Then, 25 mg / mL, 10 mL of histidine solution was prepared, and 250 mg of sodium subtilisin was added to the solution. After mixing evenly, 5 mol / L NaOH was used to adjust the pH of the mixed solution to 7.5 to obtain flavonoid medium interfering agent B.

[0060] Then, A was slowly added to B and ultrasonicated at 60°C for 30 minutes to obtain a uniform mixed solution C of flavonoid active substances; then, C was transferred to a rotary evaporator and the methanol was slowly removed under reduced pressure at room temperature;

[0061] Subsequently, the mixed solution was placed in a centrifuge tube and placed in liquid nitrogen for 30 minutes for rapid cooling and freezing, and freeze-dried to obtain a high-content flavonoid active ingredient freeze-dried powder. The freeze-dried powder can be efficiently and quickly dissolved in an aqueous solution containing a 10% polyol system, and the luteolin solubility concentration can reach 0.89 mg / mL.

[0062] For the poorly soluble quercetin and myricetin system, the crystallization characteristics of the freeze-dried powders of Example 1 and Example 3 were studied respectively, with quercetin (Que) and myricetin (Myr) powders as controls.

[0063] Freeze-dried system X-ray diffraction analysis method:

[0064] Take the freeze-dried powder of Que-Sur-Arg and Myr-Sur-Arg, press it into thin sheets and fill it into the scanning container, and use the SmartLab SEX X-ray diffractometer of Japan Rigaku Company to scan and inspect. Use copper target test, the scanning range is 5-90°, the scanning speed is 2° / min, and the lattice analysis is performed by comparing the XRD standard card through MDIJade software. Figure 1 .

[0065] Depend on Figure 1 a It can be seen that compared with quercetin itself, the diffraction peak of the freeze-dried powder Qua-Sur-Arg in Example 1 has undergone a diffuse change, and at the same time, there is The peaks of Qua's (200), (101), and (140) interplanar spacings increased from 7.06, 3.62, and 3.24 to 7.21, 3.78, and 3.42 for Qua-Sur-Arg. This result indicates that the regulation of sodium subtilisin and arginine system interfered with the crystal lattice of quercetin and inhibited its own crystallization behavior.

[0066] In addition, the XRD spectrum of the freeze-dried powder of Example 3 is as follows Figure 1 As shown in b, the results show that the diffraction peak of Myr-Sur-Arg has undergone a significant diffusion change, with The peaks of Myr (110), (202), (102) interplanar spacings increased from 4.01, 3.09, 2.14 to 4.8, 3.16, 2.25 of Myr-Sur-Arg. This result indicates that the regulation of sodium subtilisin and arginine system interfered with the crystal lattice of myricetin and inhibited its own crystallization behavior.

[0067] Determination of the solubilization effect of the purslane flavonoid active ingredients of Examples 1 to 6:

[0068] 2.2 mg of the lyophilized powder of Examples 1 to 6 were respectively dissolved in 10 mL of 5% 1,3-butanediol aqueous solution, filtered using a 0.22 μm filter membrane, and the filtrate was diluted 200 times and then measured for absorbance using an ultraviolet spectrophotometer to determine and calculate the maximum solubility of different active ingredients.

[0069] The specific determination method is as follows:

[0070] 2.2 mg of the lyophilized powder was dissolved in an aqueous solution containing 10 (v / v)% polyol and 4.5wt% L-arginine (pH adjusted to 7.5). The solution was diluted 20 times and then diluted to 2, 4, 8, 16, 32, and 64 times in a gradient manner. The absorbance was measured by an ultraviolet spectrophotometer, and a dissolution curve was drawn according to the active substance concentration and the dilution multiple.

[0071] Quercetin solubility standard curve can be found at Figure 2 a, Luteolin dissolution standard curve see Figure 2 b, Apigenin solubility standard curve see Figure 2 c, kaempferol solubility standard curve see Figure 2 d, myricetin solubility standard curve see Figure 2 e.

[0072] Five supersaturated solutions of lyophilized powders were prepared in a synergistic solubilization system. Different upper layer solutions were filtered through a 0.22 μm filter membrane. The clarified solution after filtration was diluted 200 times and its absorbance was measured with a UV spectrophotometer. The final solubility of different active ingredients was calculated using the corresponding solubility standard curve.

[0073] See Table 1 for the results.

[0074] Table 1

[0075]

[0076] Comparative Example 1

[0077] Test 1:

[0078] 25 mg of quercetin was added to 10 mL of methanol solution to obtain a uniform quercetin solution A.

[0079] Then transfer to a rotary evaporator and slowly remove methanol under reduced pressure at room temperature;

[0080] Subsequently, the mixed solution was placed in a centrifuge tube and placed in liquid nitrogen for 30 minutes for rapid freezing, and freeze-dried to obtain a freeze-dried powder with a high content of flavonoid active ingredients;

[0081] The freeze-dried powder is dissolved in an aqueous solution containing 10% of a polyol system, and the quercetin dissolution concentration is 0.09 μg / mL.

