CN1730479A - A kind of aloe dihydro-coumarin and separation method and application - Google Patents
A kind of aloe dihydro-coumarin and separation method and application Download PDFInfo
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- CN1730479A CN1730479A CN 200410070996 CN200410070996A CN1730479A CN 1730479 A CN1730479 A CN 1730479A CN 200410070996 CN200410070996 CN 200410070996 CN 200410070996 A CN200410070996 A CN 200410070996A CN 1730479 A CN1730479 A CN 1730479A
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- chloroform
- aloe
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- coumarin
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Abstract
A kind of aloe dihydro-coumarin and separation and application, its separation method comprise carries out extracting with the Chinese medicine Aloe extractum with acid chloroform, and extracting solution is concentrated the back with the aqueous ethanolic solution dissolving, carries out solvent with normal hexane, chloroform, pure water successively then and distributes.To carrying out column chromatography after the chloroform partial concentration, repeatedly behind the wash-out, obtain a neat compounds.Pharmacological evaluation confirms that it has oxidation-resistance, immunoregulation effect and anti-tumor activity.Whole extraction process process is simple, and cost is low.This extract has bigger using value.
Description
Invention field
The present invention relates to a kind of from aloe isolated compound.
The invention still further relates to the method for separating compound from aloe.
The invention still further relates to the purposes of this compound aspect pharmacology.
Background technology
Aloe is the long for many years green meat herbaceous plant of Liliaceae (Liliaceae) Aloe (Aloe), and aloe plant is used for the treatment of diseases such as wound, asthma, stomach ulcer and constipation among the people.Modern pharmacology experimental results show that: efficient part in this platymiscium or effective constituent have anticancer, anti-inflammatory, antibiotic, antiviral, desinsection is analgesic, protect the liver and effect such as immunostimulant.At present, aloe plant and goods thereof are widely used in a plurality of fields such as daily-use chemical industry, protective foods.The extractive technique of application of advanced to effective constituent in the herbal medicine extract with purifying be one of modern important development direction of Chinese materia medica.The folk prescription of some herbal medicine or the crude extract of compound have been used for clinical after rough segmentation and have obtained better therapeutic effect, in these compositions be not each all in action.Therefore, from natural drug, seek the low and evident in efficacy compound of toxic side effect and be applied to clinical significant.
Summary of the invention
The purpose of this invention is to provide a kind of from aloe the compound-aloe dihydro-coumarin of separation and Extraction.
Another purpose of the present invention provides the method for separating above-claimed cpd.
Compound provided by the invention, its structure as shown in the formula:
In the formula: a, b indicate 3 last two inequality hydrogen atoms; I, j sign 4 ' and 7 last two different phenolic hydroxyl groups.
Up to now, still find no the patent or the bibliographical information of relevant this compound.
Provided by the invention from aloe the method for extraction separation dihydro-coumarin, its key step is:
A. raw materials used is Aloe vulgaris medicinal extract;
B. at first carry out refluxing extraction (chloroform 5000ml, 20%H with acid chloroform
2SO
4100ml), Aloe vulgaris medicinal extract quality is 1 with the ratio of solvent volume: 3-5, and leaching each 2-5 hour, concentrates each extract more than 10 times;
C. extract concentrates with Rotary Evaporators, and temperature is controlled at 28-35 ℃, and reclaims chloroform to use repeatedly, obtains concentrating extract;
D. (volume ratio is 1: 1-3) above-mentioned extract is dissolved again with the mixed solution of warm (T=35-45 ℃) alcohol-water.With normal hexane, chloroform, water it is extracted successively, and respectively extraction liquid is concentrated;
F. carry out column chromatography for separation after chloroform extraction liquid being concentrated, used column chromatography filler is the polyamide powder particle, particle diameter 40-80 order.Silon at first carries out pre-treatment before using, with methylene dichloride: dimethyl formamide (volume ratio 100: 10-20) fully soak, time is no less than 24 hours, carries out drip washing with hexanaphthene, acetone, chloroform, ethanol successively then, to remove wax and other impurity wherein.Chromatography column is simple glass post (diameter: the post height is 1: 20).Chloroform extract is carried out wash-out with hexanaphthene, benzene, chloroform, acetone, methyl alcohol successively.At chloroform: acetone is that the blue look fluorescent substance of a blended appears in 2: 1.5 wash-out position.Merge this type of material, concentrate.With silica gel as parting material, with hexanaphthene and acetone (volume ratio is 1: 1-3) as eluent to this compound separation purifying, receive respectively, obtain a purified compound.Confirm that through spectrum resolution this compound is the compound that nature is found first, the application is with its called after aloe dihydro-coumarin.
