CN101756965A - Application of maca imidazole alkaloid in preparation of cardiovascular drugs - Google Patents
Application of maca imidazole alkaloid in preparation of cardiovascular drugs Download PDFInfo
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Abstract
The invention discloses an application of maca imidazole alkaloid, in particular to an application to the preparation of cardiovascular drugs. All or part of the components are prepared from one or mixture of compounds having the following chemical structural formula as shown in formula (I), wherein R1 can be H or CH3, and R2 is H or OCH3. The invention first discovers the remarkable pharmaceutical activity of the compounds on blood pressure drop and arrhythmia resistance. Therefore, the maca imidazole alkaloid prepared by the new process can be used for developing antineoplastic drugs and can be expected to act as drugs for treating corresponding cardiovascular diseases. Formula (I).
Description
Technical field
The present invention relates to the Natural Medicine Chemistry field, particularly, the present invention relates to a kind of application of imidazole alkaloid in the preparation cardiovascular drugs that obtains that from natural plants Lepidinm meyenii Walp (Lepidium meyenii Walper, also common its different name appellation Lepidiumperuvianum Chacon sp. of the same race), directly separate.
Background technology
Cardiovascular disease is first killer of human health, comprising multiple disease forms such as hypertension, arrhythmia, hyperlipidemia, coronary heart disease, according to World Health Organization's prediction, to the year two thousand twenty, noninfectious will account for 79% of China's cause of death, and wherein the cardiovascular diseases will account for the first place.Hypertension is modal cardiovascular disease, its prevalence is all the highest in various countries, the whole world, and the disease crowd has the trend of rejuvenation, China's Hypertensive Population increases swift and violent, therefore, the demand of Altace Ramipril constantly increases at present, and screen new and effective Altace Ramipril also is the emphasis and the focus of cardiovascular disease medicine research always.Arrhythmia also is a disease very common in the cardiovascular disease; usually treat with antiarrhythmic drug; but present most arrhythmia chemicals mechanism of action is single, the curative effect individual variation big, be easy to generate toxic and side effects (infringement internal organs, dizziness headache, blurred vision etc.); even cause new arrhythmia symptom, therefore from natural plants, seek the important component part that determined curative effect, the little novel anti antiarrhythmic medicament of side effect just become the cardiovascular drugs research field.
Lepidinm meyenii Walp is the medicine food dual purpose plant that originates in mountain area, Andean, South America, because its nutritional labeling is abundant, and has plurality of health care functions (for example resisting fatigue, enhance immunity, anti-anemia), and multiple medicinal efficacy (for example improving fertility, sexual function improving etc.), the FAO of FAO (Food and Agriculture Organization of the United Nation) extraordinarily praises highly it, recommend this industrial crops of a large amount of plantations in various countries (document: surplus Longjiang, inscription on ancient bronze objects is heard, Li Wei etc. the progress [J] of South America plant Lepidinm meyenii Walp. Chinese herbal medicine .2003,34 (2): attached 7-9.).Lepidinm meyenii Walp has bigger cultivated area in Peru, to various countries export in a large number as healthy food material every year, China has also carried out work that Lepidinm meyenii Walp introduces a fine variety and in nearly 2 years successful introduction, the planting scale on Yunnan Huize, Yunnan Lijing, Tibet Linzhi and other places was constantly enlarged.2003; people such as Cui BL are separated to two kinds of maca imidazole alkaloid Lepidiline A and Lepidiline B from the plant Lepidinm meyenii Walp; find that by experiment in vitro these two kinds of imidazole alkaloids have anti tumor activity in vitro (document: Cui BL; Zheng BL; He K; et al.Imidazole alkaloids from Lepidium meyenii[J] .J Nat Prod.2003; 66 (8): 1101-1103.), and the application of this compounds and anti proliferative disease (as antitumor) aspect thereof applied for patent protection (US 6878731 B2).It is by solvent extraction that people such as Cui BL prepare maca imidazole alkaloid, extractum dissolves with acidic methanol solution after reclaiming solvent, after staticly settling, supernatant by extraction, macroporous adsorption resin chromatography, purification on normal-phase silica gel column chromatography, acidic alumina column chromatography, preparative high performance liquid chromatography etc., obtains two kinds of maca imidazole alkaloid Lepidiline A and LepidilineB successively.This method step is more, loss is bigger in the maca imidazole alkaloid leaching process, 10 kilograms MAKA dry powder finally only is purified to 22 milligrams Lepidiline A and Lepidiline B still less, this defective has restricted the research and development of maca imidazole alkaloid to a certain extent, especially its application aspect screening active ingredients and pharmacology causes the application of present maca imidazole alkaloid to only limit to treatment aspect proliferative diseasees such as tumor.
Summary of the invention
The purpose of this invention is to provide the new application of maca imidazole alkaloid in the preparation cardiovascular drugs.
The purposes of a kind of maca imidazole alkaloid provided by the invention, it is characterized in that: the application of maca imidazole alkaloid in the preparation cardiovascular drugs, maca imidazole alkaloid active component all or mainly are independent or mixing formation by the chemical compound with the following chemical structure formula:
R wherein
1Be H or CH
3, R
2Be H or OCH
3
The research unit at inventor place has also carried out maca imidazole alkaloid research as long as five years, find maca imidazole alkaloid content in MAKA dry powder content than higher, kind is also many, forefathers' method still has the improved place of needs, solved separation of maca imidazole alkaloid high performance liquid chromatography and detection technique by scientific research after, improved extraction separation method, reduce separating step as far as possible, and adopt solid phase extraction to substitute the preparative high performance liquid chromatography technology, equipment requirements and purification cost are reduced, finally can be a large amount of, efficiently, from MAKA dry powder, obtain the maca imidazole alkaloid compositions of biologically active cheaply, and further obtain four kinds of maca imidazole alkaloid monomers by separation and purification in the said composition.
