Method for flying cinnabar by using sulfydryl-containing protein water

Abstract

The invention provides a method for water-flying cinnabar by using sulfydryl-containing protein, the cinnabar is further water-flying by using a sulfydryl-containing protein aqueous solution on the basis of traditional water-flying cinnabar, and the cinnabar prepared by the method is less in impurities, smaller and concentrated in particle size and larger in specific surface area. And sulfydryl (-SH) and / or disulfide (-S-S-) in the protein are combined with free Hg to form combined mercury, so that the precipitation of free Hg element in cinnabar is effectively reduced, the Hg solubility of cinnabar in artificial intestinal juice is reduced, and the problems that the content of free Hg in cinnabar is high and poisoning is easily caused by long-term and large-scale taking in the prior art are effectively reduced.

Description

Technical Field

[0001] The invention belongs to the field of purification and preparation technology of medicinal cinnabar, and particularly relates to a method for purifying cinnabar by utilizing thiol-containing protein water. Background Art

[0002] Cinnabar is a natural cinnabar ore, which belongs to the mineral Chinese medicine, and has the traditional effects of calming the mind, calming the nerves, improving eyesight, and detoxifying. Chinese medicine often uses cinnabar to treat various diseases, including madness, palpitations, upset, insomnia: dizziness, blurred vision, swelling, sores, scabies, etc.

[0003] The traditional method of preparing cinnabar is to use a magnet to absorb iron filings and grind them into fine powder, or use the water jet method (pure water as the medium) to make extremely fine powder. However, the traditional method has the following problems: (1) It contains a lot of impurities, the cinnabar particles are large, and the quality of cinnabar is low. (2) It contains a high content of trace elements including mercury (Hg), sulfur (S) and selenium (Se), which can easily lead to poisoning if taken for a long time or in large quantities.

[0004] Therefore, how to effectively reduce the content of toxic elements in cinnabar and obtain high-quality medicinal cinnabar is an urgent problem that needs to be solved. Summary of the invention

[0005] In view of the problems existing in the prior art, the purpose of the present invention is to efficiently achieve the detoxification and purification of cinnabar, and provide a method for purifying cinnabar using thiol-containing protein water.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] The first aspect of the present invention provides a method for producing cinnabar using thiol-containing protein water, comprising the following steps:

[0008] (1) Pour the standard cinnabar powder into a grinding pot, add a small amount of ultrapure water, and use a grinding rod to grind the powder into a paste;

[0009] (2) Pour 5 to 10 times the volume of cinnabar into ultrapure water, stir with a glass rod, and use a plastic spoon to remove the floating residue on the surface;

[0010] (3) Let stand for 1 to 2 minutes until the coarse particles are completely settled, then slowly pour the suspension into a beaker;

[0011] (4) Repeat the above steps (1) to (3) at least 10 times, collect all the suspension into a clean beaker, and leave it at room temperature away from light for at least 24 hours;

[0012] (5) Discard the coarse particles precipitated in the grinding bowl and wash the grinding bowl with ultrapure water for later use;

[0013] (6) pouring out all the supernatant after the static treatment in step (4), leaving the precipitate;

[0014] (7) using the thiol-containing protein aqueous solution and the precipitate obtained in step (6), repeating the treatment of steps (1) to (5);

[0015] (8) Pour out the supernatant of the thiol-containing protein aqueous solution, leaving the precipitate, add ultrapure water, stir, and stand at room temperature in the dark for 24 hours;

[0016] (9) Repeat step (8) at least twice, pour out the supernatant, pour the precipitate into a plastic dish, and dry it at room temperature away from light;

[0017] (10) Collect cinnabar powder.

[0018] Furthermore, the thiol-containing protein includes bovine serum albumin, whey protein, and papain.

[0019] Furthermore, the mass concentration of the thiol-containing protein aqueous solution is 0.5-10%.

