A method for constructing and screening an animal model of neuroinflammation intervention and effective components in curcuma drug residue

Abstract

This invention belongs to the field of traditional Chinese medicine technology and discloses a method for constructing an animal model of neuroinflammation intervention and screening effective components in turmeric residue. The method includes the following steps: S1: preparing experimental reagents for the animal model; S2: constructing a mouse model of Aβ1-42-induced neuroinflammation; S3: exploring the mechanism and effect of turmeric residue in intervening in mouse neuroinflammation. This invention also provides a method for screening effective components in turmeric residue, using an animal model of neuroinflammation intervention to screen for effective components in turmeric residue that intervene in neuroinflammation. This invention uses a specially constructed animal model, UPLC-MS / MS, HPLC, combined with non-targeted metabolomics technology to study the mechanism, effect, and effective components of turmeric residue in intervening in mouse neuroinflammation. It can be widely applied to the in-depth development of turmeric-based traditional Chinese medicine residues, effectively utilizing turmeric residue resources and providing support for the prevention and treatment of neuroinflammation-related diseases.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, specifically to a method for constructing an animal model of neuroinflammatory intervention and screening effective components in turmeric residue. Background Technology

[0002] Current research indicates that Alzheimer's disease is the leading type of neuroinflammatory disease, including dementia. Alzheimer's disease (AD) is a progressive neurodegenerative disease clinically characterized by cognitive impairment and progressive memory loss. Its main pathological features are the phosphorylation of extracellular β-amyloid beta (Aβ) and intracellular tau protein. Aβ aggregation and deposition induce neuroinflammation and hinder intercellular communication, while tau protein phosphorylation leads to entanglement between neurofibrils and disrupts neuronal material exchange. Among numerous pathogenesis mechanisms, the most widely accepted amyloid hypothesis posits that Aβ aggregation and deposition into plaques lead to AD development, accelerating processes such as neuroinflammation, neuronal damage, and synaptic loss. These pathological processes further accelerate Aβ aggregation, creating a cascade amplification effect, ultimately resulting in AD.

[0003] Turmeric is a commonly used traditional Chinese medicine, derived from the dried rhizome of *Curcuma long L.*, a plant belonging to the genus *Curcuma* in the ginger family (Zingiberaceae). It is mainly produced in Sichuan, Fujian, Guangdong, and Guangxi provinces. Current research indicates that the main chemical components of turmeric are curcuminoids and volatile oils, along with sugars, flavonoids, and sterols. Existing research, specifically patent application CN112351790 A, has clearly identified turmeric extracts in free water and hydrophilic organic solvents, as well as curcuminol A and curcuminol B, as effective components in the treatment of Alzheimer's disease. Most existing traditional Chinese medicine preparations using turmeric as a raw material also utilize aqueous extracts of turmeric as their active ingredients. For example, the production of turmeric granules mainly relies on water extraction. Since oil-soluble and cellulose components in turmeric are poorly soluble in water, these curcumin-like compounds remain in the residue. Currently, the turmeric residue after water extraction (the solid residue remaining after aqueous extraction) is generally treated as waste, resulting in the underutilization of other important components of turmeric. This leads to a huge waste of resources, serious environmental pollution, and increased costs for pharmaceutical companies. Existing research shows that various components can be extracted from turmeric residue, including volatile oils, curcumin, essential oils, and polysaccharides. However, due to various technical difficulties, no specialized research on the development and utilization of turmeric residue has been found. Existing studies do not cover which components in turmeric residue (qualitative and quantitative), which components are effective in intervening in neuroinflammatory diseases such as Alzheimer's disease (AD), and how these effective components produce their effects (effective components and pharmacological and pharmacodynamic effects). Therefore, they cannot provide technical support for promoting the comprehensive development and utilization of turmeric residue, nor can they meet the needs of enterprises to develop high-value drugs containing effective components from turmeric residue while reducing the cost of treating waste from traditional Chinese medicine processing. Summary of the Invention

[0004] To address the aforementioned shortcomings of existing technologies, the present invention aims to provide a method for constructing an animal model of neuroinflammatory intervention and screening effective components in turmeric residue. By constructing a scientific animal model and combining it with non-targeted metabolomics technology, the effective components in turmeric residue are qualitatively and quantitatively analyzed to identify the effective components in turmeric residue that can significantly intervene in neuroinflammatory diseases (including Alzheimer's disease). Simultaneously, the pharmacological and pharmacodynamic effects of these effective components in intervening in neuroinflammatory diseases are specifically studied. This provides technical support for promoting the comprehensive development and utilization of turmeric residue, meeting the needs of enterprises to develop high-value drugs containing effective components from turmeric residue, while simultaneously reducing the cost of treating waste from traditional Chinese medicine processing, thus solving the problems existing in the prior art.

[0005] To achieve the above objectives, the technical solution adopted by this invention is as follows:

[0006] A method for constructing an animal model of neuroinflammatory intervention, comprising the following steps:

[0007] S1: Experimental reagents for preparing animal models

[0008] Five types of experimental reagents were prepared: turmeric raw material, turmeric residue, low-dose curcumin, high-dose curcumin, and donepezil tablet solution; the turmeric residue is the solid residue remaining after water extraction of turmeric raw material;

[0009] S2: Construct Aβ 1-42 Mouse models of induced neuroinflammation

[0010] Multiple male mice were randomly divided into four groups: sham-operated group, model group, positive control group, herbal medicine group, herbal residue group, low-dose curcumin group, and high-dose curcumin group. All groups were fed the same diet, and the behavior of the mice in each group was assessed.

[0011] For one week, mice in each group were administered five experimental drugs via gavage: turmeric root, turmeric residue, low-dose curcumin, high-dose curcumin, and donepezil tablets. Then, Aβ was injected into the mice's brains via targeted injection. 1-42 A neuroinflammation model was established, and then mice in each group were administered five types of experimental drugs by gavage for three consecutive weeks.

[0012] Behavioral tests were performed on mice in each group to analyze whether the experimental drugs used in each group of the animal model had the effect of reducing neuroinflammation in the mouse brain;

[0013] S3: To investigate the mechanism and effect of turmeric residue in intervening in neuroinflammation in mice.

[0014] The mice obtained in step S2 were prepared as biological samples. Non-targeted metabolomics techniques were used to analyze and investigate whether turmeric residue and curcumin could improve Aβ by regulating the TLR4 / NF-κB pathway. 1-42 The mechanism by which the neuroinflammation is induced in mice to induce cognitive impairment, reduce nerve damage, regulate overactivated microglia, downregulate inflammatory factors and upregulate anti-inflammatory factors, thereby achieving the intervention effect of reducing neuroinflammation;

[0015] S4: Qualitative and quantitative determination of major curcuminoid compounds in turmeric residue

[0016] The main curcumin compounds in turmeric residue were qualitatively analyzed and quantitatively determined using UPLC-MS / MS and HPLC techniques, respectively.

[0017] S5: Testing the mechanism and effect of the main components of turmeric residue in intervening in the neuroinflammation of mice.

[0018] Using an animal model of neuroinflammation intervention, we tested the main curcuminoid compounds in turmeric residue to investigate the mechanism and effect of intervention on neuroinflammation in mice.

[0019] A method for screening effective components in turmeric residue that intervene in neuroinflammation, comprising the following steps: constructing an animal model for neuroinflammation intervention using the aforementioned method for constructing such an animal model; screening effective components in turmeric residue that intervene in neuroinflammation intervention; and other methods for screening effective components in turmeric residue that intervene in neuroinflammation intervention.

[0020] A1: Analysis of the main chemical components of turmeric residue

[0021] The main curcumin compounds in turmeric residue were qualitatively analyzed and quantitatively determined using UPLC-MS / MS and HPLC techniques, respectively, to preliminarily identify the active components to be tested.

[0022] A2: Screening of effective ingredients using animal models and mechanisms of neuroinflammatory intervention.

[0023] Using an animal model of neuroinflammation intervention, and utilizing the intervention mechanism of curcumin compounds on neuroinflammation, the intervention effects of each active ingredient on neuroinflammation were tested.

[0024] A3: Comparative testing to determine whether there is a synergistic effect among the various active components in turmeric residue.

[0025] A comparative test was conducted using an animal model of neuroinflammatory intervention. The simultaneous and individual use of the tested active ingredients were compared to determine whether there was a synergistic effect of multiple compounds in turmeric residue when used simultaneously, and whether the intervention and treatment effect of multiple active ingredients used together on neuroinflammatory diseases such as Alzheimer's disease was more significant than that of individual use.

[0026] The method for constructing an animal model of neuroinflammatory intervention and screening effective components in turmeric residue provided by this invention has at least the following beneficial effects:

[0027] 1. This invention provides a method for qualitative and quantitative analysis of the effective components in turmeric residue by constructing a scientific animal model and combining it with non-targeted metabolomics technology. This method identifies the effective components in turmeric residue that can significantly intervene in neuroinflammatory diseases such as Alzheimer's disease (AD). At the same time, it conducts specific research on the pharmacological and pharmacodynamic effects of these effective components in intervening in neuroinflammatory diseases. This provides technical support for promoting the comprehensive development and utilization of turmeric residue, meets the needs of enterprises to develop high-value drugs containing effective components from turmeric residue, and reduces the cost of treating waste from traditional Chinese medicine processing, thereby solving the problems existing in the prior art.

[0028] 2. This invention is the first to use UPLC-MS / MS technology to preliminarily identify the chemical components in turmeric residue. Furthermore, it analyzes the intervention mechanism and effects of the turmeric residue components and identifies the main effective active ingredients.

[0029] 3. This invention establishes Aβ 1-42A mouse model of neuroinflammation was established by targeted brain injection to investigate whether turmeric, turmeric residue, and curcumin (a curcuminoid compound), an important active ingredient in turmeric, can alleviate neuroinflammation in the mouse brain and to study its potential mechanism of action. In addition, non-targeted metabolomics technology was used to study the intervention of turmeric residue in mice with neuroinflammation.

[0030] 4. This invention discovers that turmeric residue and its effective components can reduce Aβ. 1-42 The effects of inducing neuroinflammation were observed; it was found that both turmeric residue and curcumin reduced Aβ by regulating the TLR4 / NF-κB pathway. 1-42 The potential mechanism of induced neuroinflammation is to regulate the overactivation state of microglia, regulate important proteins in the TLR4 / NF-κB pathway, downregulate inflammatory factors, and upregulate anti-inflammatory factors, thereby alleviating neuroinflammation in the brain.

[0031] 5. This invention constructs Aβ 1-42 The C57BL / 6 mouse model of induced neuroinflammation, combined with qualitative and quantitative analysis of the effective components in turmeric residue, ultimately revealed the effective components, mechanism of action, and indications of turmeric residue. This invention not only provides new ideas for the research on traditional Chinese medicine residue, but also provides support for the prevention and treatment of neuroinflammation-related diseases. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the process for constructing animal models and screening effective components in an embodiment of the present invention;

[0033] Figure 2 This is a schematic diagram of the main process of animal experiments in this embodiment of the invention;

[0034] Figure 3 This is a mouse brain localization injection atlas from animal experiments of this invention;

[0035] Figure 4 shows the effect of water maze turmeric on Aβ in an embodiment of the present invention. 1-42 Schematic diagram of the impact of damage on spatial learning and memory abilities in mice, including AD, wherein, Figure 4(A) shows the escape latency in the orientation navigation experiment; Figure 4(B) shows the number of times the platform was traversed 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 movement trajectory in the spatial exploration experiment;

[0036] Figure 5 shows the effect of turmeric on Aβ in an embodiment of the present invention. 1-42 Schematic diagrams of the effects of inducing pathological changes (200× and 500×), including Figure 5(1) and Figure 5(2);

[0037] Figure 6 shows the effect of turmeric on Aβ in an embodiment of the present invention. 1-42Schematic diagrams of the effects of induced neuronal damage and loss (200× and 500×), including Figure 6(1) and Figure 6(2);

[0038] Figure 7 Iba-1 and Aβ in immunofluorescence staining in embodiments of the present invention 1-42 A total of 200× diagrams are presented;

[0039] Figure 8 shows the effect of turmeric on Aβ in an embodiment of the present invention. 1-42 Schematic diagram of the effect of inducing Iba-1 proliferation, including AC; wherein, Figure 8(A) (including Figure 8(A-1) and Figure 8(A-2)) shows IHC staining of Iba-1 in the hippocampus (200× and 500×); Figure 8(B) shows the percentage of positive area of ​​Iba-1; Figure 8(C) shows the percentage of positive area of ​​Iba-1 cells.

[0040] Figure 9 is a schematic diagram of the 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 Figure 9(A) shows the mRNA expression level of IκB; Figure 9B This represents the mRNA expression level of p65. Figure 9C The mRNA expression level of TLR4;

[0041] Figure 10 is a schematic diagram of the regulation of cytokine mRNA levels in the TLR4 / NF-κB pathway by turmeric in an embodiment of the present invention, including A and H; wherein, Figure 10(A) shows the mRNA expression level of IL-6; Figure 10(B) shows the mRNA expression level of TNF-α; Figure 10(C) shows the mRNA expression level of iNOS; Figure 10(D) shows the mRNA expression level of COX-2; Figure 10(E) shows the mRNA expression level of IL-4; Figure 10(F) shows the mRNA expression level of IL-10; Figure 10(G) shows the mRNA expression level of TGF-1β; Figure 10(H) shows the mRNA expression level of Arg-1;

[0042] Figure 11 is a schematic diagram of the effect of turmeric on the content of important channel proteins in the TLR4 / NF-κB pathway in an embodiment of the present invention, including: (A) Figure 11(A) shows the relative expression level of pIKK / IKK protein; (B) Figure 11(B) shows the relative expression level of p-IκB / IκB protein; (C) Figure 11(C) shows the relative expression level of p-p65 / p65 protein; (D) Figure 11(D) shows the protein content of TLR4.