Above-mentioned composition and monomer are being carried out in the research of drug screening widely, and the inventor has also found the remarkable medical active of these materials aspect blood pressure lowering, arrhythmia first except having verified the anti tumor activity in vitro of these materials.Therefore the maca imidazole alkaloid for preparing by this new technology not only has the purposes of developing anti-tumor medicaments, and can expect as the medicine of the corresponding cardiovascular disease of treatment, finishes the present invention thus.
Description of drawings
Fig. 1 is the blood pressure lowering curative effect of maca imidazole alkaloid to kidney type hypertension model rat, has shown that maca imidazole alkaloid compositions and four kinds of monomers thereof have significant hypotensive activity.
The specific embodiment
The present invention has prepared maca imidazole alkaloid compositions and each maca imidazole alkaloid monomer thereof of have the treatment cardiovascular disease (comprising hypertension, arrhythmia) and antitumor application potential, and the maca imidazole alkaloid of mentioning can refer to have compound monomer and the compositions thereof shown in structural formula one:
Structural formula one
Wherein the difference according to the R base is divided into four kinds of maca imidazole alkaloid monomers again, is respectively:
Chemical compound 1 (R
1=H, R
2=H), i.e. LepidilineA, 1,3-dibenzyl-4,5-methylimidazole chloride;
Chemical compound 2 (R
1=CH
3, R
2=H), i.e. Lepidiline B, 1,3-dibenzyl-2,4,5-tri-methylimidazolium chloride;
Chemical compound 3 (R
1=H, R
2=OCH
3), i.e. Lepidiline C, 3-benzyl-1-(3-methoxy-benzyl)-4,5-methylimidazole chloride;
Chemical compound 4 (R
1=CH
3, R
2=OCH
3), i.e. Lepidiline D, 3-benzyl-1-(3-methoxy-benzyl)-2,4,5-tri-methylimidazolium chloride.
And from above maca imidazole alkaloid monomer, can obtain more similar substance again by sophisticated synthetic method, can obtain some compound monomers by synthesizing mean with architectural feature shown in structural formula two as also mentioning at american documentation literature US 6878731 B2:
Structural formula two
R wherein
1Can be H or CH
3, R
2～R
7Can be H, CH
3, CH
3O, OH, CHO, COOH, Cl, I, F, S, P, NO
3, NO
2, NH
2, R
2～R
7Can be identical, also can be different.
The present invention mentions directly obtains the maca imidazole alkaloid compositions and the monomeric preparation method of each maca imidazole alkaloid is specially from the plant Lepidinm meyenii Walp:
1) dry Lepidinm meyenii Walp root is crushed to 40～200 orders, MAKA dry powder is placed the rustless steel extraction vessel of Chinese herbal medicine flash extracter, according to the feed liquid weight ratio is 1: 8～1: 15 adding organic solvent, organic solvent is that mass percent concentration is that 80%～100% ethanol or mass percent concentration are 80%～100% methanol, the described organic solvent that perhaps contains volume fraction 0.5% hydrochloric acid, extracted 2～5 minutes down at 8000～20000 rev/mins, extracting solution with 400 order filter cloth sucking filtration after, filtering residue is extracted once by last method again, filtrate is merged, under 50 ℃～60 ℃ decompression rotary evaporations, reclaim extracting solution, obtain extracting extractum;
2) extracting extractum, to add isopyknic mass percent concentration 20% methanol mixed even, slowly add in the macroporous adsorptive resins, filler is a polystyrene type low pole macroporous adsorbent resin, water, mass percent concentration with 5～10 times of column volumes is that 10% methanol, mass percent concentration are 40%～80% methanol, methanol-eluted fractions successively, collect the 3rd elution fraction, reclaim solvent at 50 ℃～60 ℃ decompression rotary evaporations, obtain the maca imidazole alkaloid crude extract; The glacial acetic acid solution that wherein contains volume fraction 0.2%～0.8% in 40%～80% the methanol;
3) the maca imidazole alkaloid crude extract is dissolved with chloroform, go up the normal phase column chromatography again, the normal phase column chromatography is neutral alumina column chromatography or purification on normal-phase silica gel column chromatography, adopt the chloroform of 5～10 times of column volumes successively, chloroform: methanol (16: 1, v/v), chloroform: methanol (9: 1, v/v), chloroform: methanol (7: 3, v/v), methanol-eluted fractions, collect the 3rd, four elution fractions, 50 ℃～60 ℃ decompression rotary evaporations reclaim solvent, extractum is gone up C18 reverse phase silica gel post after with dissolve with methanol and is carried out chromatography, at first use the water of 5～10 times of column volumes, and then to utilize mass percent concentration successively be 10% methanol, 20% methanol, 40% methanol, methanol-eluted fractions, collect second, three, contain the maca imidazole alkaloid part in four elution fractions, merge the back and reclaim solvent at 50 ℃～60 ℃ decompression rotary evaporations, obtain maca imidazole alkaloid compositions (also abbreviating Lepidilines among the present invention as), detect by high performance liquid chromatography, contain four kinds of maca imidazole alkaloid monomer Lepidiline A～D, total maca imidazole alkaloid purity reaches 80%～90%;
4) the maca imidazole alkaloid compositions with dissolve with methanol after, be further purified by solid-phase extraction column, solid phase extraction column stuffing can be C18 or C8 silica filler, eluting adopts methanol: oxolane: 20 mMs/liter ammonium acetate solution (19: 1: 80v/v), detect monitoring by high performance liquid chromatography four kinds of maca imidazole alkaloids are collected merging respectively, after 50 ℃～60 ℃ decompression rotary evaporations reclaim solvent down, adopt acetone successively, acetone: methanol (9: 1v/v), oxolane, oxolane: petroleum ether (9: 1v/v) crystallization, can obtain four kinds of maca imidazole alkaloid monomers, wherein 2 kilograms of MAKA dry powder can obtain 0.21～0.48 Lepidiline A that restrains, 0.10 the Lepidiline B of～0.29 gram, 0.080 the Lepidiline C of～0.15 gram, 0.030 the Lepidiline D of～0.065 gram, each monomer purity all reaches 95.0%～99.5%.
