CN109694366B - Method for separating and purifying active ingredients of clematis filamentosa dunn - Google Patents
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Abstract
The invention discloses a method for separating and purifying the effective components of clematis filamentosa dunn, which comprises the following steps: (1) Extracting aerial parts of Clematis Filifolia with ethanol; (2) performing gradient elution on the normal phase silica gel chromatographic column; (3) Subjecting the extract A2 to normal phase silica gel column chromatography, performing gradient elution, collecting eluate, subjecting to Sephadex LH-20 column chromatography, and eluting; (4) Subjecting the extract A3 to normal phase silica gel chromatographic column, eluting, and concentrating by evaporation; (5) Subjecting the extract A5 to normal phase silica gel chromatographic column, eluting and concentrating; (6) Concentrating the extract A6, adding n-butanol, extracting for several times, mixing n-butanol phases, concentrating, purifying with reverse phase silica gel chromatographic column, eluting with methanol water solution, collecting eluate, concentrating, and drying to obtain extract. The method can purify the active substances in caulis Clematidis Armandii one by one, and the purified substances have high purity.
Description
Technical Field
The invention relates to the field of chemical purification, in particular to a method for separating and purifying active ingredients of clematis filamentosa dunn.
Background
Caulis et folium Nardostachyos (Clematis filamentosa Dunn) of Clematis, also known as ophidia, ranunculaceae, nelumbo, with its leaves and stems as raw materials, has sweet and slightly cool taste. Folk medicine is used for treating red eye headache, etc., and has effects of lowering blood pressure, tranquilizing, hypnotizing, dilating blood vessel and improving blood circulation. Is considered to be a plant medicine for treating coronary heart disease and hypertension, which has no toxicity, high safety and rich medicine sources. The single prescription Chinese patent medicine Guanxinkang tablet taking the same as the raw material is clinically used for treating coronary heart disease, hypertension and the like.
The literature reports that after the clematis filamentosa dunn is extracted, the main active ingredient in the extract is flavonoid. The flavonoid compounds are chemical frameworks with important medicinal value, and the frameworks have multiple active functions and especially have obvious treatment effect on myocardial ischemia. At present, the components in the clematis filamentosa dunn are separated and purified, and the overground part of the clematis filamentosa dunn is usually crushed and then soaked in ethanol for purification. However, this purification method still remains in the crude extraction degree, the crude extract is a mixture of a plurality of substances, which also contains a plurality of inactive impurities, and when further separation is performed by a conventional separation method, it is found that spots on TLC (thin layer chromatography) overlap, liquid chromatography also shows overlapping peaks, and it is difficult to separate the active substances one by one, so that the specific constitution and structure of each flavonoid contained therein are not clear, and the exact action mechanism thereof in the treatment of myocardial ischemia cannot be known.
Therefore, the purified clematis filamentosa dunn is subjected to fine purification to obtain the pure active substance, which has important significance for researching and analyzing the mechanism of treating myocardial ischemia, and the active substance obtained after purification can be further used for preparing medicines to treat coronary heart disease and hypertension patients.
Disclosure of Invention
The invention aims to provide a method for separating and purifying the effective components of clematis filamentosa dunn, which can be used for individually purifying the active substances in the clematis filamentosa dunn one by one and ensuring that the purified substances have high purity.
The purpose of the invention is realized by the following technical scheme:
a method for separating and purifying the effective components of clematis filamentosa dunn comprises the following steps:
(1) Extracting the aerial parts of the clematis filamentosa dunn with ethanol to obtain ethanol extract;
(2) Subjecting the ethanol extract to a normal phase silica gel chromatography column, performing gradient elution using a petroleum ether/acetone mixed solvent, acetone and methanol in a volume ratio of 11 to 9;
(3) Subjecting the extract A2 to normal phase silica gel column chromatography, gradient elution is carried out by using chloroform/methanol mixed solvent with volume ratio of 100-80 and 60-1-40 in sequence, the chloroform/methanol eluent of 60;
(4) Subjecting the extract A3 to a normal phase silica gel chromatographic column, eluting by using a chloroform/methanol mixed solvent with the volume ratio of 40 to 1-20, collecting eluents t1 and t2 in two batches in sequence, and concentrating by evaporating the solvent to obtain an extract F1 and an extract F2;
(4a) Loading the extract F1 on a Sephadex LH-20 column, eluting by using a chloroform/methanol mixed solvent with the volume ratio of 2;
(4b) Subjecting the extract F2 to Sephadex LH-20 chromatography on a Sephadex column, eluting with a chloroform/methanol mixed solvent at a volume ratio of 2:1 to 0.5, treating by HPLC preparative chromatography to obtain purified solutions t3 and t4, respectively, removing the solvent and drying to obtain larch resinol (CAS No. 27003-73-2) and dihydrodehydroconiferol (CAS No. 126253-41-6), respectively;
(5) Loading the extract A5 on a normal phase silica gel chromatographic column, eluting by using a chloroform/methanol mixed solvent with the volume ratio of 25 to 1-5, collecting eluents t5 and t6 in two batches in sequence, and concentrating to obtain extracts F3 and F4;
(5a) Loading the extract F3 on a Sephadex LH-20 column, eluting by using a chloroform/methanol mixed solvent with the volume ratio of 2;
(5b) Loading the extract F4 on MCI GEL CHP20/P120 column, eluting with 70-90% methanol, collecting eluate, concentrating, recrystallizing, and drying to obtain luteolin (CAS number: 491-70-3);
(6) Concentrating the extract A6, adding water to dissolve the extract, then adding n-butyl alcohol which has the same volume as the total volume of the liquid after adding water to perform multiple extraction, merging n-butyl alcohol phases, concentrating the n-butyl alcohol phases, applying to a reverse phase silica gel chromatographic column, sequentially eluting by using 10-20%, 20-35% and 35-50% methanol aqueous solutions, collecting eluent, concentrating and drying to obtain extracts F5, F6 and F7;
(6a) Concentrating the extract F5, then loading the concentrated extract on a Sephadex LH-20 column, eluting the concentrated extract by using a chloroform/methanol mixed solvent with the volume ratio of 2 to 0.5;
(6b) Concentrating the extract F6, then loading the concentrated extract on a Sephadex LH-20 column, eluting by using a chloroform/methanol mixed solvent with the volume ratio of 2;
(6c) Concentrating the extract F7, then loading the concentrated extract on a Sephadex LH-20 column, eluting the concentrated extract by using a chloroform/methanol mixed solvent with the volume ratio of 2.