[0043] Figure 12 is a schematic diagram of the regulation of cytokine protein expression levels in the TLR4 / NF-κB pathway by turmeric in an embodiment of the present invention, including AH; Figure 12(A) shows the protein expression level of IL-6; Figure 12(B) shows the protein expression level of TNF-α; Figure 12(C) shows the protein expression level of iNOS; Figure 12(D) shows the protein expression level of COX-2; Figure 12(E) shows the protein expression level of IL-4; Figure 12(F) shows the protein expression level of IL-10; Figure 12(G) shows the protein expression level of TGF-1β; Figure 12(H) shows the protein expression level of Arg-1;

[0044] Figure 13 In this embodiment of the invention, turmeric alleviates Aβ by regulating the TLR4 / NF-κB signaling pathway. 1-42 A diagram illustrating the biological mechanisms that induce neuroinflammation;

[0045] Figure 14 shows the total ion flow map in an embodiment of the present invention, including AD; wherein, Figure 14(A) is the total ion flow map of plasma in positive ion mode; Figure 14(B) is the total ion flow map of plasma in negative ion mode; Figure 14(C) is the total ion flow map of brain tissue in positive ion mode; Figure 14(D) is the total ion flow map of brain tissue in negative ion mode.

[0046] Figure 15 shows the spatial distribution visualization results of plasma non-targeted metabolomics data in the embodiments of the present invention, including A and B; wherein, Figure 15(A) is the PLS-DA score map in positive ion mode; Figure 15(B) is the PLS-DA score map in negative ion mode;

[0047] Figure 16 shows the spatial distribution visualization results of non-targeted metabolomics data of brain tissue in the embodiments of the present invention, including A and B; wherein, Figure 16(A) is the PLS-DA score map of positive ion mode; Figure 16(B) is the PLS-DA score map of negative ion mode;

[0048] Figure 17 shows the spatial distribution visualization results of non-targeted metabolomics data of plasma in the sham surgery group compared with the model group in the embodiment of the present invention, including AD; wherein, Figure 17(A) is the PLS-DA score map of positive ion mode; Figure 17(B) is the PLS-DA score map of negative ion mode; Figure 17(C) is the VIP map of positive ion mode; Figure 17(D) is the VIP map of negative ion mode.

[0049] Figure 18 shows the spatial distribution visualization results of non-targeted metabolomics data of brain tissue in the sham surgery group compared with the model group in the embodiment of the present invention, including AD; wherein, Figure 18(A) is the OPLS-DA score map of positive ion mode; Figure 18(B) is the OPLS-DA score map of negative ion mode; Figure 18(C) is the VIP map of positive ion mode; Figure 18(D) is the VIP map of negative ion mode.

[0050] Figure 19 This is a schematic diagram of plasma KEGG metabolic pathway scores in an embodiment of the present invention;

[0051] Figure 20 This is a schematic diagram of the KEGG metabolic pathway score in brain tissue according to an embodiment of the present invention. Detailed Implementation

[0052] The following embodiments of the present invention mainly address the problems of large quantities of turmeric residue after water extraction in pharmaceutical companies, which are treated as waste, wasting resources and increasing enterprise costs. On the one hand, the components of turmeric residue are analyzed; on the other hand, the mechanisms by which turmeric residue and its effective components intervene in neuroinflammation and whether they have a mitigating effect are investigated (pharmacological mechanism and efficacy). This aims to promote the comprehensive development and utilization of turmeric residue, reduce the cost of waste disposal in traditional Chinese medicine processing, and meet the needs of enterprises to develop high-value drugs containing the effective components of turmeric residue.

[0053] In the following embodiments of the present invention, the turmeric residue refers to the solid residue remaining after water extraction in the production of turmeric granules by pharmaceutical companies using turmeric raw materials, and is referred to as "turmeric residue," "residue," or "HR." For comparative studies, each embodiment also includes turmeric raw materials (referred to as "HM") as the research object and compares them with the turmeric residue. Unless otherwise specified, turmeric refers to curcumin compounds.

[0054] The experimental reagents used for biological sample analysis in the following embodiments of the present invention are shown in Table 1. All experimental instruments used are commercially available, and the English abbreviations used are commonly used abbreviations in this industry.

[0055] Table 1

[0056]

[0057] Basic Implementation

[0058] See Figures 1-20 The embodiments of this invention include two main parts: mutually supportive material research and pharmacological research. First, a mouse neuroinflammation model is constructed to explore the mechanism of action of turmeric on neuroinflammation. Then, modern analytical methods are used to analyze the main components of turmeric medicinal material and turmeric residue. Next, the mechanism of action and the effects of each main component in turmeric medicinal material and turmeric residue are analyzed to screen out components (or combinations of multiple components) with significant effects, providing a basis for the in-depth development and utilization of turmeric and turmeric-containing residue.

[0059] The method for constructing an animal model of neuroinflammatory intervention includes the following steps:

[0060] S1: Experimental reagents for preparing animal models

[0061] Five types of experimental reagents were prepared: turmeric raw material, turmeric residue, low-dose curcumin, high-dose curcumin, and donepezil tablet solution; the turmeric residue is the solid residue remaining after water extraction of turmeric raw material;

[0062] S2: Construct Aβ 1-42 Mouse models of induced neuroinflammation

[0063] Multiple male mice were randomly divided into four groups: sham-operated group, model group, positive control group, herbal medicine group, herbal residue group, low-dose curcumin group, and high-dose curcumin group. All groups were fed the same diet, and the behavior of the mice in each group was assessed.

[0064] For one week, mice in each group were administered five experimental drugs via gavage: turmeric root, turmeric residue, low-dose curcumin, high-dose curcumin, and donepezil tablets. Then, Aβ was injected into the mice's brains via targeted injection. 1-42 A neuroinflammation model was established, and then mice in each group were administered five types of experimental drugs by gavage for three consecutive weeks.

[0065] Behavioral tests were performed on mice in each group to analyze whether the experimental drugs used in each group of the animal model had the effect of reducing neuroinflammation in the mouse brain;

[0066] S3: To investigate the mechanism and effect of turmeric residue in intervening in neuroinflammation in mice.

[0067] The mice obtained in step S2 were prepared as biological samples. Non-targeted metabolomics techniques were used to analyze and investigate whether turmeric residue and curcumin could improve Aβ by regulating the TLR4 / NF-κB pathway. 1-42 The mechanism by which the neuroinflammation induction in mice induces cognitive impairment, reduces nerve damage, regulates overactivated microglia, downregulates inflammatory factors, and upregulates anti-inflammatory factors achieves the intervention effect of reducing neuroinflammation.

[0068] S4: Qualitative and quantitative determination of major curcuminoid compounds in turmeric residue

[0069] The main curcuminoid compounds in turmeric residue were qualitatively analyzed and quantitatively determined using UPLC-MS / MS and HPLC techniques, respectively.

[0070] S5: Testing the mechanism and effect of the main components of turmeric residue in intervening in the neuroinflammation of mice.

[0071] Using an animal model of neuroinflammation intervention, we tested the main curcuminoid compounds in turmeric residue to investigate the mechanism and effect of intervention on neuroinflammation in mice.

[0072] A method for screening effective components in turmeric residue for intervening in neuroinflammation, comprising the following steps: An animal model for neuroinflammation intervention constructed using the aforementioned method is used to screen for effective components in turmeric residue for intervening in neuroinflammation.

[0073] A1: Analysis of the main chemical components of turmeric residue

[0074] The main curcumin compounds in turmeric residue were qualitatively analyzed and quantitatively determined using UPLC-MS / MS and HPLC techniques, respectively, to preliminarily identify the active components to be tested.

[0075] A2: Screening of effective ingredients using animal models and mechanisms of neuroinflammatory intervention.

[0076] Using an animal model of neuroinflammation intervention, the intervention mechanism of the main chemical components in turmeric on neuroinflammation was utilized to test the intervention effects of each active ingredient on neuroinflammation.

[0077] A3: Comparative testing to determine whether there is a synergistic effect among the various active components in turmeric residue.

[0078] A comparative test was conducted using an animal model of neuroinflammatory intervention. The simultaneous and individual use of the tested active ingredients were compared to determine whether there was a synergistic effect of multiple compounds in turmeric residue when used simultaneously, and whether the intervention and treatment effect of multiple active ingredients used together on neuroinflammatory diseases such as Alzheimer's disease was more significant than that of individual use.

[0079] The following detailed description is provided in conjunction with the accompanying drawings and several specific embodiments.

[0080] Example 1

[0081] Referring to Figures 2-4, the method for constructing an animal model of neuroinflammatory intervention provided in this embodiment of the invention is a refinement and specific application based on the aforementioned basic embodiment. The difference lies in that the five types of experimental agents in step S1 are prepared using the following steps:

[0082] S1-1: Preparation of low-dose and high-dose solutions of curcumin reference standard

[0083] Weigh out curcumin reference standard, 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.

[0084] S1-2: Preparation of Turmeric Herb and Dregs Solution

[0085] 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 medicinal liquid in a water bath, completely evaporate the ethanol, and prepare medicinal material and medicinal residue solutions with concentrations of 0.1 g / mL and 0.12 g / mL respectively. Store at -20°C and sterilize at high temperature before use;

[0086] S1-3: Preparation of positive drug solution

[0087] Grind donepezil hydrochloride tablets (5 mg / tablet) into fine powder and dissolve in distilled water to obtain a positive drug solution with a concentration of 0.076 mg / mL.

[0088] See Figure 2 Schematic diagram of the animal experiment procedure. The specific steps of step S2 are as follows:

[0089] S2-1: Grouping of mice

[0090] Select multiple male mice. After 7 days of adaptive feeding, randomly divide them 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 at least 5 mice in each group, and feed them with the same feed; The specific experimental animals and feeding methods used are as follows:

[0091] Take 56 SPF-grade 2-month-old male C57BL / 6 mice, with a body weight of 18 - 22 g, purchased from the Guangdong Provincial Medical Experimental Animal Center, and its license number is SYXK (Guangdong) 2022-0002. All animals are raised in the Animal Center of Guangdong Pharmaceutical University, and its license number is SYXK (Guangdong) 2022-0125. They are allowed to eat and drink freely, the environmental temperature is 20 - 25°C, the humidity is 40 - 70%, and they are illuminated alternately for 12 h / 12 h every day. They are adaptively fed for 1 week;

[0092] S2-2: Administration to each group of mice before stereotaxic injection into the brain

[0093] Gavage each mouse in each group for one week. The specific drug administration for each group is as follows:<°

[0094] Sham operation group (Sham): C57BL / 6 mice injected with sterile normal saline into the bilateral hippocampal regions, gavage 0.1 mL / g distilled water;

[0095] Model group (Model): C57BL / 6 mice injected with Aβ 1-42 into the bilateral hippocampal regions, gavage 0.1 mL / 10 g distilled water;

[0096] Positive drug group (Donepezil): C57BL / 6 mice injected with Aβ 1-42C57BL / 6 mice were administered a dose of 0.76 mg / kg via gavage with 0.1 mL / 10 g donepezil hydrochloride solution.

[0097] Herbal materials (HM): Bilateral hippocampal injection of Aβ 1-42 C57BL / 6 mice were administered a dose of 1 g (crude drug) / kg via gavage with 0.1 mL / 10 g turmeric solution.

[0098] Herbal residues (HR): Aβ injected bilaterally into the hippocampus 1-42 C57BL / 6 mice were administered a dose of 1.2 g (crude drug) / kg via gavage with 0.1 mL / 10 g turmeric residue solution.

[0099] Low-dose group (L-CUR): Bilateral hippocampal injection of Aβ 1-42 C57BL / 6 mice were given a dose of 10 mg / kg via gavage with a low-dose solution of 0.1 mL / 10 g curcumin.

[0100] High-dose group (H-CUR): Bilateral hippocampal injection of Aβ 1-42 C57BL / 6 mice were administered a high-dose curcumin solution of 0.1 mL / 10 g via gavage at a dose of 20 mg / kg.

[0101] S2-3: Localized injection into the brain of mice to induce neuroinflammation.

[0102] Aβ 1-42 Solution treatment

[0103] Aβ 1-42 Dissolve in sterile physiological saline to prepare an Aβ1-42 injection solution with a concentration of 410 pmol / μL. Place the prepared solution in a 37℃ constant temperature incubator and incubate for 7 days before use.

[0104] After 7 days of acclimatization, mice were randomly divided into 7 groups. Mice were fasted for 12 hours before modeling (water was allowed during this period). For modeling, mice were anesthetized via intraperitoneal injection of 1% sodium pentobarbital solution (0.08 mL / 10 g). Gentle pinching of the tail and toes resulted in no obvious response, indicating the mice were under anesthesia. The mice were then fixed to a brain localization device, and the hair on their heads was removed. Erythromycin was thickly applied to both eyes to prevent damage from strong light. After disinfection with iodine tincture, the skin was longitudinally cut to expose the skull. The brain was then analyzed using a brain atlas (e.g., ...). Figure 3As shown in the figure, the injection site was selected as the bilateral hippocampal CA1 region (coordinates: AP: -0.22 cm, ML: ±0.2 cm, DV: -0.2 cm). Aβ was aspirated using a microsyringe. 1-42 Solution, Aβ from one side of the brain 1-42 The solution volume was 1.5 μL, the infusion time was 10 min, the needle was left in place for 15 min, and the needle was slowly withdrawn over 5 min. The wound was then sutured and disinfected. The same procedure was performed on the other side of the brain. The sham surgery group received an equal volume of sterile saline.

