Application of agilawood extract in preparation of medicine for resisting Parkinson's disease

Abstract

The invention discloses application of an agilawood extract in preparation of a medicine for treating Parkinson's disease, and belongs to the technical field of traditional Chinese medicines. According to the present invention, MPTP induced Parkinson's disease mouse subacute model research results show that the agilawood extract can improve Parkinson's disease motor coordination imbalance, cognitive impairment and gastrointestinal dysfunction, the action mechanism and the treatment mechanism of agilawood in treatment of Parkinson's disease are disclosed, and the agilawood extract can provide significant effects for treatment of behavioral diseases of Parkinson's patients, and a theoretical basis is laid for improving the life quality of the Parkinson's disease patient.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and in particular to application of agarwood extract in preparing medicines for Parkinson's disease. Background Art

[0002] Parkinson's disease (PD), as a neurodegenerative disease that seriously threatens human health, is becoming a major problem that needs to be solved in the global medical field. Whether from the perspective of improving the quality of life of patients, reducing the burden on families and society, or improving the medical treatment system, PD urgently needs more effective solutions, including the development of drugs for neuroprotection and delaying disease progression. Agarwood is a traditional precious Chinese medicine. At present, there are no reports on the intervention mechanism of agarwood on PD at home and abroad. Therefore, exploring the protective effect and potential mechanism of agarwood (CX) on PD can provide a theoretical basis for clarifying the pathological mechanism of PD and the development of new Chinese medicine anti-PD drugs. Summary of the invention

[0003] The purpose of the present invention is to provide an application of agarwood extract in the preparation of an anti-Parkinson's disease drug to solve the problems existing in the above-mentioned prior art. The present invention found through a study of the MPTP-induced subacute model of Parkinson's disease in mice that agarwood extract can improve Parkinson's disease motor coordination imbalance, cognitive impairment and gastrointestinal dysfunction, and revealed the mechanism of action and treatment mechanism of agarwood in the treatment of Parkinson's disease, which laid a theoretical foundation for treating the motor and non-motor symptoms of Parkinson's patients and improving the quality of life of Parkinson's patients.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] The present invention provides an application of an agarwood extract in preparing a drug for treating Parkinson's disease. The preparation method of the agarwood extract comprises the following steps:

[0006] The agarwood medicinal material is mixed with an ethanol solution with a volume fraction of 80%, and reflux extraction is performed twice, each time for 2 hours, and the extracts are combined and dried to obtain the agarwood extract;

[0007] The treatment of Parkinson's disease is to improve Parkinson's motor coordination imbalance, cognitive impairment and gastrointestinal dysfunction.

[0008] Furthermore, the volume ratio of the agarwood medicinal material to the ethanol solution is 1:10.

[0009] Furthermore, the temperature of the reflux extraction is 90°C.

[0010] Furthermore, the Parkinson's disease is subacute Parkinson's disease.

[0011] Furthermore, the treatment of Parkinson's disease is achieved by increasing dopaminergic neurons, reducing the aggregation of α-syn, reducing the activation levels of microglia and astrocytes, and inhibiting the activation of the NF-κB pathway.

[0012] The present invention also provides a drug for treating Parkinson's disease, wherein the active ingredient of the drug is an agarwood extract; and the preparation method of the agarwood extract comprises the following steps:

[0013] The agarwood medicinal material is mixed with an ethanol solution with a volume fraction of 80%, and reflux extraction is performed twice, each time for 2 hours, and the extracts are combined and dried to obtain the agarwood extract;

[0014] The treatment of Parkinson's disease is to improve Parkinson's motor coordination imbalance, cognitive impairment and gastrointestinal dysfunction.

[0015] Optionally, the volume ratio of the agarwood medicinal material to the ethanol solution is 1:10.

[0016] Optionally, the temperature of the reflux extraction is 90°C.

[0017] The present invention discloses the following technical effects:

[0018] The present invention establishes a subacute model of Parkinson's disease induced by MPTP in mice. By analyzing behavioral manifestations and typical pathological changes of PD, it is found that the ethanol extract of agarwood can alleviate motor coordination imbalance, cognitive impairment and gastrointestinal dysfunction in PD mice, improve dopaminergic neuron damage in the substantia nigra-striatum region of the brain, reduce abnormal aggregation of α-syn, and regulate PD neuroinflammation. The present invention reveals the mechanism of action and treatment of agarwood in the treatment of Parkinson's disease, and lays a theoretical foundation for treating the behavioral symptoms of Parkinson's patients and improving the quality of life of Parkinson's patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 Flow chart for the construction of subacute PD mouse model;

