CN101270118B - Technique for spherosinin purification with continuous column chromatography - Google Patents
Technique for spherosinin purification with continuous column chromatography Download PDFInfo
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- CN101270118B CN101270118B CN200710017542XA CN200710017542A CN101270118B CN 101270118 B CN101270118 B CN 101270118B CN 200710017542X A CN200710017542X A CN 200710017542XA CN 200710017542 A CN200710017542 A CN 200710017542A CN 101270118 B CN101270118 B CN 101270118B
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Abstract
Belonging to the field of chemical analyses on natural products, the present invention relates to a method for extracting, separating, purifying and testing natural products, in particular to a technique for extracting swainsonine by continuous column chromatography. The present invention resolves the defects of the prior art, including a complex process, high cost and low yield rate. The solution orderly includes the following steps: (1) the preprocessing of locoweed; (2) the acquisition of locoweed extract; (3) the preparation of a crude product: The extract is roughly separated by polar macroporous adsorption resin and, after TLC test, merged with eluant containing swainsonine to serve as the crude product for later use; (4) the purification of the swainsonine: The crude product is purified by silica gel column chromatography, and by the TLC test and the GC test, the parts of the swainsonine, which have relative content larger than or equal to 10 percent, are respectively collected; (5) the acquisition of a pure swainsonine product by the HPLC test; the parts collected in the step (4) undergo the HPLC test in order to collect eluate in the appearance time period of the swainsonine, and after the solvent is evaporated, the pure swainsonine product can be produced by recrystallization.
Description
One, technical field
The invention belongs to the natural product chemistry research field, be specifically related to natural product extraction, separation, purifying and detection method, relate in particular to the technology that a kind of continuous column chromatography extracts trihydroxyoctahydroindolizidine.
Two, background technology
Loco weed (Locoweed) is a kind of very common weeds in NORTHWEST CHINA portion, after domestic animal is eaten by mistake, causes poisoning easily, is often regarded as injurious weed.Research in recent years shows, main toxic component-trihydroxyoctahydroindolizidine of loco weed is under the suitable situation of consumption; Growth that not only can anticancer; Can also activate immunity of organisms, alleviate the spinoff that the cancer patients causes because of chemotherapy, thereby be a kind of natural drug that the exploitation future is arranged very much.
(swainsonine, source SW) mainly contains three kinds of approach to trihydroxyoctahydroindolizidine: 1. extraction separation from plant.At present the known plant that contains trihydroxyoctahydroindolizidine mainly is pulse family Astragalus (Astragalus) and whin genus (Oxytopis) plant (being referred to as loco weed), and this type plant is distributed widely in all over the world, especially North America and China, and resource is very abundant; Next is a darling pea, and this platymiscium only is confined to Australia.Australia scholar Colegate utilizes mannosidase from darling pea plant ass swainson pea, successfully to isolate trihydroxyoctahydroindolizidine first.2. extraction separation from beans rhizoctonia (Rhizoctonia leguminicola).Schneider etc. (1983) isolate trihydroxyoctahydroindolizidine the earliest from the beans rhizoctonia, China Yang Ming fine jade etc. are also from isolating trihydroxyoctahydroindolizidine the isolating beans rhizoctonia voluntarily.3. synthetic trihydroxyoctahydroindolizidine.Americanized scholar Yasuda (1984), Bennett (1989) and China academician Zhou Weishan successively synthetic trihydroxyoctahydroindolizidine.Canadian subsequently Toronto chemistry institute has also had sintetics.Because swainson pea have four chiral carbon atoms, even the better controlled reaction also have 16 chemical structures identical in the synthetic product, and the different isomer of three-dimensional conformation separates very difficulty, entire synthesis process had 21 steps, and productive rate is merely 6.6%.