[0105] On day 8 of the experiment, based on the mouse brain atlas, mice in each group underwent targeted brain injection. The injection site was selected as the bilateral CA1 region of the hippocampus (coordinates: AP: -0.22 cm, ML: ±0.2 cm, DV: -0.2 cm). Aβ was aspirated using a microsyringe. 1-42 Solution, Aβ from one side of the brain 1-42 The solution volume was 1.5 μL, the infusion time was 10 min, the needle was left in place for 15 min, and the needle was slowly withdrawn over 5 min. The wound was then sutured and disinfected. The same procedure was performed on the other side of the brain. The sham-operated group was injected with an equal volume of sterile saline, and then the mice were allowed to recover naturally for 2 days.

[0106] S2-4: Grouping and administration of drugs after localized brain injection in mice

[0107] Starting from day 10 of the experiment, mice in each group were administered the same grouping and dosage method as in step S2-2 by gavage for three weeks; the overall process is shown in Figure 2.

[0108] Sham-operated group: C57BL / 6 mice that were injected with sterile saline into both hippocampi were then gavaged with 0.1 mL / g distilled water.

[0109] Model group: Bilateral hippocampal injection of Aβ 1-42 C57BL / 6 mice were administered 0.1 mL / 10 g distilled water by gavage.

[0110] Positive drug group: bilateral hippocampal injection of Aβ 1-42 C57BL / 6 mice were administered a dose of 0.76 mg / kg via gavage with 0.1 mL / 10 g donepezil hydrochloride solution.

[0111] Herbal medicine group: Aβ injected into both hippocampal regions 1-42 C57BL / 6 mice were administered a dose of 1 g (crude drug) / kg via gavage with 0.1 mL / 10 g turmeric solution.

[0112] Residue group: Aβ injected bilaterally into the hippocampus 1-42C57BL / 6 mice were administered a dose of 1.2 g (crude drug) / kg via gavage with 0.1 mL / 10 g turmeric residue solution.

[0113] Low-dose group: Bilateral hippocampal injection of Aβ 1-42 C57BL / 6 mice were administered a low-dose curcumin solution of 0.1 mL / 10 g via gavage at a dose of 10 mg / kg.

[0114] High-dose group: Bilateral hippocampal injection of Aβ 1-42 C57BL / 6 mice were administered a high-dose curcumin solution of 0.1 mL / 10 g via gavage at a dose of 20 mg / kg.

[0115] Then, animal behavioral experiments were conducted on mice in each group, and the experimental results were analyzed to investigate whether turmeric, turmeric residue, and curcumin in the model group could reduce neuroinflammation in the mouse brain.

[0116] The animal behavioral experiments in steps S2-5 specifically involve the Morris water maze experiment, which includes a navigation experiment and a spatial exploration experiment. The water maze experiment begins on day 25 after drug administration, with the navigation experiment conducted from days 25 to 29 and the spatial exploration experiment on day 30. The experiment includes the following steps:

[0117] S2-5-1: Positioning and Navigation Experiment

[0118] Mice were placed into the water from the midpoint of the pool walls in four different quadrants, and the software counted down for 90 seconds. The time from entry into the water to successfully finding the water platform and remaining there for more than 10 seconds was recorded as the escape latency. If the mouse could not find the water platform within 90 seconds, its escape latency was 90 seconds. After 90 seconds, the experimenter guided the mouse to the water platform and had it stay there for 10 seconds. The escape latency time of the mice was recorded for five days. The experimental results of the escape latency time in the navigation experiment are shown in Figure 4(A).

[0119] S2-5-2: Space Exploration Experiment

[0120] 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 quadrant where the platform was located. 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 the mice 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).

[0121] S2-5-3: Analysis of Experimental Results

[0122] Analysis of the Morris water maze test results indicates that injection of Aβ... 1-42 It can induce neuroinflammation in the brain of mice, prolong the escape latency, reduce the number of platform crossings, and reduce the time spent in the target quadrant. In contrast, the drug-treated model group can shorten the escape latency, increase the number of platform crossings, and increase the time spent in the target quadrant, indicating that turmeric, turmeric residue, and curcumin can all reduce neuroinflammation in the brain of mice.

[0123] The experimental results are shown in Figures 4(A)-4(D). The results were analyzed to determine whether the turmeric, turmeric residue, and curcumin in the turmeric herb group, turmeric residue group, low-dose curcumin group, and high-dose curcumin group could alleviate neuroinflammation in the mouse brain to different degrees. In the figures, ##: p < 0.01, compared with the sham-operated group; *: p < 0.05; **: p < 0.01, compared with the model group.

[0124] In this embodiment, behavioral tests were performed on mice in each group to analyze the experimental drugs Huangyao, turmeric residue, and curcumin used in each drug administration group in the animal model. All of them had the effect of reducing neuroinflammation in the mouse brain.

[0125] Example 2

[0126] See attached Figure 4- Figure 20 In this embodiment, based on the biological samples obtained in step S2, mice from each group were prepared as biological samples. Non-targeted metabolomics technology was then used to explore the mechanism and effect of turmeric residue and curcumin in intervening in mice with neuroinflammation. The study analyzed whether turmeric residue and curcumin could improve Aβ by regulating the TLR4 / NF-κB pathway. 1-42 The mechanism by which turmeric induces cognitive impairment in mice with neuroinflammation, reduces nerve damage, regulates overactivated microglia, downregulates inflammatory factors, and upregulates anti-inflammatory factors, thereby reducing neuroinflammation, was investigated. As a comparison, this example also tested the mechanism and effect of turmeric herb and turmeric residue in intervening in mouse neuroinflammation.

[0127] The method for constructing an animal model of neuroinflammation intervention provided in this invention is a further specific application based on the aforementioned basic embodiments and Example 1. Step S3 analyzes the mechanism of turmeric intervention in mouse neuroinflammation, elucidating how turmeric, the effective component in turmeric medicinal material and turmeric residue, alleviates Aβ. 1-42 The mechanism of action in inducing neuroinflammation includes the following steps:

[0128] S3-1 Analyzes the data from the Morris water maze experiment to observe the effect of turmeric on Aβ. 1-42 Effects of damage on spatial learning and memory abilities in mice

[0129] First, investigate the effect of turmeric on Aβ. 1-42 The impact of impaired spatial learning and memory abilities in mice.

[0130] Based on the results of the Morris water maze experiment conducted in Example 1, changes in the animals' spatial memory and learning abilities were observed. The Morris water maze navigation experiment data (see Table 6-1, changes in escape latency during the navigation experiment) were compiled. (x̅) ± s, n=8 Compared with the sham-operated group, 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 of training days, the escape latency of the positive drug group, the medicinal material group, the low-dose group, and the high-dose group mice was significantly shortened (all p < 0.01) (as shown in Figure 4(A)). Starting from the 4th day of the experiment, the latency of the medicinal residue group mice to find the platform was significantly shortened (p < 0.01) (see Table 6-1). According to the spatial exploration experiment data (Table 6-1, Figure 4(BD)), compared with the sham-operated group mice, the number of times the model group mice crossed the platform and the time spent in the target quadrant were significantly reduced (all p < 0.01). Compared with the model group, the number of times the medicinal residue group mice crossed the platform showed an increasing trend but was not significant (p > 0.05), while the number of times the other drug-treated groups crossed the platform and the time spent in the target quadrant were significantly increased (all p < 0.05). In conclusion, turmeric can improve Aβ. 1-42 Impaired spatial learning and memory abilities in mice. In Figures 4(A)–4(D), ##：p＜0.01 Compared with the sham surgery group; *：p＜0.05 , **：p＜ 0.01 , compared with the model group.

[0131] Table 6-1

[0132]

[0133] Note: ##: p < 0.01, compared with the sham surgery group; **: p < 0.01, compared with the model group.

[0134] S3-2: Perform histopathological examination

[0135] The pathological changes of neurons in the hippocampus were observed using HE staining (Hematoxylin-eosin staining, HE) and Nissl staining, respectively.

[0136] 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. Immunohistochemistry (IHC) was then used to observe the changes in Iba-1 in the CA1 region of the hippocampus. 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 content of important channel proteins and cytokines in the TLR4 / NF-κB signaling pathway.

[0137] Analysis of experimental mice using HE staining, Nissl staining, immunohistochemistry, immunofluorescence staining, real-time quantitative PCR, and ELISA revealed that turmeric, turmeric residue, and curcumin in the turmeric herb group, turmeric residue group, low-dose curcumin group, and high-dose curcumin group all reduced neuroinflammation in the mouse brain to varying degrees.

[0138] S3-2-1: HE staining

[0139] (1) Collection of materials

[0140] The entire brain of the mouse was removed and placed in tissue fixative. Blood was stored at 4°C, allowed to stand for 2 hours, centrifuged for 20 minutes (3,000 ×g, 4°C), and the supernatant was collected, aliquoted, and stored at -80°C.

[0141] (2) Slicing

[0142] After fixing the tissue for 24 h, it was dehydrated with a gradient of alcohols (70% ethanol-80% ethanol-95% ethanol-anhydrous ethanol), cleared with xylene, and embedded in paraffin. The paraffin block was placed on a microtome and cut into 6 μm sections. The sections were spread in warm water, transferred to glass slides, and dried at 70 °C.

[0143] (3) Dewaxing

[0144] The sections were then immersed in xylene for 20 min, repeated twice; then in anhydrous ethanol for 5 min, repeated twice; finally, in 75% ethanol for 5 min; and rinsed with tap water. After dewaxing, the sections were stained in different ways.

[0145] (4) Staining

[0146] Follow the instructions to operate, and after mounting the slide, observe the histopathological changes under an optical microscope.

[0147] S3-2-2: Nissl staining

[0148] Samples were processed using the same steps as in (1)-(3) of S3-2-1; after adding toluidine blue staining solution 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 dried in an oven. The sections were cleared with benzoyl peroxide for 10 min and then mounted with neutral resin.

[0149] S3-2-3 Immunohistochemical analysis was performed.

[0150] The specific steps for sample processing are the same as those in (1)-(3) of S3-2-1.

[0151] (4) Antigen retrieval: Place the slide rack in a beaker containing antigen retrieval solution, put it in a pressure cooker, and heat continuously for 15 minutes. Do not dry the slides. Allow it to cool naturally to room temperature, then remove the slide rack and soak it in PBS (pH 7.4) three times, 5 minutes each time.

[0152] (5) Blocking endogenous peroxidase: Place in 3% hydrogen peroxide solution and incubate at room temperature for 25 min, taking care to avoid light. Place the slide in PBS (pH 7.4) and wash it 3 times on a shaker for 5 min each time.

[0153] (6) Serum blocking: Add 3% BSA to the histochemistry zone to cover the tissue and block for 30 min;

[0154] (7) Add primary antibody: Discard the blocking solution, add primary antibody Iba-1 (1:1000) to the slide, and incubate at 4℃.

[0155] (8) Add secondary antibody: Place the slide in PBS (pH 7.4) for decolorization, and wash three times on a shaker for 5 min each time. Shake the slide dry, add goat anti-rabbit for labeling, and incubate at room temperature for 50 min;

[0156] (9) Color development: Place the slide in PBS (pH 7.4) to decolorize, and wash 3 times on a shaker for 5 minutes each time. Shake off the water from the slide, add DAB color development solution, control the color development time under a microscope, and rinse with tap water until the color development is stopped;

[0157] (10) Counterstaining cell nuclei: Counterstain with hematoxylin for 3 min, rinse with tap water, differentiate with hematoxylin for a few seconds, rinse with tap water, use hematoxylin blue solution for blueing, and rinse with tap water.

[0158] (11) Dehydration and mounting: Place the slides in a gradient of alcohol (75% ethanol-85% ethanol-anhydrous ethanol twice) to dehydrate and clear them, each time for 5 min, then place them in xylene for 5 min, take them out and air dry them, and then mount them.

[0159] (12) Image J was used to quantitatively analyze the positive area and positive cell density of Iba-1.

[0160] S3-2-4 was subjected to immunofluorescence staining.

[0161] The specific steps for sample processing are the same as those in (1)-(6) of S3-2-3;

[0162] (7) Add the first primary antibody: Remove the blocking solution, add the prepared Iba-1 (1:1000), and incubate in a humidified chamber at 4°C overnight;

[0163] (8) Add secondary antibody: The steps are the same as in (8) of S3-2-3;

[0164] (9) Add the corresponding TSA: Wash the slide three times with PBS (pH 7.4) on a decolorizing shaker, 5 min each time. Add the corresponding type of TSA, incubate in the dark for 10 min, and then wash three times with TBST on a decolorizing shaker, 5 min each time;

[0165] (10) Heating: Place the slices in the repair box and microwave on medium for 8 min - stop for 8 min - low for 7 min;

[0166] (11) Add a second primary antibody: Add Aβ dropwise 1-42 Antibody (1:200), incubated overnight at 4°C in a humidified chamber;

[0167] (12) Add secondary antibody: Same as step (8) above;

[0168] (13) Add the corresponding TSA: Same as step (9) above;

[0169] (14) Heating: Same as step (10) above;

[0170] (15) DAPI counterstaining: Add DAPI staining solution to the circle to counterstain the cell nuclei, and incubate at room temperature in the dark for 10 min;

[0171] (16) Quenching of tissue autofluorescence: The slide was placed in PBS (pH 7.4) and washed 3 times on a shaker for 5 min each time. Autofluorescence quencher was added for 5 min, and the slide was rinsed with running water for 10 min.

[0172] (17) Mounting: Mounting anti-fluorescence quenching tablets;

[0173] (18) Take pictures under a microscope.