The overground part of the clematis filamentosa dunn comprises stem branches of the clematis filamentosa dunn.
In order to overcome the problem that the conventional separation method can not separate the effective components in the clematis filamentosa one by one, after long-term experiments, different types of chromatographic columns are combined with separation methods such as HPLC (high performance liquid chromatography) and extraction, and the like, and the separation and purification are performed step by step in an ethanol crude extract of the clematis filamentosa Dunn with complex material composition, so that the separation and purification of valuable effective components of carnosine, oleanolic acid, larch resinol, dihydrodehydroconiferol, apigenin, luteolin, caffeic acid, urolignoside, astragalin and linarin are efficiently realized, the specific composition of flavonoid compounds in the clematis filamentosa Dunn is determined, the purity of the purified materials can reach more than 95 percent, and the method has important significance in the fields of scientific research and medicine.
In the elution process of the step (2), the extract A1 is obtained by eluting with a petroleum ether/acetone mixed solvent of 11; eluting with a petroleum ether/acetone mixed solvent of 8; eluting with a petroleum ether/acetone mixed solvent of 4; eluting with a petroleum ether/acetone mixed solvent of 1.5; eluting with acetone to obtain extract A5; the extract A6 is obtained by purifying and eluting with methanol, wherein the extracts A1 and A4 are impurity components. Collecting the eluents in two batches one after the other as described in steps (4), (5) and (6 c), it is understood that the confirmation is performed by TLC, the eluents of the first eluate are collected and combined into a first batch, and the eluents of the later eluate are collected and combined into a second batch to collect two different extracts.
As an embodiment of the present invention, the process of extracting the overground part of clematis filamentosa in step (1) is specifically as follows: and (2) crushing the overground part of the dried clematis filamentosa dunn, adding 85% industrial ethanol according to the solid mass/liquid volume ratio of 1.
Preferably, step (2) is performed by gradient elution sequentially using a petroleum ether/acetone mixed solvent, acetone and methanol in a volume ratio of 10.
Preferably, in step (3), the extract A2 is subjected to normal phase silica gel column chromatography, gradient elution is sequentially performed by using chloroform/methanol mixed solvent with the volume ratio of 100.
Preferably, the step (4) is performed by eluting with a chloroform/methanol mixed solvent at a volume ratio of 30.
Preferably, step (4 a) is eluted with a chloroform/methanol mixed solvent at a volume ratio of 1.
Preferably, step (4 b) is eluted with a chloroform/methanol mixed solvent at a volume ratio of 1.
Preferably, step (5) is eluted with a chloroform/methanol mixed solvent at a volume ratio of 15.
Preferably, in the step (5 a), the extract F3 is applied to Sephadex LH-20 column, eluted by chloroform/methanol mixed solvent with volume ratio of 1.
Preferably, step (5 b) is eluted with 80% methanol.
Preferably, in the step (6 a), the extract F5 is concentrated and then applied to a Sephadex LH-20 column, eluted with a chloroform/methanol mixed solvent at a volume ratio of 1.
Preferably, in the step (6 b), the extract F6 is concentrated and then applied to a Sephadex LH-20 column, eluted with a chloroform/methanol mixed solvent at a volume ratio of 1.
Preferably, step (6 c) is eluted with a chloroform/methanol mixed solvent in a volume ratio of 1.
The invention recommends using a silica gel chromatographic column with a diameter of 3.5cm and a length of 40cm in the step (2); silica gel chromatography columns with a diameter of 3.5cm and a length of 40cm glass columns were used in steps (3), (4) and (5).
As an embodiment of the present invention:
filtering and separating powdery precipitate in the step (3), and washing with methanol;
filtering and separating the powdery precipitate in the step (4 a), and washing with acetone;
in the step (4 b) of HPLC preparative chromatography, the solvent is 30-50% methanol aqueous solution;
the organic solvent for dissolving the powdery precipitate of step (5 a) is DMSO;
the steps (5 a) and (5 b) are recrystallized by using methanol;
the drying of the invention adopts freeze drying.