[0082] Test 2:

[0083] 25 mg of quercetin was added to 10 mL of methanol solution to obtain a uniform quercetin solution A.

[0084] Then, 25 mg / mL and 10 mL of arginine solution were prepared, and after being evenly mixed, 5 mol / L NaOH was used to adjust the pH of the mixed solution to 7.5 to obtain flavonoid medium interfering agent B.

[0085] Then, A was slowly added into B and ultrasonicated at 60°C for 30 min to obtain a uniform flavonoid active ingredient mixed solution C;

[0086] Then, C was transferred to a rotary evaporator and the methanol was slowly removed under reduced pressure at room temperature;

[0087] Subsequently, the mixed solution was placed in a centrifuge tube and placed in liquid nitrogen for 30 minutes for rapid freezing, and freeze-dried to obtain a freeze-dried powder with a high content of flavonoid active ingredients;

[0088] The freeze-dried powder can be efficiently and quickly dissolved in an aqueous solution containing a 10% polyol system, and the quercetin dissolution concentration can reach 0.33 mg / mL.

[0089] Test 3:

[0090] 25 mg of quercetin was added to 10 mL of methanol solution to obtain a uniform quercetin solution A.

[0091] Then, 10 mL of 25 mg / mL sodium subtilisin solution was prepared, and after being evenly mixed, 5 mol / L NaOH was used to adjust the pH of the mixed solution to 7.5 to obtain flavonoid medium interfering agent B.

[0092] Then, A was slowly added into B and ultrasonicated at 60°C for 30 min to obtain a uniform flavonoid active ingredient mixed solution C;

[0093] Then, C was transferred to a rotary evaporator and the methanol was slowly removed under reduced pressure at room temperature;

[0094] Subsequently, the mixed solution was placed in a centrifuge tube and placed in liquid nitrogen for 30 minutes for rapid freezing, and freeze-dried to obtain a freeze-dried powder with a high content of flavonoid active ingredients;

[0095] The freeze-dried powder can be efficiently and quickly dissolved in an aqueous solution containing a 10% polyol system, and the quercetin dissolution concentration can reach 0.69 mg / mL.

[0096] It can be seen that the synergistic effect of sodium subtilisin and arginine has an excellent solubilizing effect on purslane flavonoids, thereby improving their bioavailability.

[0097] In summary, the present invention provides a method for increasing the solubility of purslane flavonoid compounds, by synergistically compounding a surfactant, arginine and a polyol system through hydrophobic interaction, cation-π and solvation to interfere with the π-π stacking degree between flavonoid molecules, ultimately achieving an excellent solubilizing effect on purslane flavonoid compounds, thereby improving their bioavailability.

[0098] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. A method for increasing the solubility of purslane flavonoid compounds, characterized in that: include, The purslane flavonoids are dissolved in methanol or ethanol, and ultrasonically dissolved to obtain a clear, transparent and uniform phase A; Mixing cationic amino acid, dissolution agent, polyol and water into a uniform phase B; Phase A is slowly added dropwise to phase B under ultrasonic conditions, and after the addition is complete, methanol is removed under reduced pressure to obtain a mixed solution; The mixed solution is placed in liquid nitrogen for rapid cooling and freezing, and freeze-dried to obtain a high-content purslane flavonoids complex freeze-dried powder; Wherein, the cationic amino acid is one or more of arginine, polyarginine, lysine and histidine; The dissolution promoter is one or more than two of sodium lauroyl glutamate, potassium lauroyl sarcosinate, sodium subtilisin, sodium cocoyl glycinate, Tween 80, polyether-26, cyclodextrin and hydroxypropyl cyclodextrin.

2. The method according to claim 1, characterized in that: The purslane flavonoid compound is any one of quercetin, myricetin, kaempferol, apigenin and luteolin.

3. The method according to claim 2, characterized in that: The polyol is one or more of butanediol, propylene glycol, pentanediol and hexanediol.

4. The method according to any one of claims 1 to 3, characterized in that: In terms of the mass percentage of each raw material, 5. The method according to claim 4, characterized in that: The pH of the homogeneous phase B was 7.

5.

6. The method according to claim 5, characterized in that: The pH of the solution was adjusted by 5 mol / L NaOH.

7. The method according to claim 1, characterized in that: The rapid cooling and freezing in liquid nitrogen is performed for 30 minutes.

8. The method according to claim 1, characterized in that: The step of slowly dropping phase A into phase B under ultrasonic conditions also includes ultrasonic treatment, wherein the ultrasonic temperature is 60° C. and the ultrasonic treatment time is 30 min.

9. The method according to claim 8, characterized in that: The removing of methanol under reduced pressure comprises slowly removing methanol under reduced pressure at room temperature using a rotary evaporator.

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