CN103242417A - Sterol derivative, and preparation method and application thereof - Google Patents
Sterol derivative, and preparation method and application thereof Download PDFInfo
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- CN103242417A CN103242417A CN2012100235239A CN201210023523A CN103242417A CN 103242417 A CN103242417 A CN 103242417A CN 2012100235239 A CN2012100235239 A CN 2012100235239A CN 201210023523 A CN201210023523 A CN 201210023523A CN 103242417 A CN103242417 A CN 103242417A
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- 0 CC(C(*)C(C1(C)*)OC(C2)C1C(C)(CCC1C3(C)CCC(*)C4)C2C11OOC34C=C1)C(C)(C)* Chemical compound CC(C(*)C(C1(C)*)OC(C2)C1C(C)(CCC1C3(C)CCC(*)C4)C2C11OOC34C=C1)C(C)(C)* 0.000 description 1
- HSENTSRDHIZQCA-UHFFFAOYSA-N CC(C(C1OC(CC(C2(C)CCC3C4(C)CCC(C5)O)C33OOC45C=C3)C2C1(C)O)O)C(C)(C)O Chemical compound CC(C(C1OC(CC(C2(C)CCC3C4(C)CCC(C5)O)C33OOC45C=C3)C2C1(C)O)O)C(C)(C)O HSENTSRDHIZQCA-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses a sterol derivative, and a preparation method and an application thereof. The sterol derivative comprises sterol compounds having a structural formula (I), or pharmaceutically acceptable salts of the sterol compounds, or extract products including the sterol compounds, or compositions including the sterol compounds. In the structural formula (I), R1 is -OH, =O, H, or a C1-C3 alkyl group; R2 is -OH, H, or a C1-C3 alkyl group; R3 is -OH, =O, H, or a C1-C3 alkyl group; R4 is -OH, H, or a C1-C3 alkyl group; and at least one of R1, R2, R3 and R4 is -OH. The compounds having the structural formula (I) can be used for preparing medicines having an HMG-CoA reductase activity inhibition effect, and the prepared medicines can effectively inhibit the activities of an HMG-CoA reductase.
Description
Technical field
The present invention relates to pharmacy field, be specifically related to a kind of sterols derivative with structural formula (I) and preparation method thereof and application.
Background technology
Red colouring agent for food, also used as a Chinese medicine (Monascus-fermented rice) is to be raw material with the rice, a kind of red-purple rice song of making through monascus ruber (Monascus) fermentation.Red colouring agent for food, also used as a Chinese medicine claims red bent ancient times, is to be that main bent mother or distiller's yeast are inoculated in the rice top fermentation and form with monascus, and its look crimson, thus have another name called red song, red rice, red rice, red wine dregs, again because of main product in Fujian etc. ground. so have another name called good fortune song, good fortune rice etc.
Red colouring agent for food, also used as a Chinese medicine is the traditional Chinese medicine that dietotherapy is simply had both.Early it has been widely used in food color, wine brewing, fermentation, traditional Chinese medicine aspect in ancient times.Principle of Correct Diet has red colouring agent for food, also used as a Chinese medicine " it is sweet, flat, nontoxic to distinguish the flavor of " " in invigorating the spleen, beneficial gas, the temperature "; Compendium of Material Medica has " sweet, warm, nontoxic ", " control woman's blood pain and postpartum extravesated blood unclean, it is good to beat beverage "; " Amplification on Materia Medica addendum " has records such as " invigorate blood circulation, help digestion, the warm stomach of invigorating the spleen, control red white diarrhea, wound ".
The seventies in last century, Japan professor Endo has isolated since the physiologically active substance Mo Nakelin K (monacolin K) from red monascus (Monascus ruber) first, numerous Chinese scholars are constantly found physiologically active substance in the monascus meta-bolites, comprise the monacolin compounds, monascorubin, some terpenoids of antihypertensive compositions GABA and antioxidant component dimerumic acid and separation recently etc.Along with modern biochemistry and pharmacological development, the step-down of red colouring agent for food, also used as a Chinese medicine, hypoglycemic, anti-obesity, anticancer, effects such as control senile dementia and osteoporosis are constantly excavated.Thereby make traditional red colouring agent for food, also used as a Chinese medicine increase new intension.But because composition is various in the red colouring agent for food, also used as a Chinese medicine, what kind of effect people bring into play to each composition in the red colouring agent for food, also used as a Chinese medicine is also understood very fewly, and this science that has limited red colouring agent for food, also used as a Chinese medicine is to a certain extent used, and has hindered the widespread use of red colouring agent for food, also used as a Chinese medicine.
Summary of the invention
The present invention successfully separates from the red colouring agent for food, also used as a Chinese medicine prepared product and has obtained a kind of phytosterin compound with structural formula (I), and this compound can have the inhibiting medicine of HMG-CoA reductase activity in order to preparation.
