CN115650915A - Method for separating and preparing alkaloid from lindera root - Google Patents
Method for separating and preparing alkaloid from lindera root Download PDFInfo
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- 229930013930 alkaloid Natural products 0.000 title claims abstract description 59
- 150000003797 alkaloid derivatives Chemical class 0.000 title claims abstract description 52
- 238000000034 method Methods 0.000 title claims abstract description 26
- 241000146029 Lindera aggregata Species 0.000 title description 25
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 48
- HORZNQYQXBFWNZ-LBPRGKRZSA-N (6as)-2,10-dimethoxy-5,6,6a,7-tetrahydro-4h-dibenzo[de,g]quinoline-1,9-diol Chemical compound N1CCC2=CC(OC)=C(O)C3=C2[C@@H]1CC1=C3C=C(OC)C(O)=C1 HORZNQYQXBFWNZ-LBPRGKRZSA-N 0.000 claims abstract description 26
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- 239000002904 solvent Substances 0.000 claims abstract description 21
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Abstract
The invention relates to a countercurrent chromatographic separation preparation method of alkaloid in lindera root, in particular to a method for separating and preparing alkaloid from an lindera root extract by adopting countercurrent chromatographic separation. The invention discloses a method for separating and preparing alkaloid from lindera root, which comprises the following steps: extracting radix Linderae powder with organic solvent to obtain alkaloid crude extract; obtaining immiscible two-phase solvents by using n-butanol, ethyl acetate and 0.5% acetic acid, and preparing to obtain corresponding alkaloid crude extract solution; separating by countercurrent chromatography to obtain norisoboldine and boldine.
Description
Technical Field
The invention relates to a countercurrent chromatographic separation preparation method of alkaloid in lindera root, in particular to a method for separating and preparing alkaloid from an lindera root extract by adopting countercurrent chromatographic separation.
Background
The combined spicebush root, called as the traditional Chinese medicinal material in China, is the best quality of Tiantai in Zhejiang province, was originally recorded in Ben Cao Shi Yi in Tang Dynasty, and is mainly used for treating syndromes of cold accumulation and qi stagnation, chest and abdomen distending pain, adverse rising of qi, dyspnea with rapid respiration, bladder deficiency and cold, enuresis and frequent micturition, hernia pain, cold channel, abdominal pain and the like. Alkaloid contained in the lindera aggregate is an alkaline nitrogenous organic substance, most of which have complex annular structures and obvious biological activity, and is an important effective component in the lindera aggregate. The alkaloids contained in radix Linderae mainly comprise isoquinoline type alkaloids, such as norisoboldine and boldine (chemical structures shown in formula I and II). As the main characteristic chemical components in the combined spicebush root, the compound spicebush root has wide activity, and comprises anti-inflammation, analgesia, anesthesia, blood pressure reduction, arrhythmia resistance, tumor resistance, depression resistance, blood sugar reduction, lipid peroxidation resistance, organic phosphorus poisoning resistance and the like.
At present, more than ten kinds of alkaloid compounds are totally found in the combined spicebush root, and related effective components and structural information are less. A common method for separating alkaloid from radix Linderae is column chromatography. However, the physicochemical properties of the alkaloid components in lindera aggregate are similar, and when the traditional separation technology such as column chromatography is adopted, tailing, pollution and loss are often caused by the adsorption of a solid carrier, so that the compound monomer with higher purity (the obtained purity is generally less than 90%) is difficult to obtain.
Counter-current Chromatography (CCC) is a continuous, efficient and fast liquid-liquid partition Chromatography technique that has been developed in nearly 30 years without any solid support, which avoids various problems caused by solid supports or carriers, i.e., the sample is easily adsorbed, lost and denatured, and when other liquid Chromatography methods are used for preparative separation, the partition efficiency is significantly reduced, the solvent consumption is high, and CCC ensures high peak resolution, the separation amount is large, the sample is free of loss, the recovery rate is high, the separation environment is mild, and the solvent is saved. The countercurrent chromatograph can directly carry out a large amount of crude extraction samples or synthesis mixtures, the separation result can reach quite high purity, even can be directly used together with instruments such as a mass spectrometer and the like, and the countercurrent chromatograph is widely applied to the preparation, separation and purification of chemical substances in the fields of biology, medicine, environmental protection and the like. One of the key points/difficulties in the use of countercurrent chromatography is the determination of optimal chromatographic parameters such as biphasic solvent systems, flow rates and rotational speeds.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for separating and preparing alkaloid from lindera root.