Aloe dihydro-coumarin, pale yellow powder.M.p.:284-286 ℃, heating is dissolved in ethyl acetate, is slightly soluble in methyl alcohol, acetone.EI-MS：394，366，351，335，323，296，278，267，186，141，119105，94，60。High resolution mass spectrum: 393.0982 (negative ion peaks), molecular composition is C
22H
18O
7
UV spectrum λ: 287.5 (0.4156), 252.0 (0.2634), 224.0 (0.0581).
Infrared spectra γ cm
-1(KBr compressing tablet): 3363.44,1619.21,1639.84,1595.01,1563.67,1514.15,1458.36,1406.69,1372.86,1251.37,1136.11,1013.75,826.40,551.14.
Proton nmr spectra (DMSO): δ ppm:10.26 (OH, OH-i); 9.38 (OH, OH-j); 6.86:d, J=8.53, (2H, H-2 ', H-6 '); 6.70:d, J=8.45, (2H, H-3 ', H-5 '); 6.60:s, (1H, H-8); 6.22:d, J=2.15, (1H, H-5 "); 5.66:d, J=2.15, (1H, H-3 "); 4.41:d, J=5.86, (1H, H-4); 3.84:s, (3H, H-7 "); 3.21:m, (1H, H-3a); 2.83:t, (1H, H-3b); 1.97:s, (3H, H-9).
Carbon-13 nmr spectra (DMSO): δ ppm:170.71 (C-5 "); 167.26 (C-2); 163.82 (C-2); 157.99 (C-6 "); (156.39 C-4 '); 170.71 (C-4 "); 155.67 (C-7); 153.13 (C-8a); 136.82 (C-5); (131.01 C-1 '); (127.75 2C, C-2 ', C-6 '); 117.33 (C-6); (115.64 2C, C-3 ', C-5 '); 115.19 (C-4a); 104.41 (C-5 "); 101.54 (C-8); 88.27 (C-3 "); 56.37 (C-7 "); 37.74 (C-3); 36.01 (C-4); 15.78 (C-9).
Only rely on H
1-NMR, C
13-NMR (seeing accompanying drawing 1,2) is comprehensive inadequately to the evaluation of this compound structure, and the application has also made relevant two-dimensional nucleus magnetic spectrum: H-H COSY, HMBC, HMQC and DEPT-135 carry out further comprehensively determining to its structure.
Through pharmaceutical research, this compound also has oxidation-resistance, stronger immunoregulation effect and anti-tumor activity.
Description of drawings
Fig. 1: compound
1The H-NMR spectrum;
Fig. 2: compound
13The C-NMR spectrum;
Fig. 3: the oxidation-resistance experimental result synoptic diagram of compound;
Fig. 4: the active test result synoptic diagram of macrophage phagocytic;
Fig. 5: scavenger cell respiratory burst result schematic diagram;
Fig. 6: induce KB apoptosis TUNEL experiment (a) and Hoechst 33258 dyeing photos (b).
Embodiment
With Aloe vulgaris medicinal extract is raw material, carries out refluxing extraction (chloroform 5000ml, 100ml 20%H with acid chloroform
2SO
4), Aloe vulgaris medicinal extract quality is 1: 5 with the ratio of solvent volume, leaches 10 times, each 3 hours, concentrates each extract;
Extract concentrates with Rotary Evaporators, and temperature is controlled at 30 ℃, and reclaims chloroform to use repeatedly, obtains concentrating extract;
Mixed solution (volume ratio is 1: 1) with 40 ℃ of alcohol-waters dissolves again to above-mentioned extract.With normal hexane, chloroform, water it is extracted successively, and respectively extraction liquid is concentrated;
Carry out column chromatography for separation after chloroform extraction liquid concentrated, used column chromatography filler is the polyamide powder particle, 60 orders.Silon at first carries out pre-treatment.With methylene dichloride: dimethyl formamide (volume ratio 100: 10) fully soaks, and the time is no less than 24 hours, carries out drip washing with hexanaphthene, acetone, chloroform, ethanol successively then, with except that wherein wax and other impurity.Chromatography column is simple glass post (diameter: the post height is 1: 20).Chloroform extract is carried out wash-out with hexanaphthene, benzene, chloroform, acetone, methyl alcohol successively.At chloroform: acetone is that the blue look fluorescent substance of a blended appears in 2: 1.5 wash-out position.Merge this type of material, concentrate.With silica gel as parting material, with hexanaphthene and acetone (volume ratio is 1: 1) as eluent to this compound separation purifying, receive respectively, obtain a purified compound.