In one aspect of the invention, provide a kind of phytosterin compound or its pharmacy acceptable salt with structural formula (I), (I) is as follows for structural formula:
Wherein, R
1For-OH ,=O (carbonyl), H or C1-C3 alkyl; R
2For-OH, H or C1-C3 alkyl; R
3For-OH ,=O, H or C1-C3 alkyl; R
4For-OH, H or C1-C3 alkyl; And R
1, R
2, R
3, R
4In at least one be-OH.
Further, above-mentioned phytosterin compound or its pharmacy acceptable salt have structural formula (II), and (II) is as follows for structural formula:
In another aspect of the present invention, a kind of above-mentioned preparation method with phytosterin compound of structural formula (I) also is provided, may further comprise the steps: get the red colouring agent for food, also used as a Chinese medicine prepared product, carry out supersound extraction behind the adding solvent, and the extracting solution concentrating under reduced pressure is got extract; Silica gel column chromatography on the extract is separated, adopt sherwood oil and ethyl acetate that extract is carried out gradient elution in the sepn process, the volume ratio of gradient elution process PetroChina Company Limited.'s ether and ethyl acetate was followed successively by 75: 25,50: 50～25: 75,0: 100; The volume ratio of getting sherwood oil and ethyl acetate is 50: 50～25: 75 o'clock resulting elutriants, be that 1: 1 methylene dichloride and the mixed solution of methyl alcohol are that moving phase is carried out sephadex LH-20 gel filtration chromatography with volume ratio, merge identical part through the TLC trace detection, obtain 6 part flow points; The 4th part flow point is carried out column chromatography separate, chromatographic column is C18 reverse phase silica gel post, and moving phase is that volume ratio is 75: 25 methyl alcohol and water mixed liquid, detects through TLC, removes to collect behind the impurity band to obtain phytosterin compound.
Further, above-mentioned preparation method further carries out the silicagel column purification process to phytosterin compound, be that the mixed solution of 20: 20: 1 methylene dichloride, ethyl acetate and methyl alcohol carries out wash-out with volume ratio, remove and collect the phytosterin compound that obtains behind the purifying behind the impurity band
Further, the solvent in the above-mentioned supersound extraction process is sherwood oil, methylene dichloride, and ethyl acetate, ethanol, one or more in methyl alcohol or the normal hexane, volume are 2-6 times of red colouring agent for food, also used as a Chinese medicine prepared product; And/or in the supersound extraction process extraction time be 2-6 time, each time is 20-40min; And/or the volume ratio of gradient elution process PetroChina Company Limited.'s ether and ethyl acetate was followed successively by 75: 25,50: 50,25: 75,0: 100, the volume ratio of getting sherwood oil and ethyl acetate is that 25: 75 o'clock resulting elutriants carry out sephadex LH-20 gel filtration chromatography.
In another aspect of the present invention, a kind of extract also is provided, this extract comprises above-mentioned phytosterin compound.
Further, the said extracted thing is the extract of red colouring agent for food, also used as a Chinese medicine prepared product.
Further, the said extracted thing comprises: the volume ratio of gradient elution process PetroChina Company Limited.'s ether and ethyl acetate is 50: 50～25: 75 o'clock resulting elutriants among the preparation method of (1) above-mentioned phytosterin compound; (2) the 4th part flow point that obtains in the TLC trace detection process among the preparation method of above-mentioned phytosterin compound; (3) TLC collects the phytosterin compound that obtains after detecting the removal impurity band among the preparation method of above-mentioned phytosterin compound; The perhaps phytosterin compound behind the purifying among the preparation method of (4) above-mentioned phytosterin compound.
In another aspect of the present invention, a kind of composition also is provided, it comprises above-mentioned phytosterin compound and/or said extracted thing; Alternatively, also comprise pharmaceutically acceptable carrier or auxiliary material.
In another aspect of the present invention, also provide a kind of above-mentioned phytosterin compound or said extracted thing or above-mentioned composition preparation reduce or regulate blood fat or prevent and/or treat hyperlipemia, hyperlipidemia, hypercholesterolemia or atherosclerosis improve function of vascular endothelium or the medicine of anticoagulant in purposes.
In another aspect of the present invention, method in vivo a kind of or vitro inhibition HMG-CoA reductase enzyme also is provided, comprise that the HMG-CoA reductase inhibitor that uses significant quantity suppresses the step of HMG-CoA reductase enzyme, the HMG-CoA reductase inhibitor is above-mentioned phytosterin compound or said extracted thing or above-mentioned composition.
Beneficial effect of the present invention: the present invention successfully separates from the red colouring agent for food, also used as a Chinese medicine prepared product and has obtained a kind of compound, and this compound has the structure in the structural formula (I), and it can suppress the HMG-CoA reductase enzyme effectively; Have as reducing or regulating blood fat or prevent and/or treat hyperlipemia, hyperlipidemia, hypercholesterolemia or atherosclerosis or improve function of vascular endothelium or the potentiality of the medicine of anticoagulant.