At present, the method for purifying spherosinin mainly is through organic solvent lixiviate, extraction both at home and abroad, passes through cation exchange column chromatography or silica gel column chromatography again, and last recrystallization or decompression distillation obtain the pure article of trihydroxyoctahydroindolizidine.Be that " 02114590.3 ", denomination of invention are that disclosed technical process is in " purifying technique of trihydroxyoctahydroindolizidine in a kind of loco weed " at number of patent application for example: (1) uses industrial methanol as extraction agent, from the loco weed grass meal, obtains loco weed medicinal extract with cold soak formulation; (2) from loco weed medicinal extract, prepare the trihydroxyoctahydroindolizidine bullion through a plurality of steps; (3) chromatographic separation of trihydroxyoctahydroindolizidine; (4) prepare pure article.Comprise steps such as acid dissolving, alkali adjusting, ion-exchange chromatography in (2) step wherein.The problem that this technology exists is: complex process, and cost is high, and yield is low.
Three, summary of the invention
The objective of the invention is to, a kind of technology that adopts spherosinin purification with continuous column chromatography is provided, to overcome the complex process that exists in the prior art, cost is high, the deficiency that yield is low.
For overcoming the deficiency that prior art exists, the technology of spherosinin purification with continuous column chromatography of the present invention comprises the steps: successively
(1) pre-treatment of loco weed: loco weed is treated to dry powdered, subsequent use;
(2) obtaining of loco weed medicinal extract: with exsiccant loco weed powder with polar solvent repeatedly cold soaking carry, loco weed medicinal extract, for use;
(3) bullion preparation: gained medicinal extract through after the simple pre-treatment, is carried out rough segmentation with polar macroporous adsorption resin, and TLC detects, and it is subsequent use as bullion to merge the elutriant that contains trihydroxyoctahydroindolizidine;
(4) purifying trihydroxyoctahydroindolizidine: above-mentioned bullion is crossed purification by silica gel column chromatography, and TLC detects, and GC detects, the part of collecting trihydroxyoctahydroindolizidine relative content >=10% respectively; All the other parts that contain a small amount of trihydroxyoctahydroindolizidine merge, run up to a certain amount of after, repeat the purification by silica gel column chromatography step again;
(5) HPLC detect to obtain the pure article of trihydroxyoctahydroindolizidine: utilize the trihydroxyoctahydroindolizidine standard specimen to judge it and the peak position when detecting carrying out HPLC; Again the part of the collection in (4) being carried out HPLC under identical chromatographic conditions detects; Collect the effluent of this RT; After volatilizing solvent, recrystallization promptly gets the pure article of trihydroxyoctahydroindolizidine; Rest part merges, run up to a certain amount of after, repeat the purification by silica gel column chromatography step again.
Used polar macroporous adsorption resin is commercially available D101, YWD-03 and YWD-06 in the above-mentioned steps (3).
Used polar macroporous adsorption resin is commercially available YWD-06 in the above-mentioned steps (3).Its result of use is best.
The preferred version of above-mentioned steps is:
(2) obtaining of loco weed medicinal extract: the polar solvent that will pulverize 5～10 times of weight of dried loco weed powder cold soaking repeatedly extracts 4～6 times, each 5～8 days, cross the leaching supernatant, merge, the reclaim under reduced pressure polar solvent, loco weed medicinal extract, kept dry is for use;
(3) bullion preparation: get the medicinal extract that makes, with the fully dissolving of polar solvent-water (4～6: 1, v/v concentration) of 2～4 times of amounts; Centrifugal, filter, discard residue; After the supernatant concentration; Carry out rough segmentation with polar macroporous adsorption resin YWD-06, TLC detects, and it is subsequent use as bullion to merge the elutriant that contains trihydroxyoctahydroindolizidine;
(4) purifying trihydroxyoctahydroindolizidine: above-mentioned bullion is volatilized solvent 50～60 ℃ of water-baths, with an amount of chloroform-acetone-methyl alcohol-ammoniacal liquor (65～75: 5～7: 18～22: 4, v/v/v/v) fully dissolving; Centrifugal, filter, remove residue; After the supernatant concentration, cross purification by silica gel column chromatography.
In above-mentioned steps (2) or (3), polar solvent is ethanol or methyl alcohol.These two kinds of polar solvents are suitable to carry out large-scale industrialization production.
In the above-mentioned steps (2), polar solvent is an edible ethanol, and in the step (3), polar solvent is a methyl alcohol.
In the above-mentioned steps (1), be after the week, to place 60 ℃ of baking ovens to dry the loco weed natural air drying of plucking, thoroughly remove moisture, be ground into powder with disintegrating machine, kept dry.
