CN111606783A - Compound 3,7, 11-cembratriene-2, 6-diol, preparation method and application thereof - Google Patents
Compound 3,7, 11-cembratriene-2, 6-diol, preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of medicines, and relates to a compound 3,7, 11-cembratriene-2, 6-diol, a preparation method and application thereof, wherein the novel tobacco leaf compound 3,7, 11-cembratriene-2, 6-diol has an obvious effect of preventing and treating neurodegenerative diseases, and has an obvious protective effect on SH-SY5Y cell oxygen sugar deprivation injury; secondly, the composition has obvious protective effect on the glutamic acid induced injury of SH-SY5Y cells. Pharmacological tests prove that the compounds can effectively inhibit the oxygen sugar deprivation and glutamic acid induced neuron damage, and have the potential of being further developed into medicines for preventing and/or treating neurodegenerative diseases such as cerebral apoplexy and the like.
Description
Technical Field
The invention belongs to the field of medicines, and relates to a compound 3,7, 11-cembratriene-2, 6-diol, a preparation method and application thereof, in particular to a novel compound 3,7, 11-cembratriene-2, 6-diol separated from fresh tobacco leaves, a preparation method thereof, a pharmaceutical composition containing the compound and application thereof in preventing and/or treating neurodegenerative diseases such as cerebral apoplexy.
Background
Stroke (Stroke) is commonly called cerebral Stroke, is a cerebrovascular disease causing brain tissue damage due to interruption of cerebral blood circulation caused by blockage or rupture of cerebral vessels, is one of the diseases with the highest morbidity, mortality and disability rate in the world, is the first cause of death of residents in China, and seriously threatens the physical health of people. With the improvement of living standard and the change of life style of people, the incidence of the disease still tends to rise. Cerebrovascular diseases are classified into hemorrhagic diseases and ischemic cerebrovascular diseases, wherein the incidence rate of cerebral ischemic stroke is the highest and accounts for about 80 percent of all strokes. Cerebral ischemia is extremely dangerous, and even if the cerebral ischemia is in an acute stage, most patients can have a plurality of sequelae. Statistically, about three-quarters of patients with a viable cerebrovascular disease are incapacitated to varying degrees, with about 40% of severely disabled patients. At present, the annual cost for treating cerebrovascular diseases in China is estimated to be more than 100 million yuan, and various indirect economic losses cause the annual expenditure of the disease to be close to 200 million yuan, which causes heavy economic burden to the nation and a plurality of families.
The recovery of ischemic penumbra around the cerebral infarction tissue is the basis of modern treatment of ischemic stroke, but research shows that the cerebral tissue injury is aggravated after the ischemic area recovers reperfusion, and more serious cerebral dysfunction appears, which is called cerebral ischemia reperfusion injury. The cerebral ischemia-reperfusion injury relates to a dynamic and complex pathophysiological process and is influenced by a plurality of physicochemical factors inside and outside cells, wherein various links and various influencing factors interact with each other, and although the pathophysiological mechanism of the cerebral ischemia and reperfusion injury is remarkably developed in recent years, the pathophysiological mechanism of the cerebral ischemia and reperfusion injury is still not completely clarified. With the progress of the research, intracellular Ca2+Overload, overproduction of free radicals, cytotoxicity of excitatory amino acids, inflammatory cascades, acidosis, increased mitochondrial permeability and apoptosis are thought to be major factors in the development of cerebral ischemia-reperfusion injury. Therefore, the research and development of the medicine for protecting ischemic brain injury have important theoretical significance and practical value.
Glutamate is the most abundant excitatory amino acid in the brain of humans and mammals and is involved in the transmission of information in the central nervous system. However, excessive glutamate can generate severe neuroexcitotoxicity, can generate toxic action on neurons, causes the functional decline of the neurons and leads to the death of nerve cells, and plays an important role in the pathogenesis of various nerve diseases such as cerebral anoxia, ischemic injury, Alzheimer disease, psychosis, neuroendocrine disorder and the like.