Description of drawings
Figure of description is used to provide further understanding of the present invention, constitutes a part of the present invention, and illustrative examples of the present invention and explanation thereof are used for explaining the present invention, do not constitute improper restriction of the present invention.In the accompanying drawings:
Fig. 1 shows the ultraviolet spectrogram of the prepared compound with structural formula (II) of embodiment 1;
Fig. 2 shows the infrared spectrogram of the prepared compound with structural formula (II) of embodiment 1;
Fig. 3 shows the high resolution mass spectrum figure of the prepared compound with structural formula (II) of embodiment 1;
Fig. 4 shows the hydrogen spectrogram of the prepared compound with structural formula (II) of embodiment 1;
Fig. 5 shows the carbon spectrogram of the prepared compound with structural formula (II) of embodiment 1;
Fig. 6 shows the DEPT signal graph of the prepared compound with structural formula (II) of embodiment 1;
Fig. 7 shows the HSQC coherent signal figure of the prepared compound with structural formula (II) of embodiment 1; And
Fig. 8 shows the HMBC coherent signal figure of the prepared compound with structural formula (II) of embodiment 1.
Embodiment
Need to prove that under the situation of not conflicting, embodiment and the feature among the embodiment among the application can make up mutually.Describe the present invention below with reference to the accompanying drawings and in conjunction with the embodiments in detail.
In a kind of typical embodiment of the present invention, a kind of phytosterin compound or its pharmacy acceptable salt with structural formula (I) is provided, (I) is as follows for structural formula:
Wherein, R
1For-OH ,=O (carbonyl), H or C1-C3 alkyl; R
2For-OH, H or C1-C3 alkyl; R
3For-OH ,=O, H or C1-C3 alkyl; R
4For-OH, H or C1-C3 alkyl; And R
1, R
2, R
3, R
4In at least one be-OH.Among the present invention, term " C1-C3 alkyl " comprises methyl, ethyl, propyl group or sec.-propyl.
Preferably, above-mentioned phytosterin compound or its pharmacy acceptable salt have structural formula (II), and (II) is as follows for structural formula:
Through studying for a long period of time, the contriver has extracted the compound with said structure formula (I) from the red colouring agent for food, also used as a Chinese medicine prepared product.Indication among the application " red colouring agent for food, also used as a Chinese medicine prepared product " refers to contain the composition, mixture of red colouring agent for food, also used as a Chinese medicine etc., and those skilled in the art can reasonably analyze according to the composition of material and whether can be used as the red colouring agent for food, also used as a Chinese medicine prepared product and use.For example Beijing WBL Peking University Biotech Co., Ltd's XUEZHIKANG JIAONANG of producing, commercially available Hongqu powder (red colouring agent) and commercially available red colouring agent for food, also used as a Chinese medicine lyophilized powder etc. all can be used as the red colouring agent for food, also used as a Chinese medicine prepared product.The compound that the present invention obtains from above-mentioned red colouring agent for food, also used as a Chinese medicine prepared product is a kind of brand-new compound, has the phytosterin compound parent nucleus in this structural formula, unsaturated link(age) and dioxygen bridged cycloalkyl structure, and this compound is not seen relevant report in Monas cuspurpureus Went extract.And the contriver makes further research the activity of this compounds, finds that pleasantly surprisedly it has HMG-CoA reductase activity restraining effect.
In a kind of typical embodiment of the present invention, the preparation method of above-claimed cpd may further comprise the steps: get the red colouring agent for food, also used as a Chinese medicine prepared product, carry out supersound extraction behind the adding solvent, and the extracting solution concentrating under reduced pressure is got extract.Silica gel column chromatography on the extract is separated, adopt sherwood oil and ethyl acetate that extract is carried out gradient elution in the column chromatography sepn process, the volume ratio of gradient elution process PetroChina Company Limited.'s ether and ethyl acetate was followed successively by 75: 25,50: 50～25: 75,0: 100, the volume ratio of getting sherwood oil and ethyl acetate is 50: 50～25: 75 o'clock resulting elutriants, preferably, the volume ratio of gradient elution process PetroChina Company Limited.'s ether and ethyl acetate was followed successively by 75: 25,50: 50,25: 75,0: 100, the volume ratio of getting sherwood oil and ethyl acetate is 25: 75 o'clock resulting elutriants, be that 1: 1 methylene dichloride and the mixed solution of methyl alcohol are that moving phase is carried out sephadex LH-20 gel filtration chromatography with volume ratio, merge identical part through the TLC trace detection, obtain 6 part flow points.The 4th part flow point is carried out column chromatography separate, chromatographic column is C18 reverse phase silica gel post, and moving phase is that volume ratio is 75: 25 methyl alcohol and water mixed liquid, detects through TLC, removes to collect behind the impurity band to obtain phytosterin compound.The TLC testing conditions is the purification on normal-phase silica gel plate: developping agent is methylene dichloride-ethyl acetate-methyl alcohol=8: 8: 1, and the Rf value of this phytosterin compound is about 0.3, and the flow point of collecting this Rf value namely obtains this phytosterin compound.
Preferably, among the above-mentioned preparation method phytosterin compound is carried out the silicagel column purification process, be that the mixed solution of 20: 20: 1 methylene dichloride, ethyl acetate and methyl alcohol carries out wash-out with volume ratio, remove and collect the described phytosterin compound that obtains behind the purifying behind the impurity band.
