Application of gaultheria yunnanensis extract in preparation of medicine for treating cerebral diseases

Abstract

The invention relates to application of a gaultheria yunnanensis extract in preparation of a medicine for treating cerebral diseases, and particularly relates to application of a gaultheria yunnanensis extract in clinical treatment of Alzheimer's disease, cerebral ischemic disease, Parkinson's disease dementia, traumatic brain injury, chronic traumatic encephalopathy and other diseases. Meanwhile, the clinical disease treatment range of the gaultheria yunnanensis extract is further expanded. Particularly, the traditional Chinese medicine composition has a remarkable curative effect in clinical application of treating the Alzheimer's disease.

Description

Technical Field

[0001] The application of an extract of Gaultheria leucocarpa var. yunnanensis in the preparation of a drug for treating brain diseases belongs to the field of new drug uses. Background Art

[0002] Alzheimer's Disease (AD) is a common neurodegenerative disease, generally related to factors such as the increase in the patient's age, gene mutation, and the deterioration of the living environment. Its onset is mainly manifested as the decline or even loss of memory over time, inattention, emotional dullness, cognitive dysfunction, aphasia. In the late stage, AD patients are even unable to take care of themselves in life. It is estimated that by 2050, the number of AD patients worldwide will increase to 132 million. The main pathological features of AD include the excessive accumulation of β-amyloid protein (Aβ) in the brain to form senile plaques (SP) and the hyperphosphorylation of Tau protein leading to neurofibrillary tangles (NFT) in neurons. The pathogenesis of AD has not been elucidated yet, but the β-amyloid cascade hypothesis is widely recognized. Amyloid precursor protein (APP) is hydrolyzed and cleaved by β-secretase (BACE1) and γ-secretase to obtain Aβ. APP can be cleaved into Aβ40 and Aβ42. Among them, Aβ42 is more likely to aggregate, so the toxicity it shows is greater. Therefore, it is often used to construct AD cell and animal models. Usually, the Aβ produced by cells is soluble and has almost no toxic effect on neurons; but when the physiological function of cells is abnormal or in the pathological state of patients, multiple Aβ monomers tend to aggregate to form Aβ polymers. At first, two or three monomers form dimers or trimers, and then they gradually aggregate to form oligomers and protofibrils with larger molecular weights, and even insoluble fibers form senile plaques that can be seen with the naked eye in daily life, resulting in Aβ showing neuronal toxicity at low concentrations, causing neuronal synaptic loss and apoptosis, inducing oxidative stress and inflammatory reactions in the body, and disrupting calcium balance. For AD patients, they may show symptoms such as memory impairment, cognitive decline, mental and behavioral abnormalities, motor ability decline, and sensory disorders, seriously affecting the daily behavior and quality of life of patients.

[0003] Gaultheria leucocarpa var. yunnanensis is a plant of the genus Gaultheria in the Ericaceae family. Its dried roots or whole plants are used as medicine, also known as Touguxiang and Xiaotougucao. It is distributed in the southwestern region of China and has traditional effects of dispelling wind and dampness, promoting blood circulation to remove blood stasis, clearing heat and detoxifying, and regulating qi and relieving asthma.