Above-claimed cpd has oxidation-resistance, immunomodulatory and anti-tumor activity:
1, anti-oxidation characteristics
Respectively by Fenton reaction and xanthine/XOD system, check aloe dihydro-coumarin in external restraining effect to hydroxyl radical free radical and ultra-oxygen anion free radical:
Fenton reaction system: H
2O
2(30%) 60 μ l, 5 * 10
-5Fe
2+-EDTA solution 80 μ l, 0.2mmol
-1DEPMPO 30 μ l, high purity water 80 μ l, 1.4mg/ml the DMSO solution 70 μ l of aloe dihydro-coumarin mix, final volume is 240 μ l, reaction mixture is transferred in the flat pond of quartz, place in ESR (the Bruker ESP 300) reference cavity (TE102), detect hydroxyl radical free radical behind the 5min.
Xanthine/XOD system: 0.8mmol
-1Xan 60 μ l, 0.2V/ml XOD 60 μ, 0.2mmol
-1DEPMPO 30 μ l, high purity water 80 μ l, 1.4mg/ml the DMSO solution 70 μ l of aloe dihydro-coumarin mix, final volume is 240 μ l, reaction mixture is transferred in the flat pond of quartz, place in ESR (the Bruker ESP 300) reference cavity (TE102), detect ultra-oxygen anion free radical behind the 5min.
As shown in Figure 3, under the 0.4mg/ml condition, demonstrate the more considerable inhibiting rate of hydroxyl and ultra-oxygen anion free radical in the above-mentioned system.The inhibiting rate of superoxide anion is about 43%; The hydroxyl free inhibiting rate is 44%.
2, immunoregulation effect
1) the active test of macrophage phagocytic
The macrophage phagocytic vigor is represented with its ability of engulfing toluylene red.2 * 10
6The cells scavenger cell in advance with FLZ 10
-6, 10
-5, 10
-4Mol.l
-1And EGB76140,80,160mg.l
-1Temperature is incubated 1h, reaction volume 1ml, and then cultivate 4h. altogether with ox-LDL (0.1mg protein) and come termination reaction by changing fresh nutrient solution, every hole adds 0.1ml 0.072% toluylene red (being dissolved in 0.9%NaCl), 37 ℃ are continued to cultivate 30min, and supernatant discarded is washed secondary with Hanks solution, (1: 1v/v) extract toluylene red color in the scavenger cell, the vigor of engulfing of scavenger cell is represented (as Fig. 4) by the absorbancy of the toluylene red of being engulfed to add 0.2ml 50% acetate.
2) aloe dihydro-coumarin is to bringing out the influence of Turnover of Mouse Peritoneal Macrophages respiratory burst through phorbol ester (PMA)
The cell of separator well is pressed every hole 5 * 10
6Individual cell is added to three holes in 24 well culture plates, after adherent, in every hole, add cell cultures balanced salt solution Hanks and corresponding Aloe extract, the extract final concentration is 50 μ g/ml, cell culture incubator incubation 2h, supernatant discarded adds Hanks liquid, complexing agent, trapping agent and PMA successively, mixing, final volume is 250 μ l, detects ultra-oxygen anion free radical behind the 5min, and the ESR spectrogram as shown in Figure 5.
Aloe dihydro-coumarin huge have a liking for to embody in the cell experiment have a liking for the distinctive activation of cytophagy ability to huge.When concentration was 200 μ g/ml, it significantly strengthened huge cytophilic phagocytic activity.Simultaneously, have a liking in the cell oxygen respiratory burst process in that the PMA inductive is huge, aloe dihydro-coumarin has significantly increased the generation of ultra-oxygen anion free radical in this process.
3. to the apoptotic effect of tumour cell
1) TUNEL experiment
In cell generation apoptotic process, the DNA of cellular genome can be cut off, and produces double-stranded fragment (mononucleosome or oligomerization nucleosome) or the breach of single-strand break occurs on big dna molecular.Under the effect of picodna transferring enzyme, DNA3 '-OH will be labeled endways.This kind of enzyme can will be aggregated to free 3 '-above the OH with the form that does not rely on template through the Nucleotide of mark.These DNA fragments can adopt fluorescent microscope or flow cytometer to detect.