Compared with the prior art, the invention has the following beneficial effects:
1. in the separation and purification process, after gradient crude separation is carried out by using a normal phase silica gel column, the method of separating by using sephadex chromatography or combining high performance liquid chromatography is adopted to realize the rapid and high-efficiency separation of the salvinorin, the oleanolic acid, the larch bark alcohol and the dihydrodehydro-coniferyl alcohol, and the purity can reach more than 95 percent;
2. the method adopts a mode of combining sephadex chromatography with MCI chromatography or separating MCI chromatographic column singly, realizes the rapid and efficient separation of flavonoid apigenin and luteolin, has purity of more than 95 percent, and overcomes the technical problem of difficult peak separation of high performance liquid chromatography components;
3. after constant elution by using sephadex chromatography or combining a normal phase silicagel column, the invention realizes the rapid and high-efficiency separation of caffeic acid, urolignoside, astragalin and linarin, the purity can reach more than 95 percent, and the technical problem of difficult peak separation of components of high performance liquid chromatography is overcome;
4. the method effectively separates the active substances in the clematis filamentosa dunn one by one, purifies the active substances to obtain pure compounds, is beneficial to scientific research, determines the basis of substances for treating cardiovascular diseases, identifies the chemical structure of monomers of the clematis filamentosa dunn, and clearly analyzes the action mechanism of the clematis filamentosa dunn, can amplify the purification process, and uses the purified active ingredients to prepare the medicine for reducing the occurrence and development of chronic diseases.
Drawings
The invention is further illustrated by the following figures.
FIG. 1 is a schematic diagram of the separation and purification process of example 3.
FIG. 2 NMR chart of Compound C1.
FIG. 3 NMR carbon spectrum of Compound C1.
FIG. 4 shows the molecular structure of Compound C1.
FIG. 5 NMR chart of Compound C2.
FIG. 6 NMR carbon spectrum of Compound C2.
FIG. 7 molecular structural formula of Compound C2.
FIG. 8 NMR chart of Compound C3.
FIG. 9 NMR carbon spectrum of Compound C3.
FIG. 10 molecular structural formula of Compound C3.
FIG. 11 NMR chart of Compound C4.
FIG. 12 NMR carbon spectrum of Compound C4.
FIG. 13 molecular structural formula of Compound C4.
FIG. 14 NMR chart of Compound C5.
FIG. 15 NMR carbon spectrum of Compound C5.
FIG. 16 molecular structural formula of Compound C5.
FIG. 17 NMR hydrogen spectrum of Compound C6.
FIG. 18 NMR carbon spectrum of Compound C6.
FIG. 19 molecular structural formula of Compound C6.
FIG. 20 NMR chart of Compound C7.
FIG. 21 molecular structural formula of Compound C7.
FIG. 22 NMR chart of Compound C8.
FIG. 23 NMR carbon spectrum of Compound C8.
FIG. 24 molecular structural formula of Compound C8.
FIG. 25 NMR chart of Compound C9.
FIG. 26 NMR carbon spectrum of Compound C9.
FIG. 27 molecular structural formula of Compound C9.
FIG. 28 NMR hydrogen spectrum of Compound C10.
FIG. 29 molecular structural formula of Compound C10.
Detailed Description
The present invention will be further described with reference to the following specific examples.
Example 1
(1) Crushing 10kg of dried overground part of clematis filamentosa dunn, adding 85% industrial ethanol according to the solid mass/liquid volume ratio of 1;
(2) Subjecting the ethanol extract to normal phase silica gel chromatography column, gradient elution sequentially using petroleum ether/acetone mixed solvent, acetone and methanol in volume ratio of 11, 8;
(3) Subjecting the extract A2 to normal phase silica gel column chromatography, performing gradient elution by using chloroform/methanol mixed solvents with volume ratios of 90 and 60 in sequence, collecting 60 chloroform/methanol eluent, evaporating the solvent, concentrating, subjecting to Sephadex LH-20 column chromatography, eluting by using chloroform/methanol mixed solvent with volume ratio of 2;
(4) Loading the extract A3 on a normal phase silica gel chromatographic column, eluting by using a chloroform/methanol mixed solvent with the volume ratio of 40;
(4a) Loading the extract F1 onto Sephadex LH-20 column, eluting with chloroform/methanol mixed solvent with volume ratio of 2;
(4b) Subjecting the extract F2 to Sephadex LH-20 column chromatography, eluting with chloroform/methanol mixed solvent at volume ratio of 2; respectively obtaining purified liquids t3 and t4, removing the solvent and drying to respectively obtain a compound C3 and a compound C4;
(5) Subjecting the extract A5 to normal phase silica gel chromatographic column, eluting with chloroform/methanol mixed solvent with volume ratio of 25;
(5a) Loading the extract F3 on a Sephadex LH-20 chromatographic column, eluting by using a chloroform/methanol mixed solvent with the volume ratio of 2;
(5b) Loading the extract F4 on MCI GEL CHP20/P120 column, eluting with 70% methanol, collecting eluate, concentrating, recrystallizing in methanol, and drying to obtain C6;
(6) Concentrating the extract A6, dissolving in water, adding n-butanol with volume equal to the total volume of the solution after adding water, extracting for multiple times, mixing n-butanol phases, concentrating, loading onto reverse phase silica gel chromatographic column, eluting with 10%, 20% and 35% methanol aqueous solutions in sequence, collecting eluate, concentrating, and drying to obtain extracts F5, F6 and F7;
(6a) Concentrating the extract F5, then loading the concentrated extract on a Sephadex LH-20 column, eluting by using a chloroform/methanol mixed solvent with the volume ratio of 2;
(6b) Concentrating the extract F6, then loading the concentrated extract on a Sephadex LH-20 column, eluting by using a chloroform/methanol mixed solvent with the volume ratio of 2;
(6c) Concentrating the extract F7, loading the concentrated extract on a Sephadex LH-20 column, eluting by using a chloroform/methanol mixed solvent with the volume ratio of 2.