It is to extract raw material that the present invention adopts loco weed, and directly extraction separation prepares trihydroxyoctahydroindolizidine from plant, and its advantage is:
1, operating process is easy: the present invention directly adopts continuous column chromatography with medicinal extract, makes operating process more easy, also is easier to production process is monitored, and is convenient to the realization that serialization is produced, and has effectively practiced thrift manpower.Especially obtain pure article through HPLC; The process of purifying and detection is united two into one; Thereby reduced produce earlier in the past, when detecting again; Error owing to personnel's operation causes also is beneficial to the raising of production efficiency and the reduction of production cost, finally for the industrialization production of trihydroxyoctahydroindolizidine at aspects such as antitumor drug and immunostimulants reliable raw material is provided.
2, cost is low: the present invention unites two into one the detection of trihydroxyoctahydroindolizidine and the two steps operation of producing of pure article; The purifying technique simple flow; Simplified the operation steps of producing in batches, also for the purity of last products obtained therefrom provides reliable result, facility investment is few; Cost is low, for its suitability for industrialized production is laid a good foundation.
3, purity is high: the present invention has carried out comprehensive consideration for guaranteeing product gas purity, and this advantage can obtain embodying from a plurality of steps of the present invention:
Because dopant species is many in the loco weed medicinal extract that step (2) obtains, composition is more complicated also, and it is not fine directly carrying out separating effect with silica gel; Damage to silica gel is also bigger; Therefore in the step (3) earlier with the polar macroporous adsorption resin rough segmentation, remove the nonpolar and low-pole impurity of major part, recycle silicon glue carries out next step purifying, such separating effect that reaches is better; It is also higher to separate the product purity that obtains, and helps final refining and obtains pure article.
Adopt in the step (4) GC to detect to be in order to choose the sample segments that next step will carry out purifying, just only in detected result, show contain trihydroxyoctahydroindolizidine part we be necessary to bring the purifying that carries out next step, and in existing known technology, do not do this step.So how, conclude in the bullion that distils of taking away and necessarily contain trihydroxyoctahydroindolizidine, how much its content can reach again, also do not get rid of the part that will contain trihydroxyoctahydroindolizidine when accepting or rejecting by rule of thumb and discard and damnous possibility.And the employing instrument detecting has at first been got rid of the possibility that this mistake is abandoned target compound, secondly, carries out next step purifying after the detection level again, has also increased the predictable of final purification result and yield.Collect the part of trihydroxyoctahydroindolizidine relative content >=10%; Be according to the repeated detection result and an empirical value of artificial regulation, regulation is because in the silica gel column chromatography process like this, and the outflow of trihydroxyoctahydroindolizidine is a successive process; There is not the hop value between " having " and " not having "; For content part seldom, because the sensitivity that GC detects is very high, the trihydroxyoctahydroindolizidine of trace also can be detected; If but took this sample that contains micro-trihydroxyoctahydroindolizidine to remove to carry out next step HPLC purification refine, production cost must be increased so; And all the other trihydroxyoctahydroindolizidine content 10% part is not to discard not yet, but merging in these segment sets; Reach a certain amount of after, carry out column chromatography purification again, repeat such step; Both avoided waste (guaranteeing yield to greatest extent); Reduced production cost again, this way also is to scale operation and customized.
Step has adopted retrocession to guarantee product gas purity in (5): can see that 1. obtaining the pure article of trihydroxyoctahydroindolizidine through decompression distillation and the present invention through the HPLC purifying in the background technology is two distinct purification route; The decompression distillation is the character that is prone to distillation according to trihydroxyoctahydroindolizidine; Make its distillation condensation and with other separating substances, and HPLC to separate be that polarity according to each material in the trihydroxyoctahydroindolizidine bullion is different and have the characteristic RT under given conditions and carry out isolating.This difference is to influence that separating effect produced: easy distillation is a general character; That is to say if in bullion, also exist other to be prone to the material of distillation; Also can come out with the trihydroxyoctahydroindolizidine condensation separation that distils together; And the introducing of this impurity will be to avoid, and also can't adopt the resublime method to remove; And separate according to the RT of compound through HPLC is that characteristic properties according to compound carries out isolating; The stratographic meliority just is that specific material has specific RT under given conditions; Therefore can therefore have uniqueness, even be both strong polar compound with the qualitative index of RT as material according to the isolating result of RT; When carrying out the HPLC separation; Also can show different RTs, thereby guarantee that at collected effluent of this time all be only to contain trihydroxyoctahydroindolizidine, further guarantee the purity of cutting.