[0174] S3-2-5: Quantitative PCR was used to determine the mRNA expression of important channel proteins and cytokines in the TLR4 / NF-κB signaling pathway; real-time quantitative PCR was performed, including the following steps:

[0175] (1) RNA extraction

[0176] Approximately 50 mg of brain tissue was placed in a centrifuge tube, a steel ball was added, and 1 mL of RNA isolater solution was added. The mixture was homogenized until powdered. 200 μL of chloroform was added to the lysis buffer, and the mixture was vigorously vortexed to form an emulsion. The emulsion was centrifuged for 15 min (12,000 × g, 4 °C). 400 μL of the supernatant was collected, and an equal volume of pre-cooled isopropanol was added. The mixture was mixed and centrifuged for 10 min (12,000 × g, 4 °C). The supernatant was discarded, and 1 mL of 75% ethanol (prepared with DEPC water) was added. The bottom of the tube was gently blown with a pipette tip to suspend the white precipitate. The tube was centrifuged for 5 min (12,000 × g, 4 °C), and this process was repeated twice. The centrifuge tube was left open for 5 min to allow the white precipitate to dry. 20 μL of sterile, enzyme-free water was added to dissolve the precipitate. 1.5 μL of the sample solution was analyzed for purity and concentration using a micro-ultraviolet analyzer. A purity OD260 / OD280 ratio between 1.9 and 2.1 indicates high RNA purity.

[0177] (2) Reverse transcription

[0178] Using the aforementioned RNA as a template, a reverse transcription reaction system was prepared according to the instructions to synthesize a cDNA template.

[0179] (3) PCR fluorescence quantitative quantification

[0180] The reaction conditions were as follows: pre-denaturation: 95℃ for 30 sec; cycling reaction: 95℃ for 10 sec, 60℃ for 30 sec, 40 cycles; melting curve: 65℃ for 5 sec, 95℃ for 5 sec, 40 cycles. Each sample was repeated three times, and quantification was performed using a real-time PCR instrument.

[0181] S3-2-6: The levels of important channel proteins and cytokines in the TLR4 / NF-κB signaling pathway were determined by enzyme-linked immunosorbent assay (ELISA).

[0182] Take an appropriate amount of brain tissue, add a certain amount of PBS (pH 7.4), homogenize, centrifuge for 10 min (4℃, 5,000 g), collect the supernatant, aliquot it, use one part as the sample to be tested, and store the rest at -80℃. Follow the instructions for operation; zero the blank well and measure the absorbance of each well at a wavelength of 450 nm.

[0183] S3-2-7 Statistical processing of experimental data

[0184] GraphPad Prism 9.4 software was used for analysis. All data are expressed as mean ± standard deviation (x̅ ± s). In the Morris water maze experiment, the escape latency was analyzed using two-way AVOVA, while other inter-group analyses used one-way ANOVA. Multiple comparisons were performed using the Turkey method. p < 0.05 was considered statistically significant between groups.

[0185] S3-3 conducts non-targeted metabolomics research

[0186] To conduct a non-targeted metabolomics study on turmeric and its residue in AD mice, plasma and brain tissue samples were prepared from various experimental mice and detected using chromatography and mass spectrometry. Then, the raw non-targeted metabolomics data of the serum samples from each group of rats were analyzed using non-targeted serum metabolomics technology.

[0187] S3-3-1 Preparation of Plasma and Brain Tissue Samples

[0188] (1) Processing of plasma samples: Take each plasma sample out of the -80℃ freezer according to the number, thaw it on ice until it is completely liquid, vortex mix for 30 s, at 4 ℃, and aspirate 40 μL into a 1.5 mL EP tube; add 200 μL of pre-cooled (-30℃) precipitant (acetonitrile:methanol=1:1), vortex for 5 min, and let stand in the -30℃ freezer for 20 min; centrifuge at 13000 rpm for 10 min at 4 ℃, and transfer 150 μL of the supernatant into the inner liner tube of the corresponding sample bottle for instrumental analysis.

[0189] (2) Brain tissue sample processing method: After thawing the brain tissue samples on ice after removing them from the -80℃ freezer, accurately weigh 50mg of tissue using a 0.01% balance and place them into homogenization tubes according to their numbers. Add pre-cooled (-30℃) extraction buffer (methanol:acetonitrile:water = 2:2:1) and 8mm magnetic beads to the homogenization tubes at a volume ratio of 1:10 (v / v). Homogenize for 5min in a cryogenic homogenizer, then centrifuge at 13000rpm for 10min at 4℃. After centrifugation, transfer the supernatant to another set of 5mL EP tubes with corresponding numbers, add the extraction buffer again for a second extraction, centrifuge at 13000rpm for 10min at 4℃, and combine the two supernatants. The supernatant is then purified using N2O. 2 After drying, add the reconstitution solution (80% methanol solution) at a volume ratio of 5:1 (mg / v), let stand in a -30 ℃ refrigerator for 30 min, vortex for 5 min, centrifuge at 13000 rpm for 10 min at 4 ℃, and transfer 180 μL of the supernatant into the inner liner tube of the corresponding numbered sample vial for instrumental analysis (the entire preparation process is carried out on ice).

[0190] (3) QC sample preparation: 10 μL of supernatant was taken from each sample of plasma and brain tissue and mixed. The mixture was vortexed for 30 s to prepare QC samples.

[0191] S3-3-2 Chromatographic and Mass Spectrometric Conditions for Plasma and Brain Tissue

[0192] (1) Blood plasma chromatography conditions: Ultimate 3000 chromatography system, using Waters ACQUITY UPLC HSS T3C18 (2.1 mm*100 mm, 1.8 μm) column, autosampler temperature 8 ℃, flow rate 0.3 mL / min, column temperature 40℃, 1 μL of sample injected for gradient elution, mobile phase 0.1% formic acid water (A) — 0.1% formic acid acetonitrile (D).

[0193] (2) Chromatographic conditions for brain tissue: The chromatographic system was Ultimate 3000, and a Waters ACQUITY UPLC HSST3 C18 (2.1 mm*100 mm, 1.8 μm) column was used. The autosampler temperature was set to 8 ℃, the flow rate was 0.3 mL / min, the column temperature was 40 ℃, and 2 μL of sample was injected for gradient elution. The mobile phase was 2 mm ammonium formate-0.1% formic acid water (A)-acetonitrile (D).

[0194] (3) Mass spectrometry conditions for plasma and brain tissue: Thermo Q Exactive Focus mass spectrometry system, ionization mode: ESI- / ESI+.

[0195] S3-3-3 Targeted determination of characteristic biomarkers - amino acids

[0196] Untargeted serum metabolomics was used to analyze the raw untargeted metabolomics data of rat serum samples from each group. Progenesis QI 3.0 and Compound Discoverer 3.3 software were used to process and analyze the raw untargeted metabolomics data, performing multivariate statistical analysis and identifying differentially expressed metabolites. Differentially expressed metabolites were screened based on a VIP (Variable Importance in the Projection) > 1, Max fold change > 2, and ANOVA P-value < 0.05. The chemical structures of these differentially expressed metabolites were identified by comparison with databases such as HMDB, m / z Cloud, and PubChem. Finally, the differentially expressed metabolites were imported into MetaboAnalyst 5.0 online analysis software for pathway enrichment, and data for targeted assays of characteristic biomarkers—amino acids—were obtained.

[0197] The research results of this embodiment include:

[0198] (a) Turmeric's effect on Aβ 1-42 Effects of inducing pathological changes and neuronal damage

[0199] (1) HE staining analysis

[0200] HE staining, used in this embodiment, is one of the commonly used staining methods in histology, providing key 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 group mice were neatly and tightly arranged, with clear cell membranes and nuclei; however, the hippocampus of the model group mice showed interstitial edema, cell deformation and deterioration, and the normal morphology and arrangement of neurons were disrupted by Aβ. 1-42Damage was observed, and the cell nuclei darkened in color (as indicated by the white arrow in Figure 5(1)). Compared with the model group mice, the pathological changes in the treated group mice were improved, most nerve cells were neatly arranged, and the normal cell shape and morphology were restored, with clear cell nuclei. In summary, turmeric alleviates Aβ. 1-42 Induced pathological changes.

[0201] (2) Nissl staining analysis

[0202] Nissl bodies are subcellular structures found in the body and dendrites of nerve cells, composed of granular endoplasmic reticulum and ribosomes. The reticulum is present throughout the nerve cell, but is more densely packed in the Nissl body region than in the rest of the cell. Nissl bodies are relatively dispersed in neurons, loosely contacting the outer edge of the endoplasmic reticulum or arranged within it. The main functions of Nissl bodies are protein synthesis, the protein structure required for organelle renewal, and the synthesis of enzymes and neurotransmitters required for neurotransmitter synthesis. As the functional state of motor neurons changes, their number, size, and arrangement also change. The neuronal state can be assessed by these small particles in different metabolic or disease states of neurons. Nissl staining showed that the neurons in the CA1 region of the hippocampus of sham-operated mice were neatly and densely arranged, with prominent nucleoli in the intercytoplasm and abundant Nissl bodies in the cytoplasm (as shown by the black arrow in Figure 6(1)). Compared with the sham-operated group mice, the model group mice showed loosely arranged cells, increased gaps, incomplete cell structure, missing nucleoli in the cytoplasm, deeply stained cytoplasm, and reduced or absent Nissl bodies in this region (as shown by the red arrows in Figure 6(1)). After drug administration, the integrity of cell structure improved, the nucleoli became more obvious and clearly visible, and the number of Nissl bodies increased. In summary, the experimental results indicate that turmeric can alleviate Aβ. 1-42 The resulting neuronal damage and loss.

[0203] (ii) Turmeric's effect on Aβ 1-42 Effects of induced overactivation of microglia

[0204] Iba-1 protein is a member of the calcium-binding proteome and is considered a marker of microglia. Microglia activate Iba-1 protein production under normal physiological or pathological conditions. Experiments were conducted on Iba-1 and Aβ... 1-42 Perform immunofluorescence co-labeling (e.g.) Figure 7 The results showed that Aβ 1-42 After injection, the expression of Iba-1 in the brain of mice was enhanced, accompanied by Aβ. 1-42 Enhanced positive expression of Iba-1. Under normal conditions, microglia in sham-operated mice produce little or no Iba-1 (as shown by the black arrow in Figure 8(A-1)); as shown in Figures 8(A)-8(C), compared with the sham-operated group, the expression of Iba-1 protein in the CA1 region of the hippocampus in the model group mice was significantly increased (as shown by the red arrow in Figure 8(A-1)).p <0.01), indicating injection of Aβ 1-42 Stimulated microglia to transform into an overactivated state; compared with the model group, Iba-1 protein expression was significantly decreased in mice in each treatment group (all p <0.01). Therefore, turmeric can reduce Aβ. 1-42 Induced Iba-1 proliferation, thereby regulating the over-activation state of microglia.

[0205] (III) Effects of Curcuma longa on the TLR4 / NF-κB signaling pathway

[0206] (1) Effects of turmeric on mRNA expression of related proteins in the TLR4 / NF-κB signaling pathway

[0207] The results are shown in Figure 9. Figure 9 shows the level of mRNA in important channel proteins in the TLR4 / NF-κB pathway regulated by turmeric ( 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 figures, ##: p <0.01, compared with the sham surgery group; *: p <0.05, **: p<0.01, compared with the model group.

[0208] This shows that, compared with the sham-operated group, the mRNA expression of IκB, p65, and TLR4 in the brain tissue of the model group mice was significantly increased (all... p <0.01); compared with the model group, the mRNA levels of p65 and TLR4 in the drug-treated mice were significantly decreased (both <0.01). p <0.01); Compared with the model group, although the mRNA level of IκB in mice decreased after drug administration, there was no significant difference ( p >0.05). The above results indicate that turmeric can regulate Aβ. 1-42 Induced expression levels of important channel proteins mRNA.

[0209] In addition to studying the effects of turmeric on the mRNA expression levels of important proteins, this invention also measures the mRNA levels of inflammatory and anti-inflammatory factors. Figure 10 (AH) shows the cytokine mRNA levels regulated by turmeric in the TLR4 / NF-κB pathway. x̅ ± s , n=3); In the subplots of Figure 10, Figure 10(A) shows the mRNA expression level of IL-6; Figure 10(B) shows the mRNA expression level of TNF-α; Figure 10(C) shows the mRNA expression level of iNOS; Figure 10(D) shows the mRNA expression level of COX-2; Figure 10(E) shows the mRNA expression level of IL-4; Figure 10(F) shows the mRNA expression level of IL-10; Figure 10(G) shows the mRNA expression level of TGF-1β; Figure 10(H) shows the mRNA expression level of Arg-1; In the figures, ##: p <0.01, compared with the sham surgery group; *: p <0.05, **: p<0.01, compared with the model group.

[0210] As shown in Figures 10(A)-10(D), compared with the sham surgery group, injection of Aβ... 1-42 The mRNA levels of IL-6, TNF-α, iNOS, and COX-2 in the mouse brain were significantly increased (all... p <0.01); the drug-treated group effectively inhibited brain-targeted injection of Aβ. 1-42 It induces the expression of mRNAs of IL-6, TNF-α, iNOS, and COX-2 (all of which are induced). p <0.01).

[0211] As shown in Figures 10(E)-10(H), compared with the sham-operated group, the mRNA levels of anti-inflammatory factors IL-4, IL-10, TGF-1β, and Arg-1 in the model group mice were significantly decreased (all... p <0.01); compared with the model group, the mRNA expression levels of IL-4, IL-10, TGF-1β and Arg-1 in mice in the positive drug group and the herbal medicine group were significantly increased (all <0.01). p <0.01); The mRNA expression levels of IL-4, TGF-1β, and Arg-1 were increased in mice in the residue group (all <0.01). p <0.05), but there was no significant difference in IL-10 mRNA expression levels between the model group and the model group. p >0.05); compared with the model group, the low-dose curcumin group significantly increased the mRNA expression level of IL-10 ( p <0.01), with no significant effect on the mRNA expression levels of other anti-inflammatory factors ( p >0.05), while the high-dose curcumin group showed a significant increase in the mRNA expression of IL-4 and IL-10 (both >0.05). p <0.05), with no significant effect on TGF-1β and Arg-1 ( p >0.05). The above results indicate that turmeric can regulate Aβ. 1-42 Induced inflammation and the mRNA expression levels of anti-inflammatory factors.

