Method for constructing neuroinflammation intervention animal model and screening effective components in turmeric dregs

Abstract

The invention belongs to the technical field of traditional Chinese medicine, and discloses a method for constructing a neuroinflammation intervention animal model and screening effective components in turmeric dregs, and the method comprises the following steps: S1, preparing an experimental medicament for an animal model; s2, constructing a mouse animal model of the A beta 1-42 induced neuroinflammation; and S3, ascertaining the mechanism and effect of the curcuma longa dregs in intervening the neuroinflammation of the mouse. The invention further provides a method for screening the effective components in the rhizoma curcumae longae dregs, and the effective components for intervening neuroinflammation in the rhizoma curcumae longae dregs are screened by adopting a neuroinflammation intervention animal model. According to the invention, a specially constructed animal model, UPLC-MS / MS and HPLC are combined with a non-targeted metabonomics technology to research the mechanism, effect and effective components of the curcuma longa dreg intervened neuroinflammation mouse, and the method can be widely applied to deep development of curcuma longa traditional Chinese medicine dregs, effectively utilizes curcuma longa dreg resources, and provides support for prevention and treatment of neuroinflammation related diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and in particular to a method for constructing an animal model for neuroinflammation intervention and screening effective ingredients in turmeric residues. Background Art

[0002] Existing studies have shown that among neuroinflammatory diseases such as dementia, Alzheimer's disease is the main type of disease. Alzheimer's disease (AD) is a neurological disease with clinical manifestations of cognitive impairment and progressive memory impairment. The main pathological features are extracellular amyloid beta (Aβ) and intracellular Tau protein phosphorylation. Aβ aggregates to form deposits, inducing neuroinflammation and hindering intercellular communication, while Tau protein phosphorylation forms entanglements between neurofibrils and disordered neuronal material exchange. Among the many pathogenesis mechanisms, the most recognized amyloid hypothesis believes that Aβ aggregation and deposition into plaques leads to the onset of AD, accelerating neuroinflammation, neuronal damage, synaptic loss and other processes, and these pathological processes further accelerate Aβ aggregation, thereby forming a cascade amplification effect, nerve damage, and ultimately leading to the onset of AD.

[0003] Turmeric is a commonly used Chinese medicinal material, derived from the dried rhizome of Curcuma long L., a plant of the genus Curcuma in the Zingiberaceae family, and is mainly produced in Sichuan, Fujian, Guangdong, Guangxi and other provinces and regions. Existing studies have shown that the chemical components of turmeric are mainly curcuminoid compounds and volatile oils, as well as sugars, flavonoid compounds, sterols, etc. In existing studies, the patent technology document with application publication number CN112351790 A has clearly stated that turmeric extracts of free water and hydrophilic organic solvents, turmeric ketones A and turmeric ketones B are effective ingredients for treating Alzheimer's disease. The existing Chinese patent medicines in my country that use turmeric as raw materials also mostly use turmeric aqueous extracts as effective ingredients. For example, the production of turmeric formula granules is mainly based on water extraction. Since oil-soluble and fiber components such as curcumin in turmeric are difficult to dissolve in water, these curcumin compounds will still remain in the residue; and the turmeric dregs after water extraction (solid residues remaining after aqueous extraction) are generally treated as waste. Other important components of turmeric are not fully utilized, which not only causes a huge waste of resources, but also causes serious environmental pollution and increases the cost of pharmaceutical companies. Existing studies have shown that a variety of components can be extracted from turmeric dregs, including volatile oils, curcumin, essential oils, polysaccharides, etc. However, due to various technical difficulties, no specialized research on the development and utilization of turmeric residues has been found. Existing research does not involve details such as which components (qualitative and quantitative) in turmeric residues, which components can be effective in intervening in neuroinflammation such as AD, and how the active ingredients produce their effects (active ingredients and pharmacology, pharmacodynamic effects). It cannot provide technical support for promoting the comprehensive development and utilization of turmeric residues, nor can it meet the needs of enterprises to develop high-value drugs containing active ingredients in turmeric residues and reduce the cost of processing waste from traditional Chinese medicine processing. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a method for constructing an animal model for neuroinflammation intervention and screening effective ingredients in turmeric residues. By constructing a scientific animal model and combining it with non-targeted metabolomics technology, the effective ingredients in turmeric residues are qualitatively and quantitatively analyzed to find out the effective ingredients in turmeric residues that can significantly intervene in neuroinflammation (including AD). At the same time, specific research is conducted on the pharmacology and pharmacodynamic effects of these effective ingredients on how to specifically intervene in neuroinflammation, so as to provide technical support for the comprehensive development and utilization of turmeric residues, meet the needs of enterprises to develop high-value drugs containing effective ingredients in turmeric residues, and at the same time reduce the cost of processing waste from traditional Chinese medicine processing, so as to solve the problems existing in the above-mentioned prior art.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is: A method for constructing an animal model for neuroinflammation intervention, comprising the following steps: S1: Preparation of experimental agents for animal models Five types of experimental agents were prepared respectively: turmeric medicinal materials, turmeric dregs, low-dose curcumin, high-dose curcumin and donepezil tablet solution; the turmeric dregs were the solid residues remaining after water extraction of turmeric medicinal materials; S2: Construction of Aβ 1-42 Mouse animal model of induced neuroinflammation Several male mice were selected and randomly divided into sham operation group, model group, positive drug group, medicinal material group, medicinal residue group, low-dose curcumin group, and high-dose curcumin group. They were fed with the same feed, and the behavior of mice in each group was tested: First, five experimental drugs were given to each group of mice by gavage for one week: turmeric, turmeric residue, low-dose curcumin, high-dose curcumin, and donepezil tablets. Then, Aβ was injected into the brain of the mice. 1-42 The neuroinflammation model was established, and then the mice in each group were given five types of experimental drugs by gavage for 3 consecutive weeks; Each group of mice was subjected to behavioral tests to analyze whether the experimental drugs used in each group in the animal model had the effect of reducing neuroinflammation in the mouse brain; S3: To explore the mechanism and effect of turmeric residue in intervening neuroinflammation in mice The mice in each group obtained in step S2 were prepared as biological samples, and non-targeted metabolomics technology was used to analyze and explore whether turmeric residues and curcumin can improve Aβ by regulating the TLR4 / NF-κB pathway. 1-42 Inducing cognitive impairment in mice with neuroinflammation, alleviating neural damage, regulating overactivated microglia, downregulating inflammatory factors and upregulating anti-inflammatory factors, thus achieving the intervention effect of alleviating neuroinflammation; S4: Qualitative and quantitative determination of major curcuminoids in turmeric residues UPLC-MS / MS and HPLC techniques were used to qualitatively analyze and quantitatively determine the main curcuminoid compounds in turmeric residues. S5: Testing the mechanism and effect of the main components of turmeric residues in intervening neuroinflammation in mice A neuroinflammation intervention animal model was used to test the main curcuminoid compounds in turmeric residues and the mechanism and effect of intervening in neuroinflammation in mice.

[0006] A method for screening effective ingredients for intervening neuroinflammation in turmeric residues, comprising the following steps: A1: Analysis of the main chemical components in turmeric residue UPLC-MS / MS and HPLC techniques were used to qualitatively analyze and quantitatively determine the main curcuminoid compounds in turmeric residues, and the active ingredients to be tested were preliminarily determined. A2: Screening of active ingredients using animal models and mechanisms of neuroinflammation intervention Using the neuroinflammation intervention animal model, and utilizing the intervention mechanism of curcuminoids on neuroinflammation, the intervention effects of each active ingredient to be tested on neuroinflammation were tested respectively; A3: Comparative test to determine whether there is synergistic effect between the active ingredients in turmeric residue A comparative test was conducted using an animal model of neuroinflammation intervention, comparing the simultaneous use and single use of the active ingredients to be tested, to determine whether there is a synergistic effect when multiple compounds in turmeric residues are used simultaneously, and whether the intervention and therapeutic effects of multiple active ingredients on neuroinflammation such as AD are more significant when used together than when used alone.

[0007] The method for constructing an animal model for neuroinflammation intervention and screening effective ingredients in turmeric residues provided by the present invention has at least the following beneficial effects: 1. The present invention provides a method for qualitatively and quantitatively analyzing the active ingredients in turmeric dregs by constructing a scientific animal model and combining non-targeted metabolomics technology to find out the active ingredients in turmeric dregs that can significantly intervene in neuroinflammation such as AD, and at the same time, to conduct specific research on the pharmacology and pharmacodynamic effects of these active ingredients on how to specifically intervene in neuroinflammation, so as to promote the comprehensive development and utilization of turmeric dregs and provide technical support, meet the needs of enterprises to develop high-value drugs containing active ingredients in turmeric dregs, and reduce the cost of processing waste from traditional Chinese medicine processing, so as to solve the problems existing in the above-mentioned prior art.

[0008] 2. The present invention uses UPLC-MS / MS technology for the first time to preliminarily identify the chemical components in turmeric residues, and further analyzes the intervention mechanism and effect of the components of turmeric residues to find out the main effective active ingredients.

[0009] 3. The present invention establishes Aβ 1-42 A mouse model of neuroinflammation was established by brain targeted injection to explore whether turmeric, turmeric residues and curcumin (curcuminoids), an important medicinal ingredient in turmeric, can reduce neuroinflammation in the mouse brain and study its potential mechanism of action; combined with non-targeted metabolomics technology, a study was conducted on mice with neuroinflammation intervened by turmeric residues.

[0010] 4. The present invention finds that turmeric residues and their effective ingredients have the effect of reducing Aβ 1-42 Inducing neuroinflammation; It was found that both turmeric residue and curcumin alleviated Aβ by regulating the TLR4 / NF-κB pathway 1-42The potential mechanism of induced neuroinflammation is to reduce neuroinflammation in the brain by regulating the over-activated state of microglia, regulating important proteins in the TLR4 / NF-κB pathway, downregulating inflammatory factors, and upregulating anti-inflammatory factors.

[0011] 5. The present invention constructs Aβ 1-42 The C57BL / 6 mouse animal model of inducing neuroinflammation, combined with the qualitative and quantitative analysis of the active ingredients in turmeric residues, ultimately explored the active ingredients, mechanism of action and indications in turmeric residues. This invention not only provides new ideas for the research on the development of traditional Chinese medicine residues, but also provides support for the prevention and treatment of neuroinflammation-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is a schematic diagram of the process of constructing an animal model and screening effective ingredients in an embodiment of the present invention; Figure 2 This is a schematic diagram of the main process of animal experiments in an embodiment of the present invention; Figure 3 This is a mouse brain localization injection atlas for the animal experiment of the embodiment of the present invention; FIG4 is a water maze of turmeric against Aβ in an embodiment of the present invention. 1-42 Schematic diagram of the effects of damaged spatial learning and memory abilities in mice, including AD, where Figure 4 (A) shows the escape latency in the orientation navigation experiment; Figure 4 (B) shows the number of platform crossings in the spatial exploration experiment; Figure 4 (C) shows the time spent in the target quadrant in the spatial exploration experiment; Figure 4 (D) shows the trajectory of movement in the spatial exploration experiment; FIG. 5 shows the effect of turmeric on Aβ in an embodiment of the present invention. 1-42 Schematic diagram of the effects of induced pathological changes (200× and 500×), including Figure 5 (1) and Figure 5 (2); FIG. 6 shows the effect of turmeric on Aβ in an embodiment of the present invention. 1-42 Schematic diagram of the effects of induced neuronal damage and loss (200× and 500×), including Figure 6 (1) and Figure 6 (2); Figure 7 Iba-1 and Aβ in immunofluorescence staining in the present invention 1-42 Schematic diagram of co-expression (200×); FIG8 shows the effect of turmeric on Aβ in an embodiment of the present invention. 1-42 Schematic diagram of the effects of inducing Iba-1 proliferation, including AC; wherein, Figure 8 (A) (including Figure 8 (A-1) and Figure 8 (A-2)) is the IHC staining of Iba-1 in the hippocampus (200× and 500×); Figure 8 (B) is the percentage of Iba-1 positive area; Figure 8 (C) is the percentage of Iba-1 positive cells in area; FIG. 9 is a schematic diagram of mRNA levels of important channel proteins in the TLR4 / NF-κB pathway regulated by turmeric in an embodiment of the present invention, including AC, wherein FIG. 9 (A) is the mRNA expression level of IκB; Fig. 9B is the mRNA expression level of p65; Fig. 9C is the mRNA expression level of TLR4; Figure 10 is a schematic diagram of turmeric regulating the mRNA levels of cytokines in the TLR4 / NF-κB pathway in an embodiment of the present invention, including AH; wherein, Figure 10 (A) is the mRNA expression level of IL-6; Figure 10 (B) is the mRNA expression level of TNF-α; Figure 10 (C) is the mRNA expression level of iNOS; Figure 10 (D) is the mRNA expression level of COX-2; Figure 10 (E) is the mRNA expression level of IL-4; Figure 10 (F) is the mRNA expression level of IL-10; Figure 10 (G) is the mRNA expression level of TGF-1β; Figure 10 (H) is the mRNA expression level of Arg-1; Figure 11 is a schematic diagram of the effect of turmeric on the protein content of important channels in the TLR4 / NF-κB pathway according to an embodiment of the present invention, including: wherein AD, Figure 11 (A) is the relative expression level of pIKK / IKK protein; Figure 11 (B) is the relative expression level of p-IκB / IκB protein; Figure 11 (C) is the relative expression level of p-p65 / p65 protein; Figure 11 (D) is the protein content of TLR4; Figure 12 is a schematic diagram of the protein expression level of cytokines in the TLR4 / NF-κB pathway regulated by turmeric in an embodiment of the present invention, including AH; Figure 12 (A) is the protein expression level of IL-6; Figure 12 (B) is the protein expression level of TNF-α; Figure 12 (C) is the protein expression level of iNOS; Figure 12 (D) is the protein expression level of COX-2; Figure 12 (E) is the protein expression level of IL-4; Figure 12 (F) is the protein expression level of IL-10; Figure 12 (G) is the protein expression level of TGF-1β; Figure 12 (H) is the protein expression level of Arg-1; Fig.13 In the embodiment of the present invention, turmeric reduces Aβ by regulating the TLR4 / NF-κB signaling pathway 1-42 Diagram of biological mechanisms that induce neuroinflammation; FIG14 is a total ion current diagram in an embodiment of the present invention, including AD; wherein FIG14 (A) is a total ion current diagram in a positive ion mode of plasma; FIG14 (B) is a total ion current diagram in a negative ion mode of plasma; FIG14 (C) is a total ion current diagram in a positive ion mode of brain tissue; FIG14 (D) is a total ion current diagram in a negative ion mode of brain tissue; FIG15 is a visualization result of the spatial distribution of plasma non-targeted metabolomics data in an embodiment of the present invention, including AB; wherein FIG15 (A) is a PLS-DA score diagram in positive ion mode; FIG15 (B) is a PLS-DA score diagram in negative ion mode; FIG. 16 is a visualization result of the spatial distribution of non-targeted metabolomics data of brain tissue in an embodiment of the present invention, including AB; wherein FIG. 16 (A) is a PLS-DA score diagram in positive ion mode; FIG. 16 (B) is a PLS-DA score diagram in negative ion mode; FIG17 is a visualization result of the spatial distribution of non-targeted metabolomics data of plasma in the sham operation group versus the model group in an embodiment of the present invention, including AD; wherein FIG17 (A) is a PLS-DA score graph in positive ion mode; FIG17 (B) is a PLS-DA score graph in negative ion mode; FIG17 (C) is a positive ion VIP graph; and FIG17 (D) is a negative ion VIP graph; FIG18 is a visualization result of the spatial distribution of non-targeted metabolomics data of brain tissues of the sham operation group and the model group in an embodiment of the present invention, including AD; wherein FIG18 (A) is a positive ion mode OPLS-DA score graph; FIG18 (B) is a negative ion mode OPLS-DA score graph; FIG18 (C) is a positive ion VIP graph; and FIG18 (D) is a negative ion VIP graph; Fig.19 This is a schematic diagram of the plasma KEGG metabolic pathway score according to an embodiment of the present invention; Fig. 20 This is a schematic diagram of the KEGG metabolic pathway score of brain tissue according to an embodiment of the present invention. DETAILED DESCRIPTION