The human neuroblastoma cell strain SH-SY5Y cells are taken as a research object, an in-vitro nerve cell injury model is established by oxygen sugar deprivation and glutamic acid induction, the protective effect of a compound on nerve cells is reflected by measuring the cell survival rate, and a preliminary experiment basis of medicines is provided for research and development of protection of ischemic brain injury.
The cembrane diterpenoid belongs to fourteen-element macrocyclic diterpenoid, and is mostly present in the plants of Pinus, Corallium scorzonerifolium and tobacco, and the modern pharmacological research shows that most cembrane diterpenoid compounds have better cytotoxic and anti-tumor activities and certain neurophysiological activities. Patent ZL201910356466.8 discloses a compound cembratriene-4-ol-6-carboxylic ester used for preparing medicines for treating and preventing tumor diseases and application thereof, wherein although the compound cembrate-4-ol-6-carboxylic ester belongs to cembrate diterpenes, the positions of double bonds and hydroxyl groups on the dodecaquaternary macrocyclic mother nucleus skeletons of the cembrate diterpenes are different, and the compound plays different roles in the medical field. At present, no research report on the application of the compounds in nerve cell protection exists, and no patent literature on the preparation method of the monomers exists.
Disclosure of Invention
The invention provides a compound 3,7, 11-cembratriene-2, 6-diol and a preparation method and application thereof, and the inventor carries out systematic research on glandular hair secretion of fresh tobacco leaves, and separates a new cembrane diterpene from the glandular hair secretion, wherein the chemical structure of the cembrane diterpene is shown in a formula (1), and the compound is novel in that the fourteen-membered macrocyclic mother nucleus skeleton double bonds and hydroxyl substitution positions are different.
The technical scheme of the invention is realized as follows:
the compound 3,7, 11-cembratriene-2, 6-diol, the formula (1) 'the compound of formula (1') includes stereoisomers and tautomers of the compound:
the preparation method of the compound 3,7, 11-cembratriene-2, 6-diol comprises the following steps:
(1) washing fresh tobacco leaves, then carrying out reduced pressure distillation and concentration to obtain brown extractum, then re-dissolving the brown extractum by using an organic solvent, washing and washing for 2-3, collecting an organic phase, and then carrying out reduced pressure distillation to obtain a crude extract containing the thujatam alkane component;
(2) separating and eluting the crude extract prepared in the step (1) by silica gel column chromatography, and collecting eluent to obtain a crude product of a new compound 3,7, 11-cembratriene-2, 6-diol;
(3) and (3) purifying the crude product prepared in the step (2) by preparative HPLC to obtain a pure product of 3,7, 11-cembratriene-2, 6-diol.
The fresh tobacco leaves in the step (1) are rinsed and then subjected to reduced pressure distillation, and the specific operation is as follows: washing fresh tobacco leaves with dichloromethane for 3 times, filtering, and concentrating the filtrate at 40-45 deg.C under reduced pressure to obtain brown extract.
In the step (1), the organic solvent is dichloromethane, and 1-1.2L of organic solvent is adopted to redissolve every 500g of brown extract; 5-8% phosphoric acid solution is adopted for washing, and the mass volume ratio of the brown extract to the phosphoric acid solution is 1 (1-1.2) g/mL.
In the step (2), the silica gel column chromatography is sequentially eluted by petroleum ether/ethyl acetate eluent with the volume ratio of 10:1, 8:1, 6:1, 4:1, 1:1 and 3:4, and then sequentially eluted by ethyl acetate and methanol.
The reaction conditions of the preparative HPLC in the step (3) are as follows: 60% acetonitrile-water, flow rate of 5mL/min, wavelength detection of 210nm, sample size of 400 μ L, and chromatography column YMC-Pack ODS-A C18 preparative column, model number 250 × 10 mm, 5 μm.
The structure of the 3,7, 11-cembratriene-2, 6-diol is analyzed and identified by means of spectroscopy such as UV, IR, NMR, MS and the like, and is shown as a formula (1).
The compound 3,7, 11-cembratriene-2, 6-diol and salt thereof and/or a pharmaceutical composition as an active ingredient are applied to preparation of medicines for preventing or/and treating neurodegenerative diseases. Pharmacological activity experiments show that the compound shown in the formula (1) has good neuroprotective effect and can increase the oxygen sugar deprivation and glutamic acid induced nerve cell survival rate.