[0021] Figure 2 The results of the test on the effect of CX on the motor coordination ability and cognitive impairment of the subacute model of PD mice; A is the test result of the climbing pole experiment of each group of mice; B is the test result of the balance beam experiment of each group of mice; C is the test result of the novel object recognition experiment of each group of mice;

[0022] Figure 3 The test results of the effect of CX on the gastrointestinal function of the subacute model of PD mice; A is the test result of the intestinal propulsion rate of mice in each group; B is the test result of the fecal water content of mice in each group; C is the test result of the weight change of mice in each group;

[0023] Figure 4 The results of the test on the typical pathological characteristics and glial cell activation of the subacute model of PD mice by CX; A is the IHC test result of TH in the brain tissue of each group of mice; B is the IHC test result of α-syn in the brain tissue of each group of mice; C is the IHC test result of Iba-1 in the brain tissue of each group of mice; D is the IHC test result of GFAP in the brain tissue of each group of mice;

[0024] Figure 5 These are the test results of the effect of CX on NF-κB activation in the subacute model of PD mice; A is the test result of NFKB1A gene mRNA expression in each group of mice; B is the test result of p65 protein expression; C is the test result of p-IκB-α expression. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] Example 1 Preparation of Agarwood Extract

[0031] The agarwood medicinal materials were tested according to the 2020 edition of the Chinese Pharmacopoeia. 17 H 18 O6) content is 0.17%, which meets the requirements of pharmacopoeia and can be used to prepare agarwood extract.

[0032] The CX crude drug was extracted twice with 10 times the volume of 80% ethanol solution at 90°C for 2 hours each time, and the two extracts were combined, concentrated, and vacuum dried to obtain the agarwood extract (CX).

[0033] Example 2 Functional verification of agarwood extract

[0034] 1. Experimental methods

[0035] The subacute model of Parkinson's disease (PD) in mice was established using MPTP, and the agarwood extract (CX) of Example 1 was administered by gavage to study the mechanism of action of CX on subacute Parkinson's disease. The construction process of the subacute model of PD in mice is as follows: Figure 1 The specific process is as follows:

[0036] SPF male C57BL / 6 mice were selected and randomly divided into 3 groups (12 mice in each group): Control group, MPTP group, CX-L group and CX-H group. During the experiment, the weight of mice in each group was recorded. Starting from the first day of the experiment, mice in the CX-L group and CX-H group were treated with CX (dissolved in saline containing 0.5% sodium carboxymethylcellulose) by gavage, where the crude drug dosage of the CX-L group was 0.225g / kg / d, and the crude drug dosage of the CX-H group was 0.45g / kg / d, for 14 consecutive days; mice in the Control and MPTP groups received the same volume of saline containing 0.5% sodium carboxymethylcellulose. From the 5th to the 9th day of the experiment, 2h after CX administration, mice in the MPTP, CX-L and CX-H groups received intraperitoneal injection of 30mg / kg / d of MPTP (dissolved in saline) for 5 consecutive days; the Control group received an intraperitoneal injection of the same amount of saline. On the 3rd day after the MPTP injection (the 12th day of the experiment), the mice were trained once a day for 3 days. The formal experiment was conducted on the 15th day. Clean feces of mice in each group were collected for fecal water content test and behavioral tests (pole climbing test, balance beam test and novel object recognition test). After the behavioral test, the mice were anesthetized and killed, and the intestinal tissue was immediately taken for intestinal propulsion rate test, and the brain tissue was immediately taken, the substantia nigra-striatum was separated, and placed in a -80℃ refrigerator for testing; the whole brain of the mouse was obtained by cardiac perfusion with 0.01mol / LPBS and 4%PFA, and frozen sections and subsequent pathological index tests were performed after fixation.

[0037] 2. Experimental results

[0038] Effects of CX on motor coordination and cognitive impairment in subacute PD mouse model

[0039] After constructing the MPTP-induced subacute PD mouse model, the MPTP group mice showed PD behavioral changes such as stiff and upright tails, involuntary body tremors, and decreased spontaneous activity. The effect of CX on movement disorders in PD mice was determined by climbing pole test and balance beam test ( Figure 2 A and B), novel object recognition experiment to investigate the effect of CX on the learning and cognitive ability of PD mice ( Figure 2 The results showed that compared with the MPTP group, CX could significantly improve the motor coordination and learning and memory abilities of PD mice.