Example 2
(1) Pulverizing overground part of 15kg dried clematis filamentosa dunn, adding 85% industrial ethanol according to the solid mass/liquid volume ratio of 1;
(2) Subjecting the ethanol extract to normal phase silica gel chromatography column, gradient elution sequentially using petroleum ether/acetone mixed solvent, acetone and methanol in a volume ratio of 9, 6;
(3) Subjecting the extract A2 to normal phase silica gel column chromatography, performing gradient elution by using chloroform/methanol mixed solvents with volume ratios of 80 and 40;
(4) Subjecting the extract A3 to normal phase silica gel chromatographic column, eluting with chloroform/methanol mixed solvent with volume ratio of 20;
(4a) Loading the extract F1 onto Sephadex LH-20 column, eluting with chloroform/methanol mixed solvent with volume ratio of 0.5;
(4b) Subjecting the extract F2 to Sephadex LH-20 column chromatography, eluting with chloroform/methanol mixed solvent at volume ratio of 0.5; respectively obtaining purified liquids t3 and t4, removing the solvent and drying to respectively obtain C3 and C4;
(5) Subjecting the extract A5 to normal phase silica gel chromatographic column, eluting with chloroform/methanol mixed solvent with volume ratio of 5;
(5a) Loading the extract F3 on Sephadex LH-20 column, eluting with chloroform/methanol mixed solvent with volume ratio of 0.5;
(5b) Loading the extract F4 on MCI GEL CHP20/P120 column, eluting with 90% methanol, collecting eluate, concentrating, recrystallizing in methanol, and drying to obtain compound C6;
(6) Concentrating the extract A6, dissolving in water, adding n-butanol with volume equal to the total volume of the solution after adding water, extracting for multiple times, mixing n-butanol phases, concentrating, loading onto reverse phase silica gel chromatographic column, eluting with 20%, 35% and 50% methanol aqueous solutions in sequence, collecting eluate, concentrating, and drying to obtain extracts F5, F6 and F7;
(6a) Concentrating the extract F5, then loading the concentrated extract on a Sephadex LH-20 column, eluting by using a chloroform/methanol mixed solvent with the volume ratio of 0.5;
(6b) Concentrating the extract F6, then loading the concentrated extract on a Sephadex LH-20 column, eluting by using a chloroform/methanol mixed solvent with the volume ratio of 0.5;
(6c) Concentrating the extract F7, loading the concentrated extract on a Sephadex LH-20 column, eluting by using a chloroform/methanol mixed solvent with the volume ratio of 0.5 to 1, collecting eluates t7 and t8 in two batches, concentrating and drying to obtain a compound C9 and a compound C10 respectively.
Example 3
(1) As shown in fig. 1, 5kg of dried caulis clematidis armandii is pulverized, 85% industrial ethanol is added according to the solid mass/liquid volume ratio of 1 to 3, the mixture is soaked and extracted for 12h and extracted for three times, and the supernatant is combined, concentrated in vacuum and refrigerated at the temperature of minus 80 ℃ to obtain an ethanol extract;
(2) Subjecting the ethanol extract to normal phase silica gel chromatography column, gradient elution sequentially using petroleum ether/acetone mixed solvent, acetone and methanol in a volume ratio of 10, 7;
(3) Subjecting the extract A2 to normal phase silica gel column chromatography, gradient elution sequentially using chloroform/methanol mixed solvents with a volume ratio of 100 and 50;
(4) Subjecting the extract A3 to normal phase silica gel chromatographic column, eluting with chloroform/methanol mixed solvent with volume ratio of 30;
(4a) Loading the extract F1 onto Sephadex LH-20 column, eluting with chloroform/methanol mixed solvent with volume ratio of 1;
(4b) Subjecting the extract F2 to Sephadex LH-20 column chromatography, eluting with chloroform/methanol mixed solvent at volume ratio of 1; respectively obtaining purified liquids t3 and t4, removing the solvent, and drying to respectively obtain a compound C3 and a compound C4;
(5) Loading the extract A5 on a normal phase silica gel chromatographic column, eluting by using a chloroform/methanol mixed solvent with the volume ratio of 15;
(5a) Loading the extract F3 on Sephadex LH-20 column, eluting with chloroform/methanol mixed solvent with volume ratio of 1;
(5b) Loading the extract F4 on MCI GEL CHP20/P120 column, eluting with 80% methanol, collecting eluate, concentrating, recrystallizing in methanol, and drying to obtain compound C6;
(6) Concentrating the extract A6, adding water for dissolving, adding n-butanol with volume equal to the total volume of the water-added liquid for multiple times of extraction, mixing n-butanol phases, concentrating, loading onto reverse phase silica gel chromatographic column, sequentially eluting with 15%, 30% and 40% methanol aqueous solutions, collecting eluate, concentrating, and drying to obtain extracts F5, F6 and F7;
(6a) Concentrating the extract F5, then loading the concentrated extract on a Sephadex LH-20 column, eluting by using a chloroform/methanol mixed solvent with the volume ratio of 1;
(6b) Concentrating the extract F6, then loading the concentrated extract on a Sephadex LH-20 column, eluting by using a chloroform/methanol mixed solvent with the volume ratio of 1;
(6c) Concentrating the extract F7, loading the concentrated extract on a Sephadex LH-20 column, eluting by using a chloroform/methanol mixed solvent with the volume ratio of 1.