[0013] The following embodiments of the present invention are mainly aimed at the problems that the turmeric residues after water extraction in pharmaceutical companies are large in quantity and are treated as waste, which wastes resources and increases corporate costs. On the one hand, the components of the turmeric residues are analyzed, and on the other hand, the mechanism of the turmeric residues and their effective ingredients in intervening in neuroinflammation and whether they have a mitigating effect (pharmacological mechanism and pharmacodynamic effect) are studied to promote the comprehensive development and utilization of the turmeric residues, reduce the cost of Chinese medicine processing waste treatment in enterprises, and meet the needs of enterprises to develop high-value drugs containing effective ingredients in turmeric residues.

[0014] The turmeric residues in the following embodiments of the present invention are all solid residues left after water extraction when pharmaceutical companies use turmeric medicinal materials to produce turmeric formula granules, and are referred to as "turmeric residues", "drug residues" or "HR". In order to conduct comparative studies, each embodiment also includes turmeric medicinal materials (abbreviated as "HM") as the research object and conducts comparative studies with turmeric residues. Unless otherwise specified, turmeric refers to curcumin compounds.

[0015] The experimental reagents used for the analysis of biological samples in the following embodiments of the present invention are shown in Table 1. The experimental instruments used are all commercially available, and the English abbreviations used are all commonly used abbreviations in the industry.

[0016] Table 1

[0017] Basic Example See also Figure 1-Figure 20 The embodiment of the present invention includes two parts: substance research and efficacy research that support each other. First, a mouse neuroinflammation model is constructed to explore the mechanism of action of turmeric on neuroinflammation, and the main components of turmeric medicinal materials and turmeric residues are analyzed using modern analytical techniques. Then, the mechanism of action and the effects of the main components in turmeric medicinal materials and turmeric residues are analyzed to screen out components (or a combination of multiple components) with significant effects, providing a basis for the in-depth development and utilization of turmeric and turmeric residues.

[0018] Among them, the method for constructing an animal model of neuroinflammation intervention includes the following steps: S1: Preparation of experimental agents for animal models Five types of experimental agents were prepared respectively: turmeric medicinal materials, turmeric dregs, low-dose curcumin, high-dose curcumin and donepezil tablet solution; the turmeric dregs were the solid residues remaining after water extraction of turmeric medicinal materials; S2: Construction of Aβ 1-42 Mouse animal model of induced neuroinflammation Several male mice were selected and randomly divided into sham operation group, model group, positive drug group, medicinal material group, medicinal residue group, low-dose curcumin group, and high-dose curcumin group. They were fed with the same feed, and the behavior of mice in each group was tested: First, five experimental drugs were given to each group of mice by gavage for one week: turmeric, turmeric residue, low-dose curcumin, high-dose curcumin, and donepezil tablets. Then, Aβ was injected into the brain of the mice. 1-42 The neuroinflammation model was established, and then the mice in each group were given five types of experimental drugs by gavage for 3 consecutive weeks; Each group of mice was subjected to behavioral tests to analyze whether the experimental drugs used in each group in the animal model had the effect of reducing neuroinflammation in the mouse brain; S3: To explore the mechanism and effect of turmeric residue in intervening neuroinflammation in mice The mice in each group obtained in step S2 were prepared as biological samples, and non-targeted metabolomics technology was used to analyze and explore whether turmeric residues and curcumin can improve Aβ by regulating the TLR4 / NF-κB pathway. 1-42Inducing cognitive impairment in mice with neuroinflammation, alleviating neural damage, regulating overactivated microglia, downregulating inflammatory factors and upregulating anti-inflammatory factors, thus achieving the intervention effect of alleviating neuroinflammation; S4: Qualitative and quantitative determination of major curcuminoids in turmeric residues UPLC-MS / MS and HPLC techniques were used to carry out qualitative analysis and quantitative determination of the main curcuminoids in turmeric residues.

[0019] S5: Testing the mechanism and effect of the main components of turmeric residues in intervening neuroinflammation in mice A neuroinflammation intervention animal model was used to test the main curcuminoid compounds in turmeric residues and the mechanism and effect of intervening in neuroinflammation in mice.

[0020] A method for screening effective ingredients for intervening neuroinflammation in turmeric dregs, which comprises the following steps: A1: Analysis of the main chemical components in turmeric residue UPLC-MS / MS and HPLC techniques were used to qualitatively analyze and quantitatively determine the main curcuminoid compounds in turmeric residues, and the active ingredients to be tested were preliminarily determined. A2: Screening of active ingredients using animal models and mechanisms of neuroinflammation intervention Using the neuroinflammation intervention animal model, the intervention mechanism of the main chemical components in turmeric on neuroinflammation was used to test the intervention effect of each active ingredient on neuroinflammation. A3: Comparative test to determine whether there is synergistic effect between the active ingredients in turmeric residue A comparative test was conducted using an animal model of neuroinflammation intervention, comparing the simultaneous use and single use of the active ingredients to be tested, to determine whether there is a synergistic effect when multiple compounds in turmeric residues are used simultaneously, and whether the intervention and therapeutic effects of multiple active ingredients on neuroinflammation such as AD are more significant when used together than when used alone.

[0021] The following is a detailed description with reference to the accompanying drawings and a number of specific embodiments.

[0022] Example 1 2 to 4 , the method for constructing an animal model for neuroinflammation intervention provided in an embodiment of the present invention is a refinement and specific application based on the aforementioned basic embodiment, the difference being that the five types of experimental agents in step S1 are specifically prepared by the following steps: S1-1: Preparation of low-dose and high-dose solutions of curcumin reference substance Weigh the curcumin reference substance, dissolve it in 2% Tween 80 and distilled water, and prepare low-dose and high-dose curcumin solutions with concentrations of 1 mg / mL and 2 mg / mL, respectively; S1-2: Preparation of Turmeric Herbs and Dregs Solution Weigh the powder, add 70% ethanol solution (1:8, w / v), shake on a shaker for 3 days, filter with gauze, place the filtrate in an evaporating dish, concentrate the drug solution in a water bath, evaporate the ethanol completely, and prepare drug material and drug residue solutions with concentrations of 0.1 g / mL and 0.12 g / mL, respectively, store at -20℃, and sterilize at high temperature before use; S1-3: Preparation of positive drug solution Donepezil hydrochloride tablets (5 mg / tablet) were ground into fine powder and dissolved in distilled water to obtain a positive drug solution with a concentration of 0.076 mg / mL.

[0023] See also Figure 2 Schematic diagram of animal experiment process, step S2 specifically includes the following steps: S2-1: Mouse grouping Several male mice were selected and after 7 days of adaptive feeding, they were randomly divided into 7 groups: sham operation group, model group, positive drug group, medicinal material group, medicinal residue group, low-dose curcumin group, and high-dose curcumin group, with no less than 5 mice in each group, and they were fed with the same feed. The specific experimental animals and feeding methods used were as follows: 56 SPF-grade 2-month-old male C57BL / 6 mice weighing 18-22 g were purchased from Guangdong Medical Experimental Animal Center with a license number of SYXK (Guangdong) 2022-0002. All animals were raised in the Animal Center of Guangdong Pharmaceutical University with a license number of SYXK (Guangdong) 2022-0125, with free access to food and water, an ambient temperature of 20-25°C, a humidity of 40-70%, and 12 h / 12 ​​h alternating lighting per day, and adaptive feeding for 1 week; S2-2: Grouping and administration of drugs before localized injection in mouse brain Each mouse in each group was first gavaged for one week, and the specific medication for each group was as follows: Sham group (Sham): C57BL / 6 mice injected with sterile saline in the bilateral hippocampus and gavaged with 0.1 mL / g distilled water; Model group: Aβ was injected into the bilateral hippocampus 1-42 C57BL / 6 mice were gavaged with 0.1 mL / 10 g distilled water; Positive drug group (Donepezil): Aβ was injected into the bilateral hippocampus 1-42C57BL / 6 mice were given 0.76 mg / kg by gavage with 0.1 mL / 10 g donepezil hydrochloride solution; Herbal materials group (HM): Aβ was injected into the bilateral hippocampus 1-42 C57BL / 6 mice were given 0.1 mL / 10 g turmeric solution by gavage at a dose of 1 g (raw drug) / kg; Herbal residues (HR): Aβ was injected into the bilateral hippocampus 1-42 C57BL / 6 mice were given a dose of 1.2 g (raw drug) / kg by intragastric administration of 0.1 mL / 10 g turmeric residue solution; Low dose of curcumin (L-CUR): Aβ was injected into the bilateral hippocampus 1-42 C57BL / 6 mice were given a dose of 10 mg / kg by gavage with 0.1 mL / 10 g of low-dose curcumin solution; High dose of curcumin (H-CUR): Aβ was injected into the bilateral hippocampus 1-42 C57BL / 6 mice were given a dose of 20 mg / kg by intragastric administration of 0.1 mL / 10 g of high-dose curcumin solution.

[0024] S2-3: Mouse brain localized injection to induce neuroinflammation Aβ 1-42 Solution treatment Aβ 1-42 Dissolve in sterile saline to prepare an Aβ1-42 injection solution with a concentration of 410 pmol / μL. Place the prepared solution in a 37°C constant temperature incubator and incubate for 7 days before use. After 7 days of adaptive feeding, the mice were randomly divided into 7 groups. The mice were fasted for 12 h before modeling (drinking was not prohibited during this period). When modeling, 1% sodium pentobarbital solution (0.08 mL / 10 g) was first injected intraperitoneally for anesthesia. There was no obvious reaction when pinching the tail and toes of the mice lightly, indicating that the mice were in an anesthetized state. The mice were fixed on the brain positioning instrument and the hair on the top of the head was removed with a shaver. Erythromycin was thickly applied to the eyes of the mice to prevent strong light from damaging the eyeballs. After disinfection with iodine tincture, the skin was cut longitudinally to expose the skull. Combined with the mouse brain atlas (such as Figure 3 The injection site was selected as the bilateral hippocampal CA1 region (coordinates: AP: -0.22 cm, ML: ±0.2 cm, DV: -0.2 cm). A microsyringe was used to draw Aβ 1-42 Solution, Aβ in one side of the brain1-42 The solution volume was 1.5 μL, and the time was 10 min. The needle was left in place for 15 min, and the needle was slowly withdrawn for 5 min. The wound was sutured and disinfected. The same operation was performed on the other side of the brain. The sham operation group was injected with an equal amount of sterile saline.

[0025] On the eighth day of the experiment, according to the mouse brain atlas, brain localization injection was performed on each group of mice. The injection site was selected as the CA1 region of the bilateral hippocampus (coordinates: AP: -0.22 cm, ML: ±0.2 cm, DV: -0.2 cm). A microsyringe was used to draw Aβ 1-42 Solution, Aβ in one side of the brain 1-42 The solution volume was 1.5 μL, and the time was 10 min. The needle was left in place for 15 min, and the needle was slowly withdrawn for 5 min. The wound was sutured and disinfected, and the same operation was performed on the other side of the brain. The sham operation group was injected with an equal amount of sterile saline, and then the mice were allowed to recover naturally for 2 days; S2-4: Grouping and dosing of mice after brain localization injection Starting from the 10th day of the experiment, each group of mice was again given the grouping and dosing method and dosage of step S2-2 by gavage and continued for three weeks; the overall process is shown in Figure 2.

[0026] Sham operation group: C57BL / 6 mice injected with sterile saline in the bilateral hippocampus and gavaged with 0.1 mL / g distilled water; Model group: Aβ was injected into the bilateral hippocampus 1-42 C57BL / 6 mice were gavaged with 0.1 mL / 10 g distilled water; Positive drug group: Aβ was injected into the bilateral hippocampus 1-42 C57BL / 6 mice were given 0.76 mg / kg by gavage with 0.1 mL / 10 g donepezil hydrochloride solution; Medicinal material group: Aβ was injected into the bilateral hippocampus 1-42 C57BL / 6 mice were given 0.1 mL / 10 g turmeric solution by gavage at a dose of 1 g (raw drug) / kg; Drug residue group: Aβ was injected into the bilateral hippocampus 1-42 C57BL / 6 mice were given a dose of 1.2 g (raw drug) / kg by intragastric administration of 0.1 mL / 10 g turmeric residue solution; Low-dose group: Aβ was injected into the bilateral hippocampus 1-42 C57BL / 6 mice were given a dose of 10 mg / kg by gavage with 0.1 mL / 10 g of low-dose curcumin solution; High-dose group: Aβ was injected into the bilateral hippocampus1-42 C57BL / 6 mice were given a dose of 20 mg / kg by intragastric administration of 0.1 mL / 10 g of high-dose curcumin solution.

[0027] Then, animal behavioral experiments were conducted on each group of mice, and the experimental results were analyzed to study whether the turmeric medicinal materials, turmeric residues and curcumin in the model group can reduce neuroinflammation in the mouse brain.