2. distil with reducing pressure and obtain after the product; And how much purity of not knowing products therefrom is on earth; Will detect once more; At this moment in sampling, sample pretreatment, analyzing and testing, all can inevitably introduce error, detecting result who obtains and the actual purity of gathering in the crops product so also has certain deviation, and very likely in sampling process, pollutes remaining sample; And adopt HPLC separate can online detection effluent in the purity of title product, and autotelic collect trihydroxyoctahydroindolizidine and flow out part, can more effectively guarantee its purity.
3. in the decompression distillation, owing to the bad control of vacuum tightness of decompression, along with the time duration of decompression distillation; SW floss compacting, closely can be made, under vacuum pressure is controlled improperly situation, trihydroxyoctahydroindolizidine bullion bumping (trihydroxyoctahydroindolizidine and be prone to moisture absorption can be made; So be difficult to guarantee the bullion complete drying), can make like this on the trihydroxyoctahydroindolizidine of other impurity components attached to condensation, pollute; Can avoid this problem fully, further guarantee product gas purity and adopt HPLC to separate.
4, extraction yield is high: the present invention has adopted spherosinin purification with continuous column chromatography; Be convenient to serialization production and monitoring, compare, saved the reagent usage quantity greatly with traditional extracting and separating; Improved separating effect; Also avoided simultaneously in the decompression distillation, because the control of temperature and pressure is improper, the problem that the extraction yield that causes descends.Extraction yield in the technology of using, is expected to be used for the large-scale production of trihydroxyoctahydroindolizidine apparently higher than at present.
Four, description of drawings
The technological process of Fig. 1 spherosinin purification with continuous column chromatography;
The ultraviolet of Fig. 2 trihydroxyoctahydroindolizidine (UV) spectrogram;
Infrared (IR) spectrogram of Fig. 3 trihydroxyoctahydroindolizidine;
The mass spectrum of Fig. 4 trihydroxyoctahydroindolizidine (MS) figure;
The gas chromatographic detection result of Fig. 5 trihydroxyoctahydroindolizidine standard specimen;
The gas chromatographic detection result of elutriant sample behind Fig. 6 purification by silica gel column chromatography;
The performance liquid chromatography detected result of Fig. 7 trihydroxyoctahydroindolizidine standard specimen;
The performance liquid chromatography detected result of elutriant sample behind Fig. 8 purification by silica gel column chromatography.
Five, embodiment
Understand the present invention for clearer, below in conjunction with accompanying drawing and embodiment the present invention is made further detailed description, but the present invention is not limited to these embodiment, the flow process of embodiment is referring to Fig. 1:
Embodiment 1: the extraction of trihydroxyoctahydroindolizidine in the Herba Oxytropis Kansuensis
(1) pre-treatment of loco weed: the Herba Oxytropis Kansuensis grass natural air drying of harvesting places 60 ℃ of baking ovens to dry after one week, thoroughly removes moisture, is ground into powder with disintegrating machine, kept dry.
(2) obtaining of loco weed medicinal extract: with Herba Oxytropis Kansuensis hay 500g, extract 4 times with 3L edible ethanol cold soaking, each 7 days, filter, merge ethanol liquid, decompression recycling ethanol gets total medicinal extract 64.8g, extraction yield 12.96%.