The dosage form of the pharmaceutical composition comprises tablets, capsules, pills, granules, oral liquid or suspensions, and the effective components account for 0.1 to 95 percent of the total weight of the pharmaceutical composition; the compound of formula (1) is typically present in an amount of 0.1 to 100 mg in a unit dosage form.
The pharmaceutical composition is prepared according to a method approved by the field of medicine and health; for this purpose, the compounds of formula (1) according to the invention can be combined, as desired, with one or more solid or pharmaceutical excipients and/or adjuvants, in administration forms or dosages for human or veterinary use.
The neurodegenerative disease comprises Parkinson, Alzheimer disease and cerebral apoplexy.
The neurodegenerative disease is neuronal damage, apoptosis-related Parkinson, Alzheimer's disease or stroke.
Furthermore, the present invention relates to a pharmaceutical composition comprising a pharmaceutically effective dose of a compound of formula (1) and a pharmaceutically acceptable carrier. According to the present invention, the compound of formula (1) has an isomeric structure, and thus, the "compound of formula (1)" according to the present invention includes stereoisomers and tautomers of the compound.
According to an embodiment of the present invention, the compound of formula (1) also includes pharmaceutically acceptable salts, hydrates, esters and prodrugs thereof.
The invention also includes pharmaceutical compositions comprising as active ingredient a compound of formula (1) together with conventional pharmaceutical excipients or adjuvants. Typically, the compound of formula (1) is present in an amount of from 0.1% to 95% by weight of the total pharmaceutical composition.
The pharmaceutical composition is prepared according to a method approved by the field of medicine and health; for this purpose, the compounds of formula (1) according to the invention can be combined, as desired, with one or more solid or pharmaceutical excipients and/or adjuvants, in administration forms or dosages for human or veterinary use. Excipients, including diluents, binders, wetting agents, disintegrants, lubricants, glidants. The tablets may also be formulated as coated tablets, e.g. sugar-coated tablets, film-coated tablets, enteric-coated tablets, or as bi-and multi-layer tablets. For making the administration units into pills, various carriers well known in the art can be widely used, such as diluents and absorbents, binders and disintegrating agents; to encapsulate the dosage unit, the compound of formula (1) is mixed with the various carriers described above and the resulting mixture is placed in a hard gelatin capsule or soft capsule, or the compound of formula (1) may be microencapsulated and suspended in an aqueous medium to form a suspension. In addition, colorants, preservatives, flavors, flavorings, sweeteners or other materials may also be added to the pharmaceutical preparation, if desired.
The compound of formula (1) of the present invention or the pharmaceutical composition containing it can be administered in unit dosage form by an administration route which may be enteral or parenteral, such as oral, intramuscular, subcutaneous, nasal, oral mucosal, ocular, pulmonary, dermal, vaginal, peritoneal, rectal, etc., preferably oral administration. The compound of formula (1) of the present invention or a pharmaceutical composition containing it may also be administered by injection. The injection includes intravenous injection, subcutaneous injection, intradermal injection, intraperitoneal injection, acupoint injection, etc. The dosage form for administration may be a liquid dosage form, a solid dosage form, or a semi-solid dosage form. The compound of formula (1) or the pharmaceutical composition containing the same can be prepared into common preparations, sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle drug delivery systems.
For the purpose of administration and enhancing the therapeutic effect, the compound of formula (1) or the pharmaceutical composition of the present invention can be administered by any known method. The dose of the pharmaceutical composition of the present invention to be administered depends on many factors such as the nature and severity of the disease to be prevented or treated, the sex, age, body weight, character and individual response of the patient or animal, the administration route, frequency and therapeutic purpose, and thus the therapeutic dose of the present invention may be widely varied.
The invention has the following beneficial effects:
1. the novel tobacco leaf compound 3,7, 11-cembratriene-2, 6-diol has an obvious effect of preventing and treating neurodegenerative diseases, and has an obvious protective effect on SH-SY5Y cell oxygen sugar deprivation injury; secondly, the composition has obvious protective effect on the glutamic acid induced injury of SH-SY5Y cells.