[0028] The animal behavior experiment of step S2-5 is specifically a Morris water maze experiment, including a positioning navigation experiment and a space exploration experiment; the water maze experiment is performed on the 25th day of the experiment after administration, wherein the positioning navigation experiment is performed on the 25th to 29th day of the experiment, and the space exploration experiment is performed on the 30th day of the experiment, and specifically includes the following steps: S2-5-1: Positioning and navigation experiment The mice were placed in the water from the midpoint of the pool wall in four different quadrants, and the software counted down for 90 seconds. If the time from entering the water to successfully finding the water platform and staying there for more than 10 seconds was recorded as the escape latency; if the water platform could not be found within 90 seconds, its escape latency was 90 seconds. After 90 seconds, the experimenter guided the mice to find the water platform and let them stay on the platform for 10 seconds. The escape latency of the mice was recorded for five days. The experimental results of the escape latency in the positioning navigation experiment are shown in Figure 4 (A); S2-5-2: Space Exploration Experiment On the 30th day of the experiment, the platform was removed and the mice were placed in the water from the diagonal quadrant of the platform. The swimming route of the mice within 90 seconds was measured, and the number of times they crossed the platform and the time they stayed in the quadrant where the water platform was placed were recorded. The experimental results of the number of times they crossed the platform in the spatial exploration experiment are shown in Figure 4 (B), the experimental results of the time they stayed in the target quadrant in the spatial exploration experiment are shown in Figure 4 (C), and the experimental results of the movement trajectory in the spatial exploration experiment are shown in Figure 4 (D). S2-5-3: Analyze the experimental results The results of Morris water maze test showed that injection of Aβ 1-42 It can cause mice to suffer from neuroinflammation in the brain, prolong the escape latency, reduce the number of platform crossings and the time spent in the target quadrant, while the drug-treated model group can shorten the escape latency, increase the number of platform crossings and the time spent in the target quadrant, indicating that turmeric, turmeric residues and curcumin can all reduce neuroinflammation in the mouse brain.

[0029] The experimental results were analyzed in Figures 4 (A) to 4 (D), to determine whether the turmeric medicinal materials, turmeric dregs and curcumin in the turmeric medicinal materials group, turmeric dregs group, low-dose curcumin group and high-dose curcumin group could respectively reduce neuroinflammation in the mouse brain to different degrees; in the figure, ##: p < 0.01, compared with the sham operation group; *: p < 0.05, **: p < 0.01, compared with the model group.

[0030] In this example, behavioral tests were performed on each group of mice to analyze the experimental drugs, including yellow medicinal materials, turmeric residues, and curcumin, used in each drug administration group in the animal model, and all of them had the effect of alleviating neuroinflammation in the mouse brain.

[0031] Example 2 See attached figure 4- Fig. 20 In this embodiment, based on the biological samples obtained in step S2, each group of mice was prepared as biological samples, and non-targeted metabolomics technology was further used to explore the mechanism and effect of turmeric residues and curcumin in intervening in mice with neuroinflammation, and to analyze whether turmeric residues and curcumin can improve Aβ by regulating the TLR4 / NF-κB pathway. 1-42 The invention can induce cognitive impairment in mice with neuroinflammation, reduce nerve damage, regulate over-activated microglia, down-regulate inflammatory factors and up-regulate anti-inflammatory factors, and achieve the effect of reducing neuroinflammation. For comparison, this embodiment also conducts comparative tests on the mechanism and effect of turmeric medicinal materials and turmeric residues in intervening neuroinflammation in mice.

[0032] The method for constructing an animal model for neuroinflammation intervention provided in the embodiment of the present invention is a further specific application based on the aforementioned basic embodiment and embodiment 1. Step S3 analyzes the mechanism of turmeric intervention in mouse neuroinflammation, and finds out how turmeric, an active ingredient in turmeric medicinal materials and turmeric dregs, can reduce Aβ 1-42 The mechanism of action of inducing neuroinflammation includes the following steps: S3-1 Analysis of Morris water maze test data to observe the effect of turmeric on Aβ 1-42 Effects of lesions on spatial learning and memory in mice First study to investigate the effect of turmeric on Aβ 1-42 Effects of lesion on spatial learning and memory in mice.

[0033] According to the results of the Morris water maze experiment in Example 1, the changes in the spatial memory and learning ability of the animals were observed. The Morris water maze positioning navigation experiment data (see Table 6-1, Changes in escape latency in the positioning navigation experiment) (x̅ ± s,n=8)), compared with the sham-operated mice, the latency of the model group mice to find the platform was significantly longer (p < 0.01); compared with the model group, with the increase in training days, the escape latency of the mice in the positive drug group, medicinal material group, low-dose group and high-dose group was significantly shortened (all p < 0.01) (as shown in Figure 4 (A)); starting from the fourth day of the experiment, the latency of the mice in the drug residue group to find the platform was significantly shortened (p < 0.01) (see Table 6-1). According to the spatial exploration experimental data (Table 6-1, Figure 4 (BD)), compared with the sham-operated mice, the number of times the model group mice crossed the platform and the time they stayed in the target quadrant were significantly reduced (both p < 0.01); compared with the model group, the number of times the mice in the drug residue group crossed the platform showed an upward trend but was not significant (p > 0.05), and the number of times the mice in the other drug-treated groups crossed the platform and the time they stayed in the target quadrant were significantly increased (all p < 0.05). In summary, turmeric can improve Aβ 1-42 Impaired spatial learning and memory abilities of mice. In Figures 4(A)-4(D), ##：p＜0.01 , compared with the sham operation group; *：p＜0.05 , **：p＜ 0.01 , compared with the model group.

[0034] Table 6-1

[0035] Note: ##: p<0.01, compared with the sham operation group; **: p<0.01, compared with the model group S3-2: Perform histopathological examination Hematoxylin-eosin staining (HE) and Nissl staining were used to observe the pathological changes of neurons in the hippocampus.

[0036] Immunofluorescence staining (IF) was used to observe the expression of Aβ1-42 and Iba-1 (Ionized calcium binding adaptor molecule 1) in the mouse brain, and immunohistochemistry (IHC) was used to observe the changes of Iba-1 in the hippocampus CA1 region (Hippocampus region 1). PCR (Polymerase chain reaction) was used to quantitatively determine the mRNA expression of important channel proteins and cytokines in the TLR4 / NF-κB signaling pathway. Enzyme linked immunosorbent assay (ELISA) was used to determine the contents of important channel proteins and cytokines in the TLR4 / NF-κB signaling pathway.

[0037] The experimental mice were subjected to HE staining analysis, Nissl staining, immunohistochemistry, immunofluorescence staining, real-time fluorescence quantitative PCR, and ELISA analysis. The experimental results showed that the turmeric medicinal materials, turmeric dregs, and curcumin in the turmeric medicinal materials group, turmeric dregs group, low-dose curcumin group, and high-dose curcumin group could reduce neuroinflammation in the mouse brain to varying degrees. The experimental results showed that the turmeric medicinal materials, turmeric dregs, and curcumin in the turmeric medicinal materials group, turmeric dregs group, low-dose curcumin group, and high-dose curcumin group could reduce neuroinflammation in the mouse brain to varying degrees.

[0038] S3-2-1: HE staining (1) Collection of materials The whole brain of the mouse was removed and placed in tissue fixative. The blood was stored at 4°C, allowed to stand for 2 h, centrifuged for 20 min (3,000 × g, 4°C), the supernatant was collected, aliquoted and frozen at -80°C; (2) Slicing After the tissue was fixed for 24 h, it was dehydrated with gradient alcohol (70% ethanol-80% ethanol-95% ethanol-absolute ethanol), transparentized with xylene, and embedded in paraffin. The wax block was placed on a microtome and cut into 6 μm slices. The slices were unfolded in warm water, transferred to a slide, and dried at 70°C. (3) Dewaxing The sections were sequentially immersed in xylene for 20 min, repeated twice, immersed in anhydrous ethanol for 5 min, repeated twice, immersed in 75% ethanol for 5 min, and rinsed with tap water. After dewaxing, the sections were stained differently; (4) Dyeing The operation was performed according to the instructions, and the tissue pathological changes were observed under an optical microscope after sealing.

[0039] S3-2-2: Nissl staining The same steps as (1) to (3) in S3-2-1 were used to process the samples; after adding toluidine blue dye for 5 min, the samples were washed with tap water, differentiated with 0.1% glacial acetic acid, rinsed with tap water until the reaction was terminated, the degree of differentiation was controlled under a microscope, and the samples were placed in an oven for drying. The slices were treated with diphenylmethane for 10 min for transparency, and sealed with neutral gum.

[0040] S3-2-3 Immunohistochemical analysis The specific steps of sample processing are the same as (1) to (3) in S3-2-1.

[0041] (4) Antigen repair: Place the slice rack in a beaker containing antigen repair solution, put it in a pressure cooker, and continue heating for 15 minutes. Do not let the slices dry. Cool naturally to room temperature, take out the slice rack and soak it in PBS (pH 7.4) 3 times, 5 minutes each time; (5) Block endogenous peroxidase: Place the slides in a 3% hydrogen peroxide solution and incubate at room temperature for 25 min. Keep away from light. Place the slides in PBS (pH 7.4) and wash on a shaker three times, 5 min each time. (6) Serum blocking: Add 3% BSA to the tissue circle to cover the tissue and block for 30 min; (7) Add primary antibody: shake off the blocking solution, add primary antibody Iba-1 (1:1000) on the slice, and incubate at 4°C; (8) Add secondary antibody: Place the slide in PBS (pH 7.4) for decolorization and wash on a shaker three times, 5 min each time. Shake the slide dry, add goat anti-rabbit for labeling, and incubate at room temperature for 50 min. (9) Color development: Place the slide in PBS (pH 7.4) for decolorization, and wash on a shaker for 3 times, 5 min each time. Shake the slides to dry, add DAB color development solution, control the color development time under a microscope, and rinse with tap water until the color development stops; (10) Counterstaining of cell nuclei: counterstain with hematoxylin for 3 min, rinse with tap water, differentiate with hematoxylin for a few seconds, rinse with tap water, reverse blue with hematoxylin bluing solution, and rinse with tap water; (11) Dehydration and sealing: Dehydrate the slices in gradient alcohol (75% ethanol-85% ethanol-absolute ethanol twice) for 5 min each, place in xylene for 5 min, take out and dry, and seal the slices; (12) Image J was used to quantitatively analyze the Iba-1 positive area and positive cell density.

[0042] S3-2-4 Immunofluorescence staining The specific steps of sample processing are the same as (1) to (6) in S3-2-3; (7) Add the first primary antibody: remove the blocking solution, add the prepared Iba-1 (1:1000), and incubate in a humidified box at 4°C overnight; (8) Adding secondary antibody: the steps are the same as those in S3-2-3 (8); (9) Add corresponding TSA: Place the slide in PBS (pH 7.4) and wash on a decolorizing shaker 3 times, 5 min each time. Add the corresponding type of TSA, incubate in the dark for 10 min, place in TBST and wash on a decolorizing shaker 3 times, 5 min each time; (10) Heating: Place the slices in a repair box and heat in a microwave oven, medium heat for 8 min, stop for 8 min, and low heat for 7 min; (11) Add the second primary antibody: Add Aβ 1-42 Antibody (1:200), incubated at 4°C in a humidified chamber, overnight; (12) Adding secondary antibody: same as above step (8); (13) Add the corresponding TSA: same as the above step (9); (14) Heating: same as the above step (10); (15) DAPI counterstaining: DAPI staining solution was added to the circle to counterstain the cell nucleus, and incubated at room temperature in the dark for 10 min. (16) Quenching tissue autofluorescence: Place the slide in PBS (pH 7.4) and wash on a decolorizing shaker three times, 5 min each time. Add autofluorescence quencher for 5 min and rinse with running water for 10 min. (17) Sealing: Sealing with anti-fluorescence quenching tablets; (18) Take photos under a microscope.

[0043] S3-2-5: Use PCR fluorescence quantitative determination to measure the mRNA expression of important channel proteins and cytokines in the TLR4 / NF-κB signaling pathway; perform real-time fluorescence quantitative PCR, including the following steps: (1) Extraction of RNA Take about 50 mg of brain tissue in a centrifuge tube, add steel beads, add 1 mL of RNA isolater solution, and grind it in a homogenizer until it becomes powder. Add 200 μL of chloroform to the above lysate, shake vigorously to form an emulsion, and centrifuge for 15 min (12,000 × g, 4°C). Take 400 μL of the upper clear layer, add an equal volume of pre-cooled isopropanol, mix well, and centrifuge for 10 min (12,000 × g, 4°C). Discard the supernatant, add 1 mL of 75% ethanol (prepared with DEPC water), blow the bottom gently with a gun tip to suspend the precipitated white precipitate, centrifuge for 5 min (12,000 × g, 4°C), and repeat twice. Open the centrifuge tube for 5 min to let the white precipitate dry, add 20 μL of sterile enzyme-free water to dissolve the precipitate. Take 1.5 μL of the sample solution and test the purity and concentration using an ultra-micro UV analyzer. Purity An OD260 / OD280 ratio between 1.9-2.1 indicates high RNA purity.

[0044] (2) Reverse transcription Using the above RNA as template, prepare the reverse transcription reaction system according to the instructions to synthesize the cDNA template.

[0045] (3) PCR fluorescence quantification The reaction conditions were as follows: pre-denaturation: 95℃ 30 sec; cycle reaction: 95℃ 10 sec, 60℃ 30 sec, 40 cycles; melting curve: 65℃ 5 sec, 95℃ 5 sec, 40 cycles. Each sample was repeated three times and quantified using a fluorescent quantitative PCR instrument.

[0046] S3-2-6: Enzyme linked immunosorbent assay (ELISA) was used to determine the contents of important channel proteins and cytokines in the TLR4 / NF-κB signaling pathway.

[0047] Take an appropriate amount of brain tissue, add a certain amount of PBS (pH 7.4), homogenize, centrifuge for 10 min (4°C, 5,000 g), collect the supernatant, divide it into portions, one portion is the sample to be tested, and the rest is stored at -80°C. Operate according to the instructions; adjust the blank well to zero, and measure the absorbance of each well at a wavelength of 450 nm.

[0048] S3-2-7 Statistical processing of experimental data GraphPad Prism 9.4 software was used for analysis, and all data were expressed as mean ± standard deviation (x̅ ± s). Two-way AVOVA was used for the escape latency in the Morris water maze test, and one-way ANOVA was used for the analysis between other experimental groups. Turkey's method was used for multiple comparisons. p < 0.05 was considered to be a significant difference between groups.

[0049] S3-3 Non-targeted metabolomics study A non-targeted metabolomics study of turmeric and its residues on AD mice was conducted. Plasma and brain tissue samples of each experimental mouse were prepared, and then detected by chromatography and mass spectrometry. Then, the non-targeted serum metabolomics technology was used to analyze the non-targeted metabolomics raw data of serum samples of each group of rats.