In order to solve the technical problems, the invention provides a method for separating and preparing alkaloid from lindera root, which comprises the following steps:
1) Extracting the lindera root powder with an organic solvent, concentrating the extracting solution by rotary evaporation to obtain an extract, and drying the obtained extract (drying for 110-130 minutes at 50 +/-5 ℃) to obtain an alkaloid crude extract (brown powder);
2) And mixing n-butyl alcohol, ethyl acetate and 0.5% acetic acid according to the ratio of 3.5-4.5: pouring the mixture into a liquid separator after the mixture is prepared according to the volume ratio of 0.5-1.5, and forming immiscible two-phase solvents;
0.5% acetic acid, which means 0.5% by volume acetic acid in water;
3) Mixing the upper phase and the lower phase in the two-phase solvent according to the volume ratio of 1; dissolving the alkaloid crude extract obtained in the step 1) in the mixing agent to obtain an alkaloid crude extract solution, wherein the feed-liquid ratio of the alkaloid crude extract to the mixing agent is 0.3-0.5 g/9-11 ml;
4) Injecting the upper phase in the two-phase solvent obtained in the step 2) into a separation column of a countercurrent chromatograph as a stationary phase, injecting the alkaloid crude extract solution obtained in the step 3) into a sample injection ring of the countercurrent chromatograph, starting the countercurrent chromatograph, injecting the lower phase in the two-phase solvent as a mobile phase, wherein the flow rate of the mobile phase is 2.0 +/-0.1 ml/min, respectively collecting components with retention time of 62-72 min and 85-95 min, and carrying out rotary evaporation (at the temperature of 45 +/-5 ℃ until the temperature is dried), so as to respectively obtain norisoboldine and boldine (2 purified alkaloid monomers).
Description of the invention: after injection of the mobile phase, the alkaloid is continuously distributed in a countercurrent chromatograph, and the effluents (the components with retention times of 62-72 min and 85-95 min, respectively) are collected and combined by on-line monitoring (for example, on-line monitoring by an ultraviolet detector).
The improvement of the method for separating and preparing the alkaloid from the lindera root of the invention is as follows:
the leaching in the step 1) is as follows: adding absolute ethyl alcohol as an organic solvent into the lindera root powder according to the material-liquid ratio of 1 kg/2-3L, and extracting for 2 +/-0.5 hours under the reflux condition.
As a further improvement of the method for separating and preparing the alkaloid from the lindera root of the invention:
in the step 1), after the first extraction, the extraction is repeated for 2 to 5 times, and when the extraction is repeated, absolute ethyl alcohol is used as an organic solvent, and the material-liquid ratio is the same as that of the first extraction; the extract obtained from each leaching is combined and concentrated.
As a further improvement of the method for separating and preparing the alkaloid from the lindera root of the invention:
the volume ratio of n-butanol, ethyl acetate and 0.5% acetic acid = 4.
As a further improvement of the method for separating and preparing the alkaloid from the lindera root of the invention:
in the step 1), the extracting solution is subjected to rotary evaporation and concentration at 45 +/-5 ℃ to obtain 5-10% of the original volume, and an extract is obtained.
The countercurrent chromatography method adopted by the invention is a continuous liquid-liquid distribution chromatography method without any solid support, avoids the problems of adsorption, loss, denaturation and the like caused by a solid support or a carrier, can ensure higher peak resolution, has large separation quantity, no sample loss, high recovery rate, mild separation environment, solvent saving, can directly separate a large amount of alkaloid crude extract samples, and the purity of the alkaloid obtained by separation is generally more than 90 percent, is an efficient and more environment-friendly separation technology, and is suitable for separating and purifying alkaloid compounds in lindera aggregate.
