阅读说明：本技术 医药黄腐酸标准品制造及其细胞实验的应用 (Preparation of medical fulvic acid standard substance and application of medical fulvic acid standard substance in cell experiment ) 是由 周霞萍 崔军彦 梁圣模 沈天瑞 崔刚彦 梁琪翊 赵日光 潘君 于 2021-08-04 设计创作，主要内容包括：本发明通过对泥炭、褐煤、风化煤腐植酸催化氧化,经立式旋流机、卧螺式分离机、碟式离心机深度分离,凝胶相变脱灰,水醇膜、均相、异相电渗析膜的连续脱盐,分子量测定,得到医药黄腐酸标准品；并通过对肝癌细胞Hep1-3B转氨酶的抑制实验；与α-葡萄糖苷酶反应,对预防胰腺抵抗降血糖的实验,对人脑胶质瘤U87细胞中存活率验证,药效达35.28-80.00％及以上,并对脑屏障有调节作用,有效提升了黄腐酸医药品附加值,应用前景广阔。(The humic acid is subjected to humic acid catalytic oxidation on peat, lignite and weathered coal, deep separation by a vertical cyclone machine, a horizontal screw separator and a disc centrifuge, gel phase change deashing, continuous desalination by a hydroalcoholic membrane and homogeneous and heterogeneous electrodialysis membranes, and molecular weight determination to obtain a medical fulvic acid standard substance; and through the inhibition experiment on hepatoma cell Hep1-3B transaminase; the compound has reaction with alpha-glucosidase, is used for experiments on preventing pancreas from resisting and reducing blood sugar, is verified for the survival rate in human brain glioma U87 cells, has the drug effect of 35.28-80.00% or more, has an adjusting effect on brain barrier, effectively improves the additional value of fulvic acid pharmaceuticals, and has wide application prospect.)

1. A method for preparing a medical fulvic acid standard substance is characterized by comprising the following steps: the method comprises the following manufacturing steps: ,

(1) peat, brown coal and weathered coal raw materials are firstly catalyzed and oxidized at the temperature of 30-150 ℃ in a reactor, the reaction time is 1.0-3.5h and the reaction pressure is 0.10-0.15 MPa, and then the raw materials are precipitated and separated to remove insoluble impurities of medical fulvic acid. .

(2) And then multi-stage liquid-solid separation is carried out by adopting an FX100370X1070 vertical cyclone machine, an LW255X765-NLWX020-N horizontal spiral separator and a DHC300 disc centrifuge to remove the insoluble fine impurities of the medical fulvic acid, wherein the effective rates are respectively 50-75%, 75-90% and 91.0-95.0%.

(3) 0.0001-0.05% of chitosan is used for deliming by gel hydrolysis phase-change method at the temperature of 20-50 ℃, the time of 0.5-1h and the pressure of 0.10-0.12MPa, and the crude fulvic acid medicine is obtained after the effective rate reaches 96.0-98.5%.

2. The manufacturing method according to claim 1, characterized in that: the catalyst is one or two of sodium percarbonate, potassium percarbonate, calcium percarbonate and hydrogen peroxide, and the concentration of the catalyst is 0.1-0.01% (by mass) of the raw materials.

3. The manufacturing method (1), (2), or (3) according to claim 1, characterized in that: the crude fulvic acid medicine is prepared by adopting a water dialysis membrane at the temperature of 20-50 ℃ and the pressure of 0.10-0.12MPa, wherein the weight ratio of the water dialysis membrane is 10-30: 70-90% (mass) alcohol water dialysis membrane, homogeneous and heterogeneous electrodialysis membrane, and gel chromatography (GPC) to determine molecular weight at 380-500Da to make the purity of refined fulvic acid medicine reach 99.00-99.99%.

4. The pharmaceutical fulvic acid according to claims 1, 2 and 3, experimentally verified using Hep1-3B transaminase cells, wherein:

the Hep1-3B cells are treated by 25% ethanol, and experiments on a transaminase standard curve confirm that the inhibition rate of fulvic acid on transaminase cells is over 55% at the concentration of 50ug/ml and reaches 70% -90% at the concentration of 200 ug/ml.