After the purification is finished, detecting and analyzing H patterns and C patterns of the purified compounds C1-C10 by using NMR with the frequency of 500MHz by using DMSO as a solvent, wherein the detection results are as follows:
1) As shown in fig. 2, the NMR hydrogen spectrum of compound C1 specifically includes: 8.07 (2H, d, J =9.0Hz, H-2', 6'), 7.12 (2H, d, J =9.0Hz, H-3', 5'), 6.94 (1H, s, H-8), 6.96 (1H, s, H-3), 12.87 (1H, s, OH-5), 3.12 (3H, s, OCH) 3 ),3.18(3H,s,OCH 3 ),3.13(3H,s,OCH 3 )；
As shown in fig. 3, the NMR carbon spectrum of compound C1 specifically includes the following data: 163.6 (C-2), 103.3 (C-3), 182.2 (C-4), 152.5 (C-5), 131.9 (C-6), 158.1 (C-7), 91.6 (C-8), 152.8 (C-5), 105.2 (C-10), 122.7 (C-1 '), 128 (C-2', 6 '), 114.6 (C-3', 5 '), 162.4 (C-4'), 60.2 (-OCH) 3 ),56.4(-OCH 3 ),55.6(-OCH 3 )。
After comprehensive analysis of the above data, the molecular structure of compound C1 was shown in FIG. 4, confirming that compound C1 is salvinorin (CAS No.: 19103-54-9).
2) As shown in fig. 5, the NMR hydrogen spectrum of compound C2 specifically includes: 12.04 (s, 1h, cooh), 5.16 (t, J =3.7,3.3hz,1h, H-12), 4.28 (d, J =5.1hz,1h, 3-OH), 2.99 (dd, J =10.6,5.1hz,1h, H-3), 2.75 (dd, J =13.9,4.0hz,1h, H-18), 1.91 (td, J =13.6,4.0hz,1h, H-16), 1.81 (dd, J =8.8,3.3hz,2h, H-11), 1.58-1.67 (m, 3h, H-2, H-15, H-19), 1.38-1.52 (m, 8h, 1, H-2, H-6, H-7, H-9, H-16, H-21), 1.07 (d, J =5.9hz,3h, H-6), 1.28-1.34 (m, 2h, H-19, H-21), 1.23 (td, J =9.9,3.3hz,1h, H-5), 1.13 (m, 1h, H-19), 1.03-1.07 (m, 1h, H-21), 0.99 (td, J =11.0,3.3hz,1h, H-15), 1.09,0.89,0.87, 0.85,0.72,0.67 (s, 3H);
as shown in fig. 6, the NMR carbon spectrum of compound C2 specifically includes the following data: 178.6 (C-28), 143.8 (C-13), 121.5 (C-12), 76.8 (C-3), 54.8 (C-5), 47.1 (C-9), 45.7 (C-19), 45.4 (C-17), 41.3 (C-18), 40.8 (C-14), 38.9 (C-8), 38.4 (C-1), 36.6 (C-10), 33.3 (C-21), 32.8 (C-29), 32.4 (C-7), 32.1 (C-22), 30.4 (C-20), 28.2 (C-23), 27.2 (C-15), 26.9 (C-27), 25.6 (C-2), 23.4 (C-30), 22.9 (C-11), 22.6 (C-16), 18.0 (C-6), 16.8 (C-26), 16.0 (C-24), 25.1 (C-1).
After the above data were comprehensively analyzed, the molecular structure of compound C2 was shown in FIG. 7, confirming that compound C2 is oleanolic acid (CAS number: 508-02-1).
