CN112851673A - Enrichment and purification method of four alkaloid components in uncaria - Google Patents
Enrichment and purification method of four alkaloid components in uncaria Download PDFInfo
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- CN112851673A CN112851673A CN202110214308.6A CN202110214308A CN112851673A CN 112851673 A CN112851673 A CN 112851673A CN 202110214308 A CN202110214308 A CN 202110214308A CN 112851673 A CN112851673 A CN 112851673A
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- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
- C07D471/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains three hetero rings
- C07D471/20—Spiro-condensed systems
Abstract
An enrichment and purification method of four alkaloid components with similar structural properties in uncaria belongs to the field of development and utilization of effective components. The main process steps for enriching and separating the compounds in uncaria are as follows: pulverizing dried ramulus Uncariae cum uncis, ultrasonic extracting with 5-10 times of 60-80% ethanol, defatting with petroleum ether, extracting with ethyl acetate, and concentrating under reduced pressure to obtain extract. The obtained extract is primarily purified by macroporous adsorption resin, and is further purified by reversed-phase semi-preparative high performance liquid chromatography, so that four alkaloid components with the content of more than 90 percent can be obtained. The invention adopts various column chromatography technologies to enrich and purify the alkaloid compounds in the uncaria, has simple process and easy operation, has high alkaloid content in the finished product, can recycle all the used resins, and is easy to industrialize.
Description
Technical Field
The invention relates to alkaloid, in particular to an enrichment and purification method of four alkaloid components with similar structural properties in uncaria.
Background
Ramulus Uncariae cum uncis (ramulus Uncariae cum uncis)Uncariarhynchophylla(Miq.) Miq.exHavil.) contains alkaloid as active ingredient, mainly including rhynchophylline, isocoubine, corynoxeine, isocoubine, etc., has wide pharmacological action, has effects of clearing heat, calming liver, calming endogenous wind, relieving spasm, etc., and also has significant curative effect on cardiovascular disease, central nervous system disease, etc. In recent years, researches show that the rhynchophylline has a protective effect on neurons, can improve the learning ability of Alzheimer Disease (AD) patients, and can treat white bloodHas special therapeutic effect on diseases and AIDS. The alkaloid compound has low content in uncaria and has an immature extraction and purification method, and is mainly prepared by chemical synthesis at present, and the chemical method has strong toxic and side effects when a compound medicine is synthesized, so that the establishment of the method for extracting and purifying the compound from natural plants has great practical significance.
Ramulus Uncariae cum uncis (ramulus Uncariae cum uncis)Uncariarhynchophylla(Miq.) Miq. exHavil.) original name ramulus Uncariae cum uncis of Rubiaceae (Rubiaceae) genus (Rubiaceae)UncariaSchred.) Perennial herbaceous plants are planted in the provinces of Fujian, Jiangxi, Hunan, Guangdong, Guangxi, Guizhou, Sichuan and the like in China. The alkaloids are main active ingredients in the uncaria rhynchophylla medicinal material, and mainly comprise indole alkaloids and oxindole alkaloids, wherein the contents of the uncarine, the isocoubine, the corynoxeine and the isocoubine are higher, the four alkaloids have similar structures and have similar pharmacological activities, and the four alkaloids which are simultaneously enriched and purified in the uncaria rhynchophylla have higher economic value.
Disclosure of Invention
The invention provides a method for enriching and purifying alkaloid compounds from uncaria so as to meet the requirements of compound standard substance preparation, pharmacological activity further research and industrial application; the extraction method of the alkaloid compounds in the uncaria rhynchophylla medicinal material is optimized firstly, and the alkaloid compounds with higher purity are obtained by separation and purification by a macroporous adsorption resin method and enrichment by a semi-preparative liquid chromatography technology.
In order to achieve the purpose, the invention adopts the technical scheme that:
an enrichment and purification method of four alkaloid components in uncaria rhynchophylla takes dry uncaria rhynchophylla as a raw material, sequentially performs extraction, separation and purification by alcohol-water extraction, ethyl acetate extraction and enrichment, macroporous adsorption resin column chromatography and reverse-phase semi-preparative high performance liquid chromatography to obtain the four alkaloid enriched components, and the total content of the four alkaloid components is detected by HPLC to reach more than 90%; four alkaloid compounds, namely rhynchophylline, isocorynine, corynoxeine and isocorynoxeine, have the structural formula shown in the specification,
rhynchophylline isocoumarin
Corynoxeine isocorynoxeine.