[0004] Through the retrieval and analysis of the existing technical literature on Gaultheria leucocarpa var. crenulata extracts, it can be seen that: ①, Gaultheria leucocarpa var. crenulata extracts have analgesic and anti-inflammatory effects. Xie Wei, Fan Dinghui, Yin Li, etc., Research on the anti-inflammatory chemical constituents of Gaultheria leucocarpa var. crenulata seeds, Chemistry and Industry of Forest Products, April 28, 2015. This article publicly records that chromatographic techniques were used to separate the chemical constituents of the ethyl acetate extract with better anti-inflammatory effects, and compounds such as physcion (1), 6-ethyl-5-hydroxy-2,7-dimethoxy-1,4-naphthoquinone (2), myricetin (3), hydroquinone (4), vanillic acid (5), β-daucosterol (6), β-sitosterol (7), and succinic acid (8) were obtained. The ethyl acetate extract of Gaultheria leucocarpa var. crenulata can significantly reduce the auricular swelling degree of mice induced by xylene. He Ting, Zhao Yicheng, Li Pengyue, etc., Research on the chemical constituents of the anti-inflammatory and analgesic active parts of Gaultheria leucocarpa var. crenulata, Chinese Traditional and Herbal Drugs, September 12, 2017. This paper discloses that the 30% ethanol extract of Gaultheria leucocarpa var. crenulata, such as methyl salicylate glycosides, monoterpene glycosides, benzoic acid compounds, ferulic acid (13), chlorogenic acid (14), etc., have anti-inflammatory and analgesic effects. ②, The antioxidant effect of Gaultheria leucocarpa var. crenulata extracts. Li Dongchen, Guo Zhiqin, Lü Haining, etc., Acta Chinese Medicine and Pharmacology, December 20, 2010. The above-ground part of Gaultheria leucocarpa var. crenulata has good in vitro antioxidant activity. ③, The protective effect on the lungs. Wang Xiaoli, Lü Jiangming. Effects of Gaultheria leucocarpa var. crenulata extracts on nuclear factor-κB and tumor necrosis factor-α in rats with chronic obstructive pulmonary disease. Guangdong Medical Journal, July 25, 2013. Gaultheria leucocarpa var. crenulata extracts can reduce the expression of NF-κB and TNF-α in lung tissues and have a preventive and therapeutic effect on chronic obstructive pulmonary disease. ④, Gastrointestinal function and antidiarrheal effect. He Fei, Wei Guining, etc., Research on the gastrointestinal function and antidiarrheal effect of Gaultheria leucocarpa var. crenulata extracts, Guiding Journal of Traditional Chinese Medicine and Pharmacy, May 15, 2016. This paper discloses that Gaultheria leucocarpa var. crenulata extracts can significantly inhibit the gastric emptying and small intestine propulsion rates during the hyperactive gastrointestinal motility induced by neostigmine in KM mice, inhibit the spastic contraction of the isolated ileum of SD rats induced by acetylcholine (Ach), increase the level of intestinal absorption of D-xylose in the mouse model of intestinal absorption dysfunction induced by reserpine, and reduce the number of diarrhea induced by castor oil. ⑤, Anti-rheumatoid arthritis effect. Xiong Yulan, Xiao Bing, Ma Xiaojun, etc., Research on the anti-rheumatic arthritis active ingredients of Gaultheria leucocarpa var. crenulata, China Journal of Chinese Materia Medica, October 1, 2009. The 30% ethanol elution part of the n-butanol extract of Gaultheria leucocarpa var. crenulata has obvious anti-rat adjuvant arthritis effect. Qiao Wenlin, Beijing University of Chinese Medicine, May 1, 2013. This article records that among the different extracts of the underground part of Gaultheria leucocarpa var. crenulata, the n-butanol and water extraction parts have better anti-rheumatic activity, providing a reference for the screening of effective ingredients in Gaultheria leucocarpa var. crenulata. ⑥, Hypoglycemic effect. CN116036148A discloses an extract of Gaultheria leucocarpa var. crenulata and its application. The extract of Gaultheria leucocarpa var. crenulata exerts multiple effects such as inhibiting α-glucosidase activity and increasing cell sensitivity to insulin to achieve the anti-diabetic effect. ⑦, Treatment of dizziness effect. CN1817352A Disclosed is the use of Gaultheria leucocarpa var. crenulata as a raw material for treating Meniere's disease, Meniere's syndrome and various types of vertigo diseases with the syndrome of phlegm and stasis blocking collaterals.

[0005] However, there is no relevant research report on the use of extracts of Gaultheria leucocarpa var. crenulata for treating Alzheimer's disease in existing technical patents and journal reports. Summary of the Invention

[0006] The application of an extract of Gaultheria leucocarpa var. crenulata in the preparation of a drug for treating brain diseases. The new use of the extract of Gaultheria leucocarpa var. crenulata in the preparation of a drug for treating brain diseases, especially the petroleum ether extract of Gaultheria leucocarpa var. crenulata, provides a new drug option for the clinical treatment of diseases such as Alzheimer's disease, cerebral ischemic diseases, Parkinson's disease dementia, traumatic brain injury and chronic traumatic encephalopathy, and at the same time further expands the scope of diseases that can be clinically treated with the extract of Gaultheria leucocarpa var. crenulata.

[0007] The technical solution provided by this invention patent application is as follows: The application of an extract of Gaultheria leucocarpa var. crenulata in the preparation of a drug for treating brain diseases.

[0008] Preferably, the brain disease is: Alzheimer's disease.

[0009] Preferably, the brain disease can be: cerebral ischemic diseases.

[0010] Preferably, the brain disease can be: Parkinson's disease dementia.

[0011] Preferably, the brain disease can be: traumatic brain injury and chronic traumatic encephalopathy.

[0012] Preferably, the preparation method of the extract of Gaultheria leucocarpa var. crenulata is: take 50 g of the whole plant of Gaultheria leucocarpa var. crenulata, use 3 times the volume of methanol, extract 2 times by the hot reflux method, with each extraction time being 30 min, combine and filter the extraction solution, evaporate to dryness to obtain the crude extract of Gaultheria leucocarpa var. crenulata, dissolve it with pure water, and perform extraction by liquid-liquid extraction method in the order of petroleum ether, ethyl acetate, n-butanol, and water, with 300 mL of each of the 4 solvents. Add the same volume of each of the three organic solvents to the remaining solution from the previous step, and each organic solvent is extracted 3 - 5 times. The solutions obtained by extracting with the same organic solvent can be collected in one container, and finally evaporate to dryness respectively to obtain extracts of 4 different polar parts.

[0013] Preferably, the extract of Gaultheria leucocarpa var. crenulata is a petroleum ether extract.

[0014] Preferably, the dosage form of the extract of Gaultheria leucocarpa var. crenulata can be: including one of granules, capsules, dripping pills, and oral liquids.

[0015] The beneficial effects of the technical solution of this invention are as follows: ⑴. The new use of the Gaultheria leucocarpa var. crenulata extract of the present invention in the preparation of drugs for treating brain diseases. In particular, the Gaultheria leucocarpa var. crenulata extract is a petroleum ether extract. The petroleum ether fraction of Gaultheria leucocarpa var. crenulata reduces the expression of AD pathogenic proteins in PC-12 cells. Under a fluorescence microscope, an increase in the number of green fluorescent cells can be observed in the well plates transfected with plasmids. After intervention with GPF (the petroleum ether fraction of Gaultheria leucocarpa var. crenulata), the number of green fluorescent cells significantly decreases, and GPF can treat the damage suffered by the cells and increase the cell survival rate. Therefore, GPF can reduce the expression of AD pathogenic proteins. The above experimental results indicate that the petroleum ether fraction of Gaultheria leucocarpa var. crenulata has the effect of treating Alzheimer's disease (AD).