[0050] S3-3-1 Preparation of plasma and brain tissue samples (1) Treatment of plasma samples: Take each plasma sample out of the -80℃ refrigerator according to the number, thaw on ice until it is fully liquid, vortex mix for 30 s, and at 4℃, pipette 40 μL into a 1.5 mL EP tube; add 200 μL of precooled (-30℃) precipitant (acetonitrile: methanol = 1:1), vortex shake for 5 min, and place in a -30℃ refrigerator for 20 min; centrifuge at 13,000 rpm for 10 min at 4℃, and transfer 150 μL of the supernatant to the inner liner of the corresponding injection bottle for analysis on the machine.

[0051] (2) Brain tissue sample processing method: After the brain tissue sample is taken out of the -80℃ refrigerator and thawed on ice, 50 mg of tissue is accurately weighed with a 1 / 10,000 balance and placed in a homogenization tube according to the number. Pre-cooled (-30℃) extraction solution (methanol: acetonitrile: water = 2:2:1) and 8mm magnetic beads are added to the homogenization tube at a volume ratio of 1:10 (v / v). Homogenize in a frozen homogenizer for 5 minutes, then centrifuge at 4℃, 13000 rpm for 10 minutes. After centrifugation, take the supernatant and put it into another set of 5 mL EP tubes with the corresponding number, add the extraction solution again for secondary extraction, centrifuge at 4℃, 13000 rpm for 10 minutes, and combine the two supernatants. The supernatant is washed with N 2 After drying, add the reconstitution solution (80% methanol solution) at a volume ratio of 5:1 (mg / v), place in a -30 ℃ refrigerator for 30 min, vortex for 5 min, centrifuge at 13000 rpm for 10 min at 4 ℃, transfer 180 μL of the supernatant to the inner liner of the corresponding numbered injection bottle for analysis on the machine (the entire preparation process is performed on ice).

[0052] (3) QC sample preparation: 10 μL of supernatant was taken from each sample of plasma and brain tissue, mixed, and vortexed for 30 s to prepare quality control samples (QC).

[0053] S3-3-2 Chromatographic and mass spectrometric conditions for plasma and brain tissue (1) Plasma chromatographic conditions: Ultimate 3000 chromatography system, using Waters ACQUITY UPLC HSS T3C18 (2.1 mm*100 mm, 1.8 μm) chromatographic column, autosampler temperature of 8 °C, flow rate of 0.3 mL / min, column temperature of 40 °C, injection of 1 μL for gradient elution, mobile phase of 0.1% formic acid in water (A) - 0.1% formic acid in acetonitrile (D).

[0054] (2) Chromatographic conditions for brain tissue: Chromatographic system Ultimate 3000, using Waters ACQUITY UPLC HSST3 C18 (2.1 mm*100 mm, 1.8 μm) chromatographic column, autosampler temperature set to 8 °C, flow rate 0.3 mL / min, column temperature 40 °C, injection of 2 μL for gradient elution, mobile phase 2 MM ammonium formate-0.1% formic acid water (A) - acetonitrile (D).

[0055] (3) Mass spectrometry conditions for plasma and brain tissue: mass spectrometry system Thermo Q Exactive Focus, ionization mode: ESI- / ESI+.

[0056] S3-3-3 Targeted determination of characteristic biomarkers - amino acids The non-targeted serum metabolomics technology was used to analyze the raw data of non-targeted metabolomics of serum samples of each group of rats. The non-targeted metabolomics raw data were processed and analyzed using Progenesis QI 3.0 and Compound Discoverer 3.3 software, and multivariate statistical analysis and differential metabolite identification were performed. The differential metabolites were screened out by comprehensively comparing VIP (Variable importance in the projection) > 1, Max fold change > 2, and ANOVA P-value < 0.05. The chemical structures of the differential metabolites were identified by comparing with the HMDB database, m / z Cloud database, PubChem database, etc. Finally, the differential metabolites were imported into MetaboAnalyst 5.0 online analysis software for pathway enrichment, and the data of targeted determination of characteristic biomarkers-amino acids were obtained after calculation.

[0057] The research results of this embodiment include: 1. Effect of turmeric on Aβ 1-42 Induced pathological changes and neuronal damage (1) HE staining analysis HE staining used in this example is one of the commonly used staining methods in histology, which provides the main information such as the morphology, structure and distribution of cells in tissue samples. As shown by the black arrow in Figure 5 (1), the neurons in the CA1 region of the hippocampus of the sham-operated mice were neatly and tightly arranged, with clear cell membranes and nuclei; however, the hippocampus of the model-operated mice had interstitial edema, cell deformation and deterioration, and the normal morphology and arrangement of neurons were damaged by Aβ. 1-42 The cell nucleus became darker (as shown by the white arrow in Figure 5 (1)). Compared with the model group, the pathological changes in the mice in the drug-treated group were improved, and most of the nerve cells were neatly arranged, with normal cell shape and morphology restored and clear cell nuclei. In summary, turmeric can reduce the Aβ 1-42 Induced pathological changes.

[0058] (2) Nissl staining analysis Nissl bodies are subcellular structures found in the cell bodies and dendrites of neurons. They are composed of granular endoplasmic reticulum and ribosomes. The reticular structure is distributed throughout the entire neuron, but is denser in the Nissl body region than in the rest of the cell. Nissl bodies are relatively dispersed in neurons. They are loosely in contact with the outer edge of the endoplasmic reticulum or arranged in the endoplasmic reticulum. The main function of Nissl bodies is to synthesize proteins, update the protein structure required for organelles, and synthesize enzymes and neurotransmitters required for neurotransmitters. As the functional state of motor neurons changes, its number, size and arrangement change accordingly. These small particles can be used to evaluate the state of neurons in different metabolic states or disease states of neurons. Nissl staining showed that the neurons in the CA1 region of the hippocampus of sham-operated mice were neatly arranged and dense, with obvious nucleoli between the cytoplasm and abundant Nissl bodies in the cytoplasm (as shown by the black arrows in Figure 6 (1)). Compared with the sham-operated mice, the cells in the model group were loosely arranged, with enlarged gaps, incomplete cell structure, missing nucleoli in the cytoplasm, darkly stained cytoplasm, and reduced or disappeared Nissl bodies (as shown by the red arrows in Figure 6 (1)). After medication, the integrity of the cell structure improved, the nucleoli were obvious and clearly visible, and the Nissl bodies increased. In summary, the experimental results show that turmeric can reduce Aβ 1-42 Resulting in neuronal damage and loss.

[0059] 2. Effect of Turmeric on Aβ 1-42 Effects of inducing an overactivated microglial state Iba-1 protein is a member of the calcium-binding protein group and is considered a marker of microglia. Microglia activate Iba-1 protein production under normal physiological or pathological conditions.1-42 Perform immunofluorescence co-labeling (eg Figure 7 ), the results showed that Aβ 1-42 After injection, Iba-1 expression in the mouse brain was enhanced and accompanied by Aβ 1-42 Under normal conditions, microglia in the sham-operated group produced little or no Iba-1 (as shown by the black arrow in Figure 8 (A-1)). As shown in Figures 8 (A) to 8 (C), compared with the sham-operated group, the expression of Iba-1 protein in the CA1 region of the hippocampus of the model group mice increased significantly (as shown by the red arrow in Figure 8 (A-1)). p <0.01), indicating that injection of Aβ 1-42 Stimulate microglia to transform into a hyperactivated state; compared with the model group, the expression of Iba-1 protein in mice in each drug-treated group was significantly decreased (all P< 0.05). p <0.01). It can be concluded that turmeric can reduce Aβ 1-42 Induced Iba-1 proliferation, thereby regulating the overactivated state of microglia.

[0060] 3. Effect of turmeric on TLR4 / NF-κB signaling pathway (1) Effect of turmeric on mRNA expression of related proteins in the TLR4 / NF-κB signaling pathway The results are shown in Figure 9 , in which turmeric regulates the mRNA levels of important channel proteins in the TLR4 / NF-κB pathway ( x̅ ± s , n =3). Figure 9 (A) shows the mRNA expression level of IκB; Figure 9 (B) shows the mRNA expression level of p65; Figure 9 (C) shows the mRNA expression level of TLR4; In the figure, ##: p <0.01, compared with the sham operation group; *: p <0.05, **: p<0.01, compared with the model group.

[0061] It can be seen that compared with the sham operation group, the mRNA expressions of IκB, p65 and TLR4 in the brain tissue of the model group mice were significantly increased (all p <0.01); compared with the model group, the mRNA levels of p65 and TLR4 in the mice in the drug-treated group were significantly decreased (both p <0.01); compared with the model group, the mRNA level of IκB in mice after administration showed a downward trend, but there was no significant difference ( p >0.05). The above results indicate that turmeric can regulate Aβ 1-42 Induced mRNA expression levels of important channel proteins.

[0062] In addition to studying the effect of turmeric on the mRNA expression level of important proteins, the present invention also measured the mRNA levels of inflammatory factors and anti-inflammatory factors. Figure 10 (AH) shows the regulation of turmeric on the mRNA levels of cytokines in the TLR4 / NF-κB pathway ( x̅ ± s , n =3); In each sub-figure of FIG10 , FIG10 (A) is the mRNA expression level of IL-6; FIG10 (B) is the mRNA expression level of TNF-α; FIG10 (C) is the mRNA expression level of iNOS; FIG10 (D) is the mRNA expression level of COX-2; FIG10 (E) is the mRNA expression level of IL-4; FIG10 (F) is the mRNA expression level of IL-10; FIG10 (G) is the mRNA expression level of TGF-1β; FIG10 (H) is the mRNA expression level of Arg-1; In the figure, ##: p <0.01, compared with the sham operation group; *: p <0.05, **: p<0.01, compared with the model group.

[0063] As shown in Figure 10 (A) to Figure 10 (D), compared with the sham operation group, the Aβ injection 1-42 The mRNA levels of IL-6, TNF-α, iNOS and COX-2 in the mouse brain increased significantly (all p <0.01); the drug group can effectively inhibit the localized injection of Aβ in the brain 1-42 The mRNA expressions of IL-6, TNF-α, iNOS and COX-2 (all p <0.01).

[0064] As shown in Figure 10 (E) to Figure 10 (H), compared with the sham operation group, the mRNA levels of anti-inflammatory factors IL-4, IL-10, TGF-1β and Arg-1 in the model group were significantly decreased (all P<0.05). p <0.01); compared with the model group, the mRNA expression levels of IL-4, IL-10, TGF-1β and Arg-1 in the positive drug group and the medicinal material group were significantly increased (all p <0.01); the mRNA expression levels of IL-4, TGF-1β and Arg-1 in the mice of the drug residue group increased (all p <0.05), but there was no significant difference in the mRNA expression level of IL-10 between the model group and the model group ( p >0.05); compared with the model group, the low-dose curcumin group could significantly increase the mRNA expression level of IL-10 ( p <0.01), and had no significant effect on the mRNA expression levels of other anti-inflammatory factors ( p>0.05), while the mRNA expression of IL-4 and IL-10 in the high-dose curcumin group was significantly increased (both p <0.05), had no significant effect on TGF-1β and Arg-1 ( p >0.05). The above results indicate that turmeric can regulate Aβ 1-42 Induced mRNA expression levels of inflammatory and anti-inflammatory factors.

[0065] (2) Effect of turmeric on the expression of related proteins in the TLR4 / NF-κB signaling pathway Effect of turmeric on the content of important channel proteins in the TLR4 / NF-κB pathway ( x̅± s,n=3 ) The results are shown in Figure 11. In each sub-figure of Figure 11, Figure 11 (A) shows the relative expression level of pIKK / IKK protein; Figure 11 (B) shows the relative expression level of p-IκB / IκB protein; Figure 11 (C) shows the relative expression level of p-p65 / p65 protein; Figure 11 (D) shows the protein content of TLR4; in the figure, ##: p < 0.01, compared with the sham operation group; *: p < 0.05, **: p < 0.01, compared with the model group.

[0066] Compared with the sham operation group, the protein levels of p-IKK / IKK, p-IκB / IκB, p-p65 / p65 and TLR4 in the brain tissue of the mice in the model group were significantly increased (all P<0.05). p <0.01); compared with the model group, the protein levels of p-IKK / IKK, p-IκB / IκB, p-p65 / p65 and TLR4 in the positive drug group were significantly decreased (all p <0.05); Compared with the model group, the protein levels of p-IκB / IκB, p-p65 / p65 and TLR4 in mice were significantly decreased after taking turmeric medicinal materials, turmeric dregs and low-dose and high-dose curcumin ( p <0.01); high dose curcumin can significantly reduce the p-IKK / IKK protein content ( p <0.01), turmeric medicinal materials, turmeric residues and low doses of curcumin had no significant effect on the expression of this protein ( p >0.05). The above results indicate that turmeric can regulate Aβ 1-42 Induced expression levels of important channel proteins.

[0067] In addition to studying the effect of turmeric on the expression level of important channel proteins, the present invention also measured the protein expression levels of inflammatory factors and anti-inflammatory factors in plasma, and the results are shown in Figure 12 (AH). Figure 12 (A)-Figure 12 (H) show that turmeric regulates the expression level of cytokine proteins in the TLR4 / NF-κB pathway ( x̅ ±s , n =3). In each sub-figure of FIG12 , FIG12 (A) is the protein expression level of IL-6; FIG12 (B) is the protein expression level of TNF-α; FIG12 (C) is the protein expression level of iNOS; FIG12 (D) is the protein expression level of COX-2; FIG12 (E) is the protein expression level of IL-4; FIG12 (F) is the protein expression level of IL-10; Figure 12G is the protein expression level of TGF-1β; FIG12 (H) is the protein expression level of Arg-1; in the figure, ##: p <0.01, compared with the sham operation group; *: p <0.05, **: p<0.01, compared with the model group.

[0068] Compared with the sham operation group, the Aβ 1-42 The mRNA levels of IL-6, TNF-α, iNOS and COX-2 in the mouse brain increased significantly (all p <0.01); positive drug group, medicinal material group and high-dose curcumin group can effectively inhibit the localized injection of Aβ in the brain 1-42 The protein expression levels of IL-6, TNF-α, iNOS and COX-2 increased (all p <0.01), the low-dose group effectively reduced the protein expression levels of IL-6, TNF-α and COX-2 (all p <0.01), the drug residue group had no significant effect on the expression level of anti-inflammatory factor protein (all p >0.05).