Through the human oral cancer epithelial cell (KB cell) of 100 μ g/ml aloe dihydro-coumarin effects, the centrifugal 10min of 1500rpm removes supernatant liquor, with PBS washing 3 times, cell concn is adjusted into 107cell/ml.Get 100 μ l cell suspending liquids, add the stationary liquid of the new preparation of 100 μ l, at 15～25 ℃ of fixing 1h.Centrifugal, repeatedly with the PBS washing, with PBS cell being adjusted at volume is 250～500 μ l.On flow cytometer, detect excitation wavelength 450～500nm, emission wavelength 515～565nm.Experimental result has the DNA of 57.43% KB cell to produce fracture shown in Fig. 6-a approximately, and apoptosis takes place.
2) Hoechst 33258 stain tests
Hoechst 33258 is a kind of and nuclear DNA bonded dyestuff, can detect apoptosis on flow cytometer according to its excitation spectrum and emission spectrum characteristics.Combine with apoptotic cell DNA because this dyestuff is easier, make it dyeing, can be in direct viewing apoptosis phenomenon under the fluorescent microscope.
Get the clean cover glass of handling well, keep flat in six well culture plates, inoculation is gone into the KB cell cultures and is spent the night irritation cell generation apoptosis (50 μ g/ml).Exhaust nutrient solution, add the 0.5ml stationary liquid, fixedly 10min.Remove stationary liquid, with PBS or 0.9%NaCl washing twice, each 3min.Add Hoechst 33258 staining fluids, dyeing 5min.Microscopy under fluorescent microscope, excitation wavelength 361nm, emission wavelength 460nm.Coloration result is shown in Fig. 6-b, and obvious shrinkage phenomenon appears in painted KB cellular form, and apoptosis takes place.
Claims (5)
1. aloe dihydro-coumarin, its structure as shown in the formula:
2. method of separating the described compound of claim 1, its key step is:
A) raw materials used is Aloe vulgaris medicinal extract;
B) at first carry out refluxing extraction with acid chloroform, this acidity chloroform is chloroform: 20%H
2SO
4=5000ml: 80-100ml, Aloe vulgaris medicinal extract quality is 1 with the ratio of solvent volume: 3-5, leaching each 2-5 hour, concentrates each extract more than 10 times;
C) extract concentrates with Rotary Evaporators, and temperature is controlled at 28-35 ℃, and reclaims chloroform to use repeatedly, obtains concentrating extract;
D) be 1 with volume ratio: the alcohol-water mixed solution of 1-3 dissolves again to above-mentioned extract, with normal hexane, chloroform, water it is extracted successively, and respectively extraction liquid is concentrated;
E) carry out column chromatography for separation after chloroform extraction liquid is concentrated, used column chromatography filler is the polyamide powder particle, and particle diameter 40-80 order, chromatography column are the simple glass post, diameter: the post height is 1: 20; Chloroform extract is carried out drip washing, and leacheate is chloroform with the volume ratio: acetone=2: 1.5, and the material of merging drip washing concentrates; As parting material, is 1 with volume ratio with silica gel: the hexane of 1-3 and acetone mixing solutions be eluent to the compound separation purifying, receive respectively, obtain aloe dihydro-coumarin.
3. the method for claim 2 is characterized in that, the alcohol-water mixeding liquid temperature in the steps d is 35-45 ℃.
4. the method for claim 2, it is characterized in that, polyamide powder particle among the step e is 100 with volume ratio before use: the methylene dichloride of 10-20 and dimethyl formamide mixed liquid dipping are more than 24 hours, carry out drip washing with hexanaphthene, acetone, chloroform, ethanol successively then, to remove wax and other impurity wherein.
5. the described compound of claim 1 has application in oxidation-resistance, immunomodulatory and the antitumor drug in preparation.
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| CN114956049A (en) * | 2022-06-17 | 2022-08-30 | 山西大学 | Long wavelength ratio fluorescent carbon dot and preparation method and application thereof |
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| CN114956049A (en) * | 2022-06-17 | 2022-08-30 | 山西大学 | Long wavelength ratio fluorescent carbon dot and preparation method and application thereof |
| CN114956049B (en) * | 2022-06-17 | 2023-07-18 | 山西大学 | Long wavelength ratio fluorescent carbon dot and preparation method and application thereof |
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