2. The novel tobacco leaf compound 3,7, 11-cembratriene-2, 6-diol has a novel structure, is not reported in documents, and has the potential of being further developed into a medicament for preventing and/or treating neurodegenerative diseases such as cerebral apoplexy. Pharmacological experiments prove that the compound can effectively inhibit the damage of the neuron cells caused by oxygen sugar deprivation and glutamic acid, has good neuroprotective effect, and the effective dose can reach 10 mu mol/L.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 shows the preparation of 3,7, 11-cembratriene-2, 6-diol1H-NMR(CDCl3)。
FIG. 2 shows the preparation of 3,7, 11-cembratriene-2, 6-diol13C-NMR(CDCl3)。
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.
Example 1
The preparation method of the compound 3,7, 11-cembratriene-2, 6-diol comprises the following steps:
1) 5 kg of fresh tobacco leaves are rinsed for 3 times by dichloromethane and filtered. At 45 ℃, dichloromethane is decompressed, distilled and concentrated to obtain 500g of brown extract. Redissolving the extract by using 1L of dichloromethane, adding 500 mL of 5% phosphoric acid solution for washing, collecting dichloromethane phase after washing for 3 times, and distilling under reduced pressure to obtain 386 g of crude extract containing the thujatamansi alkane components.
2) And (3) separating 80 g of the crude extract by silica gel column chromatography, eluting with petroleum ether/ethyl acetate eluent in a volume ratio of 10:1, 8:1, 6:1, 4:1, 1:1 and 3:4 in sequence, eluting with ethyl acetate and methanol in sequence, collecting 250 mL of the eluent once, and detecting and collecting to obtain a crude product of the new compound 3,7, 11-cembratriene-2, 6-diol.
3) And purifying the crude product of the 3,7, 11-cembratriene-2, 6-diol by preparative HPLC to obtain a pure product of the 3,7, 11-cembratriene-2, 6-diol. Wherein, the conditions of preparative HPLC are 60% acetonitrile-water, flow rate of 5ml/min, wavelength detection of 210nm, sample size of 400 μ L, and YMC-Pack ODS-A C18 preparative column (250X 10 mm, 5 μm).
4) The structure of the 3,7, 11-cembratriene-2, 6-diol is analyzed and identified by means of spectroscopy such as UV, IR, NMR, MS and the like, and is shown as a formula (1).
Process for preparing 3,7, 11-cembratriene-2, 6-diol1H-NMR(CDCl3) As shown in FIG. 1,13C-NMR(CDCl3) As shown in fig. 2.
The physicochemical properties and spectral information of 3,7, 11-cembratriene-2, 6-diol are as follows:
white amorphous powder, 5% citric acid-ethanol solution appeared green. [α]20 D+78.6 (c0.3, CH2Cl2); UV(MeOH)λ max(log) 209 (3.89) nm; HRESIMSm/z329.2451 [M + Na]+(calcd. for329.2457, C20H34NaO2);1H-NMR(600 MHz，CDCl3)H:1.25 (1H, m, H-1),4.56(1H, m, H-2), 5.38(1H, d, J = 8.4 Hz, H-3), 2.49/2.14(1H, m, H-5a/H-5b), 4.56(1H, m, H-6), 5.07(1H, d, J = 9.0 Hz, H-7), 2.11 (2H, m, H-9), 2.27/2.11 (1H, m, H-10a/H-10b),4.95(1H, ddd, J = 9.0, 4.8, 1.8 Hz, H-11), 1.92/2.09 (1H, m, H-13a/H-13b), 1.52/1.67 (1H, m, H-14a /H-14b), 1.78 (1H, m, H-15), 0.94/0.97 (2H, d,J = 7.2 Hz/7.2 Hz, H-16/H-17), 1.60 (3H, s, H-18), 1.61 (3H, s, H-19), 1.62(3H, s, H-20) .13C -NMR (150 MHz, CDCl3)C: 46.2 (C-1), 68.4 (C-2), 131.3 (C-3), 131.5 (C-4), 48.9 (C-5), 66.8 (C-6), 127.2 (C-7), 138.8 (C-8), 38.7 (C-9), 23.9 (C-10), 123.4 (C-11), 134.0 (C-12), 35.2 (C-13), 22.2 (C-14), 29.5(C-15), 21.9 (C-16), 19.0 (C-17), 16.4 (C-18), 16.9 (C-19), 17.9 (C-20).