[0016] ⑵. The petroleum ether fraction of Gaultheria leucocarpa var. crenulata inhibits the cell death of PC-12 induced by AD pathogenic proteins by activating autophagy. The experimental results show that the effect of GPF inhibiting the decrease in the fluorescence intensity of AD pathogenic proteins is reversed. Therefore, it is concluded that GPF promotes the clearance of AD pathogenic proteins by inducing autophagy, thereby exerting a neuroprotective effect. The above results also indicate that the petroleum ether fraction of Gaultheria leucocarpa var. crenulata has a certain therapeutic effect on traumatic brain injury and chronic traumatic encephalopathy.

[0017] ⑶. After the petroleum ether fraction of Gaultheria leucocarpa var. crenulata and the positive drug Rap act on DA2123 nematodes, the green fluorescent spots representing LGG-1 increase, and there is a statistically significant difference compared with the control group (P < 0.05). The BC12921 nematode strain can express the SQST-1-GFP fusion protein (homolog of mammalian p62) under the control of the sqst1 promoter. The petroleum ether fraction of Gaultheria leucocarpa var. crenulata and Rap can also reduce the p62 fluorescence expression in BC12921 nematodes (P < 0.05). In IR1631 nematodes, it can be seen that compared with the control group, the ratio of GFP / DsRed decreases after the action of the petroleum ether fraction of Gaultheria leucocarpa var. crenulata and the positive drug Urolithin A (UA) (P < 0.05). It can be seen from this that the petroleum ether fraction of Gaultheria leucocarpa var. crenulata can induce the occurrence of autophagy / mitophagy in nematodes. The above results also indicate that the petroleum ether fraction of Gaultheria leucocarpa var. crenulata has the effect of treating Parkinson's disease (PD).

[0018] ⑷. The petroleum ether fraction of Gaultheria leucocarpa var. crenulata can inhibit the cell death induced by Aβ(1-42) fibrils, improve the survival rate of PC-12 cells, and compared with the Aβ(1-42) group, it improves the survival rate of PC-12 cells (P < 0.05), and has the ability to degrade Aβ(1-42) fibrils, thereby exerting a neuroprotective effect on the brain. Description of the Drawings

[0019] The drawings described herein are used to provide a further understanding of the present invention, constitute a part of the present invention, and the schematic embodiments of the present invention and their descriptions are used to explain the present invention, and do not constitute an improper limitation to the present invention.

[0020] Figure 1 :The different polar parts of Gaultheria leucocarpa var. crenulata inhibit the death of PC-12 cells induced by Aβ(1-42) oligomers. Note: Compared with the Aβ(1-42) group; Figure 2 :The different polar parts of Gaultheria leucocarpa var. crenulata inhibit the death of PC-12 cells induced by Aβ(1-42) fibrils; Figure 3 :Four different polar parts of Gaultheria leucocarpa var. crenulata induce autophagy in U87 cells; Figure 4 :GPF degrades AD pathogenic proteins in PC-12 cells. (a) The safe concentration range of GPF on PC-12 cells; (b) GPF plays a neuroprotective role and improves cell survival rate; (c) Fluorescence microscopy was used to observe the effect of GPF on AD pathogenic proteins; (d) GPF can reduce the expression of AD pathogenic proteins; Figure 5 :GPF reduces the fluorescence intensity of AD pathogenic proteins by activating autophagy. Note: Compared with the autophagy inhibitor group, * P <0.033, ** P <0.002, *** P <0.001, n = 3; Figure 6 :GPF increases the fluorescence spots of GFP-LGG-1 in DA2123 nematodes; Figure 7 :GPF reduces the expression of p62 in BC12921 nematodes. Note: Compared with the control group, * P <0.033, ** P <0.002, n = 20, 20× scale bar: 75 μm; Figure 8 :GPF reduces the GFP / DsRed ratio in IR1631 nematodes; Figure 9 :GPF delays the paralysis of CL2006 and CL4176 nematodes. (a) GPF inhibits the paralysis of CL4176 nematodes. (b) GPF reduces the paralysis rate of CL4176 nematodes. (c) GPF prolongs the paralysis time of CL2006 nematodes; Figure 10 :GPF inhibits the oligomerization of Aβ(1-42); Figure 11 :GPF increases the expression level of LC3 II / I in PC-12 cells; The relevant terms of this study are described herein to facilitate better understanding of the present invention by those skilled in the art. AD - Alzheimer's disease, Aβ - β - amyloid protein, PBS - phosphate buffer solution, APP - amyloid precursor protein, DMSO - dimethyl sulfoxide, Rap - rapamycin, LC3 - light chain protein; 3, Baf A1 - bafilomycin A1, 3 - MA - 3 - methyladenine, Cur - curcumin, AMPK - AMP - dependent protein kinase, PI3K - phosphatidylinositol 3 - kinase, HFIP - hexafluoroisopropanol, NGM - nematode growth medium, PE - petroleum ether, EA - ethyl acetate, NBA - n - butanol. Detailed implementation manners