Furtherly, the high-efficient liquid phase chromatogram technology that is used to detect and monitor the maca imidazole alkaloid composition in the above-mentioned preparation method of the present invention can adopt following method to realize:
U.S. Agilent 1200 high performance liquid chromatographs, be equipped with G1314BVWD UV, visible light detector and Agilent LC cycle chemistry work station, mobile phase is selected methanol for use: acetonitrile: 20 mMs/liter ammonium acetate solution (75: 5: 100v/v), chromatographic column is selected 250 * 4.6 millimeters C18 post (particle diameter 5 μ m) for use, 30 ℃ of column temperatures, flow velocity 1.0 ml/min, detect wavelength 258 nanometers, occur separating four kinds of good maca imidazole alkaloid absworption peaks between retention time 7～13 minutes successively, structural confirmation is corresponding Lepidiline A～D respectively.
Four kinds of maca imidazole alkaloid structures of gained are confirmed by LC-MS technology, NMR (Nuclear Magnetic Resonance) spectrum, infrared spectrum, ultraviolet spectra in the above-mentioned preparation method of the present invention.
The present invention screens by pharmacological experiment, finds that above-mentioned maca imidazole alkaloid compositions and each monomer thereof all have anti tumor activity in vitro.
The present invention also finds above-mentioned maca imidazole alkaloid compositions and the effect of each monomer on cardiovascular disease thereof, comprise significant blood pressure lowering and antiarrhythmic effect, the medical active of these two maca imidazole alkaloids has increased such novel application of compound, expanded such application of compound frontier, can be for the cardiovascular disease medicine of development of new.This is one of important innovations of the present invention.
With some embodiment maca imidazole alkaloid compositions of the present invention and each monomeric preparation method, structure evaluation, anticancer experiment in vitro, blood pressure lowering and arrhythmia zoopery are described further below.Embodiment is used to illustrate the present invention rather than limitation of the present invention.
Embodiment one
Take by weighing 1 kilogram of rustless steel extraction vessel that places the Chinese herbal medicine flash extracter of the dry Lepidinm meyenii Walp root powder of 40 purposes, add 15 kilogram-mass percent concentrations and be 100% ethanol, extracted 5 minutes down at 8000 rev/mins, by behind the 400 order filter cloth sucking filtration filtering residue being extracted once again, filtrate merging is reclaimed extracting solution, the methanol that resultant extraction extractum is added equal-volume mass percent concentration 20% fully dissolves, add in the HP20 macroporous adsorptive resins (70 * 5.5cm i.d.), adopt 10 times of cylinder hydrops successively, 5 times of column volume mass percent concentrations are 10% methanol, 10 times of column volume mass percent concentrations are 40% methanol solution eluting (glacial acetic acid that contains volume fraction 0.8%), collect the 3rd elution fraction and reclaim solvent at 60 ℃ of rotary evaporations that reduce pressure down, extractum dissolves with chloroform, go up purification on normal-phase silica gel column chromatography (50 * 2.6cm i.d. again, filler is a 200-300 order column chromatography silica gel), adopt 10 times of column volume chloroforms successively, 5 times of column volume chloroforms: methanol (16: 1), 10 times of column volume chloroforms: methanol (9: 1), 10 times of column volume chloroforms: methanol (7: 3), 5 times of column volume methanol-eluted fractions, collect the 3rd, four elution fractions reclaim solvent at 50 ℃ of rotary evaporations that reduce pressure down, extractum is with C18 reversed-phase silica gel chromatography post (40 * 1.6cm i.d. on the dissolve with methanol, filler is an ODS-A ball-type silica gel
The aperture, particle diameter 50 μ m), use 10 times of cylinder hydrops successively, 10 times of column volume mass percent concentrations are 10% methanol, 10 times of column volume mass percent concentrations are 20% methanol, 5 times of column volume mass percent concentrations are 40% methanol, 5 times of column volume methanol-eluted fractions, adopt high-efficient liquid phase chromatogram technology that maca imidazole alkaloid is monitored, mobile phase is selected methanol for use: acetonitrile: 20 mMs/liter ammonium acetate solution (75: 5: 100v/v), chromatographic column is selected 250 * 4.6 millimeters C18 post (particle diameter 5 μ m) for use, 30 ℃ of column temperatures, flow velocity 1.0 ml/min, detect wavelength 258 nanometers, collect second, three, the part that contains maca imidazole alkaloid described in the present invention in four elution fractions, merge the back and reclaim solvent at 50 ℃ of rotary evaporations that reduce pressure down, drying obtains maca imidazole alkaloid compositions 0.556 gram, be slightly yellowy indefinite form powder, detect by high performance liquid chromatography, adopt area normalization method to calculate, total maca imidazole alkaloid purity reaches 87.5%.