(3) trihydroxyoctahydroindolizidine in polar macroporous adsorption resin (YWD-06, Cangzhou, Hebei waffle worker far away ltd produces) the initial gross separation loco weed medicinal extract:
1. the pre-treatment of loco weed medicinal extract: take by weighing the loco weed medicinal extract 10.058g of gained with the methanol-water of 30ml (5: 1, v/v) fully dissolving is centrifugal, filters, and discards residue, merges supernatant, it is for use to concentrate the back;
2. the pre-treatment of polar macroporous adsorption resin: before the use, use soaked in absolute ethyl alcohol 4h, be washed till effluent with clear water again and mix not muddyly with 3 times of clear water,, promptly can be used for general extraction with the moving phase of 3～5 times of column volumes flushing pillar;
3. polar macroporous adsorbent resin column chromatography: behind the application of sample, with methanol-water (5: 1, v/v) be at the uniform velocity drip washing of moving phase, connect portion from the post lower end with the every 50ml of volumetric flask, collect 30 parts altogether;
4. the TLC of column chromatography effluent detects: each elutriant is volatilized solvent in 60 ℃ of water-baths, fully dissolve with 2ml methyl alcohol respectively, carry out TLC and detect, merge the trihydroxyoctahydroindolizidine part.
(4) purification by silica gel column chromatography trihydroxyoctahydroindolizidine:
1. sample pretreatment: the elutriant that will contain trihydroxyoctahydroindolizidine part merges the back and in 60 ℃ of water-baths, volatilizes solvent, with chloroform-acetone-methyl alcohol-ammoniacal liquor of 10ml (70: 6: 20: 4, v/v/v/v) fully dissolve; Centrifugal, filter, remove residue; After supernatant merges, concentrate for use;
2. silica gel column chromatography: with chloroform-acetone-methyl alcohol-ammoniacal liquor (70: 6: 20: 4, v/v/v/v) be moving phase, wet method dress post, behind the application of sample, with moving phase wash-out at the uniform velocity, every 50ml collects a elutriant, collects 20 parts altogether;
3. the TLC of silica gel column chromatography effluent detects: each elutriant is volatilized solvent in 60 ℃ of water-baths, fully dissolve with 2ml methyl alcohol respectively, point sample carries out TLC and detects, and keeps through detecting the elutriant that proof contains trihydroxyoctahydroindolizidine, carries out the detection of gc (GC);
4. the GC of silica gel column chromatography effluent detects: after respectively trihydroxyoctahydroindolizidine standard specimen and sample to be checked being carried out Silanization reaction, and sample detection.Detected result shows that the RT of trihydroxyoctahydroindolizidine is: 6.161min, the sample of reservation trihydroxyoctahydroindolizidine relative content >=10%; Rest part merges, run up to a certain amount of after, repeat the purification by silica gel column chromatography step again.
(5) HPLC detects the pure article of trihydroxyoctahydroindolizidine that obtain: the trihydroxyoctahydroindolizidine standard specimen is carried out HPLC detect, judge the appearance time of trihydroxyoctahydroindolizidine.After will passing through the GC detection; The elutriant sample of trihydroxyoctahydroindolizidine relative content >=10% carries out HPLC and detects under identical chromatographic conditions, the effluent when the collection trihydroxyoctahydroindolizidine goes out the peak volatilizes solvent after the merging; Recrystallization obtains the pure article 11.021mg of trihydroxyoctahydroindolizidine, extraction yield 0.0142%.Rest part merges, run up to a certain amount of after, repeat the purification by silica gel column chromatography step again.
Embodiment 2: the extraction of trihydroxyoctahydroindolizidine in the yellowflower crazyweed herb
(1) pre-treatment of loco weed: behind the yellowflower crazyweed herb grass natural air drying of harvesting, place baking oven to dry, thoroughly remove moisture, be ground into powder with disintegrating machine, kept dry.
(2) obtaining of loco weed medicinal extract: with yellowflower crazyweed herb hay 500g, extract 5 times with the methyl alcohol cold soaking of 5L70%, each 5 days, filter, merge ethanol liquid, decompression recycling ethanol, total medicinal extract 59.3g, extraction yield 11.86%.