[0021] To more fully understand the implementation of the present invention, an experimental example is listed below. The present invention will be further described through typical examples. It should be noted that unless otherwise specified, all are obtained under conventional conditions. If the manufacturers of the reagents used are not specified, they are all conventional products that can be obtained on the market. Example 1

[0022] Experimental materials:

[0023] 1.3 Experimental methods 1.3.1 Extraction of crude extracts of Gaultheria leucocarpa var. crenulata and extraction of different parts thereof The whole dried plant of Gaultheria leucocarpa var. crenulata was purchased from the Plateau Herbal Food Business Department in Jinniu District. The surface soil was scraped clean, crushed, and extracted twice with 3 volumes of methanol by hot reflux method for 30 minutes each time. After extraction, all the extracted solutions were combined and filtered, and the crude extract was obtained by evaporation in a constant temperature water bath.

[0024] The crude extract was dissolved in pure water and extracted by liquid - liquid extraction in the order of petroleum ether, ethyl acetate, and n - butanol. The same volume of each of the three organic solvents was added to the remaining solution of the previous step, and each organic solvent was extracted 3 - 5 times. The solutions obtained by extraction with the same organic solvent were collected in one container, and finally, extracts of 4 different polarity parts were obtained by evaporation respectively.

[0025] 1.3.2 Plasmid transfection PC-12 cells were inoculated into 96-well plates at a density of 4,500 cells / well and cultured in an incubator for 24 hours. After the cells had spread out, transfection could be carried out. Prepare Opti-MEM medium, transfection reagent, plasmid, and 1.5 mL ep tubes. The volume ratio of Opti-MEM medium to complete medium and the mass ratio of transfection reagent volume to plasmid mass were both prepared according to 1:1. 1 mL of medium was added with 2 μg of plasmid. The volume of plasmid added was calculated as: (2 / plasmid concentration) × volume of plasmid solution to be prepared.

[0026] Here, the experimental method is described for preparing 1 mL of plasmid solution. Prepare two 1.5 mL ep tubes, labeled as ① and ②. Add 250 μL of Opti-MEM medium to both ① and ②. Then add 2 μL of plasmid to ① and 2 μL of transfection reagent to ②. After covering the ep tube caps, gently flick the tube body to mix the solution inside the tube. Wait for 10 min, and slowly drip the solution in ② into ①. At this time, the volume of the solution in the tube is 500 μL. Gently flick the tube body to mix the solution inside the tube. Wait for 15 min, and add 500 μL of complete medium to make it 1000 μL.

[0027] Take out the 96-well plate from the incubator, discard the medium in the wells, and then add 100 μL of the prepared plasmid solution per well into the wells. Put the plate back into the incubator and continue to culture for 6 - 8 h. Then replace it with fresh medium and continue to culture for 24 h. After 24 h, use concentrations of 200, 100, 50, 25 μg / mL for cell viability analysis and fluorescence microscopy photography.

[0028] 1.3.3 Hoechst 33842 Fluorescent Dye Staining Procedure (1) Preparation of Hoechst stock solution: Weigh 2 mg of Hoechst 33842 powder, add 100 μL of sterilized ddH20 in a laminar flow hood to make its concentration 20 mg / mL, and store it at -20°C in the dark.

[0029] When using, add 2.5 μL of 20 mg / mL Hoechst stock solution to 1 mL of basal medium. Then discard the complete medium in the well plate and replace each well with 100 μL of the diluted Hoechst solution. After placing the well plate in the incubator for 15 min, observe and take pictures under a fluorescence microscope.

[0030] 1.3.4 Intervention Method of Autophagy Inhibitor To observe the inhibitory effects of autophagy inhibitors 3-MA, Baf, and CC on GPF autophagy. After the cells were plated or transfected, 1 h before adding the specified concentration of GPF, the inhibitors were added to the wells, and then the freshly prepared medium containing the specified concentration of GPF and inhibitors was replaced. After 24 h of intervention, cell viability analysis or fluorescence microscopy was performed.

[0031] 1.4 The experimental results are as follows: 1.4.1 Extraction of crude extract of Gaultheria leucocarpa and extraction experiments of its different parts The dried whole plant of Gaultheria leucocarpa was purchased from Jinniu District Gaoyuan Baicao Food Business Department. The surface soil was scraped clean, crushed, and extracted twice with 3 volumes of methanol by hot reflux method for 30 min each time. After the extraction, all the extracted solutions were combined and filtered, and the crude extract was obtained by evaporation in a constant temperature water bath.

[0032] The crude extract was dissolved in pure water and extracted by liquid-liquid extraction in the order of petroleum ether, ethyl acetate, n-butanol, and water. The same volume of each of the four organic solvents was added to the remaining solution from the previous step, and each organic solvent was extracted 3 - 5 times. The solutions obtained by extraction with the same organic solvent were collected in one container, and finally, the extracts of 4 different polar parts were obtained by evaporation respectively.