[0069] As shown in Figure 12 (EH), compared with the sham operation group, the protein expression levels of anti-inflammatory factors IL-4, IL-10 and TGF-1β in the model group mice were significantly decreased (all P<0.05). p <0.01), and the expression level of Arg-1 protein did not change significantly ( p >0.05); compared with the model group, the protein expression levels of IL-4, IL-10, and TGF-1β in the positive drug group, low-dose curcumin, and high-dose curcumin groups were significantly increased (all p <0.05), the protein expression levels of IL-4 and TGF-1β in the mice in the medicinal material group and the medicinal residue group were significantly increased (all p <0.05), but there was no significant difference in the protein expression levels of IL-10 and Arg-1 between the model group and the control group ( p >0.05). The above results indicate that turmeric can regulate Aβ 1-42 Induced inflammatory and anti-inflammatory factor protein expression levels.

[0070] The invention discloses that turmeric reduces Aβ by regulating the TLR4 / NF-κB signaling pathway. 1-42 Biological mechanisms that induce neuroinflammation are described in Fig.13 .

[0071] (IV) Results of non-targeted metabolomics study of turmeric and its residues on AD mice 1. Metabolic profiles of plasma and brain tissue and their quality control evaluation The superimposed total ion current diagrams of the quality control samples in the positive and negative ion modes of plasma and brain tissue are shown in Figure 14 (including sub-diagrams AD). In the total ion current diagrams of Figure 14, Figure 14 (A) is the total ion current diagram of plasma in positive ion mode; Figure 14 (B) is the total ion current diagram of plasma in negative ion mode; Figure 14 (C) is the total ion current diagram of brain tissue in positive ion mode; Figure 14 (D) is the total ion current diagram of brain tissue in negative ion mode.

[0072] The results showed that the repeatability of the quality control samples was good and the stability of the entire detection process was good. In addition, 5 representative endogenous metabolites were selected for stability testing in plasma and brain tissue in positive and negative ion modes, respectively. The results are shown in Table 7-1 (Stability results of representative endogenous metabolites in plasma) and Table 7-2 (Stability results of representative endogenous metabolites in brain tissue). The results showed that the RSD of the peak area of ​​the 6 components was less than 30%, and the data quality was reliable and could be used for subsequent statistical analysis.

[0073] Table 7-1

[0074] Table 7-2

[0075] 2. Multivariate statistical analysis results of plasma and brain tissue visualization (1) As shown in Figures 15 and 16, the PLS-DA images of plasma and brain tissue are shown respectively. FIG15 (including sub-figures AB) is a visualization result of the spatial distribution of plasma non-targeted metabolomics data. In each sub-figure, FIG15 (A) is a PLS-DA score graph in positive ion mode; FIG15 (B) is a PLS-DA score graph in negative ion mode. In the figure, C: sham operation group; M: model group; D: positive drug group; Figure 16 (including sub-figures AB) is the visualization result of the spatial distribution of non-targeted metabolomics data of brain tissue. In each sub-figure, Figure 16 (A) is the PLS-DA score graph in positive ion mode; Figure 16 (B) is the PLS-DA score graph in negative ion mode. C: sham operation group; M: model group; D: positive drug group; L: low-dose curcumin group; H: high-dose curcumin group; YZ: turmeric residue group.

[0076] (2) As shown in Figures 17 and 18, the OPLS-DA and VIP images of plasma and brain tissue are respectively.

[0077] Figure 17 (including sub-figures AD) is a visualization result of the spatial distribution of non-targeted metabolomics data of plasma in the sham operation group VS the model group of an embodiment of the present invention; wherein, Figure 17 (A) is a positive ion mode OPLS-DA score graph; Figure 17 (B) is a negative ion mode OPLS-DA score graph; Figure 17 (C) is a positive ion VIP graph; and Figure 17 (D) is a negative ion VIP graph.

[0078] Figure 18 (including sub-figures AD) is a visualization result of the spatial distribution of non-targeted metabolomics data of brain tissues of the sham operation group VS the model group in an embodiment of the present invention; wherein, Figure 18 (A) is a positive ion mode OPLS-DA score graph; Figure 18 (B) is a negative ion mode OPLS-DA score graph; Figure 18 (C) is a positive ion VIP graph; and Figure 18 (D) is a negative ion VIP graph.

[0079] 3. Screening and identification of differential metabolites in plasma and brain tissue In order to screen and identify endogenous metabolites related to the therapeutic effect of turmeric on AD mice, the OPLS-DA model of the sham operation group and the model group under positive and negative ion modes was used to find the characteristic differential metabolite groups that distinguish normal mice from AD mice, as shown in Figures 17 and 18 above. After OPLS-DA analysis, the importance of the variable to the classification is measured by the size of its VIP value, and the variables are screened according to the VIP value. Variables with VIP ≥ 1 and Max fold change > 2, ANOVA P-value <0.05, are considered to have significant contributions to the model.

[0080] A total of 218 (163 positive ions and 55 negative ions) differential metabolites were screened in plasma. The chemical structures of differential metabolites were compared and identified using CompoundDiscoverer 3.3 software with the HMDB database, m / z Cloud database, and ChemSpider database, and 24 (20 positive ions and 4 negative ions) characteristic differential metabolites were identified, as shown in Table 7-3.

[0081] Table 7-3

[0082] A total of 667 (428 positive ions and 239 negative ions) differential metabolites were screened in brain tissue. The chemical structures of differential metabolites were compared and identified using CompoundDiscoverer 3.3 software with HMDB database, m / z Cloud database and ChemSpider database, and 32 (32 positive ions and 12 negative ions) characteristic differential metabolites were identified, as shown in Table 7-4.

[0083] Table 7-4

[0084] 4. Analysis of metabolic pathways in plasma and brain tissue Metabolic pathways were analyzed based on the KEGG database. Metabolic pathways were selected as potential target pathways based on the criteria of P value less than 0.05 and impact value greater than 0. Three potential metabolic pathways were enriched in plasma, as shown in Table 7-5 (Plasma KEGG enriched metabolic pathway results). Fig.19 A total of 8 potential metabolic pathways were enriched in brain tissue, as shown in Table 7-6 (KEGG enriched metabolic pathway results of brain tissue), Fig. 20 shown.

[0085] Table 7-5

[0086] Table 7-6

[0087] The above experimental results showed that the arginine succinate in the model group mice showed a downward trend, which may be caused by abnormal glutamate metabolism, while the low-dose turmeric, turmeric residue group, and donepezil group did not show a correction trend, and the high-dose turmeric successfully corrected the trend. It is speculated that abnormal aspartate metabolism, glutamate metabolism, arginine biosynthesis pathway and Aβ 1-42 There are multiple complex regulatory mechanisms between mice with induced neuroinflammation. The results show that each group of turmeric can regulate amino acid metabolism to varying degrees, thereby achieving an improvement effect. Regulating amino acid metabolism may be a treatment for Aβ 1-42 Potential mechanisms of induced neuroinflammation.

[0088] The experiment in this example detected that adenine, cholinephosphate, adenosine, hypoxanthine, guanine, guanosine, uracil, thymine, dihydrothymine, and nicotinamide adenine dinucleotide (NAD+) showed different degrees of regulation trends. Compared with the model group, adenine and cholinephosphate showed a downward trend after turmeric intervention; uracil showed an upward trend after turmeric intervention; while adenosine, hypoxanthine, guanine, guanosine, thymine, dihydrothymine, and nicotinamide adenine dinucleotide (NAD+) did not show a significant regulatory trend. This suggests that in the same stage of experimental modeling and drug administration of mice, the use of complete brain tissue homogenate extraction and detection analysis method leads to Aβ 1-42 The regional dependence of nucleotide metabolism in the early stage of induced neuroinflammation has not been clearly elucidated, but after intervention with turmeric administration, adenine, acetylcholine phosphate cytidylyltransferase, and uracil were restored, indicating that turmeric may play a protective role in neuronal cells by regulating nucleotide metabolism.

[0089] Through experiments in the embodiments of the present invention, it can be observed that sphingosine, phosphoethanolamine, and palmitate tend to be upregulated in the model group, while metabolites such as fatty acids, linoleic acid, linolenic acid, etc. tend to be downregulated. Increased or accumulated intracellular lipid content (including sphingolipids) may lead to neurotoxicity. Increased levels of sphingosine and phosphoethanolamine indicate an increased metabolic rate of ceramide. Abnormal metabolism of sphingolipids may lead to lipid metabolism disorders and activate and aggravate inflammatory responses, thereby causing damage to lipid metabolism in nerve cells. After intervention with turmeric and its residues, the concentrations of sphingosine, phosphoethanolamine, and palmitate decreased, while the concentrations of their metabolites increased, suggesting that the mechanism may be that the efflux of ceramide and sphingomyelin reduces the lipid load, thereby playing a role in Aβ. 1-42 Neuroprotection in the brain of mice induced by neuroinflammation.

[0090] Therefore, the research results of the above embodiments all show that turmeric residues can also effectively reduce Aβ 1-42Induced neuroinflammation, specifically, the results of HE staining and Nissl staining experiments showed that each drug group could improve Aβ 1-42 The pathological changes caused by the model group were reversed, and the normal shape and morphology of the cells were restored. According to the IF results, compared with the sham operation group, there was less Aβ in the brain of the mice in the model group. 1-42 A small amount of expression and increased expression of Iba-1; according to IHC results, injection of Aβ 1-42 The results of PCR fluorescence quantitative analysis showed that the mRNA expression levels of important channel proteins IκB (Inhibitorkappa B), p65 and TLR4 in the brain of mice in the model group were significantly increased (all P<0.05). p <0.01), the mRNA expression levels of inflammatory factors IL-6 (Interleukin 6), TNF-α (Tumor necrosis factoralphabet), iNOS (Inducible nitric oxide synthase) and COX-2 (Cyclooxygenase 2) were significantly increased (all p <0.01), the mRNA levels of anti-inflammatory factors IL-4 (Interleukin 4, IL-4), IL-10 (Interleukin 10), TGF-1β (Transforming growth factor 1 beta) and Arg-1 (Arginase 1) were significantly decreased (all p <0.01, the drug-treated group could down-regulate the mRNA expression levels of important channel proteins and inflammatory factors and up-regulate the mRNA expression levels of anti-inflammatory factors to a certain extent; the results of ELISA determination of protein content showed that the protein levels of p-p65 / p65 (Phosphorylated NF-κB p65 protein / NF-κB p65 protein), phosphorylated IκB / IκB (Phosphorylated inhibitor kappa B / Inhibitor kappa B), phosphorylated IKK / IKK (Phosphorylated inhibitor of kappa B kinase / Inhibitor of kappa Bkinase) and TLR4 in the model group mice were significantly increased (all P< 0.01). p <0.01), the protein levels of inflammatory factors IL-6, TNF-α, iNOS and COX-2 increased significantly (all p<0.01), while the protein contents of anti-inflammatory factors IL-4, IL-10 and TGF-1β decreased significantly (all p <0.01), the drug-treated groups could down-regulate the protein expression levels of important channel proteins and inflammatory factors and up-regulate the protein expression levels of anti-inflammatory factors to a certain extent.

[0091] In summary, this example uses non-targeted metabolomics research on AD mice using turmeric residues and finds that turmeric and curcumin reduce Aβ by regulating the TLR4 / NF-κB pathway. 1-42 The potential mechanism may be to regulate the over-activation state of microglia, regulate important proteins in the TLR4 / NF-κB pathway, down-regulate inflammatory factors, and up-regulate anti-inflammatory factors, thereby alleviating neuroinflammation in the brain; through the non-targeted metabolomics comparative study of turmeric medicinal materials and turmeric dregs on AD mice, it was found that turmeric dregs can also reduce Aβ to a certain extent. 1-42 The induced neuroinflammation was not significantly different from the intervention effect of turmeric. The experimental process and data of the embodiment of the present invention can provide new ideas for the research on the development of traditional Chinese medicine residues, and also provide theoretical support for the prevention and treatment of neuroinflammation-related diseases.

[0092] The above embodiments of the present invention are constructed by A β1-42 The C57BL / 6 mouse animal model that induces neuroinflammation is an effective tool for exploring the active ingredients, mechanism of action and indications of turmeric residues. It not only provides new ideas for the research on the development of traditional Chinese medicine residues, but also provides theoretical support for the prevention and treatment of neuroinflammation-related diseases.

[0093] Example 3 The method for constructing an animal model for neuroinflammation intervention provided in an embodiment of the present invention is a further study based on Example 1-Example 2, the difference being that the S4 analyzes the main chemical components in the turmeric residues, and first preliminarily identifies the common chemical components in the turmeric medicinal materials and the residues through UPLC-MS / MS technology, and then combines PCA and OPLS-DA models to identify potential differential substances, so as to further develop and utilize the residues.

[0094] The experimental method is as follows: S4-1: Qualitative analysis of the chemical composition of the main components in turmeric residues Qualitative analysis of the main components in turmeric residues using UPLC-MS / MS technology Qualitative analysis and quantitative determination of the differential components in turmeric residues were conducted, and it was found that turmeric residues contained 8 sesquiterpenoid compounds, 7 curcuminoid compounds (including three main components: curcumin, demethoxycurcumin, and bisdemethoxycurcumin), 6 methoxyphenolic compounds, 4 hydroxycinnamic acids and their derivatives, and other active ingredients (as shown in Table 5-2, Chemical composition identification of turmeric residues).

[0095] 1. The specific steps for analyzing the chemical composition of turmeric medicinal materials and turmeric dregs are as follows: 1.1 Preparation of test solution and reference solution Take about 1g of powder (pass through 80 mesh sieve), ultrasonicate with 5 mL 80% methanol for 15 min, centrifuge for 30 s, and take the supernatant. Add 5 mL 50% methanol and 5 mL methanol to the wet sample and repeat the above steps. Combine the three extracts. Filter with a 0.22 μm microporous membrane to obtain the test solution. Curcumin, demethoxycurcumin, bisdemethoxycurcumin, tetrahydrocurcumin, ferulic acid and coumarin were accurately weighed, and methanol was added to make up to volume to prepare a 1 mg / mL stock solution, which was then diluted to a concentration of 1 μg / mL reference solution.

[0096] 1.2 QC sample preparation Accurately pipette 8 samples respectively, shake them equally, and filter them into liquid vials with a 0.22μm microporous filter membrane to obtain QC samples. Repeat the injection 5 times, and run a QC sample every 2 samples in the sample sequence.

[0097] 1.3 Chromatographic and mass spectrometry conditions (1) Chromatographic column: Agilent SB-C18 column (100 mm × 2.1 mm, 1.8 μm); mobile phase A was acetonitrile and phase B was 0.1% formic acid. The flow rate was 0.3 mL / min, the column temperature was 40°C, the detection wavelength was 427 nm, and the injection volume was 1 μL.