Preferably, the solvent in above-mentioned preparation method's the supersound extraction process is sherwood oil, methylene dichloride, and ethyl acetate, ethanol, one or more in methyl alcohol or the normal hexane more preferably are normal hexane.The volume of the solvent that uses is 2-6 times of the red colouring agent for food, also used as a Chinese medicine prepared product.Preferably, extraction time is 2-6 time in the above-mentioned supersound extraction process, and each time is 20-40min.In this scope, can reasonably take into account time and product content.
Above-mentioned phytosterin compound with structural formula (I) provided by the present invention is except by the preparation of said extracted method, can also be by the method preparation of organic synthesis, those skilled in the art are under instruction of the present invention, have the ability according to the artificial synthesis of existing phytosterin compound, be prepared into out the have structural formula phytosterin compound of (I).Because this synthetic method is the ordinary method of this area, does not repeat them here.
In a kind of typical embodiment of the present invention, a kind of extract also is provided, this extract comprises above-mentioned phytosterin compound with structural formula (I).
Preferably, this extract is the extract of red colouring agent for food, also used as a Chinese medicine prepared product.
Preferably, this extract comprises: (1) prepares gradient elution process PetroChina Company Limited.'s ether of the phytosterin compound with structural formula (I) and the volume ratio of ethyl acetate is 50: 50～25: 75 o'clock resulting elutriants; (2) prepare the 4th part flow point that obtains in the middle TLC trace detection process of the phytosterin compound with structural formula (I); (3) TLC of preparation with phytosterin compound of structural formula (I) detect to remove to collect behind the impurity band and obtains described phytosterin compound; Perhaps (4) preparation has the described phytosterin compound behind the purifying of phytosterin compound of structural formula (I).
In a kind of typical embodiment of the present invention, a kind of composition also is provided, it comprises above-mentioned phytosterin compound and/or said extracted thing; Alternatively, also comprise pharmaceutically acceptable carrier or auxiliary material.
What usually pharmaceutical composition of the present invention contained 0.1-90 weight % has structural formula (I) or structural formula (II) phytosterin compound and/or its pharmacy acceptable salt or a said extracted thing.Pharmaceutical composition can prepare according to methods known in the art.When being used for this purpose, if desired, can be combined having structural formula (I) or structural formula (II) phytosterin compound and/or steric isomer and one or more solids or liquid medicine vehicle and/or assistant agent, make and can be used as the suitable administration form of human or dosage form.
It is of the present invention that have structural formula (I) or structural formula (II) phytosterin compound or contain its pharmaceutical composition can the unit dosage form administration, route of administration can be enteron aisle or non-enteron aisle, as oral, muscle, subcutaneous, nasal cavity, oral mucosa, skin, peritonaeum or rectum etc.Form of administration is tablet, capsule, dripping pill, aerosol, pill, pulvis, solution, suspensoid, emulsion, granule, liposome, transdermal agent, buccal tablet, suppository, lyophilized injectable powder etc. for example.Can be ordinary preparation, sustained release preparation, controlled release preparation and various particulate delivery system.For the unit form of administration is made tablet, can be extensive use of various carrier well known in the art.Example about carrier is, for example thinner and absorption agent are as starch, dextrin, calcium sulfate, lactose, N.F,USP MANNITOL, sucrose, sodium-chlor, glucose, urea, calcium carbonate, white bole, Microcrystalline Cellulose, pure aluminium silicate etc.; Wetting agent and tackiness agent are as water, glycerine, polyoxyethylene glycol, ethanol, propyl alcohol, starch slurry, dextrin, syrup, honey, glucose solution, mucialga of arabic gummy, gelatine size, Xylo-Mucine, lac, methylcellulose gum, potassiumphosphate, polyvinylpyrrolidone etc.; Disintegrating agent, for example dry starch, alginates, agar powder, laminaran, sodium bicarbonate and Citric Acid, calcium carbonate, polyoxyethylene, Sorbitol Powder fatty acid ester, sodium laurylsulfonate, methylcellulose gum, ethyl cellulose etc.; Disintegration inhibitor, for example sucrose, Tristearoylglycerol, theobroma oil, hydrogenation wet goods; Absorption enhancer, for example quaternary ammonium salt, sodium lauryl sulphate etc.; Lubricant, for example talcum powder, silicon-dioxide, W-Gum, stearate, boric acid, whiteruss, polyoxyethylene glycol etc.Tablet further can also be made coating tablet, for example sugar coated tablet, thin membrane coated tablet, ECT, or double-layer tablets and multilayer tablet.For pill is made in the administration unit, can be extensive use of various carrier well known in the art.Example about carrier is, for example thinner and absorption agent are as glucose, lactose, starch, theobroma oil, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, talcum powder etc.; Tackiness agent such as gum arabic, tragacanth gum, gelatin, ethanol, honey, liquid sugar, rice paste or batter etc.; Disintegrating agent is as agar powder, dry starch, alginates, sodium laurylsulfonate, methylcellulose gum, ethyl cellulose etc.For suppository is made in the administration unit, can be extensive use of various carrier well known in the art.Example about carrier is, for example the ester of polyoxyethylene glycol, Yelkin TTS, theobroma oil, higher alcohols, higher alcohols, gelatin, semi-synthetic glyceryl ester etc.For capsule is made in the administration unit, effective constituent is had the compound of structural formula (I) or its steric isomer mix with above-mentioned various carriers, and the mixture that will obtain thus places hard obviously capsule or soft capsule.Also compound or its steric isomer that effective constituent can be had structural formula (I) made microcapsule, is suspended in and forms suspensoid in the aqueous medium, in the hard capsule of also can packing into or make injection and use.For injection preparation is made in the administration unit, as solution, emulsion, lyophilized injectable powder and suspensoid, can use this area all thinners commonly used, for example, water, ethanol, polyoxyethylene glycol, 1, the isooctadecanol of ammediol, ethoxylation, the isooctadecanol of polyoxyization, Polyoxyethylene Sorbitol Fatty Acid Esters etc.In addition, to ooze injection liquid in order preparing etc., can in injection preparation, to add proper amount of sodium chloride, glucose or glycerine, in addition, can also add conventional solubility promoter, buffer reagent, pH regulator agent etc.