The invention has the technical advantages that:
1. a preferable two-phase solvent is obtained, so that the required alkaloid can be effectively separated and obtained;
2. the pretreatment of the sample is convenient, and the obtained crude alkaloid extract of the lindera root can be separated only by simple extraction;
3. because of adopting the liquid-liquid separation system, the irreversible adsorption of the sample is eliminated;
4. the single sample loading amount of the crude extract of the lindera root alkaloid can reach 400mg, and the preparation capacity is large.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 is a high performance liquid chromatography analysis chart of crude extract of lindera strychnifolia alkaloid in example 1;
FIG. 2 is a counter current chromatogram of the alkaloid extract of lindera strychnifolia isolated in example 1;
FIG. 3 is a high performance liquid chromatography analysis chart of isolated and purified norisoboldine in example 1;
FIG. 4 is a high performance liquid chromatography analysis of isolated and purified boldine (Bordine) in example 1;
FIG. 5 is a nuclear magnetic resonance hydrogen spectrum of the isolated and purified norisoboldine of example 1;
FIG. 6 is a nuclear magnetic resonance carbon spectrum of isolated and purified norisoboldine in example 1;
FIG. 7 is a nuclear magnetic resonance DEPT135 spectrum of the isolated and purified norisoboldine in example 1;
FIG. 8 is the NMR chart of the isolated and purified boldine in example 1;
FIG. 9 is the NMR spectrum of the isolated and purified Boldine base of example 1;
FIG. 10 is the nuclear magnetic resonance DEPT135 spectrum of the isolated and purified boldine base in example 1.
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1, a method for separating and preparing alkaloid from lindera strychnifolia, comprising the following steps:
1) 1kg of dry lindera root medicinal material powder (the water content is less than or equal to 5 percent, the particle size is 100 meshes of sieve) is heated, refluxed and extracted for 2 hours by 2.0L of absolute ethyl alcohol;
filtering to remove solvent, repeating the above extraction, i.e. adding 2.0L of anhydrous ethanol into the residue, repeating the above operation for three times, mixing the extractive solutions obtained by 4 times of extraction, and concentrating with rotary evaporator (at 45 deg.C, to 5% of the original volume) to obtain extract; oven drying the obtained extract at 50 deg.C for 120 min to obtain about 28g alkaloid crude extract, and high performance liquid chromatography analysis shows that the contents of norisoboldine and boldine are 8.5% and 2.1%, respectively.
2) Separating the alkaloid extract by a counter current chromatograph:
preparing a two-phase solvent by using n-butanol, ethyl acetate and 0.5% acetic acid according to a volume ratio of 4.
Dissolving 400mg of crude alkaloid extract in 5ml of upper phase and 5ml of lower phase to form a sample solution (i.e., crude alkaloid extract solution); the internal diameter of the countercurrent chromatography column is 2.6mm, and the column volume is 350ml. The upper phase is used as a stationary phase, the lower phase is used as a mobile phase, after the stationary phase is filled in a separation column of a countercurrent chromatograph, a sample solution is injected into a sample injection ring of the countercurrent chromatograph, the countercurrent chromatograph is started, the rotating speed is 800 revolutions, the mobile phase is injected at the flow rate of 2.0ml/min, the flow fraction is monitored by an online ultraviolet detector, the wavelength is detected to be 280nm, the flow fractions are collected and combined (the components with the retention time of 62-72 min and 85-95 min are respectively collected), the temperature is 45 ℃ until the flow fraction is dried by rotation), 28.6mg of norisoboldine and 7.2mg of boldine are respectively obtained, the purity of the two compounds is respectively 95.27% and 93.61%, the recovery rate is respectively 80.15% and 76.59% through the conventional HPLC detection, and the two compounds are shown in the figure 2, the figure 3 and the figure 4.
Recovery (%) = (mass of purified compound × purity of purified compound)/(mass of crude alkaloid extract × content of compound).
The nuclear magnetic resonance spectrum detection data of norisoboldine obtained by the separation by the method are as follows: a light brown powder of the above-mentioned nature, 1 H NMR(500MHz,D 2 O)δ:7.33(s,1H,H-11),6.54(s,1H,H-8),6.53(s,1H,H-3),3.51(s,3H,10-OCH 3 ),3.26(s,3H,2-OCH 3 ) See fig. 5. Bonding of 13 C-NMR and DEPT135 ℃ data analysis (see FIGS. 6 and 7), the compound has 18 carbon atoms and includes 3 CH 2 CH and CH 3 There were 6, and also 9 quaternary carbons (qC). 1 Delta in H-NMR H Delta 7.33 (s, 1H, H-11), 6.54 (s, 1H, H-8), 6.53 (s, 1H, H-3) show the signals of 3 independent aromatic hydrogens 13 Delta in C-NMR C :116.9The three methine signals shown by 9 (CH), 117.19 (CH) and 113.51 (CH) correspond. 1 Delta in H-NMR H :3.51(s,3H,10-OCH 3 ) And 3.26 (s, 3H, 2-OCH) 3 ) Two methoxy signals showing a high field region, and 13 delta in C-NMR C :55.25(10-OCH 3 ) And 58.04 (2-OCH) 3 ) The signals shown correspond to those with methoxy substitution on the carbon atoms at positions 2 and 10. 13 C-NMR showed a signal of 18 carbon atoms, of which delta C 151.88, 148.85, 147.18, 145.49, 129.34, 128.64, 128.34, 125.39, 122.26, 117.19, 116.99, 113.51 are the 12 aromatic ring carbons of this compound, delta C 62.21 and 43.61 are two nitrogen-connecting carbons, delta C 26.89 and 34.67 are saturated carbons on two non-substituents.