[0020] The second aspect of the present invention provides cinnabar prepared by the above method. The cinnabar has a smooth surface, a particle size range of 0.2-11.0 μm, a particle size distribution with a maximum concentration of about 4.2 μm, a specific surface area of ​​3.7-3.8, a span of 1.8-2.0, and a light shielding rate of 12.0-13.0%.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] Compared with the traditional method, the method of using thiol-containing protein water to float cinnabar in the present invention adopts a thiol-containing protein aqueous solution to replace the ultrapure water in the traditional water floating method. The cinnabar prepared by this method not only has fewer impurities, smaller and more concentrated particle size, and larger specific surface area, but also in the water floating method of the present application, the thiol (-SH) and / or disulfide (-SS-) in the protein combines with free Hg to form bound mercury, thereby effectively reducing the solubility of the free mercury Hg element in cinnabar in artificial intestinal fluid, and effectively reducing the problem that the existing cinnabar has a high free Hg content and is prone to poisoning due to long-term and large-scale use. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The present invention will be further described below in conjunction with the accompanying drawings and embodiments:

[0024] Figure 1 The properties of cinnabar and the residual rate of BSA before and after BSA water-flying are shown (mean±SEM, **P<0.01, ****P<0.0001);

[0025] Figure 2 The characteristics of cinnabar particles produced by different water-flying methods are shown (mean ± SEM, **P < 0.01, ***P < 0.001);

[0026] Figure 3 The element content of cinnabar particles prepared by different water-jet methods and the solubility of Hg, S and Se are shown. DETAILED DESCRIPTION

[0027] In order to better understand the present invention, the present invention will be further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the present invention with reference to the description text.

[0028] It should be noted that the experimental methods described in the following experiments, unless otherwise specified, are all conventional methods, and the reagents and materials, unless otherwise specified, can be obtained from commercial channels.

[0029] Main drugs:

[0030] Chinese name Cinnabar powder BSA English name Cinnabar(CNB) Bovine serum albumin (BSA) Production Company Sinopharm Lerentang Hebei Pharmaceutical Co., Ltd. BOVOGEN Origin Guizhou Australia Product batch number 19031001 BSAS1.0

[0031] Other experimental reagents:

[0032] BSA standard protein solution (Lianke Biotechnology); BCA protein quantification kit (Report Biotech); sodium carboxymethyl cellulose (CMC-Na, Tianjin Damao); artificial gastric juice (SGF, Regen Biotechnology, CZ0213); artificial intestinal fluid (SIF, Regen Biotechnology, CZ0201); nitric acid; hydrochloric acid; perchloric acid.

[0033] The detection method according to the embodiment of the present invention includes:

[0034] 1. BCA protein content determination

[0035] 1. Prepare BSA dilution (0.5% CMC-Na dissolved) protein content. Dilute BSA in a gradient, i.e. 5, 25, 50, 100, 200, 500, 1000, 2000, 3000, 4000, 5000 (μg / mL);

[0036] 2. Prepare the protein content of cinnabar test solution (0.5% CMC-Na solution). Weigh the standard cinnabar powder and dissolve it in a gradient, i.e. 0.1, 1, 10, 20, 40, 60, 80, 100 (mg / mL); weigh the BSA water-dissolved cinnabar powder and dissolve it in a gradient, i.e. 0.1, 1, 10, 20, 40, 60, 80, 100 (mg / mL);

[0037] 3. Vortex the above samples, sonicate for 15 minutes, let stand for 2 hours, centrifuge at 12000rpm and 4℃ for 20 minutes. Take the supernatant and filter it through a 0.45μm filter (Whatman), and place it on ice for later use;

[0038] 4. Prepare BCA working solution, dilute BSA standard protein solution (0.5% CMC-Na dissolved), add samples to 96-well plate, add BCA working solution, quickly cover the plate, shake gently crosswise, react at 37℃ for 30 minutes. Use microplate reader to measure: 562nm, 25 times;

[0039] 5. Draw a standard curve and calculate the sample concentration.

[0040] 2. Observation of particle characteristics

[0041] 1. Take 5 mg of cinnabar after different treatment methods and observe the SEM images using a scanning electron microscope (FEI NovaNanoSEM 450) with an accelerating voltage of 15 kV;

[0042] 2. Take 100 mg of cinnabar after different treatment methods, use a laser particle size analyzer (OMEC LS-909) to detect the particle characteristics, place it in a dry sampler, analysis mode: general mode; dispersion medium: water; sample material absorbance: 1; background sampling time: 10 seconds; single sampling time: 10 seconds; result type: volume (V).

[0043] 3. Sample preparation for element solubility determination

[0044] 1. Take 100 mg each of standard cinnabar powder, BSA water-melted cinnabar powder, and ultrapure water-melted cinnabar powder, and dissolve them in 50 mL of ultrapure water, artificial gastric juice, and artificial intestinal juice to form a suspension. Prepare another 50 mL of ultrapure water, artificial gastric juice, and artificial intestinal juice as controls, for a total of 12 samples;

[0045] 2. Mix the above samples by shaking, ultrasonicate at 37℃ for 2 hours, take out and shake once every 30 minutes, and let stand at room temperature for 1 hour after the ultrasonication.