[0212] (2) Effects of turmeric on the expression of related proteins in the TLR4 / NF-κB signaling pathway

[0213] Effects 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 the subfigures 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 figures, ##: p < 0.01, compared with the sham-operated group; *: p < 0.05, **: p < 0.01, compared with the model group.

[0214] Compared with the sham-operated group, the protein levels of p-IKK / IKK, p-IκB / IκB, p-p65 / p65, and TLR4 in the brain tissue of mice in the model group were significantly increased (all... 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 mice were significantly decreased (all <0.01). p <0.05); Compared with the model group, after administration of turmeric, turmeric residue, and low and high doses of curcumin, the protein levels of p-IκB / IκB, p-p65 / p65, and TLR4 in mice were significantly decreased ( p <0.01); High-dose curcumin can significantly decrease p-IKK / IKK protein content ( p <0.01), low doses of turmeric, turmeric residue, and 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 key channel proteins.

[0215] In addition to studying the effects of turmeric on the expression levels of important channel proteins, this invention also measured the protein expression levels of inflammatory and anti-inflammatory factors in plasma, as shown in Figure 12 (AH). Figures 12 (A)-12 (H) illustrate the regulation of cytokine protein expression levels in the TLR4 / NF-κB pathway by turmeric. x̅ ± s , n=3). In the subplots of Figure 12, Figure 12(A) shows the protein expression level of IL-6; Figure 12(B) shows the protein expression level of TNF-α; Figure 12(C) shows the protein expression level of iNOS; Figure 12(D) shows the protein expression level of COX-2; Figure 12(E) shows the protein expression level of IL-4; Figure 12(F) shows the protein expression level of IL-10. Figure 12G Figure 12(H) shows the protein expression level of TGF-1β; Figure 12(H) shows the protein expression level of Arg-1; in the figure, ##: p <0.01, compared with the sham surgery group; *: p <0.05, **: p<0.01, compared with the model group.

[0216] Compared with the sham surgery group, injection of Aβ 1-42 The mRNA levels of IL-6, TNF-α, iNOS, and COX-2 in the mouse brain were significantly increased (all... p <0.01); the positive control group, the herbal medicine group, and the high-dose curcumin group effectively inhibited brain-targeted injection of Aβ. 1-42 This caused an increase in the protein expression levels of IL-6, TNF-α, iNOS, and COX-2 (all of which...). p <0.01), the low-dose group effectively reduced the protein expression levels of IL-6, TNF-α and COX-2 (all <0.01). p <0.01), the residue group had no significant effect on the expression level of anti-inflammatory factor proteins (both <0.01). p >0.05).

[0217] See Figure 12 (EH). Compared with the sham-operated 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 <0.01), Arg-1 protein expression level showed no significant change ( p >0.05); compared with the model group, the protein expression levels of IL-4, IL-10, and TGF-1β in mice in the positive drug group, low-dose curcumin group, and high-dose curcumin group were significantly increased (all >0.05). p <0.05), the expression levels of IL-4 and TGF-1β proteins in mice in both the herbal medicine group and the dregs group were significantly increased (both <0.05). 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 model group. p >0.05). The above results indicate that turmeric can regulate Aβ. 1-42 Induced inflammation and the expression levels of anti-inflammatory factor proteins.

[0218] The embodiments of this invention have shown that turmeric alleviates Aβ by regulating the TLR4 / NF-κB signaling pathway.1-42 The biological mechanisms of inducing neuroinflammation are described in [link to relevant document]. Figure 13 .

[0219] (iv) Results of non-targeted metabolomics study on the effects of turmeric and its residue on AD mice

[0220] 1. Metabolic profiling of plasma and brain tissue and its quality control evaluation

[0221] The superimposed total ion chromatograms of quality control samples in positive and negative ion modes of plasma and brain tissue are shown in Figure 14 (including sub-plots AD). In the total ion chromatograms of Figure 14, Figure 14(A) is the total ion chromatogram of plasma in positive ion mode; Figure 14(B) is the total ion chromatogram of plasma in negative ion mode; Figure 14(C) is the total ion chromatogram of brain tissue in positive ion mode; and Figure 14(D) is the total ion chromatogram of brain tissue in negative ion mode.

[0222] The results showed that the quality control samples exhibited good repeatability and the entire detection process was stable. Furthermore, five representative endogenous metabolites from plasma and brain tissue were selected for stability studies under both positive and negative ion modes. 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 indicated that the RSD of the peak area for the six components was less than 30%, indicating reliable data quality that could be used for subsequent statistical analysis.

[0223] Table 7-1

[0224]

[0225] Table 7-2

[0226]

[0227] 2. Results of multivariate statistical analysis for visualization of plasma and brain tissue

[0228] (1) As shown in Figures 15 and 16, PLS-DA diagrams of plasma and brain tissue are respectively.

[0229] Figure 15 (including sub-figures A and B) shows the spatial distribution visualization results of plasma non-targeted metabolomics data. In each sub-figure, Figure 15(A) is the PLS-DA score map in the positive ion mode; Figure 15(B) is the PLS-DA score map in the negative ion mode. In the figure, C: sham surgery group; M: model group; D: positive drug group.

[0230] Figure 16 (including sub-figures A and B) shows the spatial distribution visualization results of non-targeted metabolomics data in brain tissue. In each sub-figure, Figure 16(A) is the PLS-DA score map in the positive ion mode; Figure 16(B) is the PLS-DA score map in the negative ion mode. C: sham surgery group; M: model group; D: positive drug group; L: low-dose curcumin group; H: high-dose curcumin group; YZ: turmeric residue group.

[0231] (2) As shown in Figures 17 and 18, the OPLS-DA and VIP diagrams represent plasma and brain tissue, respectively.

[0232] Figure 17 (including sub-figure AD) shows the spatial distribution of non-targeted metabolomics data of plasma in the sham surgery group and the model group in the embodiments of the present invention; wherein, Figure 17(A) is the OPLS-DA score map of positive ion mode; Figure 17(B) is the OPLS-DA score map of negative ion mode; Figure 17(C) is the VIP map of positive ion mode; Figure 17(D) is the VIP map of negative ion mode.

[0233] Figure 18 (including sub-figure AD) shows the spatial distribution of non-targeted metabolomics data of brain tissue in the sham surgery group and the model group in the embodiments of the present invention; wherein, Figure 18(A) is the OPLS-DA score map of positive ion mode; Figure 18(B) is the OPLS-DA score map of negative ion mode; Figure 18(C) is the VIP map of positive ion mode; Figure 18(D) is the VIP map of negative ion mode.

[0234] 3. Screening and identification of differential metabolites in plasma and brain tissue

[0235] To screen and identify endogenous metabolites related to the therapeutic effect of turmeric on Alzheimer's disease (AD) mice, an OPLS-DA model was used in both positive and negative ion modes for the sham-operated group and the model group, as shown in Figures 17 and 18 above, to identify characteristic differential metabolite groups distinguishing normal mice from AD mice. After OPLS-DA analysis, the importance of variables to the classification was measured by their VIP values, and variables were screened based on their VIP values. Variables with VIP ≥ 1, Max fold change > 2, and ANOVA p-value < 0.05 were considered to have a significant contribution to the model.

[0236] A total of 218 differential metabolites (163 positive ions and 55 negative ions) were screened from plasma. The chemical structures of the differential metabolites were identified by comparing them with the HMDB database, m / z Cloud database, and ChemSpider database using CompoundDiscoverer 3.3 software. 24 characteristic differential metabolites (20 positive ions and 4 negative ions) were identified, as shown in Table 7-3.

[0237] Table 7-3

[0238]

[0239] A total of 667 differential metabolites (428 positive ions and 239 negative ions) were screened from brain tissue. The chemical structures of these differential metabolites were identified using CompoundDiscoverer 3.3 software and compared with the HMDB, m / z Cloud, and ChemSpider databases. 32 characteristic differential metabolites (32 positive ions and 12 negative ions) were identified, as shown in Table 7-4.

[0240] Table 7-4

[0241]

[0242] 4. Analysis of metabolic pathways in plasma and brain tissue

[0243] Metabolic pathways were analyzed based on the KEGG database. Metabolic pathways were selected as potential target pathways based on a p-value less than 0.05 and an impact value greater than 0. Three potential metabolic pathways were enriched in plasma, as shown in Table 7-5 (Results of Plasma KEGG Enrichment of Metabolic Pathways). Figure 19 As shown in Table 7-6, eight potential metabolic pathways were enriched in brain tissue. Figure 20 As shown.

[0244] Table 7-5

[0245]

[0246] Table 7-6

[0247]

[0248] The above experimental results show that the arginine succinate level in the model group mice showed a decreasing trend, which may be caused by abnormal glutamate metabolism. However, the low-dose turmeric group, the turmeric residue group, and the donepezil group did not show a recovery trend, while the high-dose turmeric group successfully recovered. It is speculated that abnormalities in aspartate metabolism, glutamate metabolism, arginine biosynthesis pathways, and Aβ are involved. 1-42 Multiple complex regulatory mechanisms were observed in mice with induced neuroinflammation. The results showed that different groups of turmeric could regulate amino acid metabolism to varying degrees, thereby achieving an improvement effect. Regulating amino acid metabolism may be a therapeutic approach for Aβ. 1-42 The potential mechanisms of induced neuroinflammation.

[0249] The experiments in this embodiment detected varying degrees of regulatory trends in adenine, choline phosphate, adenosine, hypoxanthine, guanine, guanosine, uracil, thymine, dihydrothymine, and nicotinamide adenine dinucleotide (NAD+). Compared with the model group, adenine and choline phosphate were downregulated after turmeric intervention, while uracil was upregulated. However, no significant regulatory trend was observed for adenosine, hypoxanthine, guanine, guanosine, thymine, dihydrothymine, and nicotinamide adenine dinucleotide (NAD+). This suggests that the method of analyzing intact brain tissue homogenates in mice treated during the same experimental modeling phase may lead to variations in Aβ levels due to the method's homogenization properties. 1-42 The region-dependent nucleotide metabolism in the early stages of induced neuroinflammation could not be definitively elucidated, but adenine, phosphocholine cytidine transferase, and uracil were reverted after turmeric administration intervention, suggesting that turmeric may exert neuroprotective effects by regulating nucleotide metabolism.

[0250] Through experiments, this invention demonstrated that sphingosine, ethanolamine phosphate, and palmitate showed an upregulation trend in the model group, while metabolites such as fatty acids, linoleic acid, and linolenic acid showed a downregulation trend. Increased or accumulated intracellular lipid content (including sphingolipids) may lead to neurotoxicity. Elevated levels of sphingosine and ethanolamine phosphate indicate an increased rate of ceramide metabolism. Abnormal sphingolipid metabolism may lead to lipid metabolism disorders and activate and exacerbate inflammatory responses, thereby resulting in impaired lipid metabolism in nerve cells. After intervention with turmeric and its residue, the concentrations of sphingosine, ethanolamine phosphate, and palmitate decreased, while the concentrations of their metabolites increased, suggesting that the mechanism may be that the efflux of ceramides and sphingomyelin reduces the lipid load, thereby exerting an effect on Aβ. 1-42 Neuroprotective effect on the brain of mice with induced neuroinflammation.

[0251] Therefore, the research results of the above embodiments all indicate that turmeric residue can effectively alleviate Aβ to a certain extent. 1-42Induced neuroinflammation, specifically, HE staining and Nissl staining results showed that all treatment groups improved Aβ. 1-42 The pathological changes caused by the disease were reversed, and the cells were restored to their normal shape and morphology. According to the IF results, compared with the sham-operated group, the model group mice had Aβ in their brains. 1-42 Low expression of Aβ and increased expression of Iba-1; according to IHC results, injection of Aβ... 1-42 Induced proliferation of Iba-1 cells in the CA1 region of the mouse hippocampus, activating microglia; PCR quantitative PCR results showed that the mRNA expression levels of important channel proteins IκB (Inhibitorkappa B), p65, and TLR4 in the brains of the model group mice were significantly increased (all... p <0.01), the mRNA expression levels of inflammatory factors IL-6 (Interleukin 6), TNF-α (Tumor necrosis factor-alpha), iNOS (Inducible nitric oxide synthase), and COX-2 (Cyclooxygenase 2) were significantly increased (all <0.01). p <0.01), the mRNA levels of anti-inflammatory factors IL-4 (Interleukin 4), IL-10 (Interleukin 10), TGF-1β (Transforming growth factor 1 beta), and Arg-1 (Arginase 1) were significantly decreased (all <0.01). p <0.01), the drug-treated group could, to some extent, downregulate the mRNA expression levels of important channel proteins and inflammatory factors and upregulate the mRNA expression levels of anti-inflammatory factors; according to the protein content results measured by ELISA, the protein content 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 B kinase), and TLR4 in the model group mice were significantly increased (all p <0.01), the protein levels of inflammatory factors IL-6, TNF-α, iNOS, and COX-2 were significantly increased (all <0.01). p<0.01), while the protein levels of anti-inflammatory factors IL-4, IL-10, and TGF-1β were significantly decreased (all <0.01). p <0.01), the drug-treated groups were able to downregulate the protein expression levels of important channel proteins and inflammatory factors and upregulate the protein expression levels of anti-inflammatory factors to some extent.

[0252] In summary, this embodiment, through a non-targeted metabolomics study of turmeric residue in AD mice, found that turmeric and curcumin alleviate Aβ by regulating the TLR4 / NF-κB pathway. 1-42 Induced neuroinflammation. Its potential mechanism may involve regulating the overactivation state of microglia, modulating key proteins in the TLR4 / NF-κB pathway, downregulating inflammatory factors, and upregulating anti-inflammatory factors, thereby alleviating neuroinflammation in the brain. Through a non-targeted metabolomics comparative study of turmeric and turmeric residue in AD mice, it was found that turmeric residue can also alleviate Aβ to some extent. 1-42 The induced neuroinflammation was not significantly different from the intervention effect of turmeric. The experimental process and data of this 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.

