Application of cordyceps sobolifera in preparation of medicine for treating SPTLC1 gene related motor neuron disease

Abstract

The invention relates to the field of biological medicine, in particular to application of isaria cicadae miquel in preparation of medicine for treating SPTLC1 gene related motor neuron disease, and has the following beneficial effects that (1) a preparation method of a lipid extract of isaria cicadae miquel is optimized, and the content of effective components of the lipid extract is higher; (2) the cordyceps sobolifera is proved to be used for treating the SPTLC1 gene related motor neuron disease on a cellular level and a patient level; in a cell experiment, the isaria cicadae miquel extract can reduce the sphingolipid and deoxysphingolipid levels of an SPTLC1 gene mutation HEK293T cell line, which indicates that the isaria cicadae miquel extract can inhibit the activity of SPT enzyme; in the patient level, the isaria cicadae miquel can remarkably reduce the level of sphingolipid and deoxysphingolipid in the blood of a patient suffering from the SPTLC1 gene mutation related motor neuron disease, it is proved that the isaria cicadae miquel has the capacity of treating the SPTLC1 gene related motor neuron disease, a new reference is provided for developing effective medicine for treating the SPTLC1 gene related motor neuron disease, and the isaria cicadae miquel can be used for preparing a medicine for treating the SPTLC1 gene related motor neuron disease. The sources of clinical medicines are enriched.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an application of Cordyceps sinensis in preparing a medicine for treating SPTLC1 gene-related motor neuron disease. Background Art

[0002] Cordyceps sinensis is an entomogenous fungus formed by the parasitism of the fungus Cordyceps sinensis on cicada nymphs. It is an asexual form of Cordyceps sinensis. The medicinal parts are the stroma and the corpse of the host insect (sclerotium). Modern pharmacological studies have shown that Cordyceps sinensis has similar chemical components to Cordyceps sinensis, mainly including polysaccharides, amino acids, nucleosides, sugar alcohols, ergosterol and other compounds. Wang Qiong et al. reviewed the pharmacological activities of Cordyceps sinensis and found that Cordyceps sinensis has the effects of improving renal function, regulating immunity, and anti-oxidation. Its renal protective effect may be through downregulating the expression of transforming growth factor-β1 (TGF-β1), recombinant human connective tissue growth factor (CTGF) protein and its messenger RNA (mRNA), regulating sphingolipid metabolism, blocking IL-2 receptor signal transduction, and thus inhibiting the reproduction of T cells. Sun Xin et al. showed through animal experiments that Cordyceps sinensis may reduce the release of related metabolic factors by downregulating the expression of TGF-β1, α-smooth muscle actin (α-SMA) and fibronectin, thereby delaying the mesenchymal transition of renal tubular epithelial cells (HK-2), and may even reverse glomerular sclerosis and renal interstitial cell fibrosis. Xiong Wei et al. observed the effects of Cordyceps sinensis granules on inflammation and oxidative stress in MHD patients. After 12 weeks of oral Cordyceps sinensis granules, the inflammatory indicators such as IL-1, IL-6, TNF-α, and oxidative stress indicators such as MDA, myeloperoxidase (MPO), and advanced oxidation protein products (AOPP) in the treatment group were lower than those before treatment and the control group; at the same time, the anemia-related indicators such as red blood cell count, hemoglobin, and hematocrit, and the nutrition-related indicators such as albumin, prealbumin, and transferrin in the treatment group were significantly increased compared with those before treatment and the control group, and the difference was statistically significant.

[0003] Motor Neuron Disease (MND) was first described by French neurologist Jean-Martin Charcot in the late 19th century, with an incidence of approximately 2 / 100,000. MND mainly affects adults, with a typical age of onset in middle age or old age. The main characteristics of the disease are progressive muscle weakness and atrophy, which usually starts from the distal limbs or face, gradually spreads to the muscles of the whole body, and eventually leads to respiratory muscle involvement. According to pathology and clinical manifestations, MND can be divided into multiple types, including primary lateral sclerosis (PLS) with damage to upper motor neurons and amyotrophic lateral sclerosis (ALS) with both upper and lower motor neurons involved, of which ALS is the most common, accounting for about 85%-90% of all cases. In recent years, studies have found that multiple gene mutations are associated with the occurrence of MND, such as SOD1, C9orf72, TARDBP, etc. C9orf72 gene mutation is the most common cause of familial MND, accounting for about 40% of familial cases.

[0004] SPTLC1 gene mutation is a pathogenic gene that causes rare motor neuron disease. The SPTLC1 gene encodes a subunit of serine palmitoyltransferase, which plays a key role in the synthesis of sphingolipids. Sphingolipids are important components of nerve cell membranes, especially in maintaining the normal function and structure of nerve cells. SPTLC1 gene mutation usually leads to abnormal sphingolipid metabolism in nerve cells, produces neurotoxic sphingolipids, and then causes degeneration of motor neurons. Patients affected by this mutation often show early-onset motor neuron disease, whose symptoms include progressive weakness of the distal limbs, muscle atrophy, and paresthesia. These symptoms will gradually worsen, affecting the patient's mobility and quality of life. In recent years, studies have also found that SPTLC1 gene mutations are associated with a disease called "familial autonomic neuropathy" (HSAN1), suggesting that it also has a potential impact on sensory neurons. Although the current treatment for MND is mainly to relieve symptoms and improve the quality of life, the progress of genetic research provides new hope for the development of potential treatment strategies in the future. At present, the clinical treatment of this disease mainly adopts symptomatic supportive therapy, and there is no special treatment.

[0005] Prior art CN 103992416 B discloses Cordyceps sinensis polysaccharide and its application in the preparation of neuroprotective and anti-aging drugs. The Cordyceps sinensis polysaccharide is obtained according to the following method: dry Cordyceps sinensis medicinal materials, crush through the pharmacopoeia No. 2 sieve, extract the powder with water reflux twice at 90°C, each time for 2 hours, and after the extract is concentrated under reduced pressure, add anhydrous ethanol and precipitate at 4°C-25°C overnight; centrifuge at 3000r / min for 10-30min; dry the precipitate to obtain Cordyceps sinensis polysaccharide (JCHcps). The obtained Cordyceps sinensis polysaccharide can significantly inhibit the aging damage of PC12 cells induced by glutamate, prevent the release of cell LDH, improve the survival rate of cells, reduce the level of oxygen free radicals in cells, increase the activity of glutathione reductase (GSH-Px) and superoxide dismutase (SOD), and in the scavenging of DPPH - and superoxide anion (0 2- ) showed good in vitro antioxidant activity in the free radical experiment. However, the above prior art does not mention the efficacy of Cordyceps sinensis in SPTLC1 gene-related motor neuron disease. Therefore, the present invention will try to explore the efficacy of Cordyceps sinensis in the treatment of SPTLC1 gene-related motor neuron disease. Summary of the invention

[0006] In order to solve the above problems, the present invention first provides an application of Cordyceps sinensis in preparing a medicine for treating SPTLC1 gene-related motor neuron disease.

[0007] Preferably, the Cordyceps sinensis is extracted with water.

[0008] Preferably, the Cordyceps sinensis is extracted with alcohol.

[0009] Preferably, the alcohol extraction method comprises the following steps:

[0010] (1) Weigh Cordyceps sinensis, rinse with clean water, grind into powder, pass through a 100-mesh sieve, and add 200 mL of deionized water;

[0011] (2) Boil and dissolve in a water bath at 100°C for 20 min to obtain an extract, and filter to obtain a filtrate;

[0012] (3) adding an excess of 50-80% ethanol (w / v), reflux extraction at 100° C. for 3 times, each time for 2 hours; after each reflux, centrifuge at 4000 r / min for 10 minutes to obtain the supernatant, combine the three supernatants, concentrate by rotary evaporation to 1 / 10 of the original volume, and then freeze-dry to obtain a crude cicada flower extract;

[0013] (4) Dissolving the Cicadae Convolvulus extract 1 in distilled water (1:10, w / v), centrifuging at 4000 rpm for 10 min, taking the supernatant, and freeze-drying to obtain the Cicadae Convolvulus extract.

[0014] Preferably, the ethanol is 55-75% ethanol (w / v).

[0015] Preferably, the ethanol is 65% ethanol (w / v).

[0016] Compared with the prior art, the present invention has at least the following beneficial effects:

[0017] (1) The alcohol extraction method of Cordyceps sinensis was optimized, and the content of active ingredients was higher;

[0018] (2) It was demonstrated at the cellular level and in patient experiments that Cordyceps sinensis can be used to treat Charcot-Marie-Tooth disease. In cell experiments, Cordyceps sinensis extract can reduce the levels of sphingolipids and deoxysphingolipids in HEK293T cell lines with SPTLC1 gene mutations, indicating that it can inhibit SPTase activity. At the patient level, Cordyceps sinensis can significantly reduce the levels of sphingolipids and deoxysphingolipids in the blood of patients with SPTLC1 gene mutation-related motor neuron disease, demonstrating that it has the ability to treat SPTLC1 gene mutation-related motor neuron disease, providing a new reference for the development of effective drugs for the treatment of SPTLC1 gene mutation-related motor neuron disease and enriching the source of clinical drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the result chart of the content of Cordyceps sinensis extract;

[0020] Figure 2 The results are as follows: the effect of Cordyceps sinensis extract on sphingolipids and deoxysphingolipids in HEK293T cells with SPTLC1 gene knockout and overexpression of SPTLC1 mutant gene;

[0021] Figure 3 This is the result of the effect of Cordyceps sinensis extract on sphingolipids and deoxysphingolipids in the blood of patients with SPTLC1 gene mutation. DETAILED DESCRIPTION

[0022] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0023] Example 1

[0024] Weigh 3-5 g of Cordyceps sinensis, crush it with a grinding rod, mix it with 200 ml of pure water, boil it at 100° C. for 20 minutes, cool it to room temperature, and filter it through medical gauze to obtain Cordyceps sinensis extract.

[0025] Example 2

[0026] Weigh 10g of golden cicada flower, rinse with water, grind into powder, pass through a 100 mesh sieve, add 200mL of deionized water, add 20mg of cellulase (50,000 U / g), enzymolyze in a 37℃ water bath for 2h to obtain an enzymolysis solution, boil for 15 minutes to inactivate, filter, obtain the filtrate, add excess 65% ethanol (w / v), reflux extract at 100℃ for 3 times, each time for 2h. After each reflux, centrifuge at 4000r / min for 10min to take the supernatant, combine the three supernatants, rotary evaporate and concentrate to 1 / 10 of the original volume, and then freeze-dry to obtain a crude cicada flower extract. Dissolve cicada flower extract 1 (1:10, w / v) in distilled water, centrifuge at 4000r / min for 10min, and take the supernatant. Freeze-dry to obtain a cicada flower extract.

[0027] Example 3

[0028] Weigh 10g of golden cicada flower, rinse with clean water, grind into powder, pass through a 100 mesh sieve, add 200mL of deionized water, add 20mg of pectinase (50,000 U / g), enzymolyze in a 37℃ water bath for 2h to obtain an enzymolysis solution, boil for 15 minutes to inactivate, filter, obtain the filtrate, add excess 65% ethanol (w / v), reflux extract at 100℃ for 3 times, each time for 2h. After each reflux, centrifuge at 4000r / min for 10min to take the supernatant, combine the three supernatants, rotary evaporate and concentrate to 1 / 10 of the original volume, and then freeze-dry to obtain a crude cicada flower extract. Dissolve cicada flower extract 1 (1:10, w / v) in distilled water, centrifuge at 4000r / min for 10min, and take the supernatant. Freeze-dry to obtain a cicada flower extract.

[0029] Example 4

[0030] Weigh 10g of golden cicada flower, rinse with clean water, grind into powder, pass through a 100 mesh sieve, add 200mL of deionized water, add 20mg of complex enzyme (50,000 U / g cellulase and 50,000 U / g pectinase, weight ratio of 1:1), enzymolysis in a 37°C water bath for 2h to obtain an enzymolysis solution, boil for 15 minutes to inactivate, filter, obtain the filtrate, add excess 55% ethanol (w / v), reflux extract at 100°C for 3 times, each time for 2h. After each reflux, centrifuge at 4000r / min for 10min to take the supernatant, combine the three supernatants, rotary evaporate and concentrate to 1 / 10 of the original volume, and then freeze-dry to obtain a crude cicada flower extract. Dissolve cicada flower extract 1 (1:10, w / v) in distilled water, centrifuge at 4000r / min for 10min, and take the supernatant. Freeze-dry to obtain a cicada flower extract.

[0031] Example 5

[0032] Weigh 10g of golden cicada flower, rinse with clean water, grind into powder, pass through a 100-mesh sieve, add 200mL of deionized water, add 20mg of complex enzyme (50,000 U / g cellulase and 50,000 U / g pectinase, weight ratio of 1:1), enzymolysis in a 37°C water bath for 2h to obtain an enzymolysis solution, boil for 15 minutes to inactivate, filter, obtain the filtrate, add excess 75% ethanol (w / v), reflux extract at 100°C for 3 times, each time for 2h. After each reflux, centrifuge at 4000r / min for 10min to take the supernatant, combine the three supernatants, rotary evaporate and concentrate to 1 / 10 of the original volume, and then freeze-dry to obtain a crude cicada flower extract. Dissolve cicada flower extract 1 (1:10, w / v) in distilled water, centrifuge at 4000r / min for 10min, and take the supernatant. Freeze-dry to obtain a cicada flower extract.

[0033] Example 6: Cell assay

[0034] Effects of Cordyceps sinensis extract on sphingolipids and deoxysphingolipids in HEK293T cells with SPTLC1 gene knockout and overexpression of SPTLC1 mutant gene:

[0035] HEK293T cells were cultured in DMEM, which was replaced with DMEM without L-serine before the experiment. Radioactive isotope-labeled D3-15N-L-serine (1 mM) was added to this DMEM. Different percentages of water-soluble Cordyceps sinensis extract (filtered using a 0.4 um filter) were directly added to the culture medium. After 16 hours of treatment with Cordyceps sinensis extract, the cells were collected and lysed to extract lipids. Ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS / MS) was then used to detect the production levels of isotope serine-labeled sphingosine (sphingosine (SO+3)) and serine-free sphingolipids (SO) in the cell lysate, such as Figure 1 As shown, the sheath isotope serine labeled SO in HEK293T cells overexpressing SPTLC1 mutant gene and SPTLC1 knockout gene treated with Cordyceps sinensis extract +3 The levels of non-isotopic serine-labeled SO decreased, and the relevant results are shown in the attached Figure 1 .

[0036] Example 7: Patient Trial

[0037] The plasma sphingolipid profiles (dhCer (d18:0), Cer (d18:1), Cer (d18:2), dhHexCer (d18:0), HexCer (d18:1), HexCer (d18:2)) and deoxysphingolipid (1-deoxydhCer (m18:0)) levels were detected in patients with SPTLC1 gene p.L38R mutation and normal subjects. See the attached Figure 2A patient with a rare disease who was diagnosed with motor neuron disease caused by SPLTC1 gene mutation in the outpatient department of neurology of the Third Xiangya Hospital of Central South University was recruited. 3-5g of Cordyceps sinensis was weighed each time, crushed with a grinding rod, mixed with 200ml of pure water, boiled at 100℃ for 20 minutes, cooled to room temperature, filtered through medical gauze to obtain Cordyceps sinensis extract, and drunk it twice, and temporarily stored in a 4℃ refrigerator during the period.

[0038] The recruited patients took the medicine for 8 consecutive weeks. The sphingolipid profile and deoxysphingolipid levels in plasma were detected by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS / MS) before taking the Cordyceps sinensis extract, at 4 weeks and 8 weeks of taking it. It was found that sphingolipids and deoxysphingolipids decreased significantly.

[0039] The results are as follows Figure 3 Shown: Cordyceps sinensis extract can significantly improve the sphingolipid profile and deoxysphingolipid levels in patients with SPTLC1 gene mutation-related motor neuron disease.

[0040] In summary, the present invention verifies that the Cordyceps sinensis extract is a potential therapeutic drug for SPTLC1 gene-related motor neuron disease through experiments at the cell level and patient level.

[0041] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A use of Cordyceps sinensis in the preparation of a drug for treating SPTLC1 gene-related motor neuron disease.

2. The use according to claim 1, characterized in that The Cordyceps sinensis is extracted with water.

3. The use according to claim 1, characterized in that The Cordyceps sinensis is extracted with alcohol.

4. The use according to claim 3, characterized in that The method of alcohol extraction comprises the following steps: (1) Weigh the Cordyceps sinensis, crush it thoroughly, rinse it with clean water, grind it into powder, and pass it through a 100-mesh sieve. Add 200 mL of deionized water; (2) Boil and dissolve in a water bath at 100°C for 20 min to obtain an extract, and filter to obtain a filtrate; (3) adding an excess of 50-80% ethanol (w / v), reflux extraction at 100° C. for 3 times, each time for 2 hours; after each reflux, centrifuge at 4000 r / min for 10 minutes to obtain the supernatant, combine the three supernatants, concentrate by rotary evaporation to 1 / 10 of the original volume, and then freeze-dry to obtain a crude cicada flower extract; (4) Dissolving the Cicadae Convolvulus extract 1 in distilled water (1:10, w / v), centrifuging at 4000 rpm for 10 min, taking the supernatant, and freeze-drying to obtain the Cicadae Convolvulus extract.

5. The use according to claim 4, characterized in that The amount of the Cordyceps sinensis is 3-10g.

6. The use according to claim 4, characterized in that The ethanol is 55-75% ethanol (w / v).

7. The use according to claim 4, characterized in that The ethanol is 65% ethanol (w / v).

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