The method specifically comprises the following steps:
the method comprises the following steps: extracting alkaloid compounds from uncaria:
A. pulverizing dried stem and branch of ramulus Uncariae cum uncis to obtain ramulus Uncariae cum uncis raw material;
B. mixing uncaria raw materials with ethanol water solution in an amount of 1 g: mixing 5-10ml, soaking with ethanol water solution volume concentration of 60-80%, ultrasonic extracting for 0.5-1 hr, and filtering;
C. repeating the extraction process of B for 5-6 times, mixing filtrates, and concentrating under reduced pressure to remove ethanol to obtain ramulus Uncariae cum uncis crude extract;
step two: extracting and enriching alkaloid compounds in uncaria: degreasing and extracting the uncaria crude extract obtained in the step one for 3-5 times by using petroleum ether, extracting for 3-5 times by using ethyl acetate, mainly enriching four alkaloid compounds in the uncaria extract at an ethyl acetate extraction part, and concentrating the ethyl acetate extraction part under reduced pressure to obtain an extract;
step three: separating the alkaloid compounds by using a resin column;
step four: purifying a sample by using a semi-preparative high performance liquid chromatography technology:
the chromatographic conditions are as follows:
a chromatographic column: a column (5 μm, 10 mm. times.250 mm) packed with octadecylsilane bonded silica gel;
mobile phase: 0.1% triethylamine-phosphoric acid (a): acetonitrile (B) = 45: 55 (V/V);
flow rate: 5 mL/min;
elution time: 30 min;
column temperature: 30 ℃;
detection wavelength: 254 nm;
the enrichment method comprises the following steps: dissolving the four alkaloid component extracts in ramulus Uncariae cum uncis obtained in step three with 30-40% ethanol respectively, further purifying by semi-preparative high performance liquid chromatography, collecting eluate for 8-11min, concentrating under reduced pressure to remove ethanol, and freeze drying to obtain light yellow dry powder; the content of the alkaloid compounds can reach more than 90 percent by high performance liquid detection;
during extraction, the volume ratio of the uncaria crude extract to petroleum ether is 1:1-2, and the volume ratio of the uncaria crude extract to ethyl acetate is 1: 1-2; when concentrating under reduced pressure, heating the extractive solution to concentrate at 50-70 deg.C; the macroporous adsorption resin is nonpolar macroporous adsorption resin.
Dissolving the extract obtained in the step two in ethanol with the volume concentration of 20-30%, filtering, loading the extract into a column for separation by taking D3520 type macroporous adsorption resin as a filler, eluting by sequentially taking 5-10 times of column volume of water, 10% of volume concentration, 20% of volume concentration, 30% of volume concentration, 40% of volume concentration, 50% of volume concentration, 60% of volume concentration, 70% of volume concentration, 80% of volume concentration, 90% of volume concentration and 100% of ethanol solution, collecting 10-70% of fraction, concentrating under reduced pressure to obtain an extract, and checking the content of four types of rhynchophylline to be more than 20% by high performance liquid chromatography.
Dissolving the extract obtained in the step two by using ethanol with the volume concentration of 20-30%, filtering, loading the extract into a column by using D101 type macroporous adsorption resin as a filler for separation, eluting by using water with the volume of 5-10 times of the column volume and ethanol solutions with the volume concentrations of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100%, collecting the fractions of 10-60% of the part, concentrating the fractions under reduced pressure to obtain an extract, and checking the content of four types of rhynchophylline to be more than 20% by using high performance liquid chromatography.
Dissolving the extract obtained in the step two by using ethanol with the volume concentration of 20-30%, filtering, loading the extract into a column for separation by using AB8 type macroporous adsorption resin as a filler, eluting by using water with the volume of 5-10 times of the column volume and ethanol solutions with the volume concentrations of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100%, collecting the fractions of 40-60% of the part, concentrating the fractions under reduced pressure to obtain an extract, and checking the content of four types of rhynchophylline to be more than 20% by using high performance liquid chromatography.