[0046] 3. Take the upper turbid liquid separately, centrifuge at 12000rpm and 24℃ for 20 minutes, take the supernatant and filter it through a 0.45μm filter membrane, take 2mL of the filtrate, add ultrapure water to make up to 50mL, shake and mix, and store at 4℃.

[0047] 4. Sample pretreatment for element content determination

[0048] 1. Weigh the sample with an analytical balance (accurate to 0.0001g), place the sample in a clean polytetrafluoroethylene digestion cup, add 3mL of nitric acid, 9mL of hydrochloric acid, and 1mL of perchloric acid to the sample digestion container, and let it stand for about 30 minutes;

[0049] 2. Place the sample digestion container in the hot plate digester to digest the sample, digest the sample using the pre-set temperature program, and finally digest the sample continuously at 100°C for about 60 minutes;

[0050] Heating time (minutes) Heating range Temperature holding time (minutes) 10 Room temperature~50℃ 10 10 50～80℃ 10 10 80～100℃ 60

[0051] 3. After digestion, there is about 1-2 mL of digestion solution remaining, and the sample digestion procedure is completed. Remove the digestion tube and cool it naturally to room temperature in a fume hood;

[0052] 4. Transfer the digestion solution to a 10mL volumetric flask and rinse the digestion container three times with deionized water, then adjust the volume, mix well and let it stand for testing;

[0053] 5. Perform blank control in the same way;

[0054] 6. Use ICP-OES (Agilent 720) combined with ICP-MS (Agilent 7700) to detect the element content in the sample.

[0055] Example 1: BSA water-water treatment of cinnabar

[0056] This embodiment provides a method for producing cinnabar using thiol-containing protein water, wherein the protein is bovine serum albumin (BSA), and the method comprises the following steps:

[0057] (1) Pour 1-2 g of standard cinnabar powder (Guoyao Lerentang Hebei Pharmaceutical Co., Ltd.) into a grinding pot, add a small amount of ultrapure water, and use a grinding rod to grind the powder into a paste;

[0058] (2) Pour 10 times the volume of cinnabar in ultrapure water, stir with a glass rod, and use a plastic spoon to remove the floating residue on the surface;

[0059] (3) Let stand for 1 to 2 minutes until the coarse particles are completely settled, then slowly pour the suspension into a beaker;

[0060] (4) Repeat the above steps (1) to (3) at least 10 times, collect all the suspension into a clean beaker, and leave it at room temperature away from light for at least 24 hours;

[0061] (5) Discard the coarse particles precipitated in the grinding bowl and wash the grinding bowl with ultrapure water for later use;

[0062] (6) pouring out all the supernatant after the static treatment in step (4), leaving the precipitate;

[0063] (7) using a 0.5% BSA aqueous solution (dissolved in ultrapure water) to repeat the treatment of steps (1) to (5) above on the precipitate obtained in step (6);

[0064] (8) Pour out the supernatant containing 0.5% BSA aqueous solution (dissolved in ultrapure water), leaving the precipitate, add 200 mL of ultrapure water, stir, and let stand at room temperature in the dark for 24 hours;

[0065] (9) Repeat step (8) at least twice, pour out the supernatant, pour the precipitate into a plastic dish, and dry it at room temperature away from light;

[0066] (10) The cinnabar powder was collected and named BSA water-treated cinnabar (BSA).

[0067] Comparative Example 1: Treatment of cinnabar with ultrapure water

[0068] This comparative example provides traditional ultrapure water-treated cinnabar, and its preparation method is similar to that of Example 1, except that the 0.5% BSA aqueous solution in steps (7) and (8) of Example 1 is replaced by ultrapure water, and the cinnabar powder is finally collected and named ultrapure water-treated cinnabar (UP water).

[0069] Test Example 1: Properties of cinnabar before and after BSA water-flying and BSA residual rate

[0070] This test example explored the effect of the BSA water-water treatment described in Example 1 on the quality of cinnabar by observing the characteristics of cinnabar particles and measuring the BSA protein content in the sample. Figure 1 As shown, the standard sample is cinnabar powder purchased from Guoyao Lerentang Hebei Pharmaceutical Co., Ltd.

[0071] like Figure 1 As shown in A, the sample was prepared into a suspension using 0.5% sodium carboxymethylcellulose CMC-Na as a carrier and allowed to stand at 4°C for 24 hours. Compared with the standard sample, the BSA water-treated cinnabar (BSA) sample prepared in Example 1 had less impurities suspended in the solution and the solution was clear and bright.