5. The pharmaceutical fulvic acid standard according to claims 1, 2, and 3 for experimental validation of α -glucosidase cells, wherein: when PNPG is combined with galactose to form a lactose, glucose derivative, fulvic acid acts against the hypoglycemic effects of the pancreas at blank control and fulvic acid drug concentrations of 12.5ug/ml, 25ug/ml, 50ug/ml, 100ug/ml, 200 ug/ml.

6. The pharmaceutical fulvic acid standard according to claims 1, 2 or 3, for use in the survival of glioma U87 cells, wherein: compared with blank control group, the fulvic acid drugs of 12.5ug/ml, 25ug/ml, 50ug/ml, 100ug/ml and 200ug/ml are significant, p is less than 0.05, p is less than 0.01, and the half inhibition concentration is more than 35.28%.

7. The method of claims 1, 2, 3, and 6, wherein the pharmaceutical fulvic acid can also regulate blood brain barrier permeability and prevent brain inflammation, brain tumor, and stroke at 12.5ug/ml, 25ug/ml, 50ug/ml, 100ug/ml, and 200 ug/ml.

Technical Field

The invention mainly relates to the manufacture of a medical fulvic acid standard substance and application of the medical fulvic acid standard substance to transaminase, blood sugar and tumor cell experiments.

Background

Fulvic acid is one of humic acids, and the medical application of humic acid is recorded from "answer to tresomber book" written by Liuzhong Yuan of Tang dynasty, and is widened after Song, Yuan, Ming and Qing passed through the approval of medical professional committee of China humic acid industry Association by the department of Producer chemical engineering in 1995 and 5 months in 2018 after the first China humic acid medical health industry development forum is summoned by China humic acid industry Association in Shandong chat city. Tracing back from history, the safety, effectiveness and quality control research of humic acid medicine is necessary in the humic acid medicine product and market, but the humic acid fulvic acid medicine standardized product and commodity do not exist at home and abroad.

Yan Vide published a paper in Jiangxi humic acid journal in 1981, and relates to that sodium fulvate has a certain analgesic effect on malignant tumors, and 7 liver cancer cases exist. The Zhouchun Jiaojiao of Hunan university carries out surface modification on superparamagnetic Fe _3O _4 nano particles in situ by micromolecular fulvic acid (fulvic acid), synthesizes superparamagnetic Fe _3O _4 nano magnetic fluid with good water solubility, is used as an MRI liver contrast agent (medical instrument), shows that the fulvic acid is favorable for improving the detection rate of small focuses and even tiny focuses of liver tissues and is also favorable for marking, tracing and targeting cancer cells. Has different degrees of inhibition effects on 8 cancer cells such as glioma cell strain SK-N-SH, liver cancer cell strain QGY7703, gastric adenocarcinoma cell strain SGC7901, acute myelogenous leukemia cell strain K562, acute promyelocytic leukemia cell strain HL-60 and the like.

Yang light combustion, Yuanshenyuan and Zhuliangxin independently enter cells through GLUT2 glucose transporter (GLUT2 glucose transporter protein) by streptozotocin, develop the characteristic of beta-cells induced by pancreatic insulin, establish an in vitro diabetes rat animal model, measure pain threshold change by utilizing a thermal pain tester and a hot plate test, and observe peripheral nerve tissues by using a light mirror and a transmission electron microscope, thereby prompting that sodium fulvate has a certain inhibiting effect on experimental diabetic peripheral neuropathy.