In addition, as needs, also can in pharmaceutical preparation, add tinting material, sanitas, spices, correctives, sweeting agent or other material.
The present invention has structural formula (I) or structural formula (II) phytosterin compound, or the dosage of its pharmacologically acceptable salt depends on many factors, for example to prevent or treat character and the severity of disease, the sex of patient or animal, age, body weight and individual reaction, used particular compound, route of administration and administration number of times etc.Above-mentioned dosage can the single dose form or be divided into several, for example two, three or four dosage form administrations.
For composition, can be by changing the actual dose level of each activeconstituents in the pharmaceutical composition of the present invention, so that the active compound amount of gained can effectively obtain required therapeutic response at concrete patient, composition and administering mode.The dosage level fibrous root is selected according to activity, route of administration, the severity of the patient's condition for the treatment of and the patient's to be treated patient's condition and the medical history of particular compound.But the way of this area is that the dosage of compound increases dosage gradually from being lower than for obtaining the level that required result for the treatment of requires, up to obtaining required effect.
When being used for above-mentioned treat and/or prevent or during assisting therapy, a kind of The compounds of this invention that treats and/or prevents significant quantity can be used with pure form, perhaps uses with the acceptable ester of pharmacy or prodrug forms (under the situation that has these forms).Perhaps, described compound can be accepted the pharmaceutical composition administration of vehicle to contain this purpose compound and one or more medicines.Term " significant quantity " refers to realize treating, prevent, alleviate and/or alleviating the dosage of disease of the present invention or illness in the experimenter.But the total daily dosage portion that it should be understood that The compounds of this invention and composition must be examined the doctor by the master and maked decision in the medical judgment scope reliably.For any concrete patient, the concrete horizontal fibrous root for the treatment of effective dose is decided according to multiple factor, and described factor comprises the severity of the obstacle for the treatment of and this obstacle; The activity of the particular compound that adopts; The concrete composition that adopts; Patient's age, body weight, general health situation, sex and diet; The administration time of the particular compound that adopts, route of administration and excretion rate; The treatment time length; The medicine that is used in combination or uses simultaneously with the particular compound that adopts; And the known similar factor of medical field.For example, the way of this area is that the dosage of compound increases dosage gradually from being lower than for obtaining the level that required result for the treatment of requires, up to obtaining required effect.In general, the compound of the present invention with structural formula (I) is used for Mammals particularly people's dosage can be between the 0.001-1000mg/kg body weight/day, for example between the 0.01-100mg/kg body weight/day, for example between the 0.01-10mg/kg body weight/day.
In a kind of typical embodiment of the present invention, above-mentioned phytosterin compound or extract or composition preparation reduce or regulate blood fat or prevent and/or treat hyperlipemia, hyperlipidemia, hypercholesterolemia or atherosclerosis improve function of vascular endothelium or the medicine of anticoagulant in purposes.
In a kind of typical embodiment of the present invention, in vivo a kind of method of or vitro inhibition HMG-CoA reductase enzyme, comprise that the HMG-CoA reductase inhibitor that uses significant quantity suppresses the HMG-CoA reductase enzyme, the HMG-CoA reductase inhibitor is above-mentioned compound or above-mentioned extract or above-mentioned composition.
Specify the active effect that the phytosterin compound with structural formula (II) provided by the present invention suppresses the HMG-CoA reductase enzyme below with reference to embodiment 1 and 2.
Embodiment 1:
Raw material: the XUEZHIKANG JIAONANG content 2kg that the Beijing WBL Peking University Biotech Co., Ltd produces.
Embodiment 2:
Raw material: commercially available Hongqu powder (red colouring agent) 3kg.