[0253] The above embodiments of the present invention construct A β1-42 The C57BL / 6 mouse model of induced neuroinflammation serves as an effective tool for exploring the effective components, mechanisms of action, and indications of turmeric residue. It not only provides new ideas for the development of research on traditional Chinese medicine residues but also provides theoretical support for the prevention and treatment of neuroinflammation-related diseases.

[0254] Example 3

[0255] The method for constructing an animal model of neuroinflammatory intervention provided in this embodiment of the invention is a further study based on Examples 1 and 2. The difference is that in step S4, the main chemical components in the turmeric residue are analyzed. By using UPLC-MS / MS technology, the common chemical components in turmeric and the residue are first preliminarily identified. Then, PCA and OPLS-DA models are combined to identify potential differential substances, so as to further develop and utilize the residue.

[0256] The experimental method is as follows:

[0257] S4-1: Qualitative analysis of the main chemical components in turmeric residue

[0258] The main components of turmeric residue were qualitatively analyzed using UPLC-MS / MS technology.

[0259] Qualitative and quantitative analysis was conducted on the differential components in turmeric residue. Turmeric residue contains 8 sesquiterpenoids, 7 curcuminoids (including three main components: curcumin, demethoxycurcumin, and didemethoxycurcumin), 6 methoxyphenols, 4 hydroxycinnamic acid and their derivatives, and other active ingredients (as shown in Table 5-2, Chemical Composition Identification of Turmeric Residue).

[0260] 1. The specific steps for chemical composition analysis of turmeric raw material and turmeric residue are as follows:

[0261] 1.1 Preparation of test solution and reference solution

[0262] Take approximately 1 g of powder (passed through an 80-mesh sieve), sonicate with 5 mL of 80% methanol for 15 min, centrifuge for 30 s, and collect the supernatant. Repeat the above steps with 5 mL of 50% methanol and 5 mL of methanol in the wet sample. Combine the three extracts. Filter through a 0.22 μm microporous membrane to obtain the test solution.

[0263] Accurately weigh curcumin, demethoxycurcumin, bisdemethoxycurcumin, tetrahydrocurcumin, ferulic acid, and coumarin, add methanol to make up to a volume of 1 mg / mL to prepare a stock solution, and then dilute it to a standard solution with a concentration of 1 μg / mL.

[0264] 1.2 QC Sample Preparation

[0265] Eight samples were precisely pipetted, shaken evenly, and filtered through a 0.22 μm microporous membrane into a liquid chromatography vial to obtain QC samples. The injection was repeated five times initially, and then the QC sample was run every two samples in the sample sequence.

[0266] 1.3 Chromatographic and Mass Spectrometric Conditions

[0267] (1) Chromatographic column: Agilent SB-C18 column (100 mm × 2.1 mm, 1.8 μm); mobile phase A is acetonitrile, and mobile phase B is 0.1% formic acid. The flow rate is 0.3 mL / min, the column temperature is 40℃, the detection wavelength is 427 nm, and the injection volume is 1 μL.

[0268] (2) Mass spectrometry conditions

[0269] HESI ion source, ESI+ / - ion mode; full scan; scan range m / z -150.0-2000.0 Da; resolution 70000; sheath gas and auxiliary gas flow rates 40 arb and 10 arb, respectively, both high-purity nitrogen; electrospray voltage 4 kV; capillary temperature 350℃. Full MS conditions: resolution 70000; scan range 120-1000 m / z. dd-MS2 conditions: resolution 17500; collision energies NCE 20, 40, and 60 eV.

[0270] (3) The sample is tested on the instrument to obtain the test data.

[0271] 1.4 Data Processing

[0272] The obtained data were imported into Compound Discoverer 3.2 software for preliminary qualitative analysis of all compounds, compared with reference standards, and then the fragment ion peaks were compared with literature. Progenesis QI 3.0.3 software was used for peak alignment, extraction, and normalization. Principal component analysis (PCA) and partial least squares-discriminant analysis (OPLS-DA) were performed using EZinfo 2.0 software, and the model overfitting was checked using permutation tests. Compounds contributing significantly to intergroup separation were preliminarily screened based on variable importance in projection (VIP) values ​​(VIP > 2) and t-tests (p < 0.05) to identify potential differentially labeled compounds.

[0273] 2. Experimental results of chemical composition analysis of turmeric raw material and turmeric residue

[0274] The process and results of component identification were as follows: Based on the retention time of chromatographic peaks, the quasi-molecular or adduct ion peaks of primary mass spectrometry and the characteristic fragment ion information of secondary mass spectrometry, relevant literature and online databases, and in conjunction with reference standards, the chemical components in turmeric and its residue were analyzed. The results showed 47 and 36 compounds, respectively. Turmeric contained 10 sesquiterpenoids, 7 curcuminoids, 7 methoxyphenols, 4 hydroxycinnamic acid and their derivatives, and other active ingredients (as shown in Table 5-1). The residue contained 8 sesquiterpenoids, 7 curcuminoids, 6 methoxyphenols, 4 hydroxycinnamic acid and their derivatives, and other active ingredients (see Table 5-1, Chemical Component Identification Results of Turmeric).

[0275] Table 5-1

[0276]

[0277]

[0278]

[0279]

[0280]

[0281] Note: * indicates comparison with a control.

[0282] Table 5-2 Chemical composition identification of turmeric residue

[0283]

[0284]

[0285] Note: * indicates comparison with a control.

[0286] 3. Evaluation of System Approach

[0287] In the embodiments of this invention, the QC samples are tightly clustered. Throughout the entire sequence run, the retention time RSD of the compounds in the QC samples is less than 1%, and the peak intensity RSD is less than 3%, indicating that the instrument precision and repeatability meet the requirements, and the data can be further analyzed.

[0288] 4. Principal Component Analysis (PCA)

[0289] In this embodiment of the invention, four batches of medicinal materials and dregs were gathered together separately, showing a certain degree of separation. This indicates that the chemical composition of the turmeric medicinal materials and the dregs is somewhat different, while the two batches of dregs are relatively similar, indicating that the compounds have a certain degree of similarity.

[0290] 5. Orthogonal-Partial Least Squares Discriminant Analysis (OPLS-DA)

[0291] To further explore the differences between the medicinal material and the residue, an OPLS-DA analytical model was established for pairwise comparisons. In positive ion mode, R²Y = 0.999 and Q² = 0.979, indicating the model's stability and reliability. VIP ≥ 2, max fold change > 2, and Anova p < 0.05 were selected to screen for differentially expressed components. OPLS-DA analysis showed that the medicinal material and the residue were clearly divided into two groups. According to Table 5-3 (Differential Components in Turmeric Medicinal Material and Residue), eight significantly different components were found between the two, including four sesquiterpenoids, one methoxyphenol, one curcumin, one phenylnaphthalene, and one carboxylic acid and its derivative. After normalization, compared to the medicinal material, the residue showed higher average peak areas for shogaol, monodemethylcurcumin, and curcumin A / B.

[0292] Table 5-3

[0293]

[0294] Note: ↑ indicates that the average peak area of ​​the component is higher; ↓ indicates that the average peak area of ​​the component is lower.

[0295] The component identification results of turmeric raw material and residue show that curcuminoids in turmeric exhibit keto-enol tautomerism. Due to the stronger hydrophobicity of alcoholic curcuminoids, keto curcuminoids elute earlier, while alcoholic curcuminoids are eluted later. Current research indicates that enolization of curcumin derivatives is enhanced in hydrophobic media and reduced in aqueous media. Therefore, the reference solution was prepared with pure methanol, and the test solution was prepared with a non-pure methanol solution. The total ion chromatogram results showed that alcoholic curcuminoids had a higher peak response value than keto curcuminoids.

[0296] Curcuminoids are considered the most abundant metabolites in turmeric rhizomes. In positive ion mode, the main fragments of CUR, DCUR, and BDCUR are... m / z 369, m / z 339 and m / z309. The CUR, DCUR, and BDCUR in turmeric root and dregs collectively exert a therapeutic effect on Alzheimer's disease (AD). Although the mainstream view holds that curcumin is the most important component of curcuminoids and plays a dominant role in the treatment of AD with turmeric, the embodiments of this invention demonstrate through actual testing that the simultaneous use of all three compounds is more effective in treating AD than curcumin alone, and there is a synergistic enhancement effect among the three. All three curcuminoids exist as alcohol-ketone isomers; the alcohol form is the main structure for the biological activity of curcuminoids. Studies have shown that alcoholic curcumin can better bind to Aβ aggregates. The embodiments of this invention demonstrate that a gel based on tetrahydrocurcumin solid lipid nanoparticles exhibits biological activity in AD mice, with good skin permeability, reducing TNF-α and IL-6 levels, and improving histopathological changes in AD mice, and can be used to treat AD or other skin diseases with severe inflammation. Turmeric contains sesquiterpenoids such as turmeric dione, curcuminol, carvone, turmeric alcohol, and gemmaconone, which are important components of turmeric volatile oil. Existing research indicates that multiple components in turmeric volatile oil have inhibitory effects on secretases. Ferulic acid is a naturally occurring antioxidant found in plant cell walls, possessing anti-inflammatory activity and acting as a free radical scavenger, capable of inhibiting or dissociating amyloid protein structures.

[0297] Through differential analysis, this invention demonstrates that curcumin, a major dietary compound found primarily in ginger family plants, is present in medicinal residues. Curcumin inhibits Aβ... 42 Curcumin can reduce the expression of α-secretase, β-secretase, secretase subunits and COX-2, thereby alleviating AD-like pathological features in streptozotocin-induced sporadic AD mice; curcumin can also reduce the release of IL-1β, IL-6 and TNF-α, thereby reducing inflammatory responses in nucleus pulposus cells and delaying intervertebral disc degeneration.

[0298] 4-2: Determination of the chemical composition content of the main components of turmeric residue

[0299] HPLC was used to quantitatively determine the main components in turmeric residue. A method for determining the content of turmeric was established based on HPLC, and the content of curcuminoids in turmeric raw material and turmeric residue was compared.

[0300] This embodiment utilizes HPLC technology to investigate the preparation method, chromatographic conditions, and methodology of the test sample, and establishes a stable and reliable method for determining the content of turmeric. This method is applicable to the determination of turmeric medicinal materials and residues, and is used to determine the content of curcumin, demethoxycurcumin, and didemethoxycurcumin in turmeric medicinal materials and residues.

[0301] This embodiment, based on HPLC (High-performance liquid chromatography), established a method for determining the content of turmeric by selecting the sample preparation and chromatographic conditions, and determined the content of various curcuminoid compounds in different batches of turmeric raw materials and dregs. The results showed that the content of curcuminoid compounds in the raw materials was generally higher than that in the dregs, but a large amount of curcuminoid compounds still existed in the dregs, indicating high utilization value.

[0302] 4.2.1 Experimental Materials and Reagents

[0303] Four batches of turmeric medicinal materials (numbered: HM1-4) and their corresponding four batches of medicinal residue (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.

[0304] Curcumin (purity: >98.9%), demethoxycurcumin (purity: >98.5%), and bisdemethoxycurcumin (purity: >95%) were all purchased from the National Institutes for Food and Drug Control, China; chromatographic grade acetonitrile and methanol were purchased from Merck, USA; chromatographic grade glacial acetic acid, phosphoric acid, and analytical grade methanol were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0305] 4.2.2 Experimental Apparatus

[0306] The following equipment was used: H-Class UPLC system (Waters Corporation, USA); Agilent ZORBAX SB C18 (100 × 2.1 mm, 1.8 μm), Waters Acquity BEH C18 (100 × 2.1 mm, 1.7 μm), and YMC Triart C18 (100 × 2.1 mm, 1.9 μm); 0.0001g electronic balance (ME204E) and 0.000000g 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 Yongli Pharmaceutical Machinery Co., Ltd., Ruian City, Zhejiang Province; and MiliQ Direct8 ultrapure water system was manufactured by Merck, Germany.

[0307] 4.3 Experimental Methods

[0308] 4.3.1 Preparation of mixed reference solution

[0309] Accurately weigh 21.8777 mg, 7.7654 mg, and 8.5011 mg of CUR, DCUR, and BDCUR reference standards, respectively. Dissolve and dilute in methanol, and bring the volume to 10 mL in a volumetric flask. Shake well to prepare a mixed reference solution for later use. The concentrations of CUR, DCUR, and BDCUR are 2163.7043, 764.8919, and 807.6045 μg / mL, respectively.

[0310] 4.3.2 Investigation of the preparation method of the test solution

[0311] The initial method for preparing the test solution was determined as follows: 0.2 g (40 mesh) of turmeric residue powder was accurately weighed and placed in a stoppered conical flask. 25 mL of methanol was added, and the mixture was sonicated (250 W power, 40 kHz frequency) for 30 min. After cooling to room temperature, the mass was weighed again, and the lost mass was made up with methanol. The mixture was shaken well and filtered through a microporous membrane (0.22 μm) to obtain the final solution. Different powder particle sizes, extraction solvents, extraction methods, extraction times, extraction power, and extraction volumes were investigated first. Then, the content of curcuminoid compounds was used as the evaluation index. Finally, the method for preparing the test solution was determined.

[0312] The particle size of the turmeric residue powder was selected as 40, 50, 65, and 80 mesh; the extraction solvents were methanol and ethanol; the extraction methods were ultrasonic extraction and reflux extraction; the extraction times were 30, 45, and 60 min; and the extraction power was 150, 250, and 350 W. The content of curcumin-like compounds was then used as the evaluation index to determine the appropriate preparation conditions.