The functional and structural integrity of the blood-brain barrier (BBB) is critical to maintaining the homeostasis of the brain microenvironment. The blood brain barrier is a selective physical barrier formed primarily by Brain Microvascular Endothelial Cells (BMECs), glial cells (astrocytes) and pericytes (percytes). After acute ischemic stroke occurs, the Blood Brain Barrier (BBB) is quickly destroyed, the vascular permeability is obviously increased, and water and plasma protein in capillary vessels are extravasated to form angiogenetic edema and aggravate brain injury. Although the incidence of malignant brain tumor is only 1-3% of the total cancer, and 5-7% of children, the prognosis is extremely poor. This is because: 1) the cranial cavity of an adult is a hard closed bone cavity, once the intracranial growing tumor occupies space, the intracranial pressure is increased, the brain and the nerves are pressed, the central nervous system is damaged, the cerebrospinal fluid circulation and the cerebral blood flow are further influenced, the secondary cerebral ischemia, the hypoxia and the cerebral edema occur, and the brain tumor finally occurs, so that the life is threatened. 2) Brain tumor grows in infiltration, can not be eradicated completely, and is easy to relapse. 3) Brain tumors are less sensitive to radiation and have poor radiotherapy effects. 4) Most of the anticancer chemotherapeutic drugs can not enter the brain through the blood brain barrier and are difficult to take good effect. 5) Immunotherapy, gene therapy, has just started. Liuchi and the like research the influence of the sodium fulvate on the inflammatory reaction of the cerebral ischemia reperfusion injury of rats, and the result shows that: the sodium fulvate can obviously improve the permeability of a blood brain barrier, relieve the infiltration of cerebral edema and white blood cells, reduce the contents of interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) in brain tissues of an ischemic area, inhibit the activation of nuclear factor-kappa Bp65 (NF-kappa Bp65), relieve the inflammatory response of cerebral ischemia reperfusion injury, and has a protection effect on the cerebral ischemia reperfusion injury (Liu Chi, Zheng Zi Long, Zhou warship, and the like).

Application No. 202110360592.8A brain drug delivery process of carbonized polymer with ginkgolide B breaking through blood brain barrier is that carboxyl of semen Ginkgo and amino of carbonized polymer form amide bond to form drug. From the reaction mechanism, fulvic acid can also achieve the effects of lower cost and better drug resistance.

Therefore, the formation of high-quality medical fulvic acid standardized products is the key for stably and deeply researching the pharmacological and pharmacodynamic effects of fulvic acid series.

Disclosure of Invention

In order to solve the technical defects, the invention aims to creatively establish a production line of medical fulvic acid standard products in a resource manner, apply the products to cell experiments of major diseases and improve the additional value of fulvic acid medicines.

The invention 1, the method for preparing the medical fulvic acid standard substance,

(1) firstly, the screened and crushed peat, brown coal and weathered coal raw materials are catalyzed and oxidized for 1.0 to 3.5 hours at the temperature of 30 to 150 ℃ and the reaction pressure of 0.10 to 0.15MPa in a reactor, and then the raw materials are precipitated and separated to remove insoluble impurities of medical fulvic acid. .

(2) And the FX100370X1070 vertical cyclone machine, the LW255X765-NLWX020-N horizontal spiral separator and the DHC300 disc centrifuge are adopted to perform multi-stage liquid-solid separation to remove insoluble fine impurities in the medical fulvic acid, wherein the effective rates are 50-75%, 75-90% and 91.0-95.0% respectively.

(3) 0.0001-0.05% of pharmaceutical grade chitosan (provided by chemical and molecular engineering research laboratory of university of east China science) is used for deliming by gel hydrolysis phase-change method at the temperature of 20-50 ℃, the time of 0.5-1h and the pressure of 0.10-0.12MPa, and the crude fulvic acid medicine is obtained after the effective rate reaches 96.0-98.5%.

The catalyst of the preparation method is one or two of sodium percarbonate, potassium percarbonate, calcium percarbonate and hydrogen peroxide, and the concentration of the catalyst is 0.1-0.01% (by mass) of the raw materials.

The crude fulvic acid medicine prepared in the steps (1), (2) and (3) of the invention 1 adopts a water dialysis membrane at the temperature of 20-50 ℃ and the pressure of 0.10-0.12MPa, and the ratio of (10-30): 70-90% of alcohol-water dialysis membrane, homogeneous and heterogeneous electrodialysis membrane (provided by chemical engineering separation engineering research laboratory of China eastern university of science) continuously desalting, and measuring molecular weight at 380-500Da by gel chromatography (GPC) to make the purity of the refined fulvic acid medicine reach 99.00-99.99%.