(3) trihydroxyoctahydroindolizidine in polar macroporous adsorption resin (commercially available D101) the initial gross separation loco weed medicinal extract:
1. the pre-treatment of loco weed medicinal extract: take by weighing the loco weed medicinal extract 10.032g of gained with the methanol-water of 40ml (4: 1, v/v) fully dissolving is centrifugal, filters, and discards residue, merges supernatant, it is for use to concentrate the back;
2. polar macroporous adsorption resin pre-treatment: before the use, use soaked in absolute ethyl alcohol 4h, it is not muddy in 3 times of clear water mixing to be washed till effluent with clear water again, and the moving phase flushing pillar with 3～5 times of column volumes promptly can be used for general extraction;
3. polar macroporous adsorbent resin column chromatography: behind the application of sample, with methanol-water (4: 1, v/v) be at the uniform velocity drip washing of moving phase, connect portion from the post lower end with the every 50ml of volumetric flask, collect 30 parts altogether;
4. the TLC of column chromatography effluent detects: each elutriant is volatilized solvent in 60 ℃ of water-baths, fully dissolve with 2ml methyl alcohol respectively, carry out TLC and detect, merge the trihydroxyoctahydroindolizidine part.
(4) purification by silica gel column chromatography trihydroxyoctahydroindolizidine:
1. sample pretreatment: the elutriant that will contain trihydroxyoctahydroindolizidine part merges the back and in 50 ℃ of water-baths, volatilizes solvent, with chloroform-acetone-methyl alcohol-ammoniacal liquor of 10ml (75: 5: 18: 4, v/v/v/v) fully dissolve; Centrifugal, filter, remove residue; After supernatant merges, concentrate for use;
2. silica gel column chromatography: with chloroform-acetone-methyl alcohol-ammoniacal liquor (75: 5: 18: 4, v/v/v/v) be moving phase, wet method dress post, behind the application of sample, with moving phase wash-out at the uniform velocity, every 50ml collects a elutriant, collects 20 parts altogether;
3. the TLC of column chromatography effluent detects: each elutriant is volatilized solvent in 50 ℃ of water-baths, fully dissolve with 2ml methyl alcohol respectively, point sample carries out TLC and detects, and keeps through detecting the elutriant that proof contains trihydroxyoctahydroindolizidine, carries out the detection of gc (GC);
4. the GC of column chromatography effluent detects: after respectively trihydroxyoctahydroindolizidine standard specimen and sample to be checked being carried out Silanization reaction, and sample detection.Detected result shows that the RT of trihydroxyoctahydroindolizidine is: 6.157min, the sample of reservation trihydroxyoctahydroindolizidine relative content >=10%; Rest part merges, run up to a certain amount of after, repeat the purification by silica gel column chromatography step again.
(5) HPLC detects the pure article of trihydroxyoctahydroindolizidine that obtain: the trihydroxyoctahydroindolizidine standard specimen is carried out HPLC detect, judge the appearance time of trihydroxyoctahydroindolizidine.After will passing through the GC detection; The trihydroxyoctahydroindolizidine relative content is higher than 10% elutriant sample to carry out HPLC and detects under identical chromatographic conditions, the effluent when collecting trihydroxyoctahydroindolizidine and going out the peak volatilizes solvent after the merging; Recrystallization obtains the pure article 8.251mg of trihydroxyoctahydroindolizidine, extraction yield 0.0098%.Rest part merges, run up to a certain amount of after, repeat the purification by silica gel column chromatography step again.
Embodiment 3: the extraction of trihydroxyoctahydroindolizidine in the variation Radix Astragali
(1) pre-treatment of loco weed: after one week of variation Radix Astragali natural air drying of harvesting, place 60 ℃ of baking ovens to dry, thoroughly remove moisture, be ground into powder with disintegrating machine, kept dry.
(2) obtaining of loco weed medicinal extract: the Radix Astragali 500g that will make a variation extracts 6 times with 3L edible ethanol cold soaking, and each 8 days, filter, merge ethanol liquid, decompression recycling ethanol gets total medicinal extract 65.3g, extraction yield 13.06%.