[0033] 1.4.2 Inhibition of Aβ(1 - 42)-induced cell death in PC-12 by different polar parts of Gaultheria leucocarpa To further verify the effect of different polar parts of Gaultheria leucocarpa in inhibiting the formation of Aβ(1 - 42) fibrils, in this study, the aqueous solution of the total extract of Gaultheria leucocarpa was extracted with three organic solvents, petroleum ether (PE), ethyl acetate (EA), and n-butanol (NBA), to obtain four different polar parts of Gaultheria leucocarpa, hereinafter referred to as "GPF", "GEF", "GNF", and "GWF" for short. "GPF" represents the petroleum ether extraction part of Gaultheria leucocarpa, "GEF" represents the ethyl acetate extraction part of Gaultheria leucocarpa, "GNF" represents the n-butanol extraction part of Gaultheria leucocarpa, and "GWF" represents the water extraction part. The four different polar parts of Gaultheria leucocarpa at concentrations of 50, 100, and 200 μg / mL were co-incubated with 20 μM Aβ(1 - 42) monomer at 37 °C for 24 h and then applied to PC-12 cells to detect cell viability. As Figure 1 shown, the four different polar parts of Gaultheria leucocarpa could all inhibit Aβ(1 - 42)-induced cell death and improve cell viability. Compared with the Aβ(1 - 42) group, the cell viability was increased (P < 0.05). Therefore, it can be considered that they can inhibit the formation of Aβ fibrils.

[0034] Meanwhile, in order to explore whether the four different polar fractions of **Gaultheria leucocarpa** var. **crenulata** have the activity of degrading Aβ fibers, GPF at concentrations of 25, 50, and 100 μg / mL and the other three polar fractions at concentrations of 25, 50, 100, and 200 μg / mL were added to the wells containing PC-12 cells together with Aβ(1-42) fibrils at a concentration of 20 μM. After 24 h of incubation, the cell viability was statistically analyzed. As shown in Figure 2 the figure, the four different polar fractions of **Gaultheria leucocarpa** var. **crenulata** all inhibited the cell death induced by Aβ(1-42) fibrils, increased the viability of PC-12 cells, and compared with the Aβ(1-42) group, the viability of PC-12 cells was increased (P < 0.05). Therefore, it can be considered that they have the ability to degrade Aβ(1-42) fibers and thus play a role in protecting brain nerves.

[0035] 1.4.3 Autophagy induction of different polar fractions of **Gaultheria leucocarpa** var. **crenulata** in U87 cells The highest concentration of the four different polar fractions of **Gaultheria leucocarpa** var. **crenulata** was set at 400 μg / mL and then serially diluted by half to 12.5 μg / mL. After 24 h of incubation with U87 cells, fluorescence microscopy was used for image acquisition. Images of concentrations without cytotoxicity under the microscope were selected for processing, and the percentage of autophagy-positive cells in the total number of cells in the photographed field of view was statistically analyzed and plotted. As shown in Figure 3 the figure, compared with the control group, the four polar fractions of **Gaultheria leucocarpa** var. **crenulata** all activated autophagy in U87 cells (P < 0.05). The greater the polarity of the fraction, the worse the autophagy activation effect. Among them, GPF had a larger concentration range for autophagy activation and a better effect.

[0036] 1.4.4 The petroleum ether fraction of **Gaultheria leucocarpa** var. **crenulata** reduces the expression of AD pathogenic proteins in PC-12 cells As a microtubule-associated protein enriched in neuronal axons, Tau protein assists axonal growth and axonal transport, plays a role in maintaining microtubule stability and neuronal morphology. Normal or reactive phosphorylation of Tau protein has a neuroprotective effect. Under pathological conditions, persistent hyperphosphorylation of Tau protein will interfere with the physiological functions of axons, aggregate in neuronal axons to become the main component of NFT, induce neuronal damage, and thus lead to neurodegenerative diseases such as AD. Among them, Tau-P301L is more prone to misfolding and hyperphosphorylation. APP can be cleaved into Aβ40 and Aβ42, inducing neurotoxicity. Therefore, in this experiment, pRK5-EGFP-Tau-P301L, pRK5-EGFP-Tau and pEGFP-N1-APP plasmids were transiently transfected into PC-12 cells to induce cell damage, and then intervened with GPF at concentrations of 25, 50, 100 μg / mL. Hoechst 33842 fluorescent dye was used to indicate the cell nucleus to facilitate the statistical analysis of the total number of cells. Fluorescence microscopy was used for image acquisition, and the ratio of green fluorescence / blue fluorescence was statistically analyzed to reflect the effect of GPF on AD pathogenic proteins.

[0037] As Figure 4 a, GPF did not affect the growth of PC-12 cells (P>0.05). Under the fluorescence microscope, an increase in the number of green fluorescent cells was observed in the well plates transfected with plasmids. After GPF intervention, the number of green fluorescent cells decreased significantly ( Figure 4 c, d, P<0.05), and GPF could treat the damage suffered by cells and increase the cell survival rate ( Figure 4 b, P<0.05). Therefore, GPF can reduce the expression of AD pathogenic proteins.

[0038] 1.4.5 The petroleum ether fraction of Gaultheria leucocarpa Bl. var. crenulata (Kurz) T. Z. Hsu inhibits cell death of PC-12 induced by AD pathogenic proteins by activating autophagy To study whether there is a close relationship between the effect of GPF on clearing AD pathogenic proteins and the activation of autophagy, we transiently transfected pEGFP-N1-APP, pRK5-EGFP-Tau and pRK5-EGFP-Tau-P301L plasmids into PC-12 cells. After pre-intervention with autophagy inhibitors 3-MA (5 mM) and Baf A1 (10 μM) for 1 h, 100 μg / mL GPF was intervened for 24 h, and the number and intensity of green fluorescent cells were observed under a fluorescence microscope. From Figure 5 it can be seen that GPF reduced the fluorescence expression of APP, Tau and Tau-P301L. After adding autophagy inhibitors 3-MA and Baf A1, the effect of GPF on inhibiting the decrease in the fluorescence intensity of AD pathogenic proteins was reversed. Therefore, it was concluded that GPF promoted the clearance of AD pathogenic proteins by inducing autophagy, thereby exerting a neuroprotective effect.

[0039] Example 2: Extract of Gaultheria leucocarpa var. crenulata reduces Aβ aggregation in nematodes and improves their behavioral ability by activating autophagy

[0040] 2.3 Experimental methods 2.3.1 Reagent preparation 2.3.1.1 Preparation of nematode growth medium (NGM) Weigh 1.2 g of NaCl, 7.0 g of agar, and 1.0 g of peptone, add ddH2O to make up the volume to 400 mL, autoclave at 121 °C for 35 min, then add 400 μL each of CaCl2, MgSO4, and cholesterol, and 5 mL of K2HPO4 / KH2PO4 buffer solution, pour into petri dishes and dry for later use.

[0041] 2.3.1.2 Preparation of LB (Luria-Bertani) medium Weigh 3 g of peptone, 1.5 g of NaCl, and 1.5 g of yeast extract, then add ddH2O to make up the volume to 300 mL, and autoclave at high temperature for later use.

[0042] 2.3.1.3 Preparation of M9 buffer solution Weigh 2 g of NaCl, 6.05 g of Na2HPO4, and 1.2 g of KH2PO4, add ddH2O to make up the volume to 400 mL and autoclave at high temperature, then add 400 μL of MgSO4 after cooling for later use.

[0043] 2.3.1.4 Preparation of cryopreservation solution (50 mL) Weigh 0.2925 g of NaCl, 0.34 g of KH2PO4, and 15 g of glycerol; add 0.28 mL of 1 mol / L NaOH and 15 μL of 1 mol / L MgSO4, and autoclave at high temperature for later use.

[0044] 2.3.1.5 Others (1)1 mol / L CaCl2: 5.549 g of CaCl2 + 50 mL of ddH2O, autoclave at high temperature for later use.

[0045] (2)1 mol / L MgSO4: 6.0 g of MgSO4 + 50 mL of ddH2O, autoclave at high temperature for later use.

[0046] (3) 2 mol / L K2HPO4 / KH2PO4: 28.48 g of K2HPO4 + 86.64 g of KH2PO4 + 400 mL of ddH2O, sterilized by autoclaving for later use.

[0047] (4) 5 mg / mL cholesterol: 250 mg of cholesterol + 50 mL of absolute ethanol, sterilized by autoclaving for later use.

[0048] Operation methods related to bacterial culture and nematode culture 2.3.2 Cultivation of Escherichia coli OP50 Take out the frozen Escherichia coli OP50 strain from the bacterial library. Use a sterile bacterial inoculation loop to dip a small amount of bacterial liquid in the laminar flow hood. After dilution with sterilized ddH2O, streak the plate on an LB solid culture plate. Seal the plate and invert it, then place it in a 37°C constant temperature incubator overnight. Observe the plate the next day. If monoclonal colonies are formed, they can be stored in a 4°C refrigerator for later use. When in use, use a sterile pipette tip to pick a monoclonal colony in the laminar flow hood and transfer it to an Erlenmeyer flask containing LB liquid medium. Seal it and place it in a 37°C, 160 rpm constant temperature shaking incubator and shake it evenly for 16 - 24 h. After the LB medium becomes turbid, take it out, centrifuge at 2400 rpm for 10 min, discard the supernatant, add 5 - 10 mL of ddH2O to resuspend the precipitate, and store it at 4°C.

[0049] 2.3.3 Cultivation of Caenorhabditis elegans Generally, the food source of nematodes is Escherichia coli OP50. Nematodes are cultured on NGM plate medium and cultured in a constant temperature and humidity incubator at a temperature of 20°C and a humidity of 60%.

[0050] 2.3.4 Recovery of Caenorhabditis elegans Prepare NGM plates and add OP50 in advance. Then take out the frozen nematode strain from an -80°C ultra-low temperature refrigerator. Thaw it at room temperature in the laminar flow hood, gently pipette and mix it, and quickly transfer it to the plate and make marks. After the NGM plate dries naturally, transfer the plate to a 20°C constant temperature incubator and culture it for 1 - 3 days to observe whether the nematodes are successfully recovered. Before being used in experiments, nematodes need to be identified and verified for phenotype and gene line, and cultured synchronously and repeatedly for at least 3 generations or more to ensure stable and normal inheritance.

[0051] 2.3.5 Cryopreservation of Caenorhabditis elegans Keep the plate sterile and clean for 2 - 3 days until the nematodes are starved and enter the dauer stage. Use a pipette to aspirate M9 buffer to rinse the plate, and aliquot the rinsed nematodes into EP tubes. Centrifuge, discard the supernatant, add a solution prepared by mixing M9 and cryopreservation solution in a ratio of 1:1 to each tube, make marks, place them in a programmable freezing box, and store them in an ultra-low temperature freezer at -80°C for 24 h. They can be stored at -80°C for a long time.

[0052] 2.3.6 Synchronization of Caenorhabditis elegans Take a nematode culture plate that has grown at the L1 stage for 50 - 56 h at 20°C. Observe the egg-laying situation under a microscope, and try to select the period when there are more eggs in the nematodes and more eggs are discharged on the plate for synchronization. The specific operation process is as follows: (1)Nematode collection: Collect nematodes with sterilized ddH2O and make an 8 mL eluate in a 15 mL centrifuge tube.

[0053] (2)Prepare nematode lysis solution: Prepare a solution by mixing 5 mM NaOH solution (10 g NaOH + 50 mL ddH2O) and sodium hypochlorite solution in a ratio of 1:1, and use it immediately after preparation.

[0054] (3)Add 2 mL of lysis solution to the 8 mL eluate, mix well, and observe the lysis situation under a microscope. Shake intermittently until most of the nematode bodies in the tube are broken.

[0055] (4)Immediately centrifuge at 1200 rpm for 2 min, discard the supernatant, add 10 mL of ddH2O to resuspend and wash the nematodes, wash repeatedly 3 times. After the last centrifugation, discard the supernatant, add 10 mL of M9 buffer culture solution, mix well, and place it in an incubator at 20°C for overnight culture.

[0056] (5)The next day, observe under a microscope whether the nematode eggs in the culture tube have hatched into L1 larvae. If the hatching is successful, place it in an ice bath for 10 min, then centrifuge at 2000 rpm for 10 min, discard the supernatant, leave a small amount of M9 buffer, mix well again, use a pipette to aspirate an appropriate amount of M9 buffer containing nematodes, and transfer it to an NGM plate. Let it air dry and then place it in an incubator at 20°C for culture.

[0057] 2.3.7 Fluorescent quantitative detection of the expression of autophagy-related proteins Synchronized L1-stage DA2123 and BC12921 larvae were plated on NGM plates containing GPF or Rap and cultured at 20 °C for 48 h. Then, the nematodes were transferred to a glass slide containing 100 mM sodium azide (NaN3) for anesthesia, and a coverslip was gently placed on top. Observation and image acquisition were performed under an upright fluorescence microscope. Image J software was used to count the number of LGG-1 positive fluorescent dots in seam cells or intestinal cells of DA2123 nematodes in a fixed area and to quantify the p62 fluorescence intensity of BC12921 nematodes.

[0058] 2.3.8 Fluorescent quantitative detection of the aggregation of β-amyloid (3-42) Synchronize L1-stage CL2331 larvae at 15 °C. When the larvae reach the late L4 stage, transfer them to an NGM plate containing DBZ to prevent offspring hatching, and continue to culture at 25 °C for 24 h. Then, collect the nematodes with M9 buffer, transfer them to a glass slide containing 100 mM NaN3 for anesthesia, gently place a coverslip on top, and perform observation and image acquisition under an upright fluorescence microscope. Image J software was used to count the number of Aβ(3-42) positive fluorescent spots in a fixed area.

[0059] 2.3.9 Nematode paralysis assay The transgenic nematode strains CL2006 and CL4176 that specifically express human Aβ(1-42) protein in muscles were used for chronic and acute paralysis experiments.

[0060] CL2006: After maintaining L1-stage nematodes at 20 °C for 52 h, transfer them to an NGM plate containing DBZ to prevent offspring hatching, and continue to culture at 20 °C. Replace the fresh NGM plate every two days. When nematodes start to enter the paralyzed state, start recording the number of paralyzed nematodes and perform statistics every 24 h.

[0061] CL4176: Culture L1-stage nematodes at 15 °C for 36 h, then quickly transfer them to an incubator at 25 °C and continue to culture for 30 h. Observe the status of nematodes under a microscope at any time. When the nematodes do not move or only the head moves but the body cannot move under external mechanical stimulation (such as gently touching the body with a nematode needle), they can be counted as paralyzed. When many nematodes on the plate enter the paralyzed state, start taking pictures and performing statistics.

[0062] 2.4 Experimental results: 2.4.1 The petroleum ether fraction of Gaultheria leucocarpa Bl. var. crenulata (Kurz) T. Z. Hsu induces autophagy in nematodes To investigate whether GPF also activates autophagy / mitophagy in vivo, we selected DA2123, BC12921, and IR1631 nematode strains for verification. DA2123 is a transgenic nematode strain expressing LGG-1 (a homolog of the human autophagy marker gene LC3) with green fluorescence (GFP), which can reflect the activity of LGG-1. During autophagy, LGG-1 protein aggregates to form fluorescent spots, indicating the occurrence of autophagy in nematodes. As Figure 6 shown, after treatment with GPF and the positive drug Rap on DA2123 nematodes, the green fluorescent spots representing LGG-1 increased, showing a statistically significant difference compared with the control group (P < 0.05).

[0063] The BC12921 nematode strain can express the SQST-1-GFP fusion protein (a homolog of mammalian p62) under the control of the sqst1 promoter. As Figure 7 shown, GPF and Rap can also reduce the p62 fluorescence expression in BC12921 nematodes (P < 0.05).

[0064] The IR1631 nematode can express pH-sensitive mtRosella in body wall muscles or neuronal mitochondria. mtRosella is a mitochondrial-targeted dual-fluorescent probe, which includes a pH-sensitive GFP and a pH-insensitive DsRed moiety. The hallmark of mitophagy is a decrease in the ratio of GFP / DsRed fluorescence intensity. In IR1631 nematodes, it can be seen that compared with the control group, the ratio of GFP / DsRed decreased after treatment with GPF and the positive drug Urolithin A (UA) (see the attached Figure 8 instructions, P < 0.05). The above data indicate that GPF can induce autophagy / mitophagy in nematodes.

[0065] 2.4.2 GPF Petroleum Ether Fraction Alleviates Paralysis of CL-2006 and CL-4176 Nematodes To investigate the potential effect of GPF on the progression of Aβ(1-42)-induced paralysis, we selected two nematode strains, CL2006 and CL4176. The temperature-sensitive CL4176 transgenic nematode strain can express human Aβ(1-42), and Aβ accumulates in body wall muscle cells to form deposits, leading to nematode paralysis; in the CL2006 nematode strain expressing Aβ(1-42) as well, the Aβ toxicity is highly age-dependent and can be accompanied by adult paralysis with the formation of Aβ. Therefore, it is used for chronic paralysis experiments. Paralyzed nematodes have rigid and uncurved bodies. The percentage of paralyzed nematodes in the total number of nematodes was counted. As Figure 9As shown in a and b, compared with the control group, the number of paralyzed CL4176 nematodes was significantly reduced after the action of GPF (P < 0.05). From Figure 9 it can be seen that Aβ induced paralysis in CL2006 nematodes on the 10th day after adulthood. As time went by, the number of paralyzed nematodes increased more and more, and finally all the nematodes were paralyzed, while GPF delayed the time of all nematodes being paralyzed.

[0066] Example 3 Inhibitory effect of the petroleum ether fraction of Gaultheria leucocarpa Bl. var. crenulata (Kurz) T. Z. Hsu on Aβ fibrillization To verify the inhibitory effect of GPF on Aβ fiber formation, we set three concentration groups of GPF, and let it be formulated with Aβ(1-42) monomer using sterile PBS, so that the final concentrations of GPF were 50, 100, and 200 μg / mL respectively, and the working concentration of Aβ(1-42) monomer was 20 μM. They were co-incubated at 37 °C for 48 h. The protein concentration and protein denaturation of the samples did not need to be measured. After adding the loading Buffer, 10 μL was loaded into each well for WB experiment, and the Aβ antibody 6E10 was used to detect the situation of Aβ oligomerization. Figure 10 It showed that there was more formation of oligomers / fibrils in the model group and the positive control group, while the amount of oligomerized substances in the drug group decreased with the increase of concentration.

[0067] Example 4 The petroleum ether fraction of Gaultheria leucocarpa Bl. var. crenulata (Kurz) T. Z. Hsu increases the ratio of LC3 II / I To verify the effect of GPF on activating autophagy, we treated PC-12 cells with 100 μg / mL, 200 μg / mL, and 300 μg / mL of GPF and 20 μM of the positive control drug Rap. After 24 h, the proteins were extracted, and the expression level of LC3 was detected by Western Blot experiment. Similarly, the ratio of LC3 II and LC3 I was counted to reflect the autophagy activation activity of GPF. From Figure 11 it can be seen that compared with the control group, both GPF and Rap could increase the expression level of LC3 II / LC3 I (P > 0.05).

Claims

1. Use of an extract of Gaultheria leucocarpa Bl. var. crenulata (Kurz) T. Z. Hsu in the preparation of a medicament for treating brain diseases.

2. The application according to claim 1, characterized in that, The brain disease is: Alzheimer's disease.

3. The application according to claim 1, characterized in that The brain disease may be: cerebral ischemic disease.

4. The application according to claim 1, characterized in that The brain disease may be: Parkinson's disease dementia.

5. The application according to claim 1, characterized in that The brain disease may be: traumatic brain injury and chronic traumatic encephalopathy.

6. The application according to claim 1, characterized in that The preparation method of the extract of Gaultheria leucocarpa Bl. var. crenulata (Kurz) T. Z. Hsu is as follows: Take 50 g of the whole herb of Gaultheria leucocarpa Bl. var. crenulata (Kurz) T. Z. Hsu, use 3 times the volume of methanol, extract 2 times by the hot reflux method, with each extraction time being 30 min. Combine and filter the extraction solution, evaporate to dryness to obtain the crude extract of Gaultheria leucocarpa Bl. var. crenulata (Kurz) T. Z. Hsu, dissolve it with pure water, and in the order of petroleum ether, ethyl acetate, n-butanol, and water, 300 mL of each of the 4 solvents, carry out extraction by the liquid-liquid extraction method. The same volume as the remaining solution in the previous step is added to each of the three organic solvents, and each organic solvent is extracted 3 - 5 times. The solutions obtained by extraction with the same organic solvent can be collected in one container, and finally, the extracts of 4 different polar parts are obtained by evaporating to dryness respectively.

7. The application according to claim 1, characterized in that, The extract of Gaultheria leucocarpa Bl. var. crenulata (Kurz) T. Z. Hsu is the petroleum ether extract.

8. The application according to claim 1, wherein The dosage form of the extract of Gaultheria leucocarpa Bl. var. crenulata (Kurz) T. Z. Hsu may be: including one of granules, capsules, dripping pills, and oral liquids.

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