Five, embodiment
For a more clear understanding of the present invention, the present invention is described in further detail below in conjunction with drawings and Examples.
The present invention is a material with loco weed class plant-yellowflower crazyweed herb abundant on the NORTHWEST CHINA grassland, Herba Oxytropis Kansuensis, glabrous crazyweed, Astragalus strictus, the variation Radix Astragali etc., basic physicochemical property according to trihydroxyoctahydroindolizidine, adopt base exchange method to extract high polarity alkaloid crude product, use alkaline chloroform extracting again, the extracting part obtains the trihydroxyoctahydroindolizidine crude product through the silica gel column chromatography separation, and last fractional sublimation obtains the pure product of trihydroxyoctahydroindolizidine.
1. the purification principle of trihydroxyoctahydroindolizidine
Trihydroxyoctahydroindolizidine belongs to western pyridine Alkaloid in the poly-hydroxy indoles, and molecular weight is little, and polarity is big, soluble in water, methyl alcohol, ethanol, alkaline chloroform etc., and the easy moisture absorption, and have the distillation characteristic.Present technique technology is according to the basic physicochemical property of trihydroxyoctahydroindolizidine, at first adopt base exchange method to extract high polarity alkaloid crude product, use alkaline chloroform extracting again, the extracting part obtains the trihydroxyoctahydroindolizidine crude product through the silica gel column chromatography separation, and last fractional sublimation obtains the pure product of trihydroxyoctahydroindolizidine.2. the trihydroxyoctahydroindolizidine operational path of purifying
The first step: organic total composition extracts in the plant sample (loco weed grass meal).Take by weighing a certain amount of loco weed grass meal, with 5-10 doubly measure industrial methanol repeatedly cold soaking extract 4-6 time, each cold soaking 7 days filters, the merging methanol solution, reclaim under reduced pressure methyl alcohol gets medicinal extract.
Second step: the alkaloid crude product extracts.Reclaim the medicinal extract of methyl alcohol gained, with doubly (weight/volume) amount 1N hydrochloric acid repeated multiple times dissolving of 10-50, filter, to the last an acid liquid alkaloid is checked till the feminine gender.Merging filtrate is transferred pH4-5 with strong aqua, and by the hydrogen type cation exchange resin post, it is colourless earlier being eluted to effluent liquid with deionized water, uses 1N ammoniacal liquor wash-out again, collects alkaline eluant, reclaims solvent, drain the alkaloid crude product.
The 3rd step: the trihydroxyoctahydroindolizidine crude product extracts.The alkaloid crude product that second step obtained is used earlier the analytical pure dissolve with methanol, filters, and residue is abandoned or adopted, and methanol solution decompression and solvent recovery, gained medicinal extract are used alkaline chloroform extracting again, and combined chloroform liquid reclaims chloroform at last and gets the trihydroxyoctahydroindolizidine crude product.
The 4th step: trihydroxyoctahydroindolizidine separates.Adopt silica gel column chromatography, sample on the dry method, chloroform-methyl alcohol-ammoniacal liquor-water gradient elution, TLC detects, and merges the trihydroxyoctahydroindolizidine part, and decompressing and extracting gets buff powder sample trihydroxyoctahydroindolizidine.
The 5th step: trihydroxyoctahydroindolizidine purifying.The buff powder sample trihydroxyoctahydroindolizidine that the 4th step obtained adopts the fractional sublimation method to obtain the pure product of trihydroxyoctahydroindolizidine.
The contriver has provided following embodiment, but the invention is not restricted to these embodiment.
Embodiment 1: the extraction of trihydroxyoctahydroindolizidine in the Herba Oxytropis Kansuensis
The first step, medicinal extract extracts the stage: Herba Oxytropis Kansuensis hay powder 5kg, extract 4 times with 30 liters of industrial methanol cold soakings, each cold soaking 7 days filters, and merges methanol solution, and reclaim under reduced pressure methyl alcohol obtains total medicinal extract 680g, and paste-forming rate is 13.6%.
In second step, the total medicinal extract of coarse biometric alkaline extraction stage: 680g grinds molten with 6800mL1N hydrochloric acid repeated multiple times, filters, and acid liquid improvement bismuth potassium iodide inspection to the last merges acid liquid till being negative and reacting.The gained acid liquid is transferred pH to 4-5 with strong aqua, and by the hydrogen type cation exchange resin post, 100 times of amount deionized waters with applied sample amount are eluted to colourless earlier, again with 20 times of amount 1N ammoniacal liquor wash-outs, collect the basic stream fluid, decompression and solvent recovery gets 62.56g alkaloid crude product, and going out alkali content is 1.3%.
The 3rd step, the trihydroxyoctahydroindolizidine crude product extracts the stage: 62.56g alkaloid crude product is used earlier the analytical pure dissolve with methanol, filter, reclaim solvent, medicinal extract is used alkaline chloroform extracting again, till extract can't check trihydroxyoctahydroindolizidine, merges extract, the reclaim under reduced pressure chloroform extraction goes out trihydroxyoctahydroindolizidine crude product 4g, and extraction yield is 0.08%.