[0072] The BCA protein quantification method was used to detect the effective content of BSA before grinding used for water-flying treatment ( Figure 1 B and C), and the residual rate of BSA after BSA was dissolved in ultrapure water before and after water-spraying and left to stand for 24 hours ( Figure 1 D and E), it was found that when the concentration was lower than 80 mg / mL, no BSA remained in the BSA treated with water.

[0073] Through the observation of scanning electron microscope (SEM) and the detection of laser particle size analyzer, it was found that the surface of the BSA water-treated cinnabar (BSA) sample prepared in Example 1 was smoother and flatter than that of the standard sample ( Figure 2 Middle A), the particle size is smaller and more concentrated ( Figure 2 Medium BE), larger specific surface area ( Figure 2 Middle F), get a lower span and higher shading rate ( Figure 2 G, H), and the experimental residuals remain within 1.0 ( Figure 2 The results are credible.

[0074] Test Example 2: Element Content and Element Solubility of Cinnabar Particles by Different Water-Flying Methods

[0075] In order to explore the influence of different water-free methods on the content and solubility of different elements in cinnabar, this test example measured the element content and element solubility in commercial standard cinnabar (Normal), ultrapure water-free treatment (UP water) in Comparative Example 1, and BSA water-free treatment (BSA) obtained in Example 1. The results are shown in Figure 2. Figure 3 shown.

[0076] After being treated by different water-jetting methods, the contents of Hg and S in the particles of cinnabar remained basically unchanged ( Figure 3 However, after BSA hydrolysis, the contents of other trace elements such as Ca and Mg decreased, while the contents of Fe, Na, S and Zn increased ( Figure 3 The solubility of Hg, S and Se in different solutions was detected by ICP-OES combined with ICP-MS. It was found that the solubility of Hg in ultrapure water and artificial gastric juice was basically unchanged, while the solubility of free Hg in artificial intestinal juice decreased significantly after BSA was deionized. Figure 3 C). The S element dissolved in ultrapure water significantly decreased after ultrapure water and BSA water-beating, and the solubility in artificial intestinal fluid was the lowest for the sample after ultrapure water-beating ( Figure 3 (D) The solubility of Se in the sample after BSA water-freezing in ultrapure water, artificial gastric juice and artificial intestinal juice is the lowest.

[0077] Finally, it should be noted that the above is only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred arrangement scheme, a person skilled in the art should understand that the technical solution of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for producing cinnabar using thiol-containing protein water, characterized in that: The method comprises the following steps: (1) Pour the standard cinnabar powder into a grinding pot, add a small amount of ultrapure water, and use a grinding rod to grind the powder into a paste; (2) Pour 5 to 10 times the volume of cinnabar into ultrapure water, stir with a glass rod, and use a plastic spoon to remove the floating residue on the surface; (3) Let stand for 1 to 2 minutes until the coarse particles are completely settled, then slowly pour the suspension into a beaker; (4) Repeat the above steps (1) to (3) at least 10 times, collect all the suspension into a clean beaker, and leave it at room temperature away from light for at least 24 hours; (5) Discard the coarse particles precipitated in the grinding bowl and wash the grinding bowl with ultrapure water for later use; (6) pouring out all the supernatant after the static treatment in step (4), leaving the precipitate; (7) using the thiol-containing protein aqueous solution and the precipitate obtained in step (6), repeating the treatment of steps (1) to (5); (8) Pour out the supernatant of the thiol-containing protein aqueous solution, leaving the precipitate, add ultrapure water, stir, and stand at room temperature in the dark for 24 hours; (9) Repeat step (8) at least twice, pour out the supernatant, pour the precipitate into a plastic dish, and dry it at room temperature away from light; (10) Collect cinnabar powder.

2. The method for producing cinnabar using sulfhydryl protein water according to claim 1, wherein: The thiol-containing proteins include bovine serum albumin, whey protein and papain.

3. The method for producing cinnabar using sulfhydryl protein water according to claim 1, wherein: The mass concentration of the thiol-containing protein aqueous solution is 0.5-10%.

4. Cinnabar prepared by the method according to any one of claims 1 to 3.

5. Cinnabar according to claim 4, characterized in that The surface of cinnabar is smooth and flat, the particle size range is 0.2-11.0μm, the specific surface area is 3.7-3.8, the span is 1.8-2.0, and the shading rate is 12.0-13.0%.

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