One, feedstock production has the method for the phytosterin compound of structural formula (II) among the employing embodiment 1 and 2:
1) getting the about 3kg of XUEZHIKANG JIAONANG content 2kg or Hongqu powder (red colouring agent), is that solvent supersonic extracts 3 times with the normal hexane of 2-6 times of volume, and each 20-40 minute, united extraction liquid, concentrating under reduced pressure got normal hexane extract 84g.
2) get normal hexane extract 50g, last silica gel column chromatography separates, and carries out gradient elution with sherwood oil and ethyl acetate.The volume ratio of petroleum ether-ethyl acetate was followed successively by 75: 25,50: 50,25: 75,0: 100.
3) getting petroleum ether-ethyl acetate is the extract 3.0g that 25: 75 wash-outs obtain, and revolves the oily matter that steams concentrating under reduced pressure.Use micro-methylene dichloride: after methyl alcohol=dissolving in 1: 1, upper prop is moving phase with methylene chloride-methanol (1: 1), carries out sephadex LH-20 gel filtration chromatography.Received 120 flow points altogether, every flow point is 5mL.Merge identical part through the TLC trace detection, obtain 6 parts.Be respectively 1-50; 51-75; 76-80; 81-93; 94-110; The 111-120 flow point.Get the 4th part (81-93 flow point) 1.55g, revolve the oily matter that steams concentrating under reduced pressure, after micro-100% dissolve with methanol, last C18 reverse phase silica gel post is with methanol-water (75: 25) wash-out, according to the TLC detected result, collection contains the flow point of this compound, and dry concentrating obtains this phytosterin compound.The TLC testing conditions is the purification on normal-phase silica gel plate: developping agent is methylene dichloride-ethyl acetate-methyl alcohol=8: 8: 1, and the Rf value of this phytosterin compound is about 0.3, and the flow point of collecting this Rf value namely obtains this phytosterin compound.In order to be further purified this compound, can further to pass through purification by silica gel column chromatography, use methylene dichloride: ethyl acetate: methyl alcohol (20: 20: 1) wash-out can obtain the compound 5mg behind the purifying.
Two, embodiment 1 and 2 prepared compound structure authentication methods are:
1. the physicochemical data of compound
2. embodiment 1 and 2 prepared compound molecule formulas determines
As shown in Figure 3, the m/z 493.31596[M+H that provides of FT-ICR-MS]
+(calcd.493.31665, err 0.69), the molecular weight that is inferred as this compound is 492.31.In Fig. 4
1Among H-NMR figure and Fig. 5
13Shown in the C-NMR figure, have 40 hydrogen signals and 28 carbon signals.Show 6 quaternary carbons are arranged, 10 CH, 6 CH from Fig. 6 DEPT signal graph
2And 6 CH
3In Fig. 5
13Among the C-NMR figure, two olefinic carbon signal indicatings that 136.9ppm (C-6) and 131.8ppm (C-7) locate, this compound have a two key.In conjunction with Fig. 7 HSQC coherent signal figure and Fig. 5
13The C-NMR map analysis, 67.2ppm, 83.8ppm, 80.2ppm, 84.6ppm, 81.0ppm, 85.8ppm, 73.3ppm, there are 8 carbon that link to each other with Sauerstoffatom in the 75.6ppm place.Binding molecule amount and Fig. 4
1H-NMR figure, Fig. 5
13C-NMR figure and Fig. 6 DEPT signal graph surpass 492.31 if this compound has 8 Sauerstoffatom molecular weight, infer that thus this compound has 7 Sauerstoffatoms and do not embody 4 hydrogen atoms of hydrogen spectrum signal.Thereby can judge in above-mentioned 7 Sauerstoffatoms that 4 Sauerstoffatom ownership are 4 hydroxyls, all the other 3 Sauerstoffatoms exist with the non-hydroxyl form.According to above-mentioned analysis, can determine has 28 carbon atoms in this molecule, 44 hydrogen atoms and 7 Sauerstoffatoms, and molecular formula is C
28H
44O
7
3. determining of embodiment 1 and 2 prepared structural formula of compound:
Analyze this compound carbon spectrum (
13C-NMR and DEPT), have 28 carbon atoms and wherein 6 carbon be methyl and 2 olefinic carbons.And shown in the ultraviolet spectrogram of this compound among Fig. 1, the ultraviolet spectrogram of itself and ergosterol peroxide is very approaching, tentatively infer this compound have ergosterol peroxide skeleton and-O-O-peroxidation part.Can draw thus, 2 Sauerstoffatoms belong to-O-O-in 3 non-hydroxyl Sauerstoffatoms.Fig. 7 HSQC coherent signal figure analysis that combines with Fig. 8 HMBC coherent signal figure, 8 carbon that link to each other with Sauerstoffatom, ownership is 4 carbon (67.2 that 4 hydroxyls link to each other respectively, 73.3,75.6 and 81.0ppm), 2 carbon linking to each other with-2 carbon that O-O-links to each other (80.2 with 83.8ppm) and the 7th Sauerstoffatom (84.6 and 85.8ppm).Can calculate its degree of unsaturation from molecular formula is 7.Thereby can infer: this compound is except 6 unsaturated places of 1 two key and 5 rings of peroxidation sterol skeleton, also have a unsaturated place and can only be a ring herein, can judge that thus this ring is by centered by the 7th Sauerstoffatom, by C-16 (84.6ppm), C-17 (68.5ppm), 5 yuan of rings that 4 carbon atoms of C-20 (81.0ppm) and C-22 (85.8ppm) are formed.This not only satisfies degree of unsaturation, but also satisfies the carbonatoms that links to each other with Sauerstoffatom.Further analyze molecular weight and the above-mentioned molecular formula that mass spectrometry system provides, confirmed above-mentioned analysis.The above analysis can infer that this compound is: 5,8-epidioxy-16,22-epoxy ergot steroid-5,7-diene-3,20,23,25-tetrol.