In the 4th step, trihydroxyoctahydroindolizidine extracts the stage: the column chromatography silica gel mixed sample of 4g trihydroxyoctahydroindolizidine crude product and equivalent, sample on the dry method, chloroform-methyl alcohol-ammoniacal liquor-water gradient elution, every 20mL collects 1 part, collects 300 components altogether, TLC checks that merging is similar, extracts trihydroxyoctahydroindolizidine 0.5g.
The 5th step trihydroxyoctahydroindolizidine purification phase: the 0.5g trihydroxyoctahydroindolizidine vacuumizes in special sublimation pipe and draws driedly, puts oil bath, adopts the fractional sublimation method to obtain the pure product 75mg of trihydroxyoctahydroindolizidine, and extraction yield is 0.0015%.
Embodiment 2: the extraction of trihydroxyoctahydroindolizidine in the yellowflower crazyweed herb
The first step medicinal extract extracts the stage: yellowflower crazyweed herb hay powder 11.5kg, extract 6 times with 70 liters of industrial methanol cold soakings, and each cold soaking 7 days filters, and merges methanol solution, and reclaim under reduced pressure methyl alcohol obtains total medicinal extract 1600g, and paste-forming rate is 13.9%.
The second coarse biometric alkaline extraction stage in step: the total medicinal extract of 1600g grinds molten with 16000mL1N hydrochloric acid repeated multiple times, filters, and acid liquid improvement bismuth potassium iodide inspection to the last merges acid liquid till being negative and reacting.The gained acid liquid is transferred pH to 4-5 with strong aqua, and by the hydrogen type cation exchange resin post, 100 times of amount deionized waters with applied sample amount are eluted to colourless earlier, again with 20 times of amount 1N ammoniacal liquor wash-outs, collect the basic stream fluid, decompression and solvent recovery gets 127g alkaloid crude product, and going out alkali content is 1.1%.
The 3rd step trihydroxyoctahydroindolizidine crude product extracts the stage: 127g alkaloid crude product is used earlier the analytical pure dissolve with methanol, filter, reclaim solvent, medicinal extract is used alkaline chloroform extracting again, till extract can't check trihydroxyoctahydroindolizidine, merge extract, the reclaim under reduced pressure chloroform extraction goes out trihydroxyoctahydroindolizidine crude product 5g, and extraction yield is 0.0043%.
The 4th step trihydroxyoctahydroindolizidine extracts the stage: the column chromatography silica gel mixed sample of g trihydroxyoctahydroindolizidine crude product and equivalent, sample on the dry method, chloroform-methyl alcohol-ammoniacal liquor-water gradient elution, every 30mL collects 1 part, collect 400 components altogether, TLC checks that merging is similar, extracts trihydroxyoctahydroindolizidine 0.58g.
The 5th step trihydroxyoctahydroindolizidine purification phase: the 0.58g trihydroxyoctahydroindolizidine vacuumizes in special sublimation pipe and draws driedly, puts oil bath, adopts the fractional sublimation method to obtain the pure product 105mg of trihydroxyoctahydroindolizidine, and extraction yield is 0.0009%.
Embodiment 3: the extraction of trihydroxyoctahydroindolizidine in the Herba Oxytropis Kansuensis
The first step medicinal extract extracts the stage: Herba Oxytropis Kansuensis hay powder 20kg, extract 4 times with 100 liters of industrial methanol cold soakings, and each cold soaking 5 days filters, and merges methanol solution, and reclaim under reduced pressure methyl alcohol obtains total medicinal extract 2230g, and paste-forming rate is 11.2%.
The second coarse biometric alkaline extraction stage in step: the total medicinal extract of 2230g grinds molten with 15000mL1N hydrochloric acid repeated multiple times, filters, and acid liquid improvement bismuth potassium iodide inspection to the last merges acid liquid till being negative and reacting.The gained acid liquid is transferred pH to 4-5 with strong aqua, and by the hydrogen type cation exchange resin post, 100 times of amount deionized waters with applied sample amount are eluted to colourless earlier, again with 30 times of amount 1N ammoniacal liquor wash-outs, collect the basic stream fluid, decompression and solvent recovery gets 219.1g alkaloid crude product, and going out alkali content is 1.095%.
The 3rd step trihydroxyoctahydroindolizidine crude product extracts the stage: 219.1g alkaloid crude product is used earlier the analytical pure dissolve with methanol, filter, reclaim solvent, get medicinal extract 155.4g, use alkaline chloroform extracting again, till extract can't check trihydroxyoctahydroindolizidine, merge extract, the reclaim under reduced pressure chloroform extraction goes out trihydroxyoctahydroindolizidine crude product 15g, and extraction yield is 0.075%.