3) As shown in fig. 8, the NMR hydrogen spectrum of compound C3 specifically includes: 6.81 (4H, m, H-5,2',5',6 '), 6.63 (2H, m, H-2, 6), 4.72 (1H, d, J =7.2Hz, H-7 '), 4.00 (1H, dd, J =8.0,7.2Hz, H-9 α), 3.89 (1H, m, H-9' α), 3.70 (2H, m, H-9 β,9' β), 2.92 (1H, dd, J =12.6,4.8Hz, H-7 α), 2.69 (1H, m, H-8), 2.48 (1H, dd, J =12.6,10.6Hz, H-7 β), 2.39 (1H, m, H-8 '), 3.88 (3H, s,3' -OCH 3), 3.82 (3H, s,3' -OCH 3);
as shown in fig. 9, the NMR carbon spectrum of compound C3 specifically includes the following data: 147.3 (C-3 '), 146.9 (C-3), 144.9 (C-4'), 144.0 (C-4), 135.3 (C-1 '), 132.6 (C-1), 121.7 (C-6), 119.2 (C-6'), 114.7 (C-5), 114.6 (C-5 '), 111.8 (C-2), 108.9 (C-2'), 83.0 (C-7 '), 73.1 (C-9), 60.9 (C-9'), 56.1 (3, 3'-OCH 3), 52.9 (C-8'), 42.9 (C-8), 33.8 (C-7).
After the above data were comprehensively analyzed, the molecular structure of compound C3 was shown in FIG. 10, confirming that compound C3 is larch resinol (CAS number: 27003-73-2).
4) As shown in FIG. 11, the NMR spectrum of the compound C4 is shown, and the hydrogen spectrum data is specifically (C) 5 D 5 N)：7.33(1H,d,J＝1.8Hz,H-2),7.20(1H,d,J＝1.8Hz,H-5),7.25(1H,dd,J＝1.8,8.1Hz,H-6),6.06(1H,d,J＝6.8Hz,H-7),3.97(1H,m,H-8),4.21(1H,m,H-9a),4.27(1H,m,H-9b),6.92(1H,brs,H-2′),7.06(1H,brs,H-6′),2.87(2H,t,J＝7.6Hz,H-7′),2.09(2H,m,H-8′),3.92(2H,t,J＝6.4Hz,H-9′),3.63(3H,s,3-OCH3),3.84(3H,s,3′-OCH3)；
As shown in FIG. 12, the NMR spectrum of the compound C4 is shown, and the carbon spectrum data is specifically (C) 5 D 5 N)：134.4(C-1),111.4(C-2),148.6(C-3),147.9(C-4),117.0(C-5),120.3(C-6),88.9(C-7),55.6(C-8),64.9(C-9),130.7(C-1′),114.2(C-2′),145.2(C-3′),147.9(C-4′),136.7(C-5′),118.1(C-6′),33.2(C-7′),36.5(C-8′),62.0(C-9′),56.3(3-OCH3),56.8(3′-OCH3)。
After the above data were comprehensively analyzed, the molecular structure of compound C4 was shown in FIG. 13, and it was confirmed that compound C4 was Dihydrodehydrodiconiferyl Alcohol (CAS number: 126253-41-6).
5) As shown in fig. 14, the NMR hydrogen spectrum of compound C5 specifically includes: 6.78 (1h, s), 6.49 (1h, d, j = 1.8hz), 6.19 (1h, d, j = 1.8hz), 6.94 (2h, dd, j =9.0, 1.2hz), 7.94 (2h, dd, j =9.0, 1.2hz);
as shown in fig. 15, the NMR carbon spectrum of compound C5 specifically includes: 182.0 (C-4), 164.2 (C-2), 163.9 (C-7), 161.3 (C-5), 161.1 (C-4 '), 156.9 (C-9), 128.6 (C-2', 6 '), 121.0 (C-1'), 116.0 (C-3 ', 5'), 105.3 (C-10), 103.1 (C-3), 99.9 (C-6), 94.8 (C-8).
After comprehensive analysis of the above data, the molecular structure of compound C5 was shown in FIG. 16, confirming that compound C5 is apigenin (CAS No. 520-36-5).
6) As shown in fig. 17, the NMR hydrogen spectrum of compound C6 specifically includes: 6.67 (1h, s), 6.45 (1h, d, j = 1.8hz), 6.19 (1h, d, j = 1.8hz), 7.43 (1h, dd, j =8.4, 1.2hz), 7.43 (1h, d, j = 2.4hz), 6.90 (1h, d, j = 8.4hz);
as shown in fig. 18, the NMR carbon spectrum of compound C6 specifically includes the following data: 181.6 (C-4), 164.1 (C-2), 163.9 (C-7), 161.4 (C-5), 157.3 (C-9), 149.7 (C-4 '), 145.7 (C-3'), 121.5 (C-1 '), 118.9 (C-6'), 116.0 (C-5 '), 113.4 (C-2), 103.7 (C-l'), 102.8 (C-3), 98.8 (C-6), 93.8 (C-8).
After comprehensive analysis of the above data, the molecular structure of Compound C6 was shown in FIG. 19, confirming that Compound C6 is luteolin (CAS No. 491-70-3).
7) As shown in fig. 20, the NMR hydrogen spectrum of compound C7 specifically includes: 7.53 (1H, d, J =16.0Hz, H-3), 6.22 (1H, d, J =15.6Hz, H-2), 7.02 (1H, d, J =2.0Hz, H-2 '), 6.93 (1H, dd, J =8.0Hz, H-6 '), 6.77 (1H, d, J =8.0Hz, H-5 ').