[0098] (2) Mass spectrometry conditions HESI ion source, ion mode ESI+ / -; scan type full scan; scan range m / z-150.0-2000.0Da; resolution 70000; sheath gas and auxiliary gas flow rates were 40 and 10 arb, respectively, both were high-purity nitrogen; electrospray voltage 4kV; capillary temperature 350℃. Full MS conditions: resolution 70000; scan range 120-1000 m / z. dd-MS2 conditions: resolution 17500; collision energy NCE was 20, 40, 60 eV.

[0099] (3) Test the sample on the machine and obtain the test data 1.4 Data Processing The obtained data were imported into Compound Discoverer 3.2 software, and all compounds were preliminarily identified and compared with the reference substances. Then, the fragment ion peaks were compared with the literature. Progenesis QI 3.0.3 software was used to align, extract and normalize the chromatographic peaks. Principal component analysis (PCA) and partial least squares-discriminant (OPLS-DA) analysis were performed by EZinfo 2.0 software, and the permutation test method was used to test whether the model was overfitting. The compounds with large contributions to the separation between groups were preliminarily screened according to the important variable projection (VIP) value (VIP>2) and t test (p<0.05) to screen potential differential marker compounds.

[0100] 2. Experimental results of chemical composition analysis of turmeric medicinal materials and turmeric residues The component identification process and results are as follows: based on the chromatographic peak retention time, the quasi-molecular ion or adduct ion peak of the primary mass spectrometry and the characteristic fragment ion information of the secondary mass spectrometry, relevant literature and online databases, combined with reference substances for comparison, the chemical components in turmeric medicinal materials and medicinal residues were analyzed, and the results obtained 47 and 36 compounds respectively; there are 10 sesquiterpenoid compounds, 7 curcuminoid compounds, 7 methoxyphenolic compounds, 4 hydroxycinnamic acids and their derivatives and other active ingredients in turmeric medicinal materials (as shown in Table 5-1); there are 8 sesquiterpenoid compounds, 7 curcuminoid compounds, 6 methoxyphenolic compounds, 4 hydroxycinnamic acids and their derivatives and other active ingredients in the medicinal residues (see Table 5-1, chemical component identification results of turmeric medicinal materials).

[0101] Table 5-1

[0102]

[0103]

[0104]

[0105]

[0106] Note: * indicates comparison with reference product Table 5-2 Identification of chemical components of turmeric residue

[0107]

[0108] Note: * indicates comparison with reference product 3. Systematic method evaluation The QC samples in the embodiment of the present invention are closely clustered. During the entire sequence operation, the retention time RSD values ​​of the compounds in the QC samples are all less than 1%, and the peak intensity RSD is all less than 3%, indicating that the precision and repeatability of the instrument meet the requirements and the data can be further analyzed.

[0109] 4. Principal Component Analysis (PCA) The four batches of medicinal materials and the medicinal residues used in the embodiment of the present invention are respectively gathered together with a certain degree of separation, indicating that the chemical compositions of the turmeric medicinal materials and the medicinal residues are different to a certain extent, while the two batches of medicinal residues are relatively close, indicating that the compounds have certain similarities.

[0110] 5. Orthogonal-Partial Least Squares Discriminant Analysis (OPLS-DA) In order to further explore the difference between medicinal materials and medicinal residues, an OPLS-DA analysis model was established for pairwise comparison. In the positive ion mode, R²Y=0.999, Q²=0.979, indicating that the model is stable and reliable. Select VIP≥2, max fold change>2, Anova p<0.05 to screen the differential components; OPLS-DA analysis shows that the medicinal materials and medicinal residues are clearly divided into two groups. According to the obtained Table 5-3 (Differential components in turmeric medicinal materials and medicinal residues), it can be seen that there are 8 significantly different components between turmeric medicinal materials and medicinal residues, including 4 sesquiterpenoid compounds, 1 methoxyphenol, 1 curcuminoid compound, 1 phenylnaphthalene compound and 1 carboxylic acid and derivative compounds. After normalization, the average peak area of ​​-gingerdiol, monodemethylcurcumin and curcuminol A / B in the medicinal residues is higher than that of turmeric medicinal materials.

[0111] Table 5-3

[0112] Note: ↑ indicates that the average peak area of ​​the component is higher; ↓ indicates that the average peak area of ​​the component is lower From the results of component identification of turmeric medicinal materials and drug residues, it can be seen that curcumin compounds in turmeric have keto-enol interconversion. Since alcohol curcumin compounds are more hydrophobic, keto curcumin compounds elute earlier and alcohol curcumin compounds are eluted later. Existing research results show that the enolization of curcumin derivatives is enhanced in hydrophobic media and reduced in aqueous media. Therefore, the reference solution is prepared with pure methanol solution and the test solution is prepared with non-pure methanol solution. The total ion flow diagram results show that the peak response value of alcohol curcumin compounds is higher than that of keto.

[0113] Curcuminoids are considered to be the major metabolites in turmeric rhizomes. In positive ion mode, the major fragments of CUR, DCUR and BDCUR are m / z 369, m / z 339 and m / z 309. CUR, DCUR and BDCUR in turmeric medicinal materials and dregs have a therapeutic effect on AD. Although the mainstream view is that curcumin is the most important component of curcuminoids and plays a leading role in the use of turmeric to treat AD, the actual test of the embodiment of the present invention shows that the simultaneous use of the three compounds has a better therapeutic effect on AD than the use of curcumin alone, and there is a synergistic enhancement effect among the three. All three turmeric compounds have alcohol ketone isomers. The alcohol form is the main structure for the biological activity of curcuminoids. Studies have shown that alcohol curcumin can better bind to Aβ aggregates; the embodiment of the present invention shows that the gel based on tetrahydrocurcumin solid lipid nanoparticles exhibits biological activity in AD mice, has good skin permeability, reduces TNF-α and IL-6 levels, and improves AD mouse tissue pathological changes, which can be used to treat AD or other skin diseases with severe inflammation. Turmeric contains sesquiterpenoid components such as curcuma dione, curcuminol, carvone, curcumol, and germacron, which are all important components of turmeric volatile oil. Existing studies have shown that various components in turmeric volatile oil have inhibitory effects on secretase. Ferulic acid is an antioxidant naturally present in plant cell walls, has anti-inflammatory activity, can act as a free radical scavenger, and can inhibit or dissociate amyloid protein structures.

[0114] Through differential analysis, the present invention shows that turmeric ketone in the drug residue is a major dietary compound mainly found in plants of the ginger family. 42 , α-secretase, β-secretase, secretase subunits and COX-2, thereby alleviating the AD-like pathological characteristics in streptozotocin-induced sporadic AD mice; curcuminol can reduce the release of IL-1β, IL-6 and TNF-α, thereby reducing the inflammatory response in nucleus pulposus cells and delaying intervertebral disc degeneration.

[0115] 4-2: Determination of the chemical composition content of the main components of turmeric residue HPLC technology was used to quantitatively determine the main components in turmeric residues. A turmeric content determination method based on HPLC was established to compare the content of curcuminoids in turmeric medicinal materials and turmeric residues.

[0116] In this example, HPLC technology was used to investigate the sample preparation method, chromatographic conditions and methodology, and a stable and reliable method for determining the content of turmeric was established, which is suitable for the determination of turmeric medicinal materials and medicinal residues, and the contents of curcumin, demethoxycurcumin and bisdemethoxycurcumin in turmeric medicinal materials and medicinal residues were determined.

[0117] This example is based on HPLC (High performance liquid chromatography), selected sample preparation conditions and chromatographic conditions, established a method for determining the content of turmeric, and determined the content of each curcuminoid compound in different batches of turmeric medicinal materials and medicinal residues. The results show that the content of curcuminoid compounds in medicinal materials is generally higher than that in medicinal residues, but there are still a large number of curcuminoid compounds in the medicinal residues, which have a high utilization value.

[0118] 4.2.1 Experimental materials and reagents Four batches of turmeric medicinal materials (numbered: HM1-4) and their corresponding four batches of medicinal residues (numbered: HR1-R4) were all provided by Guangdong Yifang Pharmaceutical Co., Ltd. and are the dried rhizomes of Curcuma Longa L., a plant of the ginger family.

[0119] Curcumin (purity: >98.9%), demethoxycurcumin (purity: >98.5%), and bisdemethoxycurcumin (purity: >95%) were purchased from China Food and Drug Inspection Institutes; chromatographic grade acetonitrile and methanol (Merck, USA); chromatographic grade glacial acetic acid, phosphoric acid, and analytical grade methanol were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0120] 4.2.2 Experimental instruments H-Class UPLC instrument (Waters, USA); Agilent ZORBAX SB C18 (100 × 2.1 mm, 1.8 μm), Waters Acquity BEH C18 (100 × 2.1 mm, 1.7 μm), YMC Triart C18 (100× 2.1 mm, 1.9 μm); 1 / 10,000 electronic balance (ME204E) and 1 / 10,000 balance (XP26) were purchased from Mettler Toledo, Switzerland; CNC ultrasonic cleaner (KQ-500DE) was purchased from Kunshan Ultrasonic Instrument Co., Ltd.; 2-liang high-speed Chinese medicine pulverizer (111B) was from Zhejiang Ruian Yongli Pharmaceutical Machinery Co., Ltd.; MiliQ Direct8 ultrapure water machine was manufactured by Merck, Germany.

[0121] 4.3 Experimental methods 4.3.1 Preparation of mixed reference solution Take 21.8777, 7.7654, and 8.5011 mg of CUR, DCUR, and BDCUR reference substances, weigh accurately, dissolve and dilute with methanol, make up to 10 mL volumetric flask, shake well, and prepare mixed reference substance solution for use. The concentrations of CUR, DCUR, and BDCUR are 2163.7043, 764.8919, and 807.6045 μg / mL.

[0122] 4.3.2 Investigation of the preparation method of the test solution The preparation method of the test solution was initially determined as follows: take 0.2 g (40 mesh) of turmeric residue powder, accurately weigh it, place it in a stoppered conical flask, add 25 mL of methanol, ultrasonicate (power 250 W, frequency 40 kHz) for 30 min, cool to room temperature, weigh the mass again, make up the reduced mass with methanol, shake well, filter with a microporous filter membrane (0.22 μm), and obtain. First, different powder particle sizes, extraction solvents, extraction methods, extraction times, extraction powers, and extraction volumes were investigated, and then the content of curcuminoid compounds was used as the evaluation index, and finally the preparation method of the test solution was formulated.

[0123] The particle sizes of turmeric residue powder were selected as 40, 50, 65, and 80 meshes; the extraction solvents were methanol and ethanol; the extraction methods were ultrasonic extraction and heating reflux extraction; the extraction time was 30, 45, and 60 min; the extraction power was 150, 250, and 350 W, and the content of curcuminoids was used as the evaluation index to determine the appropriate preparation conditions.

[0124] 4.3.3 Investigation of liquid chromatography conditions The liquid chromatography conditions were initially determined as: Waters Acquity BEH C18 (100 × 2.1 mm, 1.7 μm), acetonitrile-0.1% acetic acid (45:55) as the mobile phase, 427 nm, flow rate 0.3 mL / min, injection volume 1 μL. The test solution was prepared according to the initial test solution preparation method, and then the detection wavelength, chromatographic column, mobile phase type and ratio, chromatographic column temperature, and flow rate were investigated. The peak shape, peak area, theoretical plate number, separation, and tailing factor of each compound were used as evaluation indicators, and finally the appropriate liquid chromatography conditions were established.

[0125] The PAD detector was used to perform full wavelength scanning in the range of 200-500 nm and select the appropriate detection wavelength; chromatographic columns with different particle sizes were selected for comparison, including YMC Triart C18 (100×2.1 mm, 1.9 μm), Agilent ZORBAX SB C18 (100×2.1 mm, 1.8 μm), and Waters Acquity BEH C18 (100×2.1 mm, 1.7 μm); The mobile phases included acetonitrile-water, acetonitrile-0.1% phosphoric acid, acetonitrile-0.1% acetic acid, acetonitrile-0.5% phosphoric acid, acetonitrile-0.5% acetic acid, and acetonitrile-1% phosphoric acid; the ratios of acetonitrile-0.5% phosphoric acid were 40:60, 45:55, 50:50, 55:45, and 60:40; The column temperature was 25, 30, 35, and 40°C; the flow rate was 0.2, 0.3, and 0.4 mL / min. The peak shape and peak area of ​​each compound were used as evaluation indicators to establish the liquid chromatography conditions.

[0126] 4.3.4 Methodological review According to the preparation method of the reference solution under "4.3.1", the determination is carried out under the chromatographic conditions under "4.3.3" to examine the linear range; the mixed reference solution is serially diluted, and the concentrations when the S / N is approximately 3 and 10 are recorded to examine the limit of quantification and the limit of detection; according to the method selected under "4.3.2", the determination is carried out under the chromatographic conditions under "4.3.3" to examine the specificity test, precision test, stability test, repeatability test, and sample recovery test.

[0127] 4.3.5 Sample content determination Prepare the test solution according to the method selected under "4.3.2", measure it according to the chromatographic conditions under "4.3.3", record the chromatogram, calculate the content of each batch of turmeric medicinal materials and medicinal residues, and perform t-test on the CUR, DCUR and BDCUR contents between the two groups.

[0128] 4.4 Experimental Results 4.4.1 Preparation of test solution Taking the content of curcuminoids as the evaluation index, a suitable test sample preparation method was established under different powder particle sizes, extraction solvents, extraction methods, extraction times, ultrasonic powers, and extraction times. The experimental results are shown in Table 3-1 (total content of curcuminoids under different preparation conditions (n=2)).

[0129] The final method for preparing the test sample was as follows: 0.2 g of turmeric powder (80 mesh) was accurately weighed and placed in a stoppered conical flask. 25 mL of methanol was added and ultrasonicated (power 250 W, frequency 40 kHz) for 30 min. The mixture was cooled to room temperature and weighed again. The reduced mass was supplemented with methanol. The mixture was shaken and filtered through a microporous filter membrane (0.22 μm).

[0130] Table 3-1

[0131] 4.4.2 Chromatographic conditions (1) Determination of detection wavelength The PDA detector was used to scan the test solution at a full wavelength of 200-500 nm. The results showed that the peak absorption of each substance was most obvious between 400-450 nm. After checking with the reference substance, the three peaks were CUR, DCUR and BDCUR. The absorption peaks of CUR, DCUR and BDCUR were observed, and the strongest absorption wavelengths were 427.2, 421.6 and 417.9 nm respectively. Since the content of curcumin in turmeric is higher than the other two components and is often used as the research object in experiments, 427 nm was selected as the absorption wavelength for determining the content of the three components.