Dissolving the extract obtained in the step two in ethanol with the volume concentration of 20-30%, filtering, loading the extract into a column by taking HP100 type macroporous adsorption resin as a filler for separation, eluting by sequentially taking 5-10 times of column volume of water, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% ethanol solution, collecting the flow of 40-60% of parts, concentrating under reduced pressure to obtain an extract, and checking the content of four types of rhynchophylline to be more than 20% by high performance liquid chromatography.
The beneficial effect of adopting above-mentioned technical scheme is:
1. the alkaloid compounds in the uncaria rhynchophylla are definite in activity, the four alkaloid compounds are similar in structural property, easy to enrich, high in enrichment purity, simple in separation operation process and low in production cost.
2. The invention can achieve the preliminary purification of the alkaloid compounds through various column chromatographs, and alkaloid components with the purity of more than 90 percent can be obtained after the enrichment by the preparative high performance liquid chromatography technology, and the product has high purity and good quality.
3. The macroporous adsorption resin used in the invention can be repeatedly regenerated and used, and is suitable for industrial application.
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FIG. 1 is a process flow chart of the enrichment and purification method of four alkaloid components in uncaria rhynchophylla of the present invention.
Detailed Description
The present invention will now be further described with reference to examples. The examples are given solely for the purpose of illustration and are not intended to be limiting.
The first embodiment is as follows:
drying and crushing stems and branches of uncaria, weighing 100g, soaking in 1000mL of 70% ethanol with volume concentration for 12h, carrying out ultrasonic treatment for 30min, filtering, and storing filtrate for later use; repeating the above extraction processes for 5-6 times, mixing filtrates, and concentrating under reduced pressure to remove ethanol to obtain ramulus Uncariae cum uncis crude extract. Extracting with petroleum ether with the same volume as the crude extract for 4 times, removing fat-soluble components, extracting with ethyl acetate with the same volume as the crude extract for 4 times, concentrating the ethyl acetate extract under reduced pressure to obtain an extract, dissolving the extract with 20% ethanol, separating with D3520 macroporous adsorbent resin column, eluting with 6 times of column volume of water, 10% ethanol, 20% ethanol, 30% ethanol, 40% ethanol, 50% ethanol, 60% ethanol, 70% ethanol, 80% ethanol, 90% ethanol and 100% ethanol, collecting 10% -70% ethanol elution part, and concentrating to obtain the extract. Dissolving the obtained extract with 40% ethanol, filtering, enriching by preparative high performance liquid chromatography, collecting eluate for 8-11min, concentrating under reduced pressure to remove ethanol, and freeze drying to obtain yellowish dry powder, wherein the content of four alkaloids compounds detected by high performance liquid chromatography can reach above 90%.
Example two:
drying and crushing stems and branches of uncaria, weighing 100g, soaking in 1000mL of 70% ethanol with volume concentration for 12h, carrying out ultrasonic treatment for 30min, filtering, and storing filtrate for later use; repeating the above extraction processes for 5-6 times, mixing filtrates, and concentrating under reduced pressure to remove ethanol to obtain ramulus Uncariae cum uncis crude extract. Extracting with petroleum ether with the same volume as the crude extract for 4 times, removing fat-soluble components, extracting with ethyl acetate with the same volume as the crude extract for 4 times, concentrating the ethyl acetate extract under reduced pressure to obtain an extract, dissolving the extract with 20% ethanol, separating with D101 macroporous adsorbent resin column, eluting with 6 times of column volume of water, 10% ethanol, 20% ethanol, 30% ethanol, 40% ethanol, 50% ethanol, 60% ethanol, 70% ethanol, 80% ethanol, 90% ethanol and 100% ethanol, collecting the 10% -60% ethanol elution part, and concentrating to obtain the extract. Dissolving the obtained extract with 40% ethanol, filtering, enriching by preparative high performance liquid chromatography, collecting eluate for 8-11min, concentrating under reduced pressure to remove ethanol, and freeze drying to obtain yellowish dry powder, wherein the content of four alkaloids compounds detected by high performance liquid chromatography can reach above 90%.