Example 2
The preparation method of the compound 3,7, 11-cembratriene-2, 6-diol comprises the following steps:
1) 5 kg of fresh tobacco leaves are rinsed for 3 times by dichloromethane and filtered. At the temperature of 43 ℃, dichloromethane is decompressed, distilled and concentrated to obtain 500g of brown extract. Redissolving the extract by 0.9L of dichloromethane, adding 550 mL of 8% phosphoric acid solution for washing, collecting dichloromethane phase after washing for 3 times, and obtaining 377 g of crude extract containing the thuja alkane components through reduced pressure distillation.
2) And (3) separating 80 g of the crude extract by silica gel column chromatography, eluting with petroleum ether/ethyl acetate eluent in a volume ratio of 10:1, 8:1, 6:1, 4:1, 1:1 and 3:4 in sequence, eluting with ethyl acetate and methanol in sequence, collecting 250 mL of the eluent once, and detecting and collecting to obtain a crude product of the new compound 3,7, 11-cembratriene-2, 6-diol.
3) And purifying the crude product of the 3,7, 11-cembratriene-2, 6-diol by preparative HPLC to obtain a pure product of the 3,7, 11-cembratriene-2, 6-diol. Wherein, the conditions of preparative HPLC are 60% acetonitrile-water, flow rate of 5ml/min, wavelength detection of 210nm, sample size of 400 μ L, and YMC-Pack ODS-A C18 preparative column (250X 10 mm, 5 μm).
4) The structure of the 3,7, 11-cembratriene-2, 6-diol is analyzed and identified by means of spectroscopy such as UV, IR, NMR, MS and the like, and is shown as a formula (1).
The physicochemical properties and spectral information of 3,7, 11-cembratriene-2, 6-diol are as follows:
white amorphous powder, 5% citric acid-ethanol solution appeared green. [α]20 D+78.6 (c0.3, CH2Cl2); UV(MeOH)λ max(log) 209 (3.89) nm; HRESIMSm/z329.2451 [M + Na]+(calcd. for329.2457, C20H34NaO2);1H-NMR(600 MHz，CDCl3)H:1.25 (1H, m, H-1),4.56(1H, m, H-2), 5.38(1H, d, J = 8.4 Hz, H-3), 2.49/2.14(1H, m, H-5a/H-5b), 4.56(1H, m, H-6), 5.07(1H, d, J = 9.0 Hz, H-7), 2.11 (2H, m, H-9), 2.27/2.11 (1H, m, H-10a/H-10b),4.95(1H, ddd, J = 9.0, 4.8, 1.8 Hz, H-11), 1.92/2.09 (1H, m, H-13a/H-13b), 1.52/1.67 (1H, m, H-14a /H-14b), 1.78 (1H, m, H-15), 0.94/0.97 (2H, d,J = 7.2 Hz/7.2 Hz, H-16/H-17), 1.60 (3H, s, H-18), 1.61 (3H, s, H-19), 1.62(3H, s, H-20) .13C -NMR (150 MHz, CDCl3)C: 46.2 (C-1), 68.4 (C-2), 131.3 (C-3), 131.5 (C-4), 48.9 (C-5), 66.8 (C-6), 127.2 (C-7), 138.8 (C-8), 38.7 (C-9), 23.9 (C-10), 123.4 (C-11), 134.0 (C-12), 35.2 (C-13), 22.2 (C-14), 29.5(C-15), 21.9 (C-16), 19.0 (C-17), 16.4 (C-18), 16.9 (C-19), 17.9 (C-20).
Example 3
The preparation method of the compound 3,7, 11-cembratriene-2, 6-diol comprises the following steps:
1) 5 kg of fresh tobacco leaves are rinsed for 3 times by dichloromethane and filtered. At 45 ℃, dichloromethane is decompressed, distilled and concentrated to obtain 500g of brown extract. Redissolving the extract by 0.8L of dichloromethane, adding 60 mL of 7% phosphoric acid solution for washing, collecting dichloromethane phase after washing for 3 times, and distilling under reduced pressure to obtain 390 g of crude extract containing the thujaplicin components.