The separated isoboldine has the following data detected by nuclear magnetic resonance spectrum: the color of the powder is brown yellow, 1 H NMR(500MHz,CDcl 3 )δ:7.90(s,1H,H-11),6.82(s,1H,H-8),6.64(s,1H,H-3),3.91(s,3H,10-OCH 3 ),3.60(s,3H,1-OCH 3 ),2.53(s,3H,-NCH 3 ) See fig. 8. Bonding of 13 C-NMR and DEPT135 ° (see FIGS. 9 and 10) data analysis revealed that this compound contained 19 carbon atoms, including 3 CH 2 CH and CH 3 There were 7, and also 9 quaternary carbons (qC). 1 Delta of H-NMR H 7.90 (s, 1H, H-11), 6.82 (s, 1H, H-8), 6.64 (s, 1H, H-3) show signals for 3 independent aromatic hydrogens, which is in contrast to 13 Delta in C-NMR C 109.94 (CH), 114.01 (CH) and 113.02 (CH) correspond to the three methine signals shown. 1 Delta in H-NMR H :3.91(s,3H,10-OCH 3 ) And 3.60 (s, 3H, 1-OCH) 3 ) Two methoxy signals showing a high field region, and 13 delta in C-NMR C :55.91(10-OCH 3 ) And 60.05 (1-OCH) 3 ) The signals shown correspond to those with methoxy substitution on the carbon atoms at positions 1 and 10. 1 Delta 2.53 (s, 3H, -NCH) in H-NMR 3 ) Shows methyl substitution on the nitrogen atom, with 13 Delta 43.86 in C-NMR (N-CH) 3 ) The displayed signals are consistent. 13 C-NMR showed a signal of 19 carbon atoms, of which delta C :147.82 145.37, 144.84, 141.83, 129.97, 126.50, 125.71, 123.39, 114.01, 113.02, 109.94 are the 12 aromatic ring carbons of this compound; delta. For the preparation of a coating C 53.12 and 62.32 are the two carbon atoms bound to the nitrogen atom on the unsubstituted radical, delta C 28.59 and 33.88 are saturated carbons on two non-substituents.
TABLE 1 isolation of two alkaloids prepared 13 C-NMR and DEPT135 ℃ data
Example 2, a method for separating and preparing alkaloid from lindera root:
the injection flow rate of the mobile phase in step 2) of example 1 was changed from 2.0ml/min to 2.5ml/min, and the rest was identical to example 1. The final purity of norisoboldine and boldine is 93.66% and 90.28%, respectively, and the recovery rate is 77.56% and 73.88%, respectively.
Example 3, a method for isolating and preparing alkaloids from lindera root:
the dosage of the crude extract of the lindera strychnifolia alkaloid in the step 2) of the embodiment 1 is changed from 400mg to 500mg, and the injection flow rate of the mobile phase is changed from 2.0ml/min to 2.5ml/min, and the rest is equal to the embodiment 1. The final purity of the obtained norisoboldine and boldine is 90.35% and 83.43%, respectively, and the recovery rate is 72.33% and 70.47%, respectively.
Comparative example 1-1, the n-butanol-ethyl acetate-0.5% acetic acid (4.
Comparative example 1-2, the n-butanol-ethyl acetate-0.5% acetic acid (4.
Comparative example 2, the two-phase solvent system consisting of n-butanol-ethyl acetate-0.5% acetic acid (4.