4. the structural formula of embodiment 1 and 2 prepared compounds is as follows:
Three, embodiment 1 prepared compound H MG-CoA reductase active is tested
1 experiment material
1.1 test specimens
The compound with structural formula (II) that embodiment 1 is prepared; Lovastatin standard substance (purchasing the company in Sigma)
1.2 enzyme
Rat liver microsome (HMG-CoA reductase enzyme): can be purchased, also can be with reference to following preparation method: take out the liver of male rat, after the flushing of KESD damping fluid, the centrifugal 15min of 1200g gets supernatant liquor.Use again after twice of the centrifugal 90min of 105000g, collect centrifugation.Add 8.3% glycerine in the centrifugation, bathe heating 1h with 37 ℃ of temperature.Rat liver microsome runic thing is with the saturated ammonium sulphate purifying and collect the 35-50% purification part.The purification part that obtains can be left in-80 ℃ refrigerators.
1.3 reagent
Repone K, potassium primary phosphate, ethylenediamine tetraacetic acid (EDTA), dithiothreitol (DTT) is purchased in Beijing chemical reagents corporation
Reduced nicotinamide-adenine dinucleotide (NADPH) is purchased the company in Merk
3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) is purchased the company in Sigma
2 experimental techniques
The compound 5.0mg with structural formula (II) and lovastatin 2.0mg that embodiment 1 is prepared, each is with 1mL 75% dissolve with ethanol solution;
Cumulative volume is 250 μ L in the mensuration system, the concentration of each composition is: Repone K 200mM, potassium primary phosphate 160mM, ethylenediamine tetraacetic acid (EDTA) 4mM, dithiothreitol (DTT) 10mM, the concentration of two substrate Reduced nicotinamide-adenine dinucleotides and 3-hydroxy-3-methylglutaryl-coenzyme A is respectively 200 μ M and 50 μ M, and pH6.8, enzyme add 30 μ L.
Test group adds the ethanolic soln that 5 μ L have the prepared compound of embodiment 1; Positive controls adds 5 μ L lovastatin ethanolic solns; The blank group adds 5 μ L, 75% ethanol.Detecting OD under 37 ℃ of conditions on the Versamax microplate reader
340Dynamic change.By detecting OD
340The speed (representing with slope value) that descended in 5 minutes is estimated the power of HMG-CoA reductase activity, and then estimates the power of inhibitor activity, the results are shown in Table 1.
3 experimental results
Table 1
Shown that by result in the table 1 the prepared compound with structural formula (II) of embodiment 1 has restraining effect to the activity of HMG-CoA reductase enzyme.Though this compound does not have the effective of lovastatin to the restraining effect of HMG-CoA reductase enzyme, its inhibiting rate has reached 31.4%, this compound can be applied in preparation fully and have in the inhibiting medicine of HMG-CoA reductase activity.
Though the compounds process for production thereof that the application only provides embodiment 1 and 2, and HMG-CoA reductase enzyme restraining effect is described in detail, each is not had the preparation method of structural formula (I) compound and the restraining effect of HMG-CoA reductase enzyme is described, but be based on the principle that the analog structure compound has similar performance, those skilled in the art can understand the preparation method of other compounds and the restraining effect of the HMG-CoA reductase enzyme that has fully.
This shows that successfully by extract obtain the having structural formula compound of (I) from the red colouring agent for food, also used as a Chinese medicine prepared product, the structural formula of this compound is not seen relevant report to the application, is new compound.Find through Related Experimental Study; this compound has the restraining effect of HMG-CoA reductase enzyme; for preparation HMG-CoA HMG-CoA Reductase Inhibitor HMG-CoA thing provides a kind of brand-new compound; have as reducing or regulating blood fat or prevent and/or treat hyperlipemia; hyperlipidemia; hypercholesterolemia; or atherosclerosis; or improve function of vascular endothelium; or the potentiality of the medicine of anticoagulant, this is undoubtedly vast hyperlipemia patient; hyperlipemia; hypercholesterolemiapatients patients; the atherosclerotic; the unusual patient of function of vascular endothelium; and platelet aggregation patient's Gospel.