(3) trihydroxyoctahydroindolizidine in polar macroporous adsorption resin (YWD-03, Cangzhou, Hebei waffle worker far away ltd produces) the initial gross separation loco weed medicinal extract:
1. the pre-treatment of loco weed medicinal extract: take by weighing the loco weed medicinal extract 10.086g of gained with the methanol-water of 20ml (5: 1, v/v) fully dissolving is centrifugal, filters, and discards residue, merges supernatant, it is for use to concentrate the back;
2. polar macroporous adsorption resin pre-treatment: before the use, use soaked in absolute ethyl alcohol 4h, it is not muddy in 3 times of clear water mixing to be washed till effluent with clear water again, and the moving phase flushing pillar with 3～5 times of column volumes promptly can be used for general extraction;
3. polar macroporous adsorbent resin column chromatography: behind the application of sample, with methanol-water (5: 1, v/v) be at the uniform velocity drip washing of moving phase, connect portion from the post lower end with the every 50ml of volumetric flask, collect 30 parts altogether;
4. the TLC of column chromatography effluent detects: each elutriant is volatilized solvent in 60 ℃ of water-baths, fully dissolve with 2ml methyl alcohol respectively, carry out TLC and detect, merge the trihydroxyoctahydroindolizidine part.
(4) purification by silica gel column chromatography trihydroxyoctahydroindolizidine:
1. sample pretreatment: the elutriant that will contain trihydroxyoctahydroindolizidine part merges the back and in 60 ℃ of water-baths, volatilizes solvent, with chloroform-acetone-methyl alcohol-ammoniacal liquor of 10ml (70: 4: 22: 4, v/v/v/v) fully dissolve; Centrifugal, filter, remove residue; After supernatant merges, concentrate for use;
2. silica gel column chromatography: with chloroform-acetone-methyl alcohol-ammoniacal liquor (70: 4: 22: 4, v/v/v/v) be moving phase, wet method dress post, behind the application of sample, with moving phase wash-out at the uniform velocity, every 50ml collects a elutriant, collects 20 parts altogether;
3. the TLC of column chromatography effluent detects: each elutriant is volatilized solvent in 60 ℃ of water-baths, fully dissolve with 2ml methyl alcohol respectively, point sample carries out TLC and detects, and keeps through detecting the elutriant that proof contains trihydroxyoctahydroindolizidine, carries out the detection of gc (GC);
4. the GC of column chromatography effluent detects: after respectively trihydroxyoctahydroindolizidine standard specimen and sample to be checked being carried out Silanization reaction, and sample detection.Detected result shows that the RT of trihydroxyoctahydroindolizidine is: 6.162min, the sample of reservation trihydroxyoctahydroindolizidine relative content >=10%; Rest part merges, run up to a certain amount of after, repeat the purification by silica gel column chromatography step again.
(5) HPLC detects the pure article of trihydroxyoctahydroindolizidine that obtain: the trihydroxyoctahydroindolizidine standard specimen is carried out HPLC detect, judge the appearance time of trihydroxyoctahydroindolizidine.After will passing through GC and detecting, the trihydroxyoctahydroindolizidine relative content is higher than 10% elutriant sample to carry out HPLC and detects, and the effluent when collecting trihydroxyoctahydroindolizidine and going out the peak volatilizes solvent after the merging, and recrystallization obtains the pure article 10.428mg of trihydroxyoctahydroindolizidine, extraction yield 0.0135%.Rest part merges, run up to a certain amount of after, repeat the purification by silica gel column chromatography step again.
Following table has been listed the technical performance index of the trihydroxyoctahydroindolizidine of the embodiment of the invention 1 extraction.
The trihydroxyoctahydroindolizidine white, needle-shaped crystals, molecular weight 173,144～145 ℃ of fusing points, purity >=99%.UV, IR, MS, NMR, GC, HPLC spectrum data and trihydroxyoctahydroindolizidine standard diagram data fit like a glove.
Trihydroxyoctahydroindolizidine UV spectroscopic data: UV λ max:289 (log4.67), 249 (log3.83) (referring to Fig. 2).
Trihydroxyoctahydroindolizidine IR spectroscopic data: IRmax (KBr compressing tablet, cm
-1): 3414.70,3376.41 (OH absorption peaks); 3060.42,2942.40,2886.32 (CH absorption peaks); 2797.95～2724.55 (Bohlmanband), 1073.78 (C-O absorption peak) (referring to Fig. 3).
Trihydroxyoctahydroindolizidine MS (m/e) data: 173 (M+), 155 (M-H2O), 138 (M-H2O-OH), 116 (138-H2O-4H); 96,84,72,43 (being parent nucleus cleaved fragment peak) (referring to Fig. 4).