After comprehensive analysis of the above data, the molecular structure of Compound C7 was shown in FIG. 21, confirming that Compound C7 is caffeic acid (CAS number: 331-39-5).
8) As shown in FIG. 22, the NMR spectrum of the compound C8 was analyzed and the hydrogen spectrum data was (CD) 3 OD)：6.71(1H,s,H-6),6.73(1H,s,H-6),2.62(2H,t,J＝7.2Hz,H-7),1.80(2H,m,H-8),3.81(2H,t,J＝6.5Hz,H-9),7.02(1H,d,J＝1.8Hz,H-2′),7.12(1H,d,J＝8.5Hz,H-5′),6.92(1H,dd,J＝8.5,1.8Hz,H-6′),5.54(1H,d,J＝5.8Hz,H-7′),3.44(1H,m,H-8′),3.81(2H,t,J＝6.5,H-9),4.88(1H,d,J＝8.0Hz,Glc H-1),3.85(3H,s,3-OCH3),3.83(3H,s,3’-OCH3)；
As shown in FIG. 23, the NMR spectrum of the compound C8 is shown, and the carbon spectrum data is specifically (CD) 3 OD)：137.1(C-1),114.1(C-2),145.2(C-3),147.6(C-4),129.5(C-5),117.9(C-6),32.9(C-7),35.8(C-8),62.1(C-9),138.3(C-1′),111.0(C-2′),150.9(C-3′),147.4(C-4′),117.8(C-5′),119.3(C-6′),88.5(C-7′),55.7(C-8′),65.0(C-9′),56.7(3-OCH3),56.6(3′-OCH3),102.7(Glc H-1),74.9(Glc H-2),78.2(Glc H-3),71.3(Glc H-4),77.8(Glc H-5),62.4(Glc H-6)。
After comprehensive analysis of the above data, the molecular structure of Compound C8 was shown in FIG. 24, confirming that Compound C8 is Urolinoside (CAS No.: 131723-83-6).
9) As shown in fig. 25, the NMR hydrogen spectrum of compound C9 has hydrogen spectrum data specifically (DMSO): 5.45 (1H, d, J =7.5Hz, H-1 "), 6.21 (1H, d, J =2.1Hz, H-6), 6.43 (1H, d, J =2.1Hz, H-8), 6.88 (2H, d, J =8.9Hz, H-3,5 '), 8.04 (2H, d, J =8.9Hz, H-2',6 '), 12.61 (1H, s, 5-OH);
as shown in fig. 26, the NMR carbon spectrum of compound C9 specifically includes: 61.0 (C-6 "), 70.0 (C-4"), 74.4 (C-2 "), 76.6 (C-3"), 77.6 (C-5 "), 93.8 (C-8), 98.8 (C-6), 101.0 (C-1"), 104.2 (C-10), 115.2 (C-3 ', 5'), 121.1 (C-1 '), 131.0 (C-2', 6 '), 133.4 (C-3), 156.4 (C-2), 156.5 (C-9), 160.1 (C-4'), 161.4 (C-5), 164.3 (C-7), 177.6 (C-4).
After comprehensive analysis of the above data, the molecular structure of compound C9 was shown in FIG. 27, confirming that compound C9 is astragalin (CAS number: 480-10-4).
10 ) as shown in fig. 28, is the NMR hydrogen spectrum of compound C9, and the hydrogen spectrum data thereof is specifically: 12.96 (1H, s, 5-OH), 8.06 (2H, d, J =8.6Hz, H-2',6 '), 7.16 (2H, d, J =8.6Hz, H-3,5 '), 6.96 (1H, s, H-3), 6.80 (1H, br s, H-8), 6.46 (1H, br s, H-6), 5.07 (1H, d, J =6.7Hz, H-1 "), 4.55 (1H, br s, H-1 '), 3.86 (3H, s,4' -OCH 3 ) 3.10-3.90 (10 sugar protons), 1.07 (3H, d, J =5.5Hz, H-6 "').
After comprehensive analysis of the above data, the molecular structure of Compound C10 was shown in FIG. 29, confirming that Compound C10 is linarin (CAS No.: 480-36-4).
From the results, the method successfully separates and purifies the effective components in the clematis filamentosa dunn one by one, and the substance obtained after separation and purification has high purity as seen from the peak pattern of the hydrogen spectrogram, so that the method is suitable for further research or used for preparing medicaments.
It should be noted that the above-mentioned embodiments are only illustrative and not restrictive, and any modifications or changes within the meaning and range of equivalents to the technical solutions of the present invention by those skilled in the art should be considered to be included in the protection scope of the present invention.