[0132] (2) Chromatographic column The turmeric spectrum of the chromatographic columns with different particle sizes is selected, and the separation results are shown in Table 3-2 (chromatographic peak separation results under different chromatographic column conditions). The YMC Trait column has obvious tailing and poor separation performance compared with the other two columns; the Waters BEH and Agilent SB columns have good peak separation, good peak shape, and high theoretical plate number; the Agilent SB column has faster peak elution and sharper peak shape, so the Agilent SB column was selected.

[0133] Table 3-2

[0134] (3) Mobile phase ratio Select the turmeric spectrum of different ratios of acetonitrile-0.1% phosphoric acid, and the separation results are shown in Table 3-3 (chromatographic peak separation effect under different mobile phase ratios). When the ratio of acetonitrile-0.1% phosphoric acid is 40:60, the peak is the slowest, and the peak shape is blunt and round; the ratios are 50:50, 55:45, and 60:40, the separation is good, the peak shape is sharp, but tailing occurs; the ratio is 45:55, the peak shape is sharp and symmetrical, and the separation is good, so the mobile phase ratio is selected as 45:55.

[0135] Table 3-3

[0136] (4) Type of mobile phase acid The turmeric spectra of different acid types were selected, and the separation results are shown in Table 3-4 (chromatographic peak separation effect under different acid conditions). When acetonitrile-water was selected as the mobile phase type, the peak shape of each compound was good, but the peak elution time was slow, and the peak area was significantly lower than the group using acid as one of the mobile phases; combined with the literature and pharmacopoeia, acetic acid is a commonly used acid for establishing the turmeric content determination method. Compared with phosphoric acid, the chromatographic peak symmetry of the acetic acid group is slightly worse, and the peak elution time is slower, so phosphoric acid is considered as one of the mobile phases; when 0.1% phosphoric acid is selected as the mobile phase, the peak area, symmetry, and theoretical plate number of the chromatographic peak are all due to 0.5% and 1% phosphoric acid, so acetonitrile-0.1% phosphoric acid is selected as the mobile phase.

[0137] In this embodiment, the content ranges of three important curcuminoid compounds, namely, curcumin, demethoxycurcumin and bisdemethoxycurcumin, contained in turmeric residues are precisely determined, and the final determined content ranges are 16.5 mg / g to 19.94 mg / g, 6.75 mg / g to 10.66 mg / g and 6.80 mg / g to 14.04 mg / g.

[0138] Table 3-4

[0139] (5) Column temperature Select the turmeric spectrum of different column temperatures, and the separation results are shown in Table 3-5 (chromatographic peak separation effect at different column temperatures). The peak shape and separation degree of the chromatographic peaks at different column temperatures are good. Except for the slight difference in the peak elution time, the three chromatographic peaks are not much different. However, when 30℃ is selected as the column temperature, the injected sample is a low-concentration mixed reference substance, and the tail of the chromatographic peak of bis-demethoxycurcumin is forked and does not separate to the baseline. When 25℃ is selected as the column temperature, the above situation does not occur, so 25℃ is selected as the column temperature.

[0140] Table 3-5

[0141] (6) Flow rate Select the turmeric spectrum with different flow rates, and the separation results are shown in Table 3-6 (chromatographic peak separation effect at different flow rates). The peak shape of the chromatographic peaks at different flow rates is good, and the separation is good; the flow rate is 0.2 mL / min, and the chromatographic peak elution time is delayed; the flow rate is 0.3 mL / min, the elution time is accelerated, the peak shape is symmetrical, and the theoretical plate number is high; the flow rate is 0.4 mL / min, the elution time is accelerated, but the chromatographic peak has a slight tailing, and the theoretical plate number decreases, so 0.3 mL / min is selected as the optimal flow rate.

[0142] Table 3-6

[0143] (7) Determine the chromatographic conditions In summary, the final chromatographic conditions were established as follows: chromatographic column Agilent ZORBAX SB C18 (100 × 2.1 mm, 1.8 μm); mobile phase acetonitrile-0.1% phosphoric acid (45:55); flow rate 0.3 mL / min; detection wavelength 427 nm; column temperature 25 °C.

[0144] 4.4.3 Methodological investigation (1) Specificity test Comparison of the HPLC chromatograms of the test solution, mixed reference solution, single reference solution and blank solution showed that the retention times of curcumin, methoxycurcumin and bismethoxycurcumin were approximately 5.44, 4.76 and 4.17 min, respectively, with good peak symmetry, and the blank solvent did not interfere with the detection of the reference solution.

[0145] (2) LOD, LOQ and linear range Linear range, LOD and LOQ results are shown in Table 3-7 (linearity, LOD and LOQ of each component). The linear range of each component shows good linear relationship within the respective concentration range.

[0146] Table 3-7

[0147] (3) Precision test The calculated RSD of retention time of each characteristic peak was 0.12%~0.15% (n=6), and the RSD of peak area was 0.12%~0.21% (n=6), indicating that the method has good precision; (4) Stability test The calculated RSD of the retention time of each characteristic peak was 0.58%~0.71% (n=6), and the RSD of the peak area was 0.45%~0.66% (n=6), indicating that the test solution had good stability within 24h.

[0148] (5) Repeatability test The calculated RSDs of retention time of each characteristic peak were 0.09%~0.12% (n=6), and the RSDs of component content were 0.72%~0.84% ​​(n=6), indicating that this method had good repeatability.

[0149] (6) Sample recovery test The average recoveries of CUR, DCUR and BDCUR were 99.66%, 97.90% and 98.44%, respectively, with RSDs of 1.75%~2.89%. The results were in line with the requirements of the 2020 edition of the Chinese Pharmacopoeia, indicating that the content determination method was stable and reliable.

[0150] 4.4.4 Determination of content of turmeric medicinal materials and residues According to the selected experimental conditions, the content of each batch was measured. The results are shown in Table 3-12 (Content of each curcuminoid compound in turmeric medicinal materials and medicinal residues (n=2)). The experimental results show that the content of CUR, DCUR and BDCUR in turmeric medicinal materials ranges from 20.1 mg / g to 30.33 mg / g, 8.86 mg / g to 14.26 mg / g and 10.25 mg / g to 18.09 mg / g, while the content of CUR, DCUR and BDCUR in medicinal residues ranges from 16.5 mg / g to 19.94 mg / g, 6.75 mg / g to 10.66 mg / g and 6.80 mg / g to 14.04 mg / g. According to the t-test results, there was no significant difference in the content of CUR, DCUR and BDCUR between the medicinal materials and medicinal residues (p>0.05). In addition, combined with the linear range of "3.5.3", all components in the medicinal materials are within the linear range, and this content determination method is also suitable for determining the content of curcumin compounds in turmeric medicinal materials.

[0151] Table 3-12

[0152] According to the prior art, the dried rhizome of Curcuma Longa L. is one of the commonly used traditional Chinese medicines, containing a variety of active ingredients. The main active ingredients in turmeric are curcuminoids, including curcumin, demethoxycurcumin and bisdemethoxycurcumin, among which curcumin has the highest content. This example determines the content of each curcuminoid in medicinal materials and medicinal residues between different batches by establishing a method for determining the content of turmeric.

[0153] First, the powder particle size, extraction solvent, extraction method, extraction time, ultrasonic power, and extraction time were investigated to establish the test sample preparation method; secondly, the detection wavelength, chromatographic column, mobile phase type and proportion, chromatographic column temperature, and flow rate were investigated to determine the chromatographic conditions.

[0154] The present embodiment not only investigates the test sample preparation conditions and chromatographic conditions, but also carries out a systematic evaluation of the content determination method. Through specificity test, stability test, repeatability test, precision test, sample recovery test, LOD, LOQ and linear range, it is confirmed that the content determination method is stable and reliable, and can be used to determine the content of each curcuminoid compound in turmeric dregs. Through t-test, because there is no significant difference in the content of each component in turmeric medicinal material and dregs and the content of each component in medicinal material is within the linear range, the content determination method is also applicable to turmeric medicinal material. Experimental results show that the content of curcuminoid compounds in medicinal material is generally higher than that of dregs, but there are still a large amount of curcuminoid compounds in dregs, which has a higher utilization value. The turmeric dregs used in this embodiment are the dregs obtained after turmeric is extracted with water to prepare turmeric formula granules. The amount of turmeric formula granules is 18.2%. After water extraction, water-soluble components and a small amount of fat-soluble components are transferred to the formula granules, while most of the fat-soluble components remain in 81.8% of the dregs. It is reported that the transfer rate of curcumin components in turmeric formula granules is only about 5%, and about 95% of curcumin components are still enriched in the residual dregs, that is, 5.5 g of turmeric medicinal materials are extracted with water to make 1 g of formula granules, and the total amount of curcumin components contained in the remaining 4.5 g of dregs is basically the same as the total amount of curcumin components in 5.5 g of turmeric medicinal materials. Because water extraction leads to the enrichment of fat-soluble components, the content of fat-soluble components in the dregs will be almost the same as that in the medicinal materials, and even the content in the dregs will be higher than that in the medicinal materials. Therefore, for the fat-soluble component curcumin, turmeric dregs have a very large potential utilization value.

[0155] This example is based on HPLC technology, and the test sample preparation conditions and chromatographic conditions are investigated. After system adaptability verification, a stable and reliable method for determining the content of curcuminoids is established, and its characteristics include the following: (1) Taking the total amount of curcuminoids as the evaluation standard, the particle size of turmeric powder was 40, 50, 65, and 80 mesh; the extraction solvents were methanol and ethanol; the extraction methods were ultrasonic extraction and heating reflux extraction; the extraction time was 30, 45, and 60 min; the extraction power was 150, 250, and 350 W. Under different test sample preparation conditions, the best test sample preparation method was selected as follows: 0.2 g of turmeric powder (80 mesh), accurately weighed, placed in a stoppered conical flask, added 25 mL of methanol, ultrasonicated (power 250 W, frequency 40 kHz) for 30 min, and filtered to obtain the test sample solution.

[0156] (2) Based on the chromatographic peak separation effect, full wavelength scanning was performed in the range of 200-500 nm to select the appropriate detection wavelength; chromatographic columns with different particle sizes, mobile phase types, mobile phase ratios, column temperatures, and flow rates were selected for investigation. After investigating different chromatographic conditions, the chromatographic conditions finally selected were: chromatographic column Agilent ZORBAX SBC18 (100 × 2.1 mm, 1.8 μm); mobile phase acetonitrile-0.1% phosphoric acid (45:55); flow rate 0.3 mL / min; detection wavelength 427 nm; column temperature 25°C; (3) After methodological investigation, the content determination method was found to be stable and reliable, and can be used for the content determination of turmeric medicinal materials and turmeric residues. The content of curcuminoid compounds in medicinal materials is generally higher than that in the residues, but there are still a large number of curcuminoid compounds in the residues, which have a high utilization value.

[0157] Through actual experiments and verification, this embodiment found that a variety of components can be extracted from the turmeric residues after water extraction, including volatile oils, curcumin, essential oils, polysaccharides, etc. These components are widely used in traditional medicine and modern health products. Combined with the current production of Chinese medicine formula granules by Guangdong Yifang Pharmaceutical Co., Ltd., water extraction is often used in the processing of turmeric Chinese medicinal materials or the production of related Chinese medicine products, resulting in high-value fat-soluble components not being fully utilized and remaining in solid waste, causing serious waste. Since curcumin in turmeric is insoluble in water, a large amount of fat-soluble components similar to curcumin may remain in the turmeric residues after extraction by traditional methods, which has extremely high development and utilization value. Therefore, in order to better develop and utilize turmeric residues, this study is divided into two parts, the component analysis of turmeric medicinal materials and residues and whether it has a mitigating effect on neuroinflammation.

[0158] Existing studies have shown that a variety of components can be extracted from turmeric residues, including volatile oils, curcumin, essential oils, polysaccharides, etc. These components are widely used in traditional medicine and modern health products. Combined with the current production of Chinese medicine formula granules by Guangdong Yifang Pharmaceutical Co., Ltd. and other companies, water extraction is often used in the processing of turmeric Chinese medicinal materials or the production of related Chinese medicine products, resulting in high-value fat-soluble components not being fully utilized and remaining in solid waste, causing serious waste. Since curcumin in turmeric is difficult to dissolve in water, a large amount of fat-soluble components similar to curcumin may remain in the turmeric residues after extraction by traditional methods, which may still have a high development and utilization value, but no specialized research on the development and utilization of turmeric residues has been found so far.

[0159] In this example, UPLC-MS / MS technology was used to preliminarily identify the components in turmeric medicinal materials and medicinal residues, and a total of 47 and 36 compounds were screened in turmeric medicinal materials and medicinal residues. There are 10 sesquiterpenoid compounds, 7 curcuminoid compounds, 6 methoxyphenolic compounds, 4 hydroxycinnamic acids and their derivatives and other active ingredients in turmeric medicinal materials. The medicinal residues contain 8 sesquiterpenoid compounds, 7 curcuminoid compounds, 5 methoxyphenolic compounds, 4 hydroxycinnamic acids and their derivatives and other active ingredients.

[0160] In this example, by comparing the components of turmeric medicinal materials and medicinal residues, it was found that there were 8 significantly different components between turmeric medicinal materials and medicinal residues, including 4 sesquiterpenoid compounds, 1 monoterpenoid compound, 1 curcuminoid compound and 2 other compounds. Among them, the average peak areas of [4]-gingerdiol, monodemethylcurcumin and curcuminol A / B contained in the 4 batches of medicinal residues were higher than those in the medicinal materials, indicating that the medicinal residues have potential development value. Developing turmeric medicinal residues can effectively reduce resource waste and improve the comprehensive utilization value of waste resources.

[0161] Example 4 The present invention further provides a method for screening effective ingredients for intervening neuroinflammation in turmeric residues, which is a further application of the aforementioned method for constructing an animal model for neuroinflammation intervention. The method is to use the aforementioned methods of Example 1 to Example 3 to construct an animal model for neuroinflammation intervention, and to screen effective ingredients for intervening neuroinflammation in turmeric residues, comprising the following steps: A1: Analysis of the main chemical components in turmeric residue UPLC-MS / MS and HPLC techniques were used to qualitatively analyze and quantitatively determine the main curcuminoid compounds in turmeric residues, and the active ingredients to be tested were preliminarily determined. A2: Screening of active ingredients using neuroinflammation intervention animal models The neuroinflammation intervention animal model was used to test the intervention effects of each active ingredient (mainly curcuminoid compounds) on neuroinflammation. A3: Comparative test to determine whether there is synergistic effect between the active ingredients in turmeric residue A comparative test was conducted using an animal model of neuroinflammation intervention, comparing the simultaneous use and single use of the active ingredients to be tested, to determine whether there is a synergistic effect when multiple compounds in turmeric residues are used simultaneously, and whether the intervention therapeutic effect of multiple active ingredients on AD when used together is more significant than when used alone.