Trihydroxyoctahydroindolizidine GC chromatographic data: hydroxyl in the trihydroxyoctahydroindolizidine molecular structure, with the monose structural similitude, can't gasify, so the GC direct injection do not go out the peak, so at first will make it derivatize, on the GC collection of illustrative plates simple spike appears.Trihydroxyoctahydroindolizidine adds 60 ℃ of water-bath 15min of silylating reagent (BSTFA) with the pyridine dissolving, forms the TMS verivate.With OV-1701 (capillary column of 0.32mm * 30m) carries out GC to be analyzed, 195 ℃ of column temperatures, 6.161min goes out the peak.
Trihydroxyoctahydroindolizidine HPLC chromatographic data: through full wavelength scanner, selecting the maximum absorption wavelength of trihydroxyoctahydroindolizidine is 288nm, under this wavelength condition; Select ODS (4.6mm * 250mm; C18,5 μ m) chromatographic column, detect trihydroxyoctahydroindolizidine; Methyl alcohol-water gradient elution, trihydroxyoctahydroindolizidine goes out the peak at 5.484min.
The technical performance index of trihydroxyoctahydroindolizidine
Claims (3)
1. the technology of a spherosinin purification with continuous column chromatography comprises the steps: successively
⑴ the pre-treatment of loco weed: loco weed is treated to dry powdered, subsequent use;
⑵ obtaining of loco weed medicinal extract: the polar solvent that will pulverize 5～10 times of weight of dried loco weed powder cold soaking repeatedly extracts 4～6 times, each 5～8 days, cross the leaching supernatant, merge, the reclaim under reduced pressure polar solvent, loco weed medicinal extract, kept dry is for use;
⑶ bullion preparation: get the medicinal extract that makes, the volume ratio of using 2～4 times of amounts is that polar solvent-water of 4～6 ︰ 1 fully dissolves, and is centrifugal; Filter; Discard residue, after the supernatant concentration, carry out rough segmentation with commercially available D101, YWD-03 or YWD-06 polar macroporous adsorption resin; TLC detects, and it is subsequent use as bullion to merge the elutriant that contains trihydroxyoctahydroindolizidine;
⑷ purifying trihydroxyoctahydroindolizidine: above-mentioned bullion is volatilized solvent 50～60 ℃ of water-baths, and using an amount of volume ratio is that chloroform-acetone-methyl alcohol-ammoniacal liquor of 65～75 ︰, 5～7 ︰, 18～22 ︰ 4 fully dissolves, centrifugal; Filter, remove residue, after the supernatant concentration; Cross purification by silica gel column chromatography; TLC detects, and GC detects, the part of collecting trihydroxyoctahydroindolizidine relative content >=10% respectively; All the other parts that contain a small amount of trihydroxyoctahydroindolizidine merge, run up to a certain amount of after, repeat the purification by silica gel column chromatography step again;
⑸ HPLC detect to obtain the pure article of trihydroxyoctahydroindolizidine: utilize the trihydroxyoctahydroindolizidine standard specimen to judge it and the peak position when detecting carrying out HPLC; Again the part of the collection in (4) being carried out HPLC under identical chromatographic conditions detects; Collect the effluent of this RT; After volatilizing solvent, recrystallization promptly gets the pure article of trihydroxyoctahydroindolizidine; Rest part merges, run up to a certain amount of after, repeat the purification by silica gel column chromatography step again;
In said step (2) or (3), polar solvent is edible ethanol or methyl alcohol.
2. the technology of spherosinin purification with continuous column chromatography as claimed in claim 1, it is characterized in that: used polar macroporous adsorption resin is YWD-06 in the said step (3).
3. the technology of spherosinin purification with continuous column chromatography as claimed in claim 1, it is characterized in that: in the said step (2), polar solvent is an edible ethanol, in the step (3), polar solvent is a methyl alcohol.
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| CN102590378B (en) * | 2012-02-05 | 2013-09-11 | 西北农林科技大学 | Method for detecting content of swainsonine in locoweed endophytic fungi |
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| 刘志滨等.甘肃棘豆中苦马豆素提取工艺改进初报.《西北农林科技大学学报(自然科学版)》.2006,第34卷(第1期),97-99,104. * |
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