[0313] 4.3.3 Investigation of Liquid Chromatography Conditions

[0314] The initial liquid chromatography conditions were determined as follows: Waters Acquity BEH C18 (100 × 2.1 mm, 1.7 μm), mobile phase acetonitrile-0.1% acetic acid (45:55), 427 nm, flow rate 0.3 mL / min, and injection volume 1 μL. The test solution was prepared according to the initially proposed method. The detection wavelength, column, mobile phase type and ratio, column temperature, and flow rate were then investigated. Peak shape, peak area, theoretical plate number, resolution, and tailing factor of each compound were used as evaluation indicators. Finally, suitable liquid chromatography conditions were established.

[0315] A PAD detector was used to perform a full wavelength scan in the 200-500 nm range, and an appropriate detection wavelength was selected. Chromatographic columns with different particle sizes were compared, 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).

[0316] The mobile phase types include 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 ratio of acetonitrile-0.5% phosphoric acid is 40:60, 45:55, 50:50, 55:45, and 60:40.

[0317] The column temperatures were 25, 30, 35, and 40℃; the flow rates were 0.2, 0.3, and 0.4 mL / min. Peak shape and peak area of ​​each compound were used as evaluation indicators to establish the liquid chromatography conditions.

[0318] 4.3.4 Methodological Examination

[0319] According to the method for preparing the reference solution under section "4.3.1", the determination was carried out under the chromatographic conditions under section "4.3.3" to examine the linear range; the mixed reference solution was serially diluted, and the concentrations when the S / N was approximately 3 and 10 were recorded to examine the limit of quantitation and the limit of detection; according to the method selected under section "4.3.2", the determination was carried out under the chromatographic conditions under section "4.3.3" to examine the specificity test, precision test, stability test, repeatability test, and recovery test.

[0320] 4.3.5 Determination of Sample Content

[0321] The test solution was prepared according to the method selected in section “4.3.2”, and the chromatographic conditions in section “4.3.3” were used for determination. The chromatogram was recorded, the content of each batch of turmeric and dregs was calculated, and the t-test was performed on the CUR, DCUR and BDCUR contents between the two groups.

[0322] 4.4 Experimental Results

[0323] 4.4.1 Preparation method of test solution

[0324] Using the content of curcumin compounds as the evaluation index, a suitable preparation method for the test sample was established under different powder particle size, extraction solvent, extraction method, extraction time, ultrasonic power, and extraction time conditions. The experimental results are shown in Table 3-1 (total content of curcumin compounds under different preparation conditions (n=2)).

[0325] The final selected method for preparing the test sample is as follows: Take 0.2 g (80 mesh) of turmeric powder, accurately weigh it, place it in a stoppered conical flask, add 25 mL of methanol, sonicate (power 250 W, frequency 40 kHz) for 30 min, cool to room temperature, weigh it again, make up the lost mass with methanol, shake well, and filter it through a microporous membrane (0.22 μm) to obtain the sample.

[0326] Table 3-1

[0327]

[0328] 4.4.2 Chromatographic conditions

[0329] (1) Determination of detection wavelength

[0330] A PDA detector was used to perform a full wavelength scan of the test solution from 200 to 500 nm. The results showed that the peak absorption of each substance was most significant in the wavelength range of 400-450 nm. After comparison with the reference standard, these three peaks were identified as CUR, DCUR, and BDCUR. Observation of the absorption peaks of CUR, DCUR, and BDCUR showed that the strongest absorption wavelengths were 427.2, 421.6, and 417.9 nm, respectively. Since curcumin in turmeric is present in higher amounts than the other two components and is often used as a research subject in experiments, 427 nm was selected as the absorption wavelength for determining the content of the three components.

[0331] (2) Chromatographic column

[0332] The turmeric-yellow spectra of chromatographic columns with different particle sizes were selected, and the separation results are shown in Table 3-2 below (chromatographic peak separation results under different column conditions). The YMC Trait column showed obvious tailing, and its resolution was worse than the other two columns; the Waters BEH and Agilent SB columns showed good peak resolution, good peak shape, and high theoretical plate number; the Agilent SB column had faster peak elution and sharper peak shape, so the Agilent SB column was selected.

[0333] Table 3-2

[0334]

[0335] (3) Mobile phase ratio

[0336] The turmeric-colored chromatograms of acetonitrile-0.1% phosphoric acid at different ratios were selected, and the separation results are shown in Table 3-3 below (chromatographic peak separation effect under different mobile phase ratios). When the acetonitrile-0.1% phosphoric acid ratio was 40:60, the peak elution was slowest, and the peak shape was relatively blunt and round. Ratios of 50:50, 55:45, and 60:40 showed good separation and sharp peaks, but all exhibited tailing. A ratio of 45:55 produced sharp and symmetrical peaks with good separation; therefore, a mobile phase ratio of 45:55 was selected.

[0337] Table 3-3

[0338]

[0339] (4) Types of acid in the mobile phase

[0340] The chromatograms of turmeric with different acid types were selected, and the separation results are shown in Table 3-4 below (chromatographic peak separation effect under different acid conditions). When acetonitrile-water was selected as the mobile phase, the peak shapes of each compound were good, but the elution time was slow, and the peak area was significantly lower than that of the group using acid as one of the mobile phases. Based on the literature and pharmacopoeia, acetic acid is a commonly used acid for establishing methods for determining turmeric content. Compared with phosphoric acid, the chromatographic peak symmetry of the acetic acid group was slightly worse, and the elution time was slower. Therefore, phosphoric acid was considered as one of the mobile phases. When 0.1% phosphoric acid was selected as the mobile phase, the peak area, symmetry, and theoretical plate number of the chromatographic peaks were all better than those of 0.5% and 1% phosphoric acid. Therefore, acetonitrile-0.1% phosphoric acid was selected as the mobile phase.

[0341] This embodiment specifically determined the content range of three important curcumin compounds in turmeric residue: curcumin, demethoxycurcumin, and didemethoxycurcumin. The final determined content ranges were 16.5 mg / g ~ 19.94 mg / g, 6.75 mg / g ~ 10.66 mg / g, and 6.80 mg / g ~ 14.04 mg / g, respectively.

[0342] Table 3-4

[0343]

[0344] (5) Column temperature

[0345] The chromatograms of curcumin at different column temperatures were selected, and the separation results are shown in Table 3-5 below (the separation effect of chromatographic peaks at different column temperatures). The peak shapes and resolutions at different column temperatures were good; apart from slight differences in elution time, the peaks were not significantly different. However, when using 30℃ as the column temperature, the peak of bis(demethoxycurcumin) showed a bifurcation at the tail of a low-concentration mixed reference sample, failing to reach the baseline. This issue did not occur when using 25℃ as the column temperature; therefore, 25℃ was selected as the column temperature.

[0346] Table 3-5

[0347]

[0348] (6) Flow velocity

[0349] The separation results of the turmeric yellow chromatograms obtained at different flow rates are shown in Table 3-6 below (the separation effect of chromatographic peaks at different flow rates). The peak shapes and resolutions were good at different flow rates. At a flow rate of 0.2 mL / min, the elution time was delayed; at 0.3 mL / min, the elution time was accelerated, the peak shape was symmetrical, and the theoretical plate number was higher; at 0.4 mL / min, the elution time was accelerated, but the chromatographic peak showed slight tailing, and the theoretical plate number decreased. Therefore, 0.3 mL / min was selected as the optimal flow rate.

[0350] Table 3-6

[0351]

[0352] (7) Determine the chromatographic conditions

[0353] In summary, the final chromatographic conditions were determined as follows: Agilent ZORBAX SB C18 column (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℃.

[0354] 4.4.3 Methodological Examination

[0355] (1) Specificity test

[0356] Comparison of the HPLC chromatograms of the test solution, mixed reference solution, single reference solution, and blank solution shows that the retention times of curcumin, methoxycurcumin, and dimethoxycurcumin are approximately 5.44, 4.76, and 4.17 min, respectively, with good peak symmetry. The blank solvent does not interfere with the detection of the reference standards.

[0357] (2) LOD, LOQ and linear range

[0358] The linear range, LOD, and LOQ results are shown in Table 3-7 (linearity of each component, LOD, and LOQ). The linear range of each component indicates good linearity within its respective concentration range.

[0359] Table 3-7

[0360]

[0361] (3) Precision test

[0362] The calculated RSD of retention time for 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.

[0363] (4) Stability test

[0364] The calculated RSDs of retention times for each characteristic peak were 0.58%–0.71% (n=6), and the RSDs of peak areas were 0.45%–0.66% (n=6), indicating that the test solution had good stability within 24 hours.

[0365] (5) Repeatability test

[0366] The calculated RSDs of retention times for 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 the method has good repeatability.

[0367] (6) Spiking recovery test

[0368] The average recoveries of CUR, DCUR, and BDCUR were 99.66%, 97.90%, and 98.44%, respectively, with RSDs ranging from 1.75% to 2.89%. The results are in accordance with the 2020 edition of the Chinese Pharmacopoeia, indicating that the content determination method is stable and reliable.

[0369] 4.4.4 Determination of Turmeric Content in Raw Material and Residue

[0370] The contents of each batch were determined under the selected experimental conditions. The results are shown in Table 3-12 below (content of curcuminoids in turmeric raw materials and dregs (n=2)). The experimental results show that the contents of CUR, DCUR, and BDCUR in turmeric raw materials ranged 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, respectively, while the contents of CUR, DCUR, and BDCUR in dregs ranged 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, respectively. According to the t-test results, there was no significant difference in the contents of CUR, DCUR, and BDCUR between the two groups (p>0.05). In addition, considering the linear range in "3.5.3", all components in the medicinal material are within the linear range, so this content determination method is also applicable to the determination of curcuminoid compounds in turmeric.

[0371] Table 3-12

[0372]

[0373] According to existing technology, the dried rhizome of Curcuma Longa L. is a commonly used traditional Chinese medicine containing various active ingredients. The main medicinal components of turmeric are curcuminoids, including curcumin, demethoxycurcumin, and didemethoxycurcumin, with curcumin having the highest content. This embodiment establishes a method for determining the content of turmeric, measuring the content of various curcuminoids in different batches of the medicinal material and its residue.

[0374] First, the powder particle size, extraction solvent, extraction method, extraction time, ultrasonic power, and extraction time were investigated to establish the sample preparation method. Second, the detection wavelength, chromatographic column, type and ratio of mobile phase, column temperature, and flow rate were investigated to determine the chromatographic conditions.

[0375] This embodiment not only examined the sample preparation and chromatographic conditions but also systematically evaluated the content determination method. Specificity, stability, repeatability, precision, recovery, LOD, LOQ, and linear range tests confirmed that the method is stable and reliable, and can be used to determine the content of various curcuminoids in turmeric residue. The t-test showed no significant difference in the content of components between turmeric root material and residue, and the content of each component in the root material was within the linear range; therefore, the method is also applicable to turmeric root material. Experimental results indicate that the overall content of curcuminoids in the root material is higher than that in the residue, but a large amount of curcuminoids still exist in the residue, indicating high utilization value. The turmeric residue used in this embodiment is the residue obtained after turmeric is extracted with water to prepare turmeric formula granules. The yield 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 the 81.8% residue. It is reported that the transfer rate of curcuminoid components in turmeric formula granules is only about 5%, and about 95% of the curcuminoid components are still enriched in the residual residue. That is, 5.5 g of turmeric herb is extracted with water to make 1 g of formula granules, and the total amount of fat-soluble curcuminoid components in the remaining 4.5 g of residue is basically the same as the total amount of fat-soluble curcuminoid components in 5.5 g of turmeric herb. Because water extraction leads to the enrichment of fat-soluble components, the content of fat-soluble components in the residue may be similar to that in the herb, or even higher. Therefore, turmeric residue has great potential utilization value for fat-soluble curcuminoid components.

[0376] This embodiment, based on HPLC technology, investigates the preparation and chromatographic conditions of the test sample. After system adaptability verification, a stable and reliable method for the determination of curcumin-like compounds is established, characterized by the following features:

[0377] (1) The total amount of curcumin compounds was used as the evaluation standard. The particle size of the curcumin powder was 40, 50, 65 and 80 mesh. The extraction solvent was 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 sample preparation conditions, the optimal sample preparation method was selected as follows: take 0.2 g (80 mesh) of curcumin powder, accurately weigh it, put it in a stoppered conical flask, add 25 mL of methanol, sonicate (power 250 W, frequency 40 kHz) for 30 min, filter, and obtain the sample solution.

[0378] (2) Using the chromatographic peak separation effect as the evaluation, a full wavelength scan was performed in the range of 200-500 nm, and a suitable detection wavelength was selected. Different particle size chromatographic columns, mobile phase types, mobile phase ratios, column temperatures, and flow rates were selected for investigation. Under different chromatographic conditions, the final chromatographic conditions were as follows: Agilent ZORBAX SBC18 column (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℃.

[0379] (3) After methodological investigation, the content determination method is stable and reliable and can be used to determine the content of turmeric medicinal material and turmeric residue. The content of curcumin compounds in the medicinal material is generally higher than that in the residue, but there are still a large amount of curcumin compounds in the residue, which has high utilization value.

[0380] This embodiment, through actual experiments and verification, found that various components can be extracted from the turmeric residue after water extraction, including volatile oils, curcumin, essential oils, and polysaccharides. These components have wide applications in traditional medicine and modern health products. Considering the current production status of traditional Chinese medicine granules at Guangdong Yifang Pharmaceutical Co., Ltd., water extraction is commonly used in the processing of turmeric medicinal materials or the production of related traditional Chinese medicine products. This results in the underutilization of high-value fat-soluble components, which remain in solid waste, causing serious waste. Since curcumin in turmeric is poorly soluble in water, the turmeric residue after traditional extraction methods may contain a large amount of fat-soluble components similar to curcumin, possessing extremely high development and utilization value. Therefore, to better develop and utilize turmeric residue, this study is divided into two parts: component analysis of turmeric medicinal materials and residue, and whether it has a mitigating effect on neuroinflammation.