[0162] This example tested the neuroinflammation intervention animal model and found that the simultaneous use of the three compounds in the residue, namely, curcumin CUR, demethoxycurcumin DCUR and bisdemethoxycurcumin BDCUR, had a synergistic enhancement effect on the treatment of AD, which was better than using curcumin alone.

[0163] The specific technical means of experiment, test and analysis adopted in this embodiment are the same as those of the aforementioned embodiments 1-3, and will not be repeated here.

[0164] The methods and applications provided in the above embodiments of the present invention focus on constructing Aβ 1-42 Induce neuroinflammation in C57BL / 6 mouse models to explore the effective ingredients, mechanism of action and indications of turmeric residues; including: preliminary identification of the chemical composition of turmeric medicinal materials and residues by UPLC-MS / MS technology, and further, differential analysis of the components of turmeric medicinal materials and residues to identify potential differential substances; establish Aβ 1-42 A mouse model of neuroinflammation was established by brain localization injection to explore whether turmeric, turmeric residues, and curcumin, an important active ingredient in turmeric, can reduce neuroinflammation in the mouse brain and study its potential mechanism of action; finally, combined with non-targeted metabolomics technology, a study was conducted on mice with neuroinflammation treated with turmeric residues. By analyzing the changes in metabolites, the metabolic pathways and metabolites in the body were revealed, and then an in-depth understanding of Aβ was obtained. 1-42 Therefore, the present invention not only provides new ideas for the research on the development of traditional Chinese medicine residues, but also provides theoretical support for the prevention and treatment of neuroinflammatory diseases.

[0165] It should be noted that the above embodiments describe only a part of the embodiments of the present invention, rather than all the embodiments. In other embodiments, within the scope of the present invention, the technical solutions obtained by selecting other components, proportions and process parameters can also achieve the technical effects recorded in the present invention, so they are not listed one by one.

[0166] The specific embodiments described above are only an introduction to the optimization method of the present invention, and are not intended to limit the scope of the present invention. It should be noted that, without departing from the spirit of the present invention, those skilled in the art may make appropriate adjustments or modifications to the technical solution of the present invention, which shall fall within the scope of protection determined by the claims of the present invention.

Claims

1. A method for constructing an animal model for neuroinflammation intervention, characterized in that: It includes the following steps: S1: Preparation of experimental agents for animal models Five types of experimental agents were prepared respectively: turmeric medicinal materials, turmeric dregs, low-dose curcumin, high-dose curcumin and donepezil tablet solution; the turmeric dregs were the solid residues remaining after water extraction of turmeric medicinal materials; S2: Construction of Aβ 1-42 Mouse animal model of induced neuroinflammation Several male mice were selected and randomly divided into sham operation group, model group, positive drug group, medicinal material group, medicinal residue group, low-dose curcumin group, and high-dose curcumin group. They were fed with the same feed, and the behavior of mice in each group was tested: First, five experimental drugs were given to each group of mice by gavage for one week: turmeric, turmeric residue, low-dose curcumin, high-dose curcumin, and donepezil tablets. Then, Aβ was injected into the brain of the mice. 1-42 A neuroinflammation model was established, and then five types of experimental drugs were given to each group of mice by gavage for several consecutive days. Each group of mice was subjected to behavioral tests to analyze whether the experimental drugs used in each group in the animal model had the effect of reducing neuroinflammation in the mouse brain; S3: To explore the mechanism and effect of turmeric residue in intervening neuroinflammation in mice The mice in each group obtained in step S2 were prepared as biological samples, and non-targeted metabolomics technology was used to analyze and explore whether turmeric residues and curcumin can improve Aβ by regulating the TLR4 / NF-κB pathway. 1-42 The mechanism of inducing cognitive impairment in mice with neuroinflammation, alleviating neural damage, regulating over-activated microglia, downregulating inflammatory factors and upregulating anti-inflammatory factors can achieve the intervention effect of alleviating neuroinflammation.

2. The method for constructing an animal model for neuroinflammation intervention according to claim 1, characterized in that: It also includes the following steps: S4: Qualitative and quantitative determination of major curcuminoids in turmeric residues UPLC-MS / MS and HPLC techniques were used to carry out qualitative analysis and quantitative determination of the main curcuminoids in turmeric residues.

3. The method for constructing an animal model for neuroinflammation intervention according to claim 2, characterized in that: It also includes the following steps: S5: Testing the mechanism and effect of the main components of turmeric residues in intervening neuroinflammation in mice A neuroinflammation intervention animal model was used to test the main curcuminoid compounds in turmeric residues and the mechanism and effect of intervening in neuroinflammation in mice.

4. The method for constructing an animal model for neuroinflammation intervention according to claim 1, characterized in that: The five types of experimental agents in step S1 are prepared specifically by the following steps: S1-1: Preparation of low-dose and high-dose solutions of curcumin reference substance Weigh the curcumin reference substance, dissolve it in 2% Tween 80 and distilled water, and prepare a low-dose curcumin solution with a concentration of 1 mg / mL and a high-dose curcumin solution with a concentration of 2 mg / mL respectively; S1-2: Preparation of turmeric medicinal materials and turmeric residue solution Weigh the powder, add 70% ethanol solution, shake on a shaker, filter with gauze, place the filtrate in an evaporating dish, concentrate the drug solution in a water bath, evaporate the ethanol completely, and prepare a 0.1 g / mL turmeric drug solution and a 0.12 g / mL turmeric drug residue solution, respectively, and store them at -20°C; S1-3: Preparation of positive drug solution Donepezil hydrochloride tablets were ground into fine powder and dissolved in distilled water to obtain a positive drug solution with a concentration of 0.076 mg / mL.

5. The method for constructing an animal model for neuroinflammation intervention according to claim 1, characterized in that: The step S2 comprises the following steps: S2-1: Mouse grouping Several male mice were selected and after 7 days of adaptive feeding, they were randomly divided into 7 groups: sham operation group, model group, positive drug group, medicinal material group, medicinal residue group, low-dose curcumin group, and high-dose curcumin group, with no less than 5 mice in each group, and they were fed with the same feed. S2-2: Grouping and administration of drugs before localized injection in mouse brain Each mouse in each group was first gavaged for one week, and the specific medication for each group was as follows: Sham operation group: C57BL / 6 mice injected with sterile saline in the bilateral hippocampus and gavaged with 0.1 mL / g distilled water; Model group: Aβ was injected into the bilateral hippocampus 1-42 C57BL / 6 mice were gavaged with 0.1 mL / 10 g distilled water; Positive drug group: Aβ was injected into the bilateral hippocampus 1-42 C57BL / 6 mice were given a dose of 0.76 mg / kg by intragastric administration of 0.1 mL / 10 g donepezil hydrochloride solution; Drug group: Aβ was injected into the bilateral hippocampus 1-42 C57BL / 6 mice were given 0.1 mL / 10 g turmeric solution by gavage at a crude drug dose of 1 g / kg. Drug residue group: Aβ was injected into the bilateral hippocampus 1-42 C57BL / 6 mice were given a crude drug dose of 1.2 g / kg by intragastric administration of 0.1 mL / 10 g of turmeric residue solution; Low-dose group: Aβ was injected into the bilateral hippocampus 1-42 C57BL / 6 mice were given a dose of 10 mg / kg by gavage with 0.1 mL / 10 g of low-dose curcumin solution; High-dose group: Aβ was injected into the bilateral hippocampus 1-42 C57BL / 6 mice were given 20 mg / kg by gavage with 0.1 mL / 10 g of high-dose curcumin solution; S2-3: Neuroinflammation induced by localized injection in the mouse brain Aβ 1-42 Solution treatment Aβ 1-42 Dissolve in sterile saline to prepare an Aβ1-42 injection solution with a concentration of 410 pmol / μL. Place the prepared solution in a 37°C constant temperature incubator and incubate for 7 days before use. On the eighth day of the experiment, the mice in each group were injected with brain localization according to the mouse brain atlas. The injection sites were selected as the CA1 regions of the bilateral hippocampi with the coordinates: AP: -0.22 cm, ML: ±0.2 cm, DV: -0.2 cm. Aβ was drawn with a microsyringe. 1-42 Solution, Aβ in one side of the brain 1-42 The solution volume was 1.5 μL, and the operation lasted for 10 min. The needle was left in place for 15 min, and the needle was slowly withdrawn for 5 min. The wound was sutured and disinfected, and the same operation was performed on the other side of the brain. The sham operation group was injected with an equal amount of sterile saline, and then the mice were allowed to recover naturally for 2 days. S2-4: Grouping and dosing of mice after brain localization injection Starting from the 10th day of the experiment, the grouping and dosing method of step S2-2 were repeated for each group of mice, and the mice were gavaged for three weeks; S2-5: Animal behavior experiments on mice in each group Animal behavioral experiments were performed on each group of mice. The effects of turmeric, turmeric residues and curcumin on the learning and memory ability of mice with Aβ1-42 damage were evaluated through the results of animal behavioral experiments, and whether the turmeric, turmeric residues and curcumin in the model group could reduce neuroinflammation in the mouse brain was determined.

6. The method for constructing an animal model for neuroinflammation intervention according to claim 5, characterized in that: The animal behavior experiment of step S2-5 is specifically a Morris water maze experiment, including a positioning navigation experiment and a space exploration experiment; the water maze experiment is started on the 25th day after the start of the drug administration, wherein the positioning navigation experiment is performed on the 25th to 29th day of the experiment, and the space exploration experiment is performed on the 30th day of the experiment, and specifically includes the following steps: S2-5-1: Positioning and navigation experiment The mice were placed in the water from the midpoint of the pool wall in four different quadrants. The software counted down for 90 seconds. If the time from entering the water to successfully finding the water platform and staying there for more than 10 seconds was recorded as the escape latency; if the water platform could not be found within 90 seconds, its escape latency was 90 seconds. After 90 seconds, the experimenter guided the mice to find the water platform and let them stay on the platform for 10 seconds. The escape latency of the mice was recorded for five days. S2-5-2: Space Exploration Experiment On the 30th day of the experiment, the platform was removed and the mice were placed in the water from the diagonal quadrant of the platform. The swimming route of the mice within 90 s was measured, and the number of times they crossed the platform and the time they stayed in the quadrant where the water platform was placed were recorded. S2-5-3: Analyze the experimental results The experimental results were analyzed to determine whether the turmeric medicinal materials, turmeric residues and curcumin in the turmeric medicinal materials group, turmeric residues group, low-dose curcumin group and high-dose curcumin group could alleviate neuroinflammation in the mouse brain to different degrees.

7. The method for constructing an animal model for neuroinflammation intervention according to claim 1, characterized in that: The step S3 is to perform immunohistochemical analysis based on the experimental data and biological samples obtained in step S2, and further use non-targeted metabolomics technology to explore the mechanism of turmeric dregs and curcumin intervening in mice with neuroinflammation, and how turmeric dregs and curcumin improve cognitive impairment, reduce neural damage, regulate overactivated microglia, downregulate inflammatory factors and upregulate anti-inflammatory factors in mice with Aβ1-42-induced neuroinflammation by regulating the TLR4 / NF-κB pathway, and finally reduce the mechanism of neuroinflammation, which specifically includes the following steps: S3-1: Analysis of Morris water maze test data to observe the effect of turmeric on Aβ 1-42 Effects of lesions on spatial learning and memory in mice S3-2: Perform histopathological examination S3-2-1: HE staining and Nissl staining were used to observe the pathological changes of neurons in the hippocampus; S3-2-2: Immunofluorescence staining IF was used to observe the expression of Aβ1-42 and Iba-1 in the mouse brain, and then immunohistochemistry was used to observe the changes of Iba-1 in the CA1 region of the hippocampus; S3-2-3: PCR fluorescence quantitative determination of mRNA expression of important channel proteins and cytokines in the TLR4 / NF-κB signaling pathway; S3-2-4: Enzyme-linked immunosorbent assay (ELISA) was used to determine the contents of important channel proteins and cytokines in the TLR4 / NF-κB signaling pathway; S3-3: Conducting untargeted metabolomics studies Plasma and brain tissue samples of each experimental mouse were prepared and tested using chromatography and mass spectrometry. Then, non-targeted serum metabolomics technology was used to analyze the raw data of non-targeted metabolomics of serum samples of each group of rats.

8. The method for constructing an animal model for neuroinflammation intervention according to claim 2, characterized in that: The step S4 analyzes the main curcuminoid compounds in the turmeric residues, comprising the following steps: S4-1: Qualitative analysis of the chemical composition of the main components in turmeric residues UPLC-MS / MS technology was used to qualitatively analyze the main components in turmeric residues. Through qualitative analysis and quantitative determination of the differential components in turmeric residues, it was determined that turmeric residues contain 8 sesquiterpenoid compounds, 7 curcuminoid compounds, 6 methoxyphenolic compounds, 4 hydroxycinnamic acids and their derivatives. S4-2: Determination of the chemical composition content of the main components of turmeric residue HPLC technology was used to quantitatively determine the main components in turmeric residues, and the content ranges of three important curcuminoids, namely curcumin, demethoxycurcumin and bisdemethoxycurcumin, in turmeric residues were accurately determined.

9. A method for screening effective ingredients for intervening neuroinflammation in turmeric residues, characterized in that: The method for constructing an animal model for neuroinflammation intervention according to any one of claims 1 to 8 is used to construct an animal model for neuroinflammation intervention, and effective ingredients for neuroinflammation intervention in turmeric residues are screened, comprising the following steps: A1: Analysis of the main chemical components in turmeric residue UPLC-MS / MS and HPLC techniques were used to qualitatively analyze and quantitatively determine the main curcuminoid compounds in turmeric residues, and the active ingredients to be tested were preliminarily determined. A2: Screening of active ingredients using animal models and mechanisms of neuroinflammation intervention A neuroinflammation intervention animal model was adopted, and the intervention mechanism of curcumin compounds on neuroinflammation was utilized to test the intervention effect of each active ingredient on neuroinflammation.

10. The method for screening effective ingredients for intervening neuroinflammation in turmeric residues according to claim 9, characterized in that: It also includes the following steps: A3: Comparative test to determine whether there is synergistic effect between the active ingredients in turmeric residue A comparative test was conducted using an animal model for neuroinflammation intervention, comparing the simultaneous use and single use of the active ingredients to be tested, to determine whether there is a synergistic effect when multiple compounds in turmeric residues are used simultaneously, and whether the intervention therapeutic effect of multiple active ingredients on neuroinflammation when used together is more significant than when used alone.

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