The medical fulvic acid standard substance is verified by an experiment of a Hep1-3B transaminase cell:

(1) on the basis of a transaminase calibration curve, the experiment proves that the fulvic acid has the inhibition rate of over 55 percent on transaminase cells under the concentration of 50ug/ml and the inhibition rate of 70 to 90 percent under the concentration of 200ug/ml when the Hep1-3B cells are treated by 25 percent ethanol. The experimental verification is carried out at college of pharmacy of university of eastern China as shown in figure 1.

(2) The medical fulvic acid standard substance is used for experimental verification of alpha-glucosidase cells, and is characterized in that: when PNPG is combined with galactose to form a lactose and glucose derivative, fulvic acid has the effect of resisting blood sugar reduction of pancreas under the conditions of blank control and concentrations of fulvic acid drugs of 12.5ug/ml, 25ug/ml, 50ug/ml, 100ug/ml and 200ug/ml, and the result is shown in figure 2, and experimental verification is carried out at the college of pharmacy of the university of eastern China.

(3) The medical fulvic acid standard substance is used for the survival rate of glioma U87 cells, and is characterized in that: compared with the blank control group, the fulvic acid drugs have significance at p, p <0.05, p <0.01, and the half inhibition concentration is more than 35.28%. As shown in figure 3, the experimental verification is carried out at college of medicine of university of eastern China

The medical fulvic acid standard substance can regulate the permeability of a blood brain barrier and prevent the occurrence of cerebral inflammation, brain tumor and cerebral apoplexy when the concentration is 12.5ug/ml, 25ug/ml, 50ug/ml, 100ug/ml and 200 ug/ml.

The present invention has the following advantageous effects

Compared with the prior art, the applicant creatively establishes a sanitary and environment-friendly medical fulvic acid standard product production line with independent intellectual property rights in a resource place, applies the product to cell experiments of major diseases, can be deeply clinically applied, improves the added value of the fulvic acid medical standard product, and has important practical significance for promoting the health of people and flourishing economy.

Description of the drawings:

FIG. 1 is a graph showing the relationship between the inhibition ratio of Hep1-3B cells to 25% ethanol of the fulvic acid standard prepared in example 1 and transaminase.

FIG. 2 is a graph showing the effect of fulvic acid standards prepared in example 2 on pancreatic resistance to hypoglycemic events.

FIG. 3 is a graph showing the relationship between the survival of the fulvic acid standard prepared in example 3 and human glioma U87 cells.

The specific embodiment is as follows:

example 1

Taking 100 parts of lignite raw material, screening and crushing, carrying out catalytic oxidation reaction for 3 hours by adopting sodium percarbonate and calcium percarbonate with the concentration of 0.05 percent (mass) of the raw material at the temperature of 130 ℃ and the reaction pressure of 0.13MPa, and then carrying out primary precipitation separation in a reactor with a sieve plate to remove insoluble impurities of medical fulvic acid; then, an FX100370X1070 vertical cyclone machine and an LW255X765-NLWX020-N horizontal spiral separator are adopted for secondary separation, and a DHC300 disc centrifuge is adopted for tertiary liquid-solid separation to remove insoluble fine impurities in the medical fulvic acid; 0.05 percent of medical grade chitosan (provided by chemical and molecular engineering research laboratory of university of eastern China) is applied to perform gel hydrolysis phase-change deashing at the temperature of 50 ℃, the time of 1h and the pressure of 0.12MPa, the crude fulvic acid medicine is obtained after the effective rate reaches 96.0 to 98.5 percent, and a water dialysis membrane is adopted at the temperature of 30 ℃ and the pressure of 0.10MPa, 30: continuously desalting 70% (by mass) alcohol-water dialysis membrane and homogeneous and heterogeneous electrodialysis membrane (provided by separation engineering research laboratory of chemical engineering of university of eastern science and technology), measuring molecular weight of 400Da, purity of fulvic acid medicine to 99.99%, sampling 10 parts, treating Hep1-3B cell supernatant glutamic-pyruvic transaminase standard curve with 25% ethanol, observing cell morphology change under an inverted microscope, and observing time effect and dose effect of MTT method under concentration of 50ug/ml, wherein the inhibition activity is 55%; the inhibitory activity reaches 70% at a concentration of 200 ug/ml.