[0381] Existing research indicates that various components can be extracted from turmeric residue, including volatile oils, curcumin, essential oils, and polysaccharides. These components have wide applications in traditional medicine and modern health products. Considering the current production of traditional Chinese medicine granules by companies such as Guangdong Yifang Pharmaceutical Co., Ltd., water extraction is commonly used in the processing of turmeric medicinal materials or the production of related traditional Chinese medicine products. This results in the underutilization of valuable fat-soluble components, which remain in solid waste, causing significant waste. Because curcumin in turmeric is poorly soluble in water, the turmeric residue after extraction using traditional methods may still contain a large amount of fat-soluble components similar to curcumin, potentially possessing high development and utilization value. However, no specific research on the development and utilization of turmeric residue has yet been found.

[0382] In this embodiment, UPLC-MS / MS technology was used to preliminarily identify the components in turmeric medicinal material and its residue. A total of 47 and 36 compounds were screened in turmeric medicinal material and its residue, respectively. Turmeric medicinal material contained 10 sesquiterpenoids, 7 curcuminoids, 6 methoxyphenols, 4 hydroxycinnamic acid and its derivatives, and other active ingredients. The residue contained 8 sesquiterpenoids, 7 curcuminoids, 5 methoxyphenols, 4 hydroxycinnamic acid and its derivatives, and other active ingredients.

[0383] This embodiment compares the components of turmeric medicinal material and its residue, and finds eight significantly different components between the two, including four sesquiterpenoids, one monoterpene, one curcuminoid, and two other compounds. Among them, the average peak areas of [4]-gingeradiol, monodemethylcurcumin, and curcumin A / B contained in the four batches of residue are higher than those in the medicinal material, indicating that the residue has potential development value. Developing turmeric residue can effectively reduce resource waste and improve the comprehensive utilization value of waste resources.

[0384] Example 4

[0385] This invention further provides a method for screening effective components in turmeric residue that intervene in neuroinflammation. This method is a further application of the aforementioned method for constructing an animal model for neuroinflammation intervention. It uses the methods described in Examples 1-3 to construct an animal model for neuroinflammation intervention, and screens for effective components in turmeric residue that intervene in neuroinflammation. The method includes the following steps:

[0386] A1: Analysis of the main chemical components of turmeric residue

[0387] The main curcumin compounds in turmeric residue were qualitatively analyzed and quantitatively determined using UPLC-MS / MS and HPLC techniques, respectively, to preliminarily identify the active components to be tested.

[0388] A2: Screening of effective ingredients using an animal model of neuroinflammatory intervention.

[0389] An animal model of neuroinflammation intervention was used to test the intervention effects of each active ingredient (mainly curcuminoids) on neuroinflammation.

[0390] A3: Comparative testing to determine whether there is a synergistic effect among the various active components in turmeric residue.

[0391] A comparative test was conducted using an animal model of neuroinflammatory intervention to compare the simultaneous and individual use of the tested active ingredients. This study aimed to determine whether there was a synergistic effect between the simultaneous use of multiple compounds in turmeric residue and whether the interventional therapeutic effect of multiple active ingredients on AD was more significant when used together than when used alone.

[0392] This embodiment, through testing in an animal model of neuroinflammatory intervention, found that the combined use of three compounds from the drug residue—curcumin CUR, demethoxycurcumin DCUR, and bisdemethoxycurcumin BDCUR—exhibited a synergistic effect on the treatment of Alzheimer's disease (AD), demonstrating better therapeutic efficacy for AD than curcumin alone.

[0393] The specific technical means of experimentation, testing and analysis used in this embodiment are the same as those in the aforementioned embodiments 1-3, and will not be repeated here.

[0394] The methods and applications provided in the above embodiments of the present invention focus on constructing Aβ 1-42 A C57BL / 6 mouse model of induced neuroinflammation was used to investigate the effective components, mechanism of action, and indications of turmeric residue. This included: preliminary identification of the chemical components of turmeric root and residue using UPLC-MS / MS technology; further, differential analysis of the components of turmeric root and residue to identify potential differential substances; and establishing an Aβ... 1-42 A mouse model of neuroinflammation was established through targeted brain injection to investigate whether turmeric root, turmeric residue, and curcumin, a key active ingredient in turmeric, could alleviate neuroinflammation in the mouse brain and to explore its potential mechanism of action. Finally, non-targeted metabolomics technology was used to study the effects of turmeric residue intervention on mice with neuroinflammation. By analyzing changes in metabolites, metabolic pathways and metabolites in the body were revealed, leading to a deeper understanding of Aβ. 1-42 The induced pathogenesis and therapeutic effects of Alzheimer's disease. Therefore, this invention application not only provides new ideas for the research of traditional Chinese medicine residues, but also provides theoretical support for the prevention and treatment of neuroinflammatory diseases.

[0395] It should be noted that the above embodiments are only some embodiments of the present invention, and not all embodiments. In other embodiments, within the scope of the present invention, other technical solutions can be obtained by selecting other components, proportions, and process parameters, etc., to achieve the technical effects described in the present invention, and therefore they will not be listed one by one.

[0396] The specific embodiments described above are merely illustrative of the optimized methods of the present invention and are not intended to limit the scope of the invention. It should be noted that any appropriate adjustments or modifications made to the technical solutions of the present invention by those skilled in the art without departing from the spirit of the invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for screening effective components in turmeric residue that intervene in neuroinflammation, characterized in that, The screening process for effective components in turmeric residue that intervene in neuroinflammation includes the following steps: A1: Analysis of the main chemical components of turmeric residue The main curcumin compounds in turmeric residue were qualitatively analyzed and quantitatively determined by UPLC-MS / MS and HPLC combined with non-targeted metabolomics technology, respectively, and the target active components were preliminarily identified. A2: Screening of effective ingredients using animal models and mechanisms of neuroinflammatory intervention. Aβ1₋ injected into the lateral ventricle 42 A mouse model of neuroinflammation was constructed using peptides. The intervention mechanism of curcumin-like compounds on neuroinflammation was utilized to test the intervention effects of each active ingredient on neuroinflammation. A3: Comparative testing to determine whether there is a synergistic effect among the various active ingredients in turmeric residue. Repeat step A2 and conduct comparative tests using an animal model of neuroinflammation intervention. Compare the simultaneous use and single use of the tested active ingredients to determine whether there is a synergistic effect of multiple compounds in turmeric residue when used simultaneously, and whether the intervention and treatment effect of multiple active ingredients used together on neuroinflammation is more significant than that of single use. In step A1, based on HPLC technology, the preparation conditions and chromatographic conditions of the test sample are determined. After system adaptability verification, a stable and reliable method for the determination of curcumin-like compounds is established, including the following steps: (1) Using the total amount of curcumin compounds as the evaluation standard, prepare the test sample: Take 0.2 g of 80 mesh curcumin powder, weigh it accurately, put it in a stoppered conical flask, add 25 mL of methanol, sonicate it for 30 min at a power of 250 W and a frequency of 40 kHz, cool it to room temperature, weigh it again, make up the lost mass with methanol, shake it well, filter it through a 0.22 μm microporous membrane to obtain the test sample solution; (2) The separation effect of the chromatographic peak was used as the evaluation. 427 nm was selected as the absorption wavelength for determining the content of the component. The selected chromatographic conditions were: Agilent ZORBAX SBC18 column, mobile phase of 45:55 acetonitrile-0.1% phosphoric acid, flow rate of 0.3 mL / min, detection wavelength of 427 nm, and column temperature of 25℃.

2. The method for screening effective components in turmeric residue for intervening in neuroinflammation according to claim 1, characterized in that, The method for constructing the neuroinflammatory intervention animal model in step A2 includes the following steps: S1: Experimental reagents for preparing animal models Five types of experimental reagents were prepared: turmeric raw material, turmeric residue, low-dose curcumin, high-dose curcumin, and donepezil tablet solution; the turmeric residue is the solid residue remaining after water extraction of turmeric raw material; S2: Construct Aβ 1-42 Mouse models of induced neuroinflammation Multiple male mice were randomly divided into four groups: sham-operated group, model group, positive control group, herbal medicine group, herbal residue group, low-dose curcumin group, and high-dose curcumin group. All groups were fed the same diet, and the behavior of the mice in each group was assessed. For one week, mice in each group were administered five experimental drugs via gavage: turmeric root, turmeric residue, low-dose curcumin, high-dose curcumin, and donepezil tablets. Then, Aβ was injected into the lateral ventricle of the mice. 1-42 A neuroinflammation model was established using peptides, and then each group of mice was administered five types of experimental drugs by gavage for several consecutive days. Behavioral tests were performed on mice in each group to analyze whether the experimental drugs used in each group of the animal model had the effect of reducing neuroinflammation in the mouse brain; S3: To investigate the mechanism and effect of turmeric residue in intervening in neuroinflammation in mice. The mice obtained in step S2 were prepared as biological samples. UPLC-MS / MS and HPLC combined with non-targeted metabolomics techniques were used to analyze and investigate whether turmeric residue and curcumin could improve Aβ by regulating the TLR4 / NF-κB pathway. 1-42 The mechanism by which the neuroinflammation is induced in mice to induce cognitive impairment, reduce nerve damage, regulate overactivated microglia, downregulate inflammatory factors and upregulate anti-inflammatory factors, thereby achieving the intervention effect of reducing neuroinflammation; S4: Qualitative and quantitative determination of major curcuminoid compounds in turmeric residue The main curcuminoid compounds in turmeric residue were qualitatively analyzed and quantitatively determined using UPLC-MS / MS and HPLC techniques, respectively.

3. The method for screening effective components in turmeric residue for intervening in neuroinflammation according to claim 2, characterized in that, The five types of experimental reagents in step S1 are prepared using the following steps: S1-1: Preparation of low-dose and high-dose solutions of curcumin reference standard Weigh out curcumin reference standard, dissolve it in 2% Tween 80 and distilled water, and prepare low-dose curcumin solutions with a concentration of 1 mg / mL and high-dose curcumin solutions with a concentration of 2 mg / mL, respectively. S1-2: Preparation of Turmeric Herb 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 medicinal liquid in a water bath, completely evaporate the ethanol, and prepare turmeric medicinal material solution with a concentration of 0.1 g / mL and turmeric residue solution with a concentration of 0.12 g / mL respectively, and store at -20℃. S1-3: Preparation of Positive Drug Solution Donepezil hydrochloride tablets were ground into a fine powder and dissolved in distilled water to obtain a positive control solution with a concentration of 0.076 mg / mL.

4. The method for screening effective components in turmeric residue for intervening in neuroinflammation according to claim 2, characterized in that, Step S2 includes the following steps: S2-1: Mouse grouping Multiple male mice were selected and, after 7 days of adaptive feeding, were randomly divided into 7 groups: sham operation group, model group, positive drug group, medicinal material group, drug residue group, low-dose curcumin group, and high-dose curcumin group. Each group had no fewer than 5 mice and were fed the same feed. S2-2: Grouping and administration of drugs to mice before brain localization injection Each mouse in each group was first administered the drug via gavage for one week, and then the mice were divided into groups to receive the drug. S2-3: Inducing neuroinflammation in mice by targeted injection into the brain Aβ 1-42 Solution treatment Aβ 1-42 Dissolve in sterile physiological saline to prepare an Aβ1-42 injection solution with a concentration of 410 pmol / μL. Place the prepared solution in a 37℃ constant temperature incubator and incubate for 7 days before use. On day 8 of the experiment, based on the mouse brain atlas, mice in each group underwent targeted brain injection. The injection site was selected as the bilateral hippocampal CA1 region, with coordinates: AP: -0.22 cm, ML: ±0.2 cm, DV: -0.2 cm. Aβ was aspirated using a microsyringe. 1-42 Solution, Aβ from one side of the brain 1-42 The solution volume was 1.5 μL, the procedure was performed over 10 min, the needle was left in place for 15 min, and the needle was slowly withdrawn over 5 min. The wound was then sutured and disinfected. The same procedure was performed on the other side of the brain. The sham-operated group was injected with an equal volume of sterile saline and then allowed to recover naturally for 2 days. S2-4: Grouping and administration of drugs after localized brain injection in mice Starting from day 10 of the experiment, the grouping and dosage of drugs in step S2-2 were repeated for each group of mice by gavage for three weeks. S2-5: Conduct animal behavioral experiments on mice in each group. Animal behavioral experiments were conducted on mice in each group. The results of these experiments were used to evaluate the effects of turmeric, turmeric residue, and curcumin on the learning and memory abilities of mice with Aβ1-42 damage, and to determine whether turmeric, turmeric residue, and curcumin in the model group could alleviate neuroinflammation in the mouse brain.

5. The method for screening effective components in turmeric residue for intervening in neuroinflammation according to claim 2, characterized in that, Step S3 involves performing immunohistochemical analysis based on the experimental data and biological samples obtained in step S2, and further employing non-targeted metabolomics technology to elucidate the mechanism by which turmeric residue and curcumin intervene in mice with neuroinflammation. The study examines how turmeric residue and curcumin improve cognitive impairment, reduce nerve damage, regulate overactivated microglia, downregulate inflammatory factors, and upregulate anti-inflammatory factors in Aβ1-42-induced neuroinflammation mice by regulating the TLR4 / NF-κB pathway, ultimately alleviating neuroinflammation.

6. The method for screening effective components in turmeric residue for intervening in neuroinflammation according to claim 2, characterized in that, Step S4, analyzing the main curcuminoid compounds in the turmeric residue, includes the following steps: S4-1: Qualitative analysis of the main chemical components in turmeric residue The main components of turmeric residue were qualitatively analyzed using UPLC-MS / MS technology. Through qualitative and quantitative analysis of the differential components in turmeric residue, the active components of turmeric residue were identified as 8 sesquiterpenoids, 7 curcuminoids, 6 methoxyphenols, 4 hydroxycinnamic acid 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 residue, and to accurately determine the content range of three important curcumin compounds, namely curcumin, demethoxycurcumin and didemethoxycurcumin, in turmeric residue.

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