Embodiment two
Precision takes by weighing among the embodiment one maca imidazole alkaloid compositions (Lepidilines) 0.2 gram dissolve with methanol, by the C18 solid-phase extraction column to the maca imidazole alkaloid compositions be further purified (extraction column is 10g/60ml, and filler is an ODS-A indefinite form silica gel,
The aperture, particle diameter 40-60 μ m), eluting adopts methanol: oxolane: 20 mMs/liter ammonium acetate solution (19: 1: 80v/v), adopt the high-efficient liquid phase chromatogram technology monitoring that four kinds of maca imidazole alkaloids are collected, after 50 ℃ the decompression rotary evaporation reclaims solvent down, can obtain four kinds of maca imidazole alkaloid monomers by crystallization, dry back precision is weighed, Lepidiline A is 0.0863 gram (purity 98.5%), Lepidiline B is 0.0402 gram (purity 98.2%), 0.0192 gram (purity 96.8%), 0.0117 gram (purity 95.6%).These four kinds of monomers are carried out microexamination and adopt LC-MS, nuclear magnetic resonance, NMR, infrared spectrum, ultraviolet spectra that its structure is identified, some physicochemical properties of gained and important structural information are as follows, can confirm four kinds of monomeric compounds of gained of the present invention 1～4 corresponding maca imidazole alkaloid Lepidiline A, Lepidiline B, Lepidiline C and Lepidiline D respectively according to these information:
1) Lepidiline A, molecular formula C
19H
21N
2Cl, white needles, the Dragendorff's reagent colour developing is positive, and fusing point is 236 ℃～240 ℃; UV (methanol) λ
Max(log ε) 200 (3.29), 258 (2.24), 278 (2.12) nm; Infrared spectrum IR (KBr tabletting) v
Max3439,3030,2955,1632,1560,1453,1362,1219,1199,742,725,701cm
-1Holotype ESI-MS m/z277[M-Cl]
+, 185[M-Cl-benzyl]
+, 91[benzyl]
+1H NMR (CD
3OD: D
2O=1: 3v/v, 400MHz) δ 8.69 (1H, s, H-2), δ 7.22～7.35 (6H, m, H-3 ', H-4 ', H-5 '), δ 7.10～7.15 (4H, dd, J=7.6,1.2Hz, H-2 ', H-6 '), δ 5.18 (4H, s, PhCH
2N), δ 1.98 (6H, s, CCH
3-4/5);
13C NMR (CD
3OD: D
2O=1: 3v/v, 400MHz) δ 7.51 (CH
3, C-6, C-7), δ 47.31～48.60 (CH
2, PhCH
2N), δ 127.55 (CH, C-4 '), δ 127.89 (C, C-4, C-5), δ 128.81 (CH, C-3 ', C-5 '), δ 129.21 (CH, C-2 ', C-6 '), δ 133.40 (C, C-1 '), δ 133.97 (CH, C-2).
2) Lepidiline B, molecular formula C
20H
23N
2Cl, white plates, the Dragendorff's reagent colour developing is positive, and fusing point is 223 ℃～226 ℃; UV (methanol) λ
Max(log ε) 201 (3.31), 258 (2.25), 278 (2.11) nm; Infrared spectrum IR (KBr tabletting) v
Max3437,3029,2955,2360,1648,1555,1454,1217,1195,740,725,700cm
-1Holotype ESI-MS m/z291[M-Cl]
+, 199[M-Cl-benzyl]
+, 91[benzyl]
+ 1H NMR (CD
3OD: D
2O=1: 3v/v, 400MHz) δ 7.24～7.32 (6H, m, H-3 ', H-4 ', H-5 '), δ 6.99～6.98 (4H, d, J=6.8Hz, H-2 ', H-6 '), δ 5.26 (4H, s, PhCH
2N), δ 2.40 (3H, s, CH
3-2), δ 2.06 (6H, s, CH
3-4/5);
13C NMR (CD
3OD: D
2O=1: 3v/v, 400MHz) δ 7.69 (CH
3, C-7, C-8), δ 9.56 (CH
3, C-6), δ 47.28～48.58 (CH
2, PhCH
2N), δ 125.99 (CH, C-4 '), δ 126.47 (C, C-4, C-5), δ 128.34 (CH, C-3 ', C-5 '), δ 129.17 (CH, C-2 ', C-6 '), δ 133.81 (C, C-1 '), δ 143.17 (CH, C-2).