Comparative example 3, the two-phase solvent system consisting of n-butanol-ethyl acetate-0.5% acetic acid (4, 1, v/v) in example 1 was changed to isobutanol-ethyl acetate-0.5% formic acid (4.
Comparative example 4, the two-phase solvent system consisting of n-butanol-ethyl acetate-0.5% acetic acid (4.
Comparative example 5-1, the two-phase solvent system consisting of n-butanol-ethyl acetate-0.5% acetic acid (4.
Comparative example 5-2, the two-phase solvent system consisting of n-butanol-ethyl acetate-0.5% acetic acid (4, 1, v/v) in example 1 was changed to n-butanol-ethyl acetate-1% acetic acid (4, 1.
The purity of the two alkaloids prepared in all the above comparative examples is shown in table 2.
TABLE 2
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by the person skilled in the art from the present disclosure are to be considered within the scope of the present invention.
Claims (6)
1. A method for separating and preparing alkaloid from lindera root is characterized by comprising the following steps:
1) Extracting the lindera root powder by using an organic solvent, carrying out rotary evaporation and concentration on the extracting solution to obtain an extract, and drying the obtained extract to obtain an alkaloid crude extract;
2) And mixing n-butanol, ethyl acetate and 0.5% acetic acid according to the weight ratio of 3.5-4.5: pouring the mixture into a liquid separator after the mixture is prepared according to the volume ratio of 0.5-1.5, and forming immiscible two-phase solvents;
3) Mixing an upper phase and a lower phase in the two-phase solvent according to a volume ratio of 1; dissolving the alkaloid crude extract obtained in the step 1) in the mixing agent to obtain an alkaloid crude extract solution, wherein the feed-liquid ratio of the alkaloid crude extract to the mixing agent is 0.3-0.5 g/9-11 ml;
4) Injecting the upper phase in the two-phase solvent obtained in the step 2) into a separation column of a countercurrent chromatograph as a stationary phase, injecting the alkaloid crude extract solution obtained in the step 3) into a sample injection ring of the countercurrent chromatograph, starting the countercurrent chromatograph, taking the lower phase injected into the two-phase solvent as a mobile phase, wherein the flow rate of the mobile phase is 2.0 +/-0.1 ml/min, collecting components with retention time of 62-72 min and 85-95 min respectively, and performing rotary evaporation to obtain norisoboldine and boldine respectively.
2. The method for separating and preparing alkaloid from radix linderae root as claimed in claim 1, wherein:
the leaching in the step 1) is as follows: adding absolute ethyl alcohol as an organic solvent into the lindera root powder according to the material-liquid ratio of 1 kg/2-3L, and extracting for 2 +/-0.5 hours under the reflux condition.
3. The method for separating and preparing alkaloid from radix linderae according to claim 2, wherein:
in the step 1), after the first extraction, the extraction is repeated for 2-5 times, and when the extraction is repeated, absolute ethyl alcohol is used as an organic solvent, and the ratio of material to liquid is the same as that of the material to liquid of the first extraction; mixing the extractive solutions obtained by each extraction, and concentrating.
4. The method for separating and preparing alkaloid from radix linderae according to any one of claims 1 to 3, wherein:
the n-butanol: ethyl acetate: 0.5% acetic acid = 4.
5. The method for separating and preparing alkaloid from radix linderae according to any one of claims 1 to 4, wherein the alkaloid is extracted from the root of radix linderae, and the method comprises the following steps:
in the step 1), the extracting solution is subjected to rotary evaporation concentration at 45 +/-5 ℃ to obtain 5-10% of the original volume, and an extract is obtained.
6. The method for separating and preparing alkaloid from radix linderae according to any one of claims 1 to 5, wherein:
the countercurrent chromatograph is started, and the rotating speed is set to 800 +/-50 revolutions.
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| CN102659656A (en) * | 2012-05-22 | 2012-09-12 | 广州中医药大学 | Method for separating dangshen alkaloid monomers from dangshen through high-speed counter-current chromatography |
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| CN101671346A (en) * | 2009-10-13 | 2010-03-17 | 中国科学院昆明植物研究所 | Lindenrane-type dimerization sesquiterpenoids, preparation method and applications thereof in pharmacy |
| CN102659656A (en) * | 2012-05-22 | 2012-09-12 | 广州中医药大学 | Method for separating dangshen alkaloid monomers from dangshen through high-speed counter-current chromatography |
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