2) And (3) separating 80 g of the crude extract by silica gel column chromatography, eluting with petroleum ether/ethyl acetate eluent in a volume ratio of 10:1, 8:1, 6:1, 4:1, 1:1 and 3:4 in sequence, eluting with ethyl acetate and methanol in sequence, collecting 250 mL of the eluent once, and detecting and collecting to obtain a crude product of the new compound 3,7, 11-cembratriene-2, 6-diol.
3) And purifying the crude product of the 3,7, 11-cembratriene-2, 6-diol by preparative HPLC to obtain a pure product of the 3,7, 11-cembratriene-2, 6-diol. Wherein, the conditions of preparative HPLC are 60% acetonitrile-water, flow rate of 5ml/min, wavelength detection of 210nm, sample size of 400 μ L, and YMC-Pack ODS-A C18 preparative column (250X 10 mm, 5 μm).
4) The structure of the 3,7, 11-cembratriene-2, 6-diol is analyzed and identified by means of spectroscopy such as UV, IR, NMR, MS and the like, and is shown as a formula (1).
The physicochemical properties and spectral information of 3,7, 11-cembratriene-2, 6-diol are as follows:
white amorphous powder, 5% citric acid-ethanol solution appeared green. [α]20 D+78.6 (c0.3, CH2Cl2); UV(MeOH)λ max(log) 209 (3.89) nm; HRESIMSm/z329.2451 [M + Na]+(calcd. for329.2457, C20H34NaO2);1H-NMR(600 MHz，CDCl3)H:1.25 (1H, m, H-1),4.56(1H, m, H-2), 5.38(1H, d, J = 8.4 Hz, H-3), 2.49/2.14(1H, m, H-5a/H-5b), 4.56(1H, m, H-6), 5.07(1H, d, J = 9.0 Hz, H-7), 2.11 (2H, m, H-9), 2.27/2.11 (1H, m, H-10a/H-10b),4.95(1H, ddd, J = 9.0, 4.8, 1.8 Hz, H-11), 1.92/2.09 (1H, m, H-13a/H-13b), 1.52/1.67 (1H, m, H-14a /H-14b), 1.78 (1H, m, H-15), 0.94/0.97 (2H, d,J = 7.2 Hz/7.2 Hz, H-16/H-17), 1.60 (3H, s, H-18), 1.61 (3H, s, H-19), 1.62(3H, s, H-20) .13C -NMR (150 MHz, CDCl3)C: 46.2 (C-1), 68.4 (C-2), 131.3 (C-3), 131.5 (C-4), 48.9 (C-5), 66.8 (C-6), 127.2 (C-7), 138.8 (C-8), 38.7 (C-9), 23.9 (C-10), 123.4 (C-11), 134.0 (C-12), 35.2 (C-13), 22.2 (C-14), 29.5(C-15), 21.9 (C-16), 19.0 (C-17), 16.4 (C-18), 16.9 (C-19), 17.9 (C-20).
EXAMPLES Effect example 1
Protective effect of 3,7, 11-cembratriene-2, 6-diol on SH-SY5Y cell oxygen sugar deprivation damage
In the experiment, a cell screening model of oxygen deprivation induced damage is established by using a neuroblastoma SH-SY-5Y cell strain, and an effective neuron protective agent is screened by measuring the change of the cell survival rate.