The 4th step trihydroxyoctahydroindolizidine extracts the stage: the column chromatography silica gel mixed sample of 15g trihydroxyoctahydroindolizidine crude product and equivalent, sample on the dry method, chloroform-methyl alcohol-ammoniacal liquor-water gradient elution, every 50mL collects 1 part, collect 350 components altogether, TLC checks that merging is similar, extracts trihydroxyoctahydroindolizidine 2.1g.
The 5th step trihydroxyoctahydroindolizidine purification phase: the 2.1g trihydroxyoctahydroindolizidine vacuumizes in special sublimation pipe and draws driedly, puts oil bath, adopts the fractional sublimation method to obtain the pure product 236mg of trihydroxyoctahydroindolizidine, and extraction yield is 0.0012%.
Embodiment 4: the extraction of trihydroxyoctahydroindolizidine in the variation Radix Astragali
The first step medicinal extract extracts the stage: variation Radix Astragali hay powder 2kg, extract 6 times with 10 liters of industrial methanol cold soakings, and each cold soaking 7 days filters, and merges methanol solution, and reclaim under reduced pressure methyl alcohol obtains total medicinal extract 219g, and paste-forming rate is 10.95%.
The second coarse biometric alkaline extraction stage in step: the total medicinal extract of 219g grinds molten with 2500mL1N hydrochloric acid repeated multiple times, filters, and acid liquid improvement bismuth potassium iodide inspection to the last merges acid liquid till being negative and reacting.The gained acid liquid is transferred pH to 4-5 with strong aqua, and by the hydrogen type cation exchange resin post, 100 times of amount deionized waters with applied sample amount are eluted to colourless earlier, again with 30 times of amount 1N ammoniacal liquor wash-outs, collect the basic stream fluid, decompression and solvent recovery gets 22.797g alkaloid crude product, and going out alkali content is 1.14%.
The 3rd step trihydroxyoctahydroindolizidine crude product extracts the stage: 22.797g alkaloid crude product is used earlier the analytical pure dissolve with methanol, filter, reclaim solvent, medicinal extract is used alkaline chloroform extracting again, till extract can't check trihydroxyoctahydroindolizidine, merge extract, the reclaim under reduced pressure chloroform extraction goes out trihydroxyoctahydroindolizidine crude product 2.6g, and extraction yield is 0.13%.
The 4th step trihydroxyoctahydroindolizidine extracts the stage: the column chromatography silica gel mixed sample of 2.6g trihydroxyoctahydroindolizidine crude product and equivalent, sample on the dry method, chloroform-methyl alcohol-ammoniacal liquor-water (70: 26: 2: 2) wash-out, every 10mL collects 1 part, collect 200 components altogether, TLC checks that merging is similar, extracts trihydroxyoctahydroindolizidine 0.3g.
The 5th step trihydroxyoctahydroindolizidine purification phase: the 0.3g trihydroxyoctahydroindolizidine is placed special sublimation pipe, vacuumize and draw driedly, adopt the fractional sublimation method to obtain the pure product 35mg of trihydroxyoctahydroindolizidine, extraction yield is 0.0018%.
Institute of the present invention produce an effect is:
1. spherosin recovery rate
China loco weed class plant has 44 kinds, and distribution area surpasses 4,000,000 hectares, because its ecoenvironment of growth difference, so that the contained spherosin amount of loco weed class plant not of the same race is also variant. Adopt the technology of the present invention that spherosin in the following loco weed is extracted, extract and the results are shown in Table 1.
Spherosin extracts the result in table 1 China main loco weed class plant
Paste-forming rate goes out alkali content spherosin recovery rate
Botanical name sampling position
(%) (%) (%) kansu crazyweed herb (Qinghai) herb 13.6 1.3 0.0015 yellowflower crazyweed herbs (Ningxia) 13.9 1.1 0.0009 maos of lobe whins of herb (Tibet) herb 8.95 0.78 0.0008 wide luxuriant whins (Qinghai) herb 9.95 0.7 0.0012 on the ground on the ground on the ground on the ground
The ground herb
12.6 0.95 0.007 sharp turn whin (Qinghai) glacier whin (Tibet) is herb 8.96 0.85 0.0007 Astragalus strictus (Tibet) the ground herb 10.2 1.0 0.0006 variation Radixs Astragali (Ningxia) ground herbs 10.95 1.14 0.0018 on the ground
2. spherosin technical indicator
Table 2 has been listed the technical performance index of the trihydroxyoctahydroindolizidine of the present invention's extraction.
Trihydroxyoctahydroindolizidine white, needle-shaped crystals, molecular weight are 173,144~145 ℃ of fusing points, purity 〉=98%.UV, IR, GC, MS, NMR spectroscopic data and trihydroxyoctahydroindolizidine standard spectrum 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: IR
Max(KBr) cm
-1: 3431,3370 (OH), 2946,2806 (CH), 2806~2725 (Bohlman band), 1074 absorption peaks (referring to Fig. 3) such as (C-O).
Trihydroxyoctahydroindolizidine GC spectroscopic data: contain hydroxyl in the trