CN103980198B - Alkaloid Casuarinine H and the purposes in preparation treatment nerve degenerative diseases medicine thereof - Google Patents

Abstract

The invention belongs to the technical field of medicine, and relates to the alkaloid Casuarinine H in the extract of rattan stone pine and its new application in medicine, and specifically relates to the use of the alkaloid Casuarinine H in the extract of rattan rock pine in the preparation of medicines for treating neurodegenerative diseases. Stress in vitro screening model, carried out activity research on the alkaloid Casuarinine H in the extract of rattan stone pine, the experiment shows that the alkaloid Casuarinine H has obvious protective effect on the cell damage caused by oxidative stress, and the alkaloid Casuarinine H can Used alone or in combination, or combined with suitable excipients, made into oral or non-oral dosage forms according to conventional methods, such as tablets, capsules, granules or injections, used to prepare drugs for the treatment of neurodegenerative diseases, including but Not limited to Alzheimer's disease or Parkinson's disease, etc.

Description

Alkaloid Casuarinine H and its use in the preparation of medicines for treating neurodegenerative diseases

technical field

The present invention relates to the technical field of medicine, and relates to the alkaloid Casuarinine H in the extract of rattan stone pine and its new application in medicine, in particular to the use of the alkaloid Casuarinine H in the extract of rattan rock pine in the preparation of medicines for treating neurodegenerative diseases, and in particular to rattan rock pine The use of the alkaloid Casuarinine H in the extract in the preparation of drugs for the treatment of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. The present invention also relates to the extraction and preparation of Casuarinine H from vine stone pine method.

Background technique

Studies have shown that neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Huntington's disease (Huntington's disease) sdisease, HD), etc. not only seriously affect the quality of life of patients, but also bring a great economic burden to the society. Although the pathogenesis of these different diseases cannot be clearly explained at present, a large amount of research data shows that in these neurodegenerative diseases, nerve cells in specific brain regions are exposed to reactive oxygen species (Reactive Oxygen Species, ROS) The attack causes the cells to undergo apoptosis, and then deteriorates until the dysfunction of the neuronal network, and eventually neurodegenerative diseases occur. Studies have also shown that ROS-oxidative stress (Oxidative Stress, OS) plays an important role in the occurrence and development of apoptosis in neurodegenerative diseases. Therefore, eliminating or reducing the oxidation of free radicals will effectively alleviate or delay the symptoms of neurodegenerative patients.

The prior art discloses that Chinese herbal medicines have the unique advantages of multi-components, multi-targets and low toxicity in the treatment of neurodegenerative diseases. For example, some classic and proven prescriptions of Chinese herbal medicines have been successfully applied clinically.

Lycopodiastrum casuarinoides (Spring) Holub. is a plant of the genus Lycopodiastrum casuarinoides (Spring) Holub. It is mainly distributed in South China, Southwest China, Hubei, Hunan, Fujian, Taiwan and other places. The whole herb is used as medicine in the folk, mainly for the treatment of rheumatic joint pain , bruises, musculoskeletal pain, etc. In the 1990s, Chinese researchers studied the chemical composition of the plant for the first time, and found three lycoperzine compounds with a skeleton structure similar to Huperzine A: Huperzine ( Huperzine), Huperzine C and Huperzine D, and the acetylcholinesterase inhibitory effect of Huperzine C is comparable to Huperzine A.

So far, there has been no report about the use of the alkaloid Casuarinine H in the extract of vine stone pine in the preparation of drugs for treating neurodegenerative diseases.

Contents of the invention

The object of the present invention is to provide the alkaloid Casuarinine H in the extract of rattan stone pine and its new medical application, specifically related to the alkaloid Casuarinine H in the extract of rattan rock pine and its use in the preparation of medicines for treating neurodegenerative diseases, especially related to rattan rock pine The use of the alkaloid Casuarinine H in the extract in the preparation of drugs for treating neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis.

The present invention conducts activity research on a variety of chemical components derived from Chinese herbal medicines through an in vitro screening model of oxidative stress. Experiments show that the alkaloid Casuarinine H in vine stone pine has obvious biological significance: the alkaloid has an effect on oxidative stress. The induced cell damage has obvious protective effect. At 10μM concentration, Casuarinine H can produce obvious protective effect on H2O2 _- induced human neuroblastoma cell (SHSY _- 5Y) damage; further, the biological The base Casuarinine H can be used alone or in combination, or combined with suitable excipients, and made into oral or non-oral dosage forms according to conventional methods, such as tablets, capsules, granules or injections, for the preparation of drugs for the treatment of neurodegenerative diseases. drug.

The invention provides a preparation method of the alkaloid Casuarinine H in the extract of vine stone pine,

The active ingredient Casuarinine H is prepared from the above-ground part of Fujishipine or its whole herb through a conventional method in the field, which includes the following steps: firstly, using the above-ground part of Fujishipine or the whole grass of Fujishipine as raw material, it is prepared by a solvent extraction method Concentrate the extract; in the solvent extraction method adopted, a single solvent such as water, methanol, ethanol, acetone or a mixed solvent of multiple solvents can be selected, with 50-95% ethanol as the best extraction solvent, soaking or extracting at room temperature Reflux extraction under heating conditions, the number of extractions is 2 to 3 times, the extract is concentrated and suspended in an appropriate amount of 3% tartaric acid aqueous solution, extracted 2 to 3 times with an equal volume of ethyl acetate; the tartaric acid aqueous solution is adjusted to pH with Na ₂ CO ₃ to 10, and then extracted with chloroform to obtain the total alkaloids (chloroform extract). After column chromatography, the monomer compound Casuarinine H was prepared from the chloroform extract of Fujishipine pine, named Casuarinine H. The biological activity research showed that the compound was The direct active substance of the vine stone pine extract against oxidative stress, through ¹ H-NMR, 13C-NMR, 2D-NMR and MS, the chemical structure of the compound is determined as formula Ⅰ:

Compound: Casuarinine H, [α] ^22.5 D-17.1° (c0.65, CHCl ₃ ). ¹ H-NMR (400MHz, CDCl ₃ ): δ6.44(1H,d,J=9.2Hz,H-2),7.76(1H,d,J=9.2Hz,H-3),3.06(1H,dd ,J=18.8,7.1Hz,H-6α),2.51(1H,br d,J=18.8Hz,H-6β),2.30(1H,m,H-7),1.29(1H,ddd,J=12.8 ,12.7,3.7Hz,H-8α),1.75(1H,br d,J=12.8Hz,H-8β),5.23(1H,dd,J=16.8,1.8Hz,H-10a),5.14(1H, dd,J=10.4,2.0Hz,H-10b),5.62(1H,ddd,J=16.8,10.4,9.6Hz,H-11),2.14(1H,dd,J=9.6,2.9Hz,H-12 ),1.09(1H,dd,J=12.0,12.0Hz,H-14a),1.64(1H,dd,J=12.0,3.9Hz,H-14b),1.40(1H,m),0.85(3H,d , J=6.4, Me-16), 13.30 (1H, br s, NH). ¹³ C-NMR (100MHz, CDCl3): δ164.9(C-1), 118.9(C-2), 140.3(C-3), 120.5(C-4), 143.7(C-5), 29.9(C -6), 34.2(C-7), 42.5(C-8), 117.2(C-10), 137.3(C-11), 49.9(C-12), 54.8(C-13), 52.2(C- 14),26.3(C-15),21.7(C-16);(+)ESI-MS m/z245[M+H] ⁺ ,267[M+Na] ⁺ ,511[2M+Na] ⁺ ;( +)HR-ESI-MS m/z245.1649[M+H] ⁺ (calcd for C ₁₅ H ₂₁ N ₂ O,245.1648).

The present invention further conducts in vitro oxidative stress screening model research and shows that the alkaloid has obvious protective effect on cell damage caused by oxidative stress. Therefore, Casuarinine H can be used alone or in combination with other drugs, or combined with appropriate excipients, and made into oral or non-oral dosage forms according to conventional methods for the treatment of neurodegenerative diseases.

The advantages of the present invention are: Casuarinine H is a new compound discovered for the first time and has a novel chemical structure; Casuarinine H has application to neurodegenerative diseases, especially Alzheimer's disease and Parkinson's disease that seriously affect the quality of life of the elderly Prospects: rattan stone pine is widely distributed in my country, rich in natural resources, cheap and easy to get; rattan stone pine is a herbaceous plant with strong regeneration ability, and even large-scale production will not cause resource depletion.

Description of drawings

Figure 1 is the result of the neuroprotective activity data of the alkaloid Casuarinine H,

Among them, the value on the vertical axis is the cell survival rate, which is represented by the mean value (Mean) ± standard deviation (SD), the survival rate of the normal control group is set as 100%, and the rest of the groups are the percentage values compared with the normal group, ^## p<0.01 compared to the normal control group; *p<0.05, **p<0.01 compared to the H ₂ O ₂ injury group, N-acetyl-L-cysteine (n-acetyl-L-cysteine, L-NAC) as a positive control.

detailed description

The present invention will be further described below with examples, but these examples are by no means any limitation to the present invention. Any changes made by those skilled in the art in the implementation of the present invention under the inspiration of this specification will fall within the scope of the claims.

Example 1

1 kg of the whole plant of Fujishipine, crushed, and cold-soaked with 3 times the amount of 90% methanol at room temperature for 24 hours, a total of 3 times. The extracts were combined three times and concentrated under reduced pressure to obtain 90 g of extract. Suspend the extract with 2 times the amount of 3% tartaric acid aqueous solution, extract three times with ethyl acetate equal to the volume of the suspension, then use Na ₂ CO ₃ to adjust the tartaric acid aqueous solution to pH = 10, and then extract three times with chloroform , the chloroform extraction part was concentrated under reduced pressure to obtain 1.1 g of the total alkaloid extract, and then the sample was mixed with equal-fold silica gel (100-200 mesh), and column chromatography was carried out with 50-fold amount of silica gel (200-300 mesh). Chloromethane: Methanol is used as mobile phase gradient elution (volume ratio 1:0-0:1). Collect dichloromethane:methanol (15:1, volume ratio) eluted fractions, which continue to pass through the silica gel column, and use dichloromethane:methanol 50:1, 30:1, 10:1 for elution. The fraction eluted at 30:1 was purified by preparative HPLC, eluted isocratically with methanol:water:diethylamine (35:65:0.05%), and the fraction was collected for 39.524 minutes to obtain 15 mg of white amorphous powder Casuarinine H.

Example 2

1 kg of the whole herb of Fujishipine was crushed, refluxed with 80% ethanol at room temperature for 3 hours, repeated 3 times, the extracts were combined, concentrated under reduced pressure to obtain 110 g of extract. The extract was suspended with 2 times the amount of 3% tartaric acid aqueous solution, extracted three times with ethyl acetate equal to the volume of the suspension, then the tartaric acid aqueous solution was adjusted to pH = ₁₀ with _Na2CO3 , and extracted three times with equal volume of chloroform. The chloroform extraction part was concentrated under reduced pressure to obtain 1.3 g of total alkaloid extract. The macroporous resin HP-20 on the extract was eluted with water, 50% ethanol, and 90% ethanol respectively, and the eluted part of 50% ethanol was applied to Sephadex LH-20, which was eluted with dichloromethane:methanol 2:1, and passed TLC detection combined to obtain 13 mg of Casuarinine H.

The neuroprotective effect test of embodiment 3 Casuarinine H

SHSY-5Y cells were digested and suspended in MEM/F12 medium containing 10% fetal bovine serum. Seed SHSY-5Y cells on 96-well culture plate at a density of 2×10 ⁵ cells/ml, with a seeding volume of 100 μl/well, and then place them in a 37°C constant temperature incubator containing 5% CO ₂ for 24 hours . Replace the culture solution of each well with fresh MEM/F12 culture solution, add the corresponding concentration of the compound to be tested (10 microliters/well) in the administration group, add the compound solvent control to the normal control group and the H ₂ O ₂ damage group (10 µl/well). After incubation for 2 hours, 1 mM H ₂ O ₂ (10 μl/well) was added to the administration group and the H ₂ O ₂ injury group respectively, with a final concentration of 100 μM. After continuing to culture for 24 hours, add 5 mg/mL MTT (10 μl/well) for live cell staining. After incubation for 3 hours, discard the culture medium, add 100% DMSO (100 μl/well), and shake on a shaker to fully dissolve it. The OD value of each group was measured at a wavelength of 490nm, and the test results showed that Casuarinine H had a significant protective effect on SHSY-5Y oxidative stress damage. The activity data of Casuarinine H is shown in Figure 1, wherein, Hu JF-8 represents the active extract Casuarinine H obtained in Example 1.

Claims (6)

1. the purposes of the alkaloid Casuarinine H compound of formula I in the preparation treatment neurodegenerative disease medicine, 2. The use according to claim 1, wherein said neurodegenerative disease is Alzheimer's disease or Parkinson's disease or amyotrophic lateral sclerosis. 3. A preparation method of alkaloid Casuarinine H, wherein said alkaloid Casuarinine H is prepared by the following method: using the above-ground part of vine stone pine or the whole herb of vine stone pine as raw material, the concentrated extract is made by solvent extraction; the solvent used is selected From water, methanol, ethanol or acetone single solvent or a mixed solvent of multiple solvents, immersion extraction at room temperature or reflux extraction under heating conditions, the number of extractions is 2 to 3 times, the extract is concentrated and suspended in an appropriate amount of 3% tartaric acid aqueous solution, Extract with an equal volume of ethyl acetate for 2 to 3 times; adjust the pH value of the tartaric acid aqueous solution to 10 with Na ₂ CO ₃ , then extract with chloroform to obtain a chloroform extract, and obtain a monomer compound from the chloroform extract by column chromatography Casuarinine H, determine the chemical structure of this compound as formula I: 4. according to the described preparation method of claim 3, it is characterized in that, the extraction solvent that adopts in the described preparation method is 50～95% ethanol. 5. The use according to claim 1, characterized in that said Casuarinine H is used alone or in combination with other drugs. 6. according to the purposes of claim 1, it is characterized in that, described Casuarinine H is combined with excipient to make tablet, capsule, granule or injection.