SH-SY5Y cells cultured in DMEM containing 10% Fetal Bovine Serum (FBS), 100U/mL cyan, streptomycin based on 5% CO2After 80% of cells fused, subculturing was performed in an incubator at 37 ℃, 100 μ L per well was inoculated in a 96-well plate at a density of 1 × 104/mL, overnight, then, the old medium was aspirated, washed 3 times with PBS, each well of the blank control group was added with serum-free normal DMEM medium, and the remaining groups were added with low-sugar medium containing 10 mM sodium dithionate2，95% N2And cultured at 37 ℃. After 3 h, the stock solution was discarded and washed 3 times with PBS. The model group and the blank control group are respectively added with a high-sugar culture medium containing calf serum. And respectively administering 10 mu M of levo-butylphthalide and a compound to be detected to the positive drug group and the sample group, and reoxidizing for 24 h. After 24 h of reoxygenation, 0.5 mg/ml ofMTT, after 5 h of reaction, absorbing supernatant, adding 200 muL DMSO into each hole, slightly oscillating until particles in the holes are completely dissolved, and measuring an optical density value (OD) at 570nm by using an enzyme labeling instrument after the DMSO is adjusted to zero. Repeating each experiment for 3 times, increasing the cell survival rate to be greater than that of the positive medicine, and performing rescreening; the results are shown in Table 1.
Table 1: protection effect of compound 3,7, 11-cembratriene-2, 6-diol on SH-SY5Y cell oxygen sugar deprivation damage
As shown in Table 1, the SH-SY5Y cell survival rate can be obviously reduced under the condition of oxygen sugar deprivation, while the 10 mu M compound 3,7, 11-cembratriene-2, 6-diol has a good improvement effect on SH-SY5Y cell damage caused by oxygen sugar deprivation, the increase cell survival rate is 41.26 percent, which is higher than that of the positive drug levo-butylphthalide.
Experiments show that the compound 3,7, 11-cembratriene-2, 6-diol shown in the formula (1) has stronger effect than the marketed drugs for treating cerebral apoplexy.
EXAMPLES Effect example 2
Protection effect of 3,7, 11-cembratriene-2, 6-diol on SH-SY5Y cells against glutamic acid-induced neuronal damage
In the experiment, a cell screening model of glutamic acid induced damage is established by using a neuroblastoma SH-SY-5Y cell strain, and an effective neuron protective agent is screened by measuring the change of the cell survival rate. SH-SY5Y cells cultured in DMEM containing 10% Fetal Bovine Serum (FBS), 100U/mL cyan, streptomycin based on 5% CO2Culturing in an incubator at 37 ℃, carrying out passage after 80% of cells are fused, inoculating 100 muL of cells into a 96-well plate at the density of 1 × 104/mL, standing overnight, then sucking out the old culture medium, washing for 3 times by PBS (phosphate buffer solution), adding serum-free normal DMEM culture medium into each hole of a blank control group, replacing the new culture medium for the blank group after 4 hours, establishing a glutamic acid damage model after pretreatment of other groups by 5 mM glutamic acid, adding 10 muM of corresponding drugs into the positive drug group and the compound group to be screened, supplementing the culture medium with corresponding volume into the model group, and adding 10 muM of corresponding volume into each hole after 4 hours of glutamic acid damagemu.L of MTT (5 mg/ml) was allowed to react for 4 hours until crystallization was complete, after which 150. mu.L of DMSO was added to each well to dissolve the crystals and the absorbance values of each group were measured at 570nm to calculate the cell viability of each well and the increased viability relative to the model group. Repeating each experiment for 3 times, increasing the cell survival rate to be greater than that of the positive medicine, and performing rescreening; the results are shown in Table 2.
Table 2: protection effect of compound 3,7, 11-cembratriene-2, 6-diol on SH-SY5Y cell glutamic acid induced injury
As shown in Table 2, the cell survival rate of SH-SY5Y cells can be obviously reduced under the condition of glutamic acid induction, while the 10 mu M compound 3,7, 11-cembratriene-2, 6-diol has a good improving effect on SH-SY5Y cell injury induced by glutamic acid, the increase of the cell survival rate is 49.68 percent, and is higher than that of the positive drug nimodipine.
