A method for increasing the solubility of disodium pyrroloquinoline quinone and a disodium pyrroloquinoline quinone gel

Abstract

This invention discloses a method for increasing the solubility of disodium pyrroloquinoline quinone. By adding an amine compound, such as aminomethylpropanol or tromethamine, to water, the solubility of disodium pyrroloquinoline quinone in water can be significantly improved. Therefore, this invention can obtain an ionic salt solution with good antioxidant properties. Furthermore, this invention provides a disodium pyrroloquinoline quinone jelly and its preparation method. The disodium pyrroloquinoline quinone jelly of this invention has a high content of added disodium pyrroloquinoline quinone, good stability, and low loss rate of disodium pyrroloquinoline quinone.

Description

Technical Field

[0001] This invention relates to the field of cosmetics and skincare technology, and more specifically, to a method for increasing the solubility of disodium pyrroloquinoline quinone and a disodium pyrroloquinoline quinone gel. Background Technology

[0002] Pyrroloquinoline quineone (PQQ), officially 4,5-dihydro-4,5-dioxo-1-hydropyrrolo(2,3-f)quinoline-2,7,9-tricarboxylic acid, was discovered as a coenzyme for a novel oxidoreductase in methyltrophic bacteria. Subsequent studies have confirmed its widespread presence in Gram-negative bacteria and higher plants and animals, exhibiting a variety of physiological functions. Research indicates that PQQ possesses multiple physiological functions, including protecting the nervous and cardiovascular systems, promoting growth and proliferation, and, as an oxidoreductase coenzyme, protecting cells from damage caused by oxidative stress.

[0003] Because the protonated form of PQQ is only slightly soluble in water, while pyrroloquinoline quinone disodium salt (PQQ disodium salt) has relatively higher solubility in water, intermediate PQQ is often obtained through PQQ disodium salt. Currently, PQQ disodium salt is the main commercial form of PQQ. Although PQQ disodium salt has comprehensive functions, its low solubility in water limits its application in cosmetic formulations. Summary of the Invention

[0004] To solve the above-mentioned technical problems, the present invention provides a method for increasing the solubility of disodium pyrroloquinoline quinone, which can significantly improve the solubility of disodium pyrroloquinoline quinone in water.

[0005] Furthermore, the present invention needs to provide an antioxidant ionic salt solution.

[0006] Furthermore, the present invention needs to provide a disodium salt pyrroloquinoline quinone jelly.

[0007] Furthermore, the present invention also provides a method for preparing the above-mentioned pyrroloquinoline quinone disodium salt jelly.

[0008] The technical solution provided by this invention is as follows:

[0009] A method for increasing the solubility of disodium pyrroloquinoline quinone.

[0010] The solubility of pyrroloquinoline ...

[0011] The amine compound is selected from either aminomethylpropanol or tromethamine.

[0012] The weight ratio of disodium pyrroloquinoline quinone to amine compounds is 1:(0.01-0.4).

[0013] This invention provides an antioxidant ionic salt solution.

[0014] This includes amine compounds and disodium pyrroloquinoline quinone.

[0015] The amine compound is selected from either aminomethylpropanol or tromethamine.

[0016] The weight ratio of disodium pyrroloquinoline quinone to amine compounds is 1:(0.01-0.4).

[0017] In addition, the present invention also provides a disodium salt pyrroloquinoline quinone jelly, comprising the following components by weight percentage:

[0018] Disodium pyrroloquinoline quinone 0.02%-0.2%;

[0019] Amine compounds: 0.008%-0.2%;

[0020] Butanediol 4%-7%;

[0021] Yuefuning 0.5%-1%;

[0022] 1,2-Hexanediol 0.8%;

[0023] Carbomer 0.4%;

[0024] Hydroxyethyl cellulose 0-0.05%;

[0025] Squalane 0.2%-0.5%;

[0026] Phenoxyethanol 0.2%;

[0027] Sodium hydroxide 0.07%

[0028] Water balance.

[0029] Preferably, the pyrroloquinoline quinone disodium salt gel of the present invention comprises the following components by weight percentage:

[0030] Disodium pyrroloquinoline quinone 0.02%-0.2%;

[0031] Aminomethylpropanol 0.008%-0.9%;

[0032] Butanediol 4%-7%;

[0033] Yuefuning 0.5%-1%;

[0034] 1,2-Hexanediol 0.8%;

[0035] Carbomer 0.4%;

[0036] Hydroxyethyl cellulose 0-0.05%;

[0037] Squalane 0.2%-0.5%;

[0038] Phenoxyethanol 0.2%;

[0039] Sodium hydroxide 0.07%

[0040] Water balance.

[0041] Preferably, the pyrroloquinoline quinone disodium salt gel of the present invention comprises the following components by weight percentage:

[0042] Disodium pyrroloquinoline quinone 0.02%-0.2%;

[0043] Tromethamine 0.2%;

[0044] Butanediol 4%-7%;

[0045] Yuefuning 0.5%-1%;

[0046] 1,2-Hexanediol 0.8%;

[0047] Carbomer 0.4%;

[0048] Hydroxyethyl cellulose 0-0.05%;

[0049] Squalane 0.2%-0.5%;

[0050] Phenoxyethanol 0.2%;

[0051] Sodium hydroxide 0.07%

[0052] Water balance.

[0053] Furthermore, the present invention also provides a method for preparing the disodium pyrroloquinoline quinone jelly as described above, comprising the following steps:

[0054] (1) Add carbomer and hydroxyethyl cellulose to pure water in proportion, stir and homogenize until uniform, heat to 80℃-85℃, and homogenize until completely uniform;

[0055] (2) Cool down to 60-65℃, add sodium hydroxide, stir until evenly dispersed, and continue stirring and cooling;

[0056] (3) Cool down to 40-45℃, add disodium PQQ salt, amine compounds, butanediol, glutenin, 1,2-hexanediol, squalane and phenoxyethanol, and stir until evenly dispersed.

[0057] Compared to existing technologies, this invention provides a method for increasing the solubility of disodium pyrroloquinoline quinone. By adding amine compounds, such as aminomethylpropanol or tromethamine, to water, the solubility of disodium pyrroloquinoline quinone in water can be significantly improved. Therefore, this invention can yield an ionic salt solution with good antioxidant properties.

[0058] Furthermore, the present invention provides a disodium pyrroloquinoline quinone salt gel and a method for preparing the same. The disodium pyrroloquinoline quinone salt gel of the present invention has a high content of added disodium pyrroloquinoline quinone salt, good stability, and low loss rate of disodium pyrroloquinoline quinone salt. Detailed Implementation

[0059] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions in the embodiments of this invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0060] The embodiments of this invention are written in a progressive manner.

[0061] Source of materials:

[0062] Yuefuning: Shanghai Jiaye;

[0063] DCFH-DA Manufacturer: MACRK;

[0064] Fetal bovine serum (FBS) manufacturer: GIBCO;

[0065] MEM culture medium manufacturer: PROCELL.

[0066] All embodiments of the present invention are derived from commercially available products.

[0067] Experiment Example 1: Experiment on the effect of amine compounds on the solubility of PQQ disodium salt

[0068] Example 1

[0069] At room temperature, add 1 part by weight of aminomethylpropanol (purity 95%) to 100 parts by weight of water and stir until homogeneous. Add excess PQQ disodium salt and stir until a precipitate forms. Filter the solution using a 0.2 μm sterile filter cartridge or ceramic membrane to obtain the precipitate and prepare a saturated ionic salt solution. Calculate the solubility of the ionic salt using the following method: [(Amount of PQQ disodium salt added - Amount of PQQ disodium salt precipitate) + Amount of aminomethylpropanol added * 0.95] / 100 water.

[0070] Example 2

[0071] At room temperature, 1 part by weight of tromethamine is added to 100 parts by weight of water and stirred until homogeneous. When excess PQQ disodium salt is added and stirred directly, a precipitate is formed. The precipitate is obtained by filtration using a 0.2 μm sterile filter cartridge or ceramic membrane, thus preparing a saturated ionic salt solution. The solubility of the ionic salt is calculated as follows: [(amount of PQQ disodium salt added - amount of PQQ disodium salt precipitate) + amount of tromethamine added] / 100 water.

[0072] Comparative Example 1

[0073] At room temperature, 1 part by weight of arginine was added to 100 parts by weight of water and stirred until homogeneous. Excess PQQ disodium salt was added and stirred until precipitation occurred. The precipitate was obtained by filtration using a 0.2 μm sterile filter cartridge or ceramic membrane, thus preparing a saturated ionic salt solution. The solubility of the ionic salt was calculated as follows: [(Amount of PQQ disodium salt added - Amount of PQQ disodium salt precipitate) + Amount of arginine added] / 100 water.

[0074] Comparative Example 2

[0075] At room temperature, add 1 part by weight of matrine to 100 parts by weight of water and stir until homogeneous. Add excess PQQ disodium salt and stir until a precipitate forms. Filter the solution using a 0.2 μm sterile filter cartridge or ceramic membrane to obtain the precipitate and prepare a saturated ionic salt solution. Calculate the solubility of the ionic salt using the following method: [(PQQ disodium salt added - PQQ disodium salt precipitate) + matrine added] / 100 water.

[0076] Comparative Example 3

[0077] At room temperature, adding excess PQQ disodium salt to 100 parts by weight of water and stirring directly produces a precipitate. The precipitate is then filtered using a 0.2 μm sterile filter cartridge or ceramic membrane to obtain a saturated ionic salt solution. The solubility of this ionic salt is calculated as follows: (amount of PQQ disodium salt added - amount of PQQ disodium salt precipitate) / 100 water.

[0078] The solubility of PQQ disodium salt was calculated using the following method and is listed in Table 1.

[0079] Table 1. Raw material ratio and experimental results for Experiment Example 1

[0080]

[0081] In Comparative Example 3, the solubility of PQQ disodium salt in water at room temperature was 0.1%. In Comparative Example 1, arginine did not improve its solubility. In Comparative Example 2, the solubility of matrine was increased by 15 times. In Example 1, the addition of aminomethylpropanol and in Example 2, the addition of tromethamine both effectively dissolved PQQ disodium salt, increasing its solubility by 25 times.

[0082] The ionic salt solutions of Examples 1-2 prepared in Experiment 1 and the pure PQQ disodium salt solution were tested for their antioxidant efficacy. The lower the average fluorescence intensity of the cells, the better the antioxidant effect. The results are listed in Table 2.

[0083] The test method for assessing the antioxidant efficacy of ionic salts is as follows:

[0084] Test method:

[0085] 1. HaCat cell complete culture medium: MEM medium (Pronosel) + 15% fetal bovine serum (FBS, Gibco) + 1% penicillin-streptomycin mixture

[0086] 2. Cell seeding: After adjusting the density of the human immortalized keratinocyte Hacat suspension to 20,000 cells / well, the cells were seeded into 96-well plates at 100 μL / well and incubated at 37°C in a 5% CO2 incubator for 24 h.

[0087] 3. Grouping: Comparative Example 4 negative group, Comparative Example 5 positive group, Example 1 sample group, and Example 2 sample group.

[0088] 4. Sample dilution: Dilute the sample of Example 1 to 10 μg / ml with MEM medium and add it to the wells of the Example 1 sample group at a volume of 100 μl / well; dilute the sample of Example 2 to 10 μg / ml with MEM medium and add it to the wells of the Example 2 sample group at a volume of 100 μl / well; add an equal volume of medium to the negative group of Comparative Example 4 and the positive group of Comparative Example 5, and incubate at 37°C in a 5% CO2 incubator for 24 hours.

[0089] 5. ROS test: Remove the cell plate from the CO2 incubator, discard the supernatant, and wash three times with PBS.

[0090] 6. Dilute DCFH-DA 1000 times with PBS and add 100 μL / well to the negative control group of Comparative Example 4, the positive control group of Comparative Example 5, and the sample groups of Example 1 and Example 2. Place in the dark at room temperature for 30 min.

[0091] 7. Discard the supernatant and wash three times with PBS. Dilute hydrogen peroxide to 500 μM with PBS and add 100 μL / well to the positive group of Comparative Example 5, the sample group of Example 1, and the sample group of Example 2. Add an equal amount of PBS to the negative group of Comparative Example 4. Incubate at room temperature in the dark for 30 min.

[0092] 8. Discard the supernatant and wash three times with PBS. Keep 50 μL of the solution in each well and observe and photograph it under an inverted fluorescence microscope.

[0093] Table 2. Antioxidant effects of PQQ disodium salt ion salt dissolution

[0094]

[0095] Generally, P<0.05 is considered statistically significant, P<0.01 is considered statistically significant, and P<0.001 is considered extremely statistically significant.

[0096] Experimental Example 2: Properties of Gel

[0097] Prepare a gel containing disodium pyrroloquinoline quinone as follows:

[0098] 1. Add carbomer and hydroxyethyl cellulose to pure water according to the proportions shown in Table 3, stir until homogenized, heat to 80℃-85℃, and homogenize until completely homogenized;

[0099] 2. Cool to 60-65℃, add sodium hydroxide, stir until evenly dispersed, and continue stirring while cooling;

[0100] 3. Cool down to 40-45℃, add other ingredients, and stir until evenly dispersed.

[0101] Table 3. Formulation of gels containing disodium pyrroloquinoline quinone (unit: parts by weight)

[0102]

[0103] PQQ disodium salt gel stability

[0104] 1. Stability assessment methods

[0105] The above samples were subjected to stability tests at room temperature, -18℃, 4℃, 40℃, and 48℃. Samples at room temperature, 4℃, and 40℃ were tested for 3 months, and the physicochemical properties and appearance changes were tested on the 30th, 60th, and 90th days. Samples at -18℃ and 48℃ were tested for 1 month under the same conditions. The results are shown in Table 4.

[0106] Table 4. Stability test results of PQQ disodium salt gel

[0107]

[0108] The results showed that there were no abnormalities in the physicochemical indicators and appearance of Examples 3-6 during the 3-month observation period. However, Comparative Example 6 showed abnormalities in physicochemical indicators and appearance after a 3-month stability observation.

[0109] PQQ disodium salt loss rate detection

[0110] 1. Detection Method

[0111] 1. Principle

[0112] PQQ disodium salt was dissolved, separated by reversed-phase high-performance liquid chromatography, detected by ultraviolet detector, and quantified by standard curve method.

[0113] 2. Reagents and Materials

[0114] Unless otherwise stated, all reagents used in this method are of analytical grade, and the water is Grade I water as specified in GB / T6682.

[0115] 2.1 Dipotassium hydrogen phosphate trihydrate.

[0116] 2.2 Tetrabutylammonium bromide.

[0117] 2.3 Phosphoric acid.

[0118] 2.4 Acetonitrile: chromatographic grade.

[0119] 2.5PQQ disodium salt standard: purity ≥97.0%.

[0120] 2.6 Aqueous phase microporous filter membrane: 0.22μm.

[0121] 2.710 mmol / L dipotassium hydrogen phosphate-15 mmol / L tetrabutylammonium bromide mixed solution (pH 7.4): Weigh 2.28 g of dipotassium hydrogen phosphate trihydrate and 4.84 g of tetrabutylammonium bromide into a beaker, add 800 mL of water and sonicate to dissolve. Adjust the pH to 7.4 with phosphoric acid, transfer to a 1000 mL volumetric flask, dilute to volume with water, and shake well. Filter under vacuum through a 0.22 μm aqueous microporous membrane, and then sonicate until no bubbles remain.

[0122] 2.8 Acetonitrile aqueous solution (volume ratio 1:3): Measure 250 mL of acetonitrile and 4750 mL of water, add them to the reagent bottle, and mix well.

[0123] 3. Instruments and Equipment

[0124] 3.1 High performance liquid chromatograph, equipped with ultraviolet detector and autosampler.

[0125] 3.2 Electronic balance: sensitivity is 0.01 mg.

[0126] 4. Analysis Steps

[0127] 4.1 Preparation of Standard Solutions: Weigh 40 mg (accurate to 0.01 mg) of PQQ disodium salt standard and place it in a 100 mL volumetric flask. Add acetonitrile aqueous solution to dissolve and dilute to the mark. Shake well to obtain a stock solution of PQQ disodium salt standard with a concentration of 0.4 mg / mL. Prepare a series of PQQ disodium salt standard solutions with concentrations of 0.04 mg / mL, 0.08 mg / mL, 0.12 mg / mL, 0.16 mg / mL, 0.20 mg / mL, and 0.24 mg / mL from this solution.

[0128] 4.2 Sample Preparation Weigh 20 mg (accurate to 0.01 mg) of each of the PQQ disodium salt samples from Examples 9-12 and place them in a 50 mL volumetric flask. Add acetonitrile aqueous solution to dissolve and dilute to the mark, then shake well. Then accurately transfer 5.0 mL of the above solution into a 10 mL volumetric flask, dilute to the mark with acetonitrile aqueous solution, shake well, and use the filtrate after vacuum filtration through a 0.22 μm aqueous microporous membrane for determination.

[0129] 4.3 Reference conditions for liquid chromatography

[0130] 4.3.1 Chromatographic column: PFchromEPC1888031205-4625 (4.6mm×250mm, 5μm, 120A) or equivalent column.

[0131] 4.4 Measurement

[0132] Under specified chromatographic conditions, 20 μL of each standard solution and sample solution were injected into the liquid chromatograph for chromatographic analysis. The peak area of ​​disodium PQQ in the chromatograms of the standard and sample solutions was recorded. A standard curve was plotted with the peak area of ​​disodium PQQ in the standard solution as the Y-axis and the corresponding concentration of the standard solution as the X-axis, thus obtaining the disodium PQQ standard curve. Based on the peak area of ​​disodium PQQ in the sample solution chromatogram, the concentration of disodium PQQ in the sample solution was determined from the standard curve.

[0133] 5. Calculation of Results

[0134] 5.1 Calculation of PQQ disodium salt content

[0135] The content of PQQ disodium salt in the sample is expressed as its mass fraction W1, and the value is expressed as a percentage (%), calculated according to formula (1):

[0136]

[0137] (1) Where: C—concentration of pyrroloquinoline quinone disodium salt in the sample solution obtained from the standard curve, in milligrams per milliliter (mg / mL); V—sample dilution volume, in milliliters (mL); f—purity of PQQ disodium salt standard; m—mass of the sample, in milligrams (mg); W2—mass fraction of water in the sample, expressed as a percentage (%). The arithmetic mean of parallel determinations is taken as the determination result, and the result is expressed to two decimal places.

[0138] 5.2 Calculation of loss rate of PQQ disodium salt

[0139] PQQ disodium salt loss rate = (Initial sample PQQ disodium salt content - Stable sample PQQ disodium salt content) / Initial sample PQQ disodium salt content * 100%

[0140] 6 Precision

[0141] The relative standard deviation of two independent determinations obtained under repeatability conditions is no greater than 2.0%.

[0142] 2. Test results of PQQ disodium salt loss rate

[0143] The maximum loss rate of each sample under each condition during the 3-month stability study period was calculated and listed in Table 5.

[0144] Table 5. Results of PQQ disodium salt loss rate test

[0145]

[0146] The data above show that the PQQ disodium salt gel prepared by this invention in Examples 3-4 exhibits good stability and low loss rate, significantly superior to the PQQ disodium salt gel without the addition of aminomethylpropanol and tromethamine. In Comparative Example 6, arginine reacts with PQQ disodium salt, resulting in a high loss rate of PQQ disodium salt, reaching as high as 83.85% at 48°C.

[0147] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for increasing the solubility of disodium pyrroloquinoline quinone, characterized in that: The solubility of pyrroloquinoline ... The amine compound is selected from one of aminomethylpropanol or tromethamine; The weight ratio of disodium pyrroloquinoline quinone to amine compounds is 1:0.4.

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