The above is the preferred embodiments of the present invention only, is not limited to the present invention, and for a person skilled in the art, the present invention can have various changes and variation.Within the spirit and principles in the present invention all, any modification of doing, be equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (11)
1. phytosterin compound or its pharmacy acceptable salt with structural formula (I) is characterized in that (I) is as follows for described structural formula:
Wherein, R
1For-OH ,=O, H or C1-C3 alkyl; R
2For-OH, H or C1-C3 alkyl; R
3For-OH ,=O, H or C1-C3 alkyl; R
4For-OH, H or C1-C3 alkyl; And R
1, R
2, R
3, R
4In at least one be-OH.
2. phytosterin compound according to claim 1 or its pharmacy acceptable salt is characterized in that, described phytosterin compound has structural formula (II), and (II) is as follows for described structural formula:
3. the preparation method of a phytosterin compound as claimed in claim 1 or 2 is characterized in that, may further comprise the steps:
Get the red colouring agent for food, also used as a Chinese medicine prepared product, add solvent and carry out supersound extraction, and the extracting solution concentrating under reduced pressure is got extract;
Described extract is carried out silica gel column chromatography to be separated, adopt sherwood oil and ethyl acetate that described extract is carried out gradient elution in the sepn process, the volume ratio of gradient elution process PetroChina Company Limited.'s ether and ethyl acetate was followed successively by 75: 25,50: 50～25: 75,0: 100;
The volume ratio of getting sherwood oil and ethyl acetate is 50: 50～25: 75 o'clock resulting elutriants, be that 1: 1 methylene dichloride and the mixed solution of methyl alcohol are that moving phase is carried out sephadex LH-20 gel filtration chromatography with volume ratio, merge identical part through the TLC trace detection, obtain 6 part flow points;
The 4th part flow point is carried out column chromatography separate, chromatographic column is C18 reverse phase silica gel post, and moving phase is that volume ratio is 75: 25 methyl alcohol and water mixed liquid, detects through TLC, removes to collect behind the impurity band to obtain described phytosterin compound.
4. preparation method according to claim 3, it is characterized in that, further described phytosterin compound is carried out the silicagel column purification process, be that the mixed solution of 20: 20: 1 methylene dichloride, ethyl acetate and methyl alcohol carries out wash-out with volume ratio, remove and collect the described phytosterin compound that obtains behind the purifying behind the impurity band.
5. according to claim 3 or 4 described preparation methods, it is characterized in that the solvent in the described supersound extraction process is sherwood oil, methylene dichloride, ethyl acetate, ethanol, one or more in methyl alcohol or the normal hexane, volume are 2-6 times of described red colouring agent for food, also used as a Chinese medicine prepared product; And/or in the described supersound extraction process extraction time be 2-6 time, each time is 20-40mi;
And/or the volume ratio of gradient elution process PetroChina Company Limited.'s ether and ethyl acetate was followed successively by 75: 25,50: 50,25: 75,0: 100; The volume ratio of getting sherwood oil and ethyl acetate is that 25: 75 o'clock resulting elutriants carry out sephadex LH-20 gel filtration chromatography.
6. an extract is characterized in that, described extract comprises claim 1 or 2 described phytosterin compounds.
7. extract according to claim 6 is characterized in that, described extract is the extract of red colouring agent for food, also used as a Chinese medicine prepared product.
8. according to claim 6 or 7 described extracts, it is characterized in that, comprising:
(1) volume ratio of gradient elution process PetroChina Company Limited.'s ether and ethyl acetate is 50: 50～25: 75 o'clock resulting elutriants in the claim 3; Perhaps
(2) the 4th part flow point that obtains in the TLC trace detection process in the claim 3; Perhaps
(3) collection obtained described phytosterin compound after TLC detected the removal impurity band in the claim 3; Perhaps
(4) the described phytosterin compound behind the purifying in the claim 4.
9. composition, it comprises each described extract in claim 1 or 2 described phytosterin compounds and/or the claim 6 to 8; Alternatively, also comprise pharmaceutically acceptable carrier or auxiliary material.
In claim 1 or 2 described phytosterin compounds or the claim 6 to 8 each described extract or the described composition of claim 9 preparation reduce or regulate blood fat or prevent and/or treat hyperlipemia, hyperlipidemia, hypercholesterolemia or atherosclerosis improve function of vascular endothelium or the medicine of anticoagulant in purposes.
11. one kind in vivo or the method for vitro inhibition HMG-CoA reductase enzyme, comprise that the HMG-CoA reductase inhibitor that uses significant quantity suppresses described HMG-CoA reductase enzyme, it is characterized in that described HMG-CoA reductase inhibitor is each described extract or the described composition of claim 9 in claim 1 or 2 described compounds or the claim 6 to 8.
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| US14/376,481 US10093695B2 (en) | 2012-02-02 | 2013-02-04 | Sterol derivative, preparation method therefor and use thereof |
| CN201380008026.4A CN104159910B (en) | 2012-02-02 | 2013-02-04 | Sterol derivative, preparation method therefor and use thereof |
| TW102104276A TWI580689B (en) | 2012-02-02 | 2013-02-04 | A sterol derivatives, preparation method and use thereof |
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