Example three:
drying and crushing stems and branches of uncaria, weighing 100g, soaking in 1000mL of 70% ethanol with volume concentration for 12h, carrying out ultrasonic treatment for 30min, filtering, and storing filtrate for later use; repeating the above extraction processes for 5-6 times, mixing filtrates, and concentrating under reduced pressure to remove ethanol to obtain ramulus Uncariae cum uncis crude extract. Extracting with petroleum ether with the same volume as the crude extract for 4 times, removing fat-soluble components, extracting with ethyl acetate with the same volume as the crude extract for 4 times, concentrating the ethyl acetate extract under reduced pressure to obtain an extract, dissolving the extract with 20% ethanol, separating with AB8 macroporous adsorbent resin column, eluting with 6 times of column volume of water, 10% ethanol, 20% ethanol, 30% ethanol, 40% ethanol, 50% ethanol, 60% ethanol, 70% ethanol, 80% ethanol, 90% ethanol and 100% ethanol, collecting the ethanol elution part with 40% -60% ethanol, and concentrating to obtain the extract. Dissolving the obtained extract with 40% ethanol, filtering, enriching by preparative high performance liquid chromatography, collecting eluate for 8-11min, concentrating under reduced pressure to remove ethanol, and freeze drying to obtain yellowish dry powder, wherein the content of four alkaloids compounds detected by high performance liquid chromatography can reach above 90%.
Claims (9)
1. The enrichment and purification method of four alkaloid components in uncaria is characterized by comprising the following steps: taking dried uncaria as a raw material, sequentially carrying out extraction, separation and purification by alcohol-water extraction, ethyl acetate extraction and enrichment, macroporous adsorption resin column chromatography and reverse phase semi-preparative high performance liquid chromatography to obtain four alkaloid enriched components, and detecting the total content of the four alkaloid components to be more than 90% by HPLC; four alkaloid compounds, namely rhynchophylline, isocorynine, corynoxeine and isocorynoxeine, have the structural formula shown in the specification,
rhynchophylline isocoumarin
Corynoxeine isocorynoxeine.
2. The method for enriching and purifying four alkaloid components in uncaria according to claim 1, which is characterized in that: the method specifically comprises the following steps:
the method comprises the following steps: extracting alkaloid compounds from uncaria:
A. pulverizing dried stem and branch of ramulus Uncariae cum uncis to obtain ramulus Uncariae cum uncis raw material;
B. mixing uncaria raw materials with ethanol water solution in an amount of 1 g: mixing 5-10ml, soaking with ethanol water solution volume concentration of 60-80%, ultrasonic extracting for 0.5-1 hr, and filtering;
C. repeating the extraction process of B for 5-6 times, mixing filtrates, and concentrating under reduced pressure to remove ethanol to obtain ramulus Uncariae cum uncis crude extract;
step two: extracting and enriching alkaloid compounds in uncaria: degreasing and extracting the uncaria crude extract obtained in the step one for 3-5 times by using petroleum ether, extracting for 3-5 times by using ethyl acetate, mainly enriching four alkaloid compounds in the uncaria extract at an ethyl acetate extraction part, and concentrating the ethyl acetate extraction part under reduced pressure to obtain an extract;
step three: separating the alkaloid compounds by using a resin column;
step four: purifying a sample by using a semi-preparative high performance liquid chromatography technology:
the chromatographic conditions are as follows:
a chromatographic column: a column (5 μm, 10 mm. times.250 mm) packed with octadecylsilane bonded silica gel;
mobile phase: 0.1% triethylamine-phosphoric acid (a): acetonitrile (B) = 45: 55 (V/V);
flow rate: 5 mL/min;
elution time: 30 min;
column temperature: 30 ℃;
detection wavelength: 254 nm;
the enrichment method comprises the following steps: dissolving the four alkaloid component extracts in ramulus Uncariae cum uncis obtained in step three with 30-40% ethanol respectively, further purifying by semi-preparative high performance liquid chromatography, collecting eluate for 8-11min, concentrating under reduced pressure to remove ethanol, and freeze drying to obtain light yellow dry powder; the content of the alkaloid compounds can reach more than 90 percent by high performance liquid detection;
during extraction, the volume ratio of the uncaria crude extract to petroleum ether is 1:1-2, and the volume ratio of the uncaria crude extract to ethyl acetate is 1: 1-2; when concentrating under reduced pressure, heating the extractive solution to concentrate at 50-70 deg.C; the macroporous adsorption resin is nonpolar macroporous adsorption resin.