Experiments show that the compound 3,7, 11-cembratriene-2, 6-diol shown in the formula (1) has stronger effect than the marketed drug for treating ischemic cerebrovascular diseases.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. The compound 3,7, 11-cembratriene-2, 6-diol has stereoisomers and tautomers shown in the formula (1):
2. a process for the preparation of the compound 3,7, 11-cembratriene-2, 6-diol according to claim 1, characterized by the following steps:
(1) washing fresh tobacco leaves, then carrying out reduced pressure distillation and concentration to obtain brown extractum, then re-dissolving the brown extractum by using an organic solvent, washing and washing for 2-3, collecting an organic phase, and then carrying out reduced pressure distillation to obtain a crude extract containing the thujatam alkane component;
(2) separating and eluting the crude extract prepared in the step (1) by silica gel column chromatography, and collecting eluent to obtain a crude product of a new compound 3,7, 11-cembratriene-2, 6-diol;
(3) and (3) purifying the crude product prepared in the step (2) by preparative HPLC to obtain a pure product of 3,7, 11-cembratriene-2, 6-diol.
3. The preparation method of the compound 3,7, 11-cembratriene-2, 6-diol according to claim 1, wherein the fresh tobacco leaves in the step (1) are rinsed and then subjected to reduced pressure distillation, and the method comprises the following specific operations: washing fresh tobacco leaves with dichloromethane for 3 times, filtering, and concentrating the filtrate at 40-45 deg.C under reduced pressure to obtain brown extract.
4. The preparation method of the compound 3,7, 11-cembratriene-2, 6-diol according to claim 2, wherein the organic solvent in the step (1) is dichloromethane, and 0.8-1L of the organic solvent is used for redissolving every 500g of brown extractum; the washing adopts 5-8% phosphoric acid solution by volume fraction, and the mass volume ratio of the brown extract to the phosphoric acid solution is 1 (1-1.2) g/mL.
5. The method for preparing 3,7, 11-cembratriene-2, 6-diol compound according to claim 2, wherein the silica gel column chromatography in step (2) is performed by sequentially eluting with petroleum ether/ethyl acetate eluent in a volume ratio of 10:1, 8:1, 6:1, 4:1, 1:1 and 3:4, and then sequentially eluting with ethyl acetate and methanol.
6. The method for preparing 3,7, 11-cembratriene-2, 6-diol compound according to claim 2, wherein the reaction conditions of preparative HPLC in step (3) are as follows: 60% acetonitrile-water, flow rate of 5mL/min, wavelength detection of 210nm, sample size of 400 μ L, and chromatography column YMC-Pack ODS-A C18 preparative column, model number 250 × 10 mm, 5 μm.
7. The use of the compound 3,7, 11-cembratriene-2, 6-diol and salts thereof according to claim 2 and/or pharmaceutical compositions thereof as an active ingredient for the preparation of a medicament for the prevention or/and treatment of neurodegenerative diseases.
8. Use according to claim 7, characterized in that: the dosage form of the pharmaceutical composition comprises tablets, capsules, pills, granules, oral liquid or suspensions, and the effective ingredients account for 0.1 to 95 percent of the total weight of the pharmaceutical composition.
9. Use according to claim 7, characterized in that: the neurodegenerative disease comprises Parkinson, Alzheimer disease and cerebral apoplexy.
10. Use according to claim 7, characterized in that: the neurodegenerative disease is neuronal damage, apoptosis-related Parkinson, Alzheimer's disease or stroke.
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| CN105001052A (en) * | 2015-07-03 | 2015-10-28 | 中国农业科学院烟草研究所 | Method used for extracting cembrenediols from tobacco inflorescence |
| CN108042547A (en) * | 2017-12-12 | 2018-05-18 | 中山大学 | Cholesteric -4- alkene -3,6- diketone is preparing the application in treating or preventing neure damage drug |
| CN109970558A (en) * | 2019-04-29 | 2019-07-05 | 郑州轻工业学院 | Western cypress triolefin -4- alcohol -6- carboxylate and its preparation method and application |
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| CN105001052A (en) * | 2015-07-03 | 2015-10-28 | 中国农业科学院烟草研究所 | Method used for extracting cembrenediols from tobacco inflorescence |
| CN108042547A (en) * | 2017-12-12 | 2018-05-18 | 中山大学 | Cholesteric -4- alkene -3,6- diketone is preparing the application in treating or preventing neure damage drug |
| CN109970558A (en) * | 2019-04-29 | 2019-07-05 | 郑州轻工业学院 | Western cypress triolefin -4- alcohol -6- carboxylate and its preparation method and application |
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