Example 2

Taking 100 parts of peat raw material, screening and crushing, carrying out catalytic oxidation reaction for 3 hours at the reaction temperature of 130 ℃ and the reaction pressure of 0.13MPa by adopting sodium percarbonate and calcium percarbonate with the concentration of 0.01% (mass) of the raw material, and then carrying out primary precipitation separation to remove insoluble coarse impurities; further adopting an FX100370X1070 vertical cyclone machine, an LW255X765-NLWX020-N horizontal spiral machine for liquid-solid separation and a DHC300 disc machine for re-separation to remove insoluble fine impurities in the fulvic acid; 0.05 percent of medical grade chitosan (provided by chemical and molecular engineering research laboratory of university of eastern China) is applied to perform gel hydrolysis phase-change deashing at the temperature of 50 ℃, the time of 1h and the pressure of 0.12MPa, the crude fulvic acid medicine is obtained after the effective rate reaches 96.0 to 98.5 percent, and a water dialysis membrane is adopted at the temperature of 30 ℃ and the pressure of 0.10MPa, 30: 70 percent (mass) alcohol-water dialysis membrane, homogeneous and heterogeneous electrodialysis membrane (provided by chemical engineering separation engineering research laboratory of China eastern university), continuously desalting, measuring the molecular weight of 380Da, and after the purity of the medical standard product of fulvic acid reaches 99.99 percent, sampling 10 parts, combining PNPG and galactose through the action of chromogenic group binding enzyme to form a lactose (or glucose) analogue, detecting the inhibition of GXAY on the enzyme under different concentrations, measuring the OD value of the combination of the enzyme and PNPG by a spectrophotometer, and enabling the fulvic acid to have more than 80 percent of effect on pancreas to resist blood sugar reduction under the conditions of blank control, 12.5ug/ml, 25ug/ml, 50ug/ml, 100ug/ml and 200 ug/ml.

Example 3

Selecting 100 parts of weathered lignite raw material, screening and crushing, carrying out catalytic oxidation reaction for 3 hours by adopting sodium percarbonate and calcium percarbonate with the concentration of 0.1 percent (mass) of the raw material at the temperature of 130 ℃ and the reaction pressure of 0.13MPa, and then carrying out primary precipitation separation in a reactor with a sieve plate to remove insoluble impurities of medical fulvic acid; then, an FX100370X1070 vertical cyclone machine and an LW255X765-NLWX020-N horizontal spiral separator are adopted for secondary separation, and a DHC300 disc centrifuge is adopted for tertiary liquid-solid separation to remove insoluble fine impurities in the medical fulvic acid; 0.05 percent of medical grade chitosan (provided by chemical and molecular engineering research laboratory of university of eastern China) is applied to perform gel hydrolysis phase-change deashing at the temperature of 50 ℃, the time of 1h and the pressure of 0.12MPa, the crude fulvic acid medicine is obtained after the effective rate reaches 96.0 to 98.5 percent, and a water dialysis membrane is adopted at the temperature of 30 ℃ and the pressure of 0.10MPa, 30: 70 percent (mass) alcohol-water dialysis membrane, homogeneous and heterogeneous electrodialysis membrane (provided by chemical engineering separation engineering research laboratory of east China university) are desalted continuously, 10 parts of the samples are taken after the purity of the medical standard product of the fulvic acid reaches 99.99 percent after the molecular weight is measured to be 500Da, and the MTT experiment is adopted to screen the influence of the drug on the survival condition of human brain glioma U87 cells (namely survival rate) under the concentration conditions of blank control, 12.5ug/ml, 25ug/ml, 50ug/ml, 100ug/ml and 200ug/ml, compared with the blank control group, the half inhibition concentration is 35.28 percent or more, and the half inhibition concentration is respectively significant when p is less than 0.05, p is less than 0.01, p is less than 0.001.