Claims (10)
1. The method for separating and purifying the active ingredients of the clematis filamentosa dunn is characterized by comprising the following steps:
(1) Extracting the aerial parts of the clematis filamentosa dunn with ethanol to obtain ethanol extract;
(2) Subjecting the ethanol extract to a normal phase silica gel chromatography column, performing gradient elution using a petroleum ether/acetone mixed solvent, acetone and methanol in a volume ratio of 11 to 9;
(3) Subjecting the extract A2 to normal phase silica gel column chromatography, performing gradient elution by using a chloroform/methanol mixed solvent with a volume ratio of 100 to 80 and 60 to 1 to 40;
(4) Subjecting the extract A3 to a normal phase silica gel chromatographic column, eluting by using a chloroform/methanol mixed solvent with the volume ratio of 40 to 1-20, collecting eluents t1 and t2 in two batches in sequence, and concentrating by evaporating the solvent to obtain an extract F1 and an extract F2;
(4a) Loading the extract F1 on a Sephadex LH-20 column, eluting by using a chloroform/methanol mixed solvent with the volume ratio of 2;
(4b) Loading the extract F2 on a Sephadex LH-20 column, eluting by using a chloroform/methanol mixed solvent with the volume ratio of 2;
(5) Loading the extract A5 on a normal phase silica gel chromatographic column, eluting by using a chloroform/methanol mixed solvent with the volume ratio of 25 to 1-5, collecting eluents t5 and t6 in two batches in sequence, and concentrating to obtain extracts F3 and F4;
(5a) Loading the extract F3 on a Sephadex LH-20 chromatographic column, eluting by using a chloroform/methanol mixed solvent with the volume ratio of 2-0.5;
(5b) Loading the extract F4 on an MCI GEL CHP20/P120 column, eluting by using 70-90% methanol, collecting eluent, concentrating, recrystallizing and drying to obtain luteolin;
(6) Concentrating the extract A6, adding water for dissolving, then adding n-butyl alcohol with the volume equal to the total volume of the liquid after adding water for multiple times of extraction, merging n-butyl alcohol phases, concentrating, then loading on a reverse phase silica gel chromatographic column, sequentially eluting by using 10-20%, 20-35% and 35-50% methanol aqueous solutions, collecting eluent, concentrating and drying to obtain extracts F5, F6 and F7;
(6a) Concentrating the extract F5, then loading the concentrated extract on a Sephadex LH-20 column, eluting the concentrated extract by using a chloroform/methanol mixed solvent with the volume ratio of 2 to 0.5;
(6b) Concentrating the extract F6, then loading the concentrated extract on a Sephadex LH-20 column, eluting the concentrated extract by using a chloroform/methanol mixed solvent with the volume ratio of 2 to 0.5;
(6c) Concentrating the extract F7, loading the concentrated extract on a Sephadex LH-20 column, eluting by using a chloroform/methanol mixed solvent with the volume ratio of 2.
2. The method for separating and purifying the active ingredients of clematis filamentosa dunn according to claim 1, wherein the step (1) of extracting the overground parts of clematis filamentosa dunn is implemented by the following steps: and (2) crushing the overground part of the dried clematis filamentosa dunn, adding 85% industrial ethanol according to the solid mass/liquid volume ratio of 1.
3. The method for separating and purifying the active ingredient of clematis filamentosa Dunn according to claim 1 or 2, wherein the step (2) is performed by gradient elution sequentially using a petroleum ether/acetone mixed solvent, acetone and methanol in a volume ratio of 10.
4. The method for separating and purifying the active ingredient of clematis filamentosa Dunn according to claim 3, wherein the step (3) comprises subjecting the extract A2 to normal phase silica gel column chromatography, gradient elution is carried out by using chloroform/methanol mixed solvents with a volume ratio of 100 to 1 and 50 in sequence, collecting 50.
5. The method for separating and purifying the active ingredient of clematis filamentosa Dunn according to claim 4, wherein the step (4) is carried out by eluting with a chloroform/methanol mixed solvent in a volume ratio of 30; step (4 a) eluting with a chloroform/methanol mixed solvent in a volume ratio of 1; step (4 b) eluting with a chloroform/methanol mixed solvent with the volume ratio of 1; and (5) eluting with a chloroform/methanol mixed solvent with a volume ratio of 15.
6. The method for separating and purifying the active ingredients of clematis filamentosa Dunn according to claim 5, wherein the step (5 a) comprises the steps of loading the extract F3 on Sephadex LH-20 column, eluting with a chloroform/methanol mixed solvent with a volume ratio of 1; step (5 b) was eluted with 80% methanol.
7. The method for separating and purifying the active ingredient of clematis filamentosa Dunn as claimed in claim 6, wherein the step (6 a) is that the extract F5 is concentrated and then applied to Sephadex LH-20, and then eluted by using a chloroform/methanol mixed solvent in a volume ratio of 1; concentrating the extract F6, then loading the concentrated extract on a Sephadex LH-20 column, eluting the Sephadex LH-20 column by using a chloroform/methanol mixed solvent with the volume ratio of 1; step (6 c) was eluted with a chloroform/methanol mixed solvent at a volume ratio of 1.
8. The method for separating and purifying the active ingredient of clematis filamentosa Dunn according to claim 1 or 7, wherein the powdery precipitate is separated by filtration in the step (3), and then washed with methanol; in the step (4 a), the powdery precipitate is separated by filtration and washed with acetone.
9. The method for separating and purifying the active ingredients of clematis filamentosa Dunn according to claim 8, wherein the solvent used in the HPLC preparative chromatography of the step (4 b) is 30-50% methanol in water.
10. The method for separating and purifying clematis filamentosa Dunn effective ingredient according to claim 9, wherein the organic solvent for dissolving the powdery precipitate in the step (5 a) is DMSO; in the steps (5 a) and (5 b), recrystallization is performed using methanol.
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