3. The method for enriching and purifying four alkaloid components in uncaria according to claim 2, which is characterized in that: dissolving the extract obtained in the step two in ethanol with the volume concentration of 20-30%, filtering, loading the extract into a column for separation by taking D3520 type macroporous adsorption resin as a filler, eluting by sequentially taking 5-10 times of column volume of water, 10% of volume concentration, 20% of volume concentration, 30% of volume concentration, 40% of volume concentration, 50% of volume concentration, 60% of volume concentration, 70% of volume concentration, 80% of volume concentration, 90% of volume concentration and 100% of ethanol solution, collecting 10-70% of fraction, concentrating under reduced pressure to obtain an extract, and checking the content of four types of rhynchophylline to be more than 20% by high performance liquid chromatography.
4. The method for enriching and purifying four alkaloid components in uncaria according to claim 2, which is characterized in that: dissolving the extract obtained in the step two by using ethanol with the volume concentration of 20-30%, filtering, loading the extract into a column by using D101 type macroporous adsorption resin as a filler for separation, eluting by using water with the volume of 5-10 times of the column volume and ethanol solutions with the volume concentrations of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100%, collecting the fractions of 10-60% of the part, concentrating the fractions under reduced pressure to obtain an extract, and checking the content of four types of rhynchophylline to be more than 20% by using high performance liquid chromatography.
5. The method for enriching and purifying four alkaloid components in uncaria according to claim 2, which is characterized in that: dissolving the extract obtained in the step two by using ethanol with the volume concentration of 20-30%, filtering, loading the extract into a column for separation by using AB8 type macroporous adsorption resin as a filler, eluting by using water with the volume of 5-10 times of the column volume and ethanol solutions with the volume concentrations of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100%, collecting the fractions of 40-60% of the part, concentrating the fractions under reduced pressure to obtain an extract, and checking the content of four types of rhynchophylline to be more than 20% by using high performance liquid chromatography.
6. The method for enriching and purifying four alkaloid components in uncaria according to claim 2, which is characterized in that: dissolving the extract obtained in the step two in ethanol with the volume concentration of 20-30%, filtering, loading the extract into a column by taking HP100 type macroporous adsorption resin as a filler for separation, eluting by sequentially taking 5-10 times of column volume of water, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% ethanol solution, collecting the flow of 40-60% of parts, concentrating under reduced pressure to obtain an extract, and checking the content of four types of rhynchophylline to be more than 20% by high performance liquid chromatography.
7. The method for enriching and purifying four alkaloid components in uncaria according to claim 2, which is characterized in that: during the extraction operation, the volume ratio of the uncaria crude extract to the petroleum ether is 1:1-2, and the volume ratio of the uncaria crude extract to the ethyl acetate is 1: 1-2.
8. The method for enriching and purifying four alkaloid components in uncaria according to claim 2, which is characterized in that: when the vacuum concentration is carried out, the temperature for heating and concentrating the extracting solution is 50-60 ℃.
9. The method for enriching and purifying four alkaloid components in uncaria rhynchophylla according to claim 3, 4, 5 or 6, wherein the method comprises the following steps: the macroporous adsorption resin is nonpolar macroporous adsorption resin.
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| CN116854690A (en) * | 2023-06-28 | 2023-10-10 | 河南中医药大学 | Indole uncaria alkaloid substance extraction method and application thereof |
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| CN101342259A (en) * | 2008-06-18 | 2009-01-14 | 沈阳药科大学 | Method for extracting alkaloid valid target from hooked uncaria leaf, with hook stem branch or full grass and uses thereof |
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