[0040] Effects of 2.2CX on gastrointestinal function in subacute PD mouse model

[0041] After constructing the MPTP-induced subacute PD mouse model, it was clearly observed that the gastrointestinal function of the mice in the MPTP group was impaired. The effect of CX on the gastrointestinal function of PD mice was determined by intestinal propulsion rate, fecal water content and mouse body weight changes ( Figure 3 ), the results showed that compared with the MPTP group, CX could significantly improve the gastrointestinal function of PD mice.

[0042] Effects of 3CX on typical pathological features and glial cell activation in the subacute model of PD mice

[0043] The loss of dopaminergic neurons and the accumulation of α-syn are typical histological features of PD. TH is the rate-limiting enzyme in dopamine biosynthesis, and its expression level can indicate the changes in dopaminergic neurons and dopamine. The test found that the number of TH-positive dopaminergic neurons in the substantia nigra compacta of mice in the MPTP group was significantly reduced (P<0.0001), while CX treatment significantly restored the loss of neurons (P<0.0001) ( Figure 4 A).

[0044] α-syn accumulation is another important histological hallmark of PD in the substantia nigra. The expression of α-syn in the MPTP group was found to be increased by about 400% compared with the control group. However, CX treatment significantly reduced the aggregation of α-syn (P<0.0001) ( Figure 4 B).

[0045] In addition, studies have shown that the activation of microglia and astrocytes may lead to neuroinflammation and dopaminergic neuron death in the substantia nigra compacta during the development of PD. Iba-1 and GFAP are widely used as specific markers of microglia and astrocyte reactivity, respectively. By detecting Iba-1 and GFAP, it was found that the positive density of Iba-1 and GFAP in the brain tissue of PD mice was significantly increased (P < 0.0001). Low-dose CX treatment significantly reduced the reactivity of Iba-1 and GFAP (P < 0.0001), and this effect was further enhanced in the high-dose CX treatment group (P < 0.0001). Morphologically, microglia and astrocytes in the brain tissue of PD mice had round, enlarged cell bodies, accompanied by pseudopodia, and were in a reactive state ( Figure 4 C and D).

[0046] Effect of 2.4CX on NF-κB activation in subacute PD mouse model

[0047] NF-κB is a key transcription factor in the activation process of microglia. When microglia are stimulated by inflammatory stimulation and other activation signals, the NF-κB pathway is activated. Through IHC experiments, it was found that microglia in PD mice in the MPTP group were significantly activated, and CX could effectively reduce the activation of microglia. Therefore, in order to further evaluate the effect of CX on microglial activation, the degree of NF-κB activation was determined by qPCR and Western-blot experiments. The results showed that compared with the Control group, the mRNA level of the NFKB1A gene in the MPTP group increased significantly, the expression of p65 protein increased, and the expression of p-IκB-α decreased significantly (P < 0.001), indicating that NF-κB in MPTP-induced PD mice was significantly activated, showing more severe neuroinflammation, while CX could significantly inhibit the activation of NF-κB (P < 0.001), and was dose-dependent ( Figure 5 AC).

[0048] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. The use of agarwood extract in the preparation of a drug for treating Parkinson's disease, characterized in that: The preparation method of the agarwood extract comprises the following steps: The agarwood medicinal material is mixed with an ethanol solution with a volume fraction of 80%, and reflux extraction is performed twice, each time for 2 hours, and the extracts are combined and dried to obtain the agarwood extract; The treatment of Parkinson's disease is to improve Parkinson's motor coordination imbalance, cognitive impairment and gastrointestinal dysfunction.

2. The use according to claim 1, characterized in that: The volume ratio of the agarwood medicinal material to the ethanol solution is 1:

10.

3. The use according to claim 1, characterized in that: The temperature of the reflux extraction is 90°C.

4. The use according to claim 1, characterized in that: The Parkinson's disease is subacute Parkinson's disease.

5. The use according to claim 1, characterized in that: The treatment of Parkinson's disease is achieved by increasing dopaminergic neurons, reducing the aggregation of α-syn, reducing the activation degree of microglia and astrocytes, and inhibiting the activation of NF-κB pathway.

6. A drug for treating Parkinson's disease, characterized in that: The active ingredient of the drug is agarwood extract; the preparation method of the agarwood extract comprises the following steps: The agarwood medicinal material is mixed with an ethanol solution with a volume fraction of 80%, and reflux extraction is performed twice, each time for 2 hours, and the extracts are combined and dried to obtain the agarwood extract; The treatment of Parkinson's disease is to improve Parkinson's motor coordination imbalance, cognitive impairment and gastrointestinal dysfunction.

7. The drug according to claim 6, characterized in that The volume ratio of the agarwood medicinal material to the ethanol solution is 1:

10.

8. The drug according to claim 6, characterized in that The temperature of the reflux extraction is 90°C.

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