3) Lepidiline C, molecular formula C
20H
23N
2OCl, white column glomerocryst, the Dragendorff's reagent colour developing is positive, and fusing point is 225 ℃～228 ℃; UV (methanol) λ
Max(log ε) 205 (3.34), 275 (2.27), 282 (2.15) nm; Infrared spectrum IR (KBr tabletting) v
Max3415,3030,2958,1633,1602,1560,1456,1352,1265,1209,1188,1036,836,793,745,730,703cm
-1Holotype ESI-MS m/z307[M-Cl]
+, 215[M-Cl-benzyl]
+, 185[M-Cl-methoxybenzyl]
+, 91[benzyl]
+, 121[methoxybenzyl]
+1H NMR (CD
3OD: D
2O=1: 3v/v, 400MHz) δ 8.59 (1H, s, H-2), δ 7.23～7.34 (4H, m, H-3 ", H-4 ", H-5 ", H-5 '), δ 7.13～7.16 (2H; dd, J=7.6,2.2Hz, H-2 ", H-6 "), δ 6.86～6.88 (1H, dd, J=8.4,2.4Hz; H-4 '), δ 6.72～6.74 (1H, d, J=7.6Hz, H-6 '), δ 6.68 (1H; s, H-2 '), δ 5.21 (2H, s, PhCH
2N-3), δ 5.18 (2H, s, PhCH
2N-1), δ 3.66 (3H, s, OCH
3), δ 2.02 (3H, s, CH
3-4), δ 2.00 (3H, s, CH
3-5);
13C NMR (CD
3OD: D
2O=1: 3v/v, 400MHz) δ 7.48 (CH
3, C-7), δ 7.51 (CH
3, C-6), δ 47.32～48.61 (CH
2, PhCH
2N), δ 55.29 (CH
3, OCH
3), δ 113.07 (CH, C-2 '), δ 114.23 (CH, C-4 '), δ 120.07 (CH, C-6 '), δ 127.55 (CH, C-4 "), δ 127.99 (C, C-4), δ 128.04 (C; C-5), δ 128.85 (CH, C-3 ", C-5 "), δ 129.24 (CH; C-2 ", C-6 "), δ 130.58 (CH, C-5 '), δ 133.47 (C; C-1 "), δ 134.06 (CH, C-2), δ 135.17 (C, C-1 '), δ 159.50 (C, C-3 ').
4) Lepidiline D, molecular formula C
21H
25N
2OCl, white plates, the Dragendorff's reagent colour developing is positive, and fusing point is 211 ℃～214 ℃; UV (methanol) λ
Max(log ε) 208 (3.35), 274 (2.27), 282 (2.14) nm; Infrared spectrum IR (KBr tabletting) v
Max3419,3030,2957,2367,1635,1604,1560,1456,1265,1213,1186,1038,832,793,742,733,703cm
-1Holotype ESI-MS m/z321[M-Cl]
+, 229[M-Cl-benzyl]
+, 91[benzyl]
+, 199[M-Cl-methoxybenzyl]
+, 121[methoxybenzyl]
+ 1H NMR (CD
3OD: D
2O=1: 3v/v, 400MHz) δ 7.18～7.31 (4H, m, H-3 "; H-4 ", H-5 ", H-5 '), δ 7.00～7.02 (2H, d; J=7.2Hz, H-2 ", H-6 "), δ 6.81～6.79 (1H, dd; J=8.0,2.2Hz, H-4 '), δ 6.53～6.54 (1H, d; J=5.6Hz, H-6 '), δ 6.51 (1H, s; H-2 '), δ 5.29 (2H, s, PhCH
2N-3), δ 5.27 (2H, s, PhCH
2N-1), δ 3.66 (3H, s, OCH
3), δ 2.48 (3H, s, CH
3-2), δ 2.13 (3H, s, CH
3-4), δ 2.08 (3H, s, CH
3-5);
13C NMR (CD
3OD: D
2O=1: 3v/v, 400MHz) δ 7.48 (CH
3, C-8), δ 7.51 (CH
3, C-7), δ 9.19 (CH
3, C-6), δ 46.98～48.25 (CH
2, PhCH
2N), δ 54.45 (CH
3, OCH
3), δ 111.84 (CH, C-2 '), δ 113.29 (CH, C-4 '), δ 117.70 (CH, C-6 '), δ 125.93 (CH, C-4 "), δ 126.57 (C, C-4), δ 126.63 (C; C-5), δ 128.24 (CH, C-3 ", C-5 "), δ 129.07 (CH; C-2 ", C-6 "), δ 130.27 (CH, C-5 '), δ 133.87 (C; C-1 "), δ 135.35 (C, C-1 '), δ 143.71 (CH, C-2), δ 160.57 (C, C-3 ').
Embodiment three
Maca imidazole alkaloid compositions and four kinds of monomeric antitumor actions thereof:
Maca imidazole alkaloid compositions and the various imidazole alkaloid monomer got among embodiment one and the embodiment two are mixed with 1 mcg/ml, 10 mcg/ml, 20 mcg/ml, 40 mcg/ml, the 80 mcg/ml liquid to be measured of totally 5 Concentraton gradient, adopt mtt assay to study the vitro cytotoxicity of these materials to human lung carcinoma cell NCI-H460, human liver cancer cell HepG-2, human bladder cancer cell UMUC3, human pancreatic cancer cell PACA2, people's thymic carcinoma cell MDA231 and Proliferation of Human Ovarian Cell FDIGROV.The phase tumor cell of taking the logarithm is made cell suspension (20000/milliliter), be inoculated in the culture plate, every hole 200 microlitres, cultivate and pour out culture fluid after 48 hours, add the various liquid to be measured of 20 microlitres (establishing blank), cultivate and add MTT solution 20 microlitres after 24 hours again, continue to cultivate 4 hours, pour out culture fluid, the DMSO that adds 200 microlitres measures each hole A value on microplate reader, detecting wavelength is 490 nanometers, calculate maca imidazole alkaloid compositions and half suppression ratio (IC50 value) of various imidazole alkaloid monomers by testing result, gained IC50 value such as table 1 to different tumor cell proliferations:
Table 1 maca imidazole alkaloid is to half suppression ratio of different tumor cell proliferations
* annotate: show do not have a little less than cytotoxicity or the effect during IC50＞10 μ g/ml; Show that remarkable cytotoxicity is arranged during IC50＜4 μ g/ml, have the antitumor drug potentiality to be exploited.
Result of the test by table 1, can find no matter be maca imidazole alkaloid compositions or each monomer, in-vitro multiplication activity to kinds of tumor cells all has inhibitory action, as being Lepidiline D, the Lepidiline B cytotoxicity to FDIGROV, MDA231, PACA2 more significantly, Lepidilines is to the cytotoxicity of FDIGROV.Experimental result shows the natural plant composition for preparing by the present invention, and multiple human body tumour cell is had vitro cytotoxicity, possesses the potentiality of further developing anti-tumor medicaments.
Embodiment four
Take by weighing 2 kilograms of rustless steel extraction vessels that place the Chinese herbal medicine flash extracter of the dry Lepidinm meyenii Walp root powder of 200 purposes, the mass percent concentration that adds 16 kilograms is 80% methanol (hydrochloric acid that contains volume fraction 0.5%), adopt the Chinese herbal medicine flash extracter to extract 2 minutes down at 20000 rev/mins, behind the sucking filtration filtering residue is extracted once again, press embodiment one operation with HP20 post on the gained extractum, use 10 times of cylinder hydrops successively, 10 times of column volume mass percent concentrations are 10% methanol, 8 times of column volume mass percent concentrations are 60% methanol solution (glacial acetic acid that contains volume fraction 0.2%) eluting, collect the 3rd elution fraction and reclaim solvent at 50 ℃ of rotary evaporations that reduce pressure down, extractum dissolves with chloroform, go up neutral alumina column chromatography (50 * 2.6cm i.d. again, filler is a 200-300 order chromatography neutral alumina powder), all the other operations are with embodiment one, obtain maca imidazole alkaloid compositions 1.010 grams, total maca imidazole alkaloid purity reaches 90.0%.Get 0.2 gram maca imidazole alkaloid composition powder and be further purified and crystallization according to embodiment two operations, wherein solid-phase extraction column is selected Cctyl (C8) filler of bonding octane on the silica gel for use,
The aperture, 45 micron grain sizes, eluting adopts methanol: oxolane: 20 mMs/liter ammonium acetate solution (19: 1: 80v/v), the decompression rotary evaporation reclaims solvent and crystallization down, dry back results Lepidiline A is 0.0568 gram (purity 99.5%) by collecting maca imidazole alkaloid monomer component, 60 ℃, Lepidiline B is 0.0574 gram (purity 98.7%), 0.0297 gram (purity 97.2%), 0.0089 gram (purity 96.3%).
Embodiment five
Maca imidazole alkaloid compositions and four kinds of monomeric hypotensive activities thereof:
It is an amount of to press embodiment four preparation maca imidazole alkaloid compositionss and four kinds of imidazole alkaloid monomers, be mixed with 1.5 mg/ml liquid to be measured with normal saline solution respectively, other joins captopril (Altace Ramipril commonly used) solution 0.5 mg/ml as positive control, and normal saline is as blank.
Get 120 of male Wister rats (220～250 gram), injection pentobarbital sodium (50 mg/kg), anesthesia back surgical ligation left renal artery, choose the rat that two week backs blood pressures (systolic pressure) are elevated to 150 millimetress of mercury and be used for test, select 84 altogether, be divided into 7 groups, every group 12, irritate stomaches with the capacity of 2 milliliters/100 grams, continuous 14 days, adopt the rat tail to press the heart rate measurement instrument to observe the blood pressure of respectively organizing rat during the administration.Stop after the administration, continue to observe the blood pressure (systolic pressure) 7 days of respectively organizing rat.Record the results are shown in Table 2 and Fig. 1:
Table 2 maca imidazole alkaloid to the influence of kidney type Hypertensive Rats blood pressure lowering effect (X ± SD, n=10)
Fig. 1 maca imidazole alkaloid is to the blood pressure lowering curative effect of kidney type hypertension model rat
Compare with the blank group, administration recorded the blood pressure (p＜0.01) that each maca imidazole alkaloid group all can extremely significantly reduce kidney type Hypertensive Rats in 7 days, 14 days, especially Lepidiline D organizes, its blood pressure lowering effect is suitable with the effect of positive control Captopril group, and the bounce-back of 7 days blood pressures also is weaker than Captopril group after the drug withdrawal, and the Lepidilines group also has the same blood pressure unconspicuous phenomenon that rebounds.This illustrates that maca imidazole alkaloid compositions provided by the invention and monomeric blood pressure lowering effect thereof are definite, and the side effect aspect the bounce-back of drug withdrawal blood pressure is smaller, has the purposes of the good Altace Ramipril of exploitation.
Embodiment six
Maca imidazole alkaloid compositions and four kinds of monomeric antiarrhythmic effects thereof:
It is an amount of to press embodiment four preparation maca imidazole alkaloid compositionss and four kinds of imidazole alkaloid monomers, be mixed with five kind of 1.5 mg/ml liquid to be measured with normal saline solution respectively, other joins 0.7 mg/ml quinidine (antiarrhythmic drug commonly used), and solution is as positive control, and normal saline is as blank.Get 84 (male and female half and half of Wister rat, 220～250 grams), be divided into 7 groups, every group 12 (male and female half and half), irritate stomaches with the capacity of 2 milliliters/100 grams, continuous 5 days, behind the last gastric infusion 30 minutes, with the urethane anesthetized rat of 1.2 gram/kilograms and to face upward the position fixing, observe and write down II with BL-420F biological function experimental system and lead ECG.Angular vein was injected aconitine 30 micrograms/kg body weight (aconitine is mixed with 10 mcg/ml) in 5 seconds, write down the time of occurrence of ventricular premature contraction (premature beat) in 35 minutes, ventricular tachycardia (chamber speed), ventricular fibrillation (quivering in the chamber), asystole (stopping fighting).Record the results are shown in Table 3:
Table 3 maca imidazole alkaloid to aconitine cause ARR influence (X ± SD, n=10)
The liquid various to be measured that contains maca imidazole alkaloid is compared with matched group, the time of occurrence (P＜0.01) of all kinds of arrhythmia symptoms such as equal energy significant prolongation rat ventricular premature contraction, ventricular tachycardia, wherein remarkable with the effect of Lepidiline B and Lepidiline D, Lepidiline D is suitable with the effect of quinidine positive controls.As seen the maca imidazole alkaloid for preparing among the present invention has the arrhythmia symptom that good anti-aconitine brings out, and can expect that the exploitation medical product is used for the treatment of cardiac arrhythmia.
Embodiment seven
Maca imidazole alkaloid compositions blood pressure lowering and antiarrhythmic dose relationship:
It is an amount of to press embodiment four preparation maca imidazole alkaloid compositionss, be mixed with 1.5 mg/ml, 0.5 mg/ml, high, medium and low three the dosage groups of 0.15 mg/ml, according to the maca imidazole alkaloid compositions of the pharmacological testing method research various dose of embodiment five, six to rat hypertension, ARR therapeutical effect.The results are shown in Table 4,5:
The dose-effect relationship of table 4 maca imidazole alkaloid compositions blood pressure lowering (X ± SD, n=10)
Lepidilines by the visible various dose of table 4 all has certain blood pressure lowering effect to Hypertensive Rats, there is tangible dosage effect simultaneously, (it is identical to irritate the stomach amount for the Lepidilines of high dose group, concentration is the highest) data analysis administration the 7th day and 14 days shows to have extremely significant blood pressure lowering effect (p＜0.01), the Lepidilines of middle dosage group also has remarkable blood pressure lowering effect (0.05＜p＜0.01), the Lepidilines of low dose group then only has more weak hypotensive activity from data, but effect not significantly (p＞0.05).Therefore the maca imidazole alkaloid of sufficient dosage is important to the performance of drug effect in the Altace Ramipril design, but does not represent that also the maca imidazole alkaloid of low dosage can not be applied to Altace Ramipril.
Table 5 maca imidazole alkaloid compositions antiarrhythmic dose-effect relationship (X ± SD, n=10)
All comparatively significant delay action appears in the arrhythmia symptom that the Lepidilines by the visible various dose of table 5 brings out aconitine, wherein three kinds of dosage all has significant curative effect to the delay of premature beat, chamber speed symptom, in the dosage group symptom of stopping fighting is had certain effect, but effect is not remarkable, low dose group to the chamber quiver, stop fighting the influence not remarkable.As seen maca imidazole alkaloid also shows dose relationship on the arrhythmia disease, and the maca imidazole alkaloid compositions of low dosage also has certain curative effect aspect arrhythmia.
When using maca imidazole alkaloid combination treatment cardiovascular disease described in the present invention, can be made into capsule or tablet, took preceding 3 days, 30 milligrams of everyone every days of being grown up, can divide one after each meal 3 times, visual afterwards curative effect increases dose to 120 milligrams of adult everyone every days.
In order to ensure the safety of long-term prescription, the present invention carries out safety overall merit by national medicine food Surveillance Authority, national I kind new medicine chronic toxicity test standard to the compositions of maca imidazole alkaloid described in the present invention.The maca imidazole alkaloid compositions does not make significant difference for rat continuous irrigation stomach 3 months, 6 months and 7 months convalescent periods by 10,50,100 times various dose of clinical consumption to rat body weight change, feed situation and behavioral activity etc., the hematology of rat, blood biochemical are learned and every index such as electrocardiogram does not also have tangible adverse effect, and 22 kinds of internal organs are observed with the histopathology of tissue and blank relatively there is no unusually.The result shows that the long-term heavy dose of oral medication of maca imidazole alkaloid compositions does not have obvious damage to animal, finds to be a kind of safe and reliable medicine because of accumulating the toxic reaction to the body generation.
Claims (5)
1. the purposes of a maca imidazole alkaloid, it is characterized in that: the application of maca imidazole alkaloid in the preparation cardiovascular drugs, the active component of maca imidazole alkaloid all or mainly are independent or mixing formation by the chemical compound with the following chemical structure formula:
R wherein
1Be H or CH
3, R
2Be H or OCH
3
2. the purposes of the maca imidazole alkaloid described in the claim 1 is characterized in that: the active component of maca imidazole alkaloid all or mainly be by having the following chemical structure formula chemical compound separately or mix and constitute:
R wherein
1Be H or CH
3, R
2～R
7Be H, CH
3, CH
3O, OH, CHO, COOH, Cl, I, F, S, P, NO
3, NO
2Or NH
2, R
2～R
7Identical or different.
3. the purposes of the maca imidazole alkaloid described in the claim 1 or 2 is characterized in that: the application of maca imidazole alkaloid in the preparation antiarrhythmic drug.
4. the purposes of the maca imidazole alkaloid described in the claim 1 or 2 is characterized in that: the application of maca imidazole alkaloid in the preparation Altace Ramipril.
5. the purposes of the maca imidazole alkaloid described in the claim 1, it is characterized in that: maca imidazole alkaloid is from natural plants Lepidinm meyenii Walp (Lepidium meyenii Walper, also common its different name appellation Lepidium peruvianum Chacon sp. of the same race) separate obtaining in, its process is:
The 1st step was crushed to 40～200 orders with dry Lepidinm meyenii Walp root, MAKA dry powder is placed the rustless steel extraction vessel of Chinese herbal medicine flash extracter, according to the feed liquid weight ratio is 1: 8～1: 15 adding organic solvent, organic solvent is that mass percent concentration is that 80%～100% ethanol or mass percent concentration are 80%～100% methanol, the described organic solvent that perhaps contains volume fraction 0.5% hydrochloric acid, extracted 2～5 minutes down at 8000～20000 rev/mins, extracting solution with 400 order filter cloth sucking filtration after, filtering residue is extracted once by last method again, filtrate is merged, under 50 ℃～60 ℃ decompression rotary evaporations, reclaim extracting solution, obtain extracting extractum;
The 2nd step was extracted extractum, and to add isopyknic mass percent concentration 20% methanol mixed even, slowly add in the macroporous adsorptive resins, filler is a polystyrene type low pole macroporous adsorbent resin, water, mass percent concentration with 5～10 times of column volumes is that 10% methanol, mass percent concentration are 40%～80% methanol, methanol-eluted fractions successively, collect the 3rd elution fraction, reclaim solvent at 50 ℃～60 ℃ decompression rotary evaporations, obtain the maca imidazole alkaloid crude extract; The glacial acetic acid solution that wherein contains volume fraction 0.2%～0.8% in 40%～80% the methanol;
The 3rd step dissolved the maca imidazole alkaloid crude extract with chloroform, go up the normal phase column chromatography again, the normal phase column chromatography is neutral alumina column chromatography or purification on normal-phase silica gel column chromatography, adopt the chloroform of 5～10 times of column volumes successively, chloroform: methanol (16: 1, v/v), chloroform: methanol (9: 1, v/v), chloroform: methanol (7: 3, v/v), methanol-eluted fractions, collect the 3rd, four elution fractions, 50 ℃～60 ℃ decompression rotary evaporations reclaim solvent, extractum is gone up C18 reverse phase silica gel post after with dissolve with methanol and is carried out chromatography, at first use the water of 5～10 times of column volumes, and then to utilize mass percent concentration successively be 10% methanol, 20% methanol, 40% methanol, methanol-eluted fractions, collect second, three, contain the maca imidazole alkaloid part in four elution fractions, merge the back and reclaim solvent, obtain the maca imidazole alkaloid compositions at 50 ℃～60 ℃ decompression rotary evaporations, detect by high performance liquid chromatography, contain whole four kinds of maca imidazole alkaloid monomers described in the claim 1;
The 4th step maca imidazole alkaloid compositions with dissolve with methanol after, be further purified by solid-phase extraction column, solid phase extraction column stuffing is C18 or C8 silica filler, eluting adopts methanol: oxolane: 20 mMs/liter ammonium acetate solution (19: 1: 80v/v), high performance liquid chromatography detection monitor mode according to the 3rd step is collected merging respectively to four kinds of maca imidazole alkaloids, after 50 ℃～60 ℃ decompression rotary evaporations reclaim solvent down, adopt acetone successively, acetone: methanol (9: 1v/v), oxolane, oxolane: (9: 1v/v) crystallization promptly obtains four kinds of maca imidazole alkaloid monomers to petroleum ether.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103044335A (en) * | 2012-12-28 | 2013-04-17 | 武汉华士特工业生物技术开发有限公司 | Synthesis method of 3-benzyl-1-(3-methoxy benzyl)-2,4,5-trimethylimidazole chloride |
| CN103275012A (en) * | 2013-06-08 | 2013-09-04 | 中国科学院过程工程研究所 | Preparation method and application of Maca alkaloid |
| CN104513173A (en) * | 2013-09-30 | 2015-04-15 | 中国科学院过程工程研究所 | MACA extract, preparation method and application thereof |
| CN106668115A (en) * | 2017-02-10 | 2017-05-17 | 烟台新时代健康产业有限公司 | Method for extracting active ingredients from maca |
| CN114394931A (en) * | 2022-01-30 | 2022-04-26 | 西安交通大学 | Monoterpene alkaloid with vasodilatation activity and extraction method and application thereof |
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2010
- 2010-01-29 CN CN201010104626A patent/CN101756965A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103044335A (en) * | 2012-12-28 | 2013-04-17 | 武汉华士特工业生物技术开发有限公司 | Synthesis method of 3-benzyl-1-(3-methoxy benzyl)-2,4,5-trimethylimidazole chloride |
| CN103044335B (en) * | 2012-12-28 | 2015-12-23 | 武汉华士特工业生物技术开发有限公司 | The muriatic synthetic method of a kind of 3-benzyl-1-(3-methoxy-benzyl)-2,4,5-tri-methylimidazolium |
| CN103275012A (en) * | 2013-06-08 | 2013-09-04 | 中国科学院过程工程研究所 | Preparation method and application of Maca alkaloid |
| CN103275012B (en) * | 2013-06-08 | 2015-07-01 | 中国科学院过程工程研究所 | Preparation method and application of Maca alkaloid |
| CN104513173A (en) * | 2013-09-30 | 2015-04-15 | 中国科学院过程工程研究所 | MACA extract, preparation method and application thereof |
| CN104513173B (en) * | 2013-09-30 | 2018-03-06 | 中国科学院过程工程研究所 | A kind of Maca extract, Its Preparation Method And Use |
| CN106668115A (en) * | 2017-02-10 | 2017-05-17 | 烟台新时代健康产业有限公司 | Method for extracting active ingredients from maca |
| CN114394931A (en) * | 2022-01-30 | 2022-04-26 | 西安交通大学 | Monoterpene alkaloid with vasodilatation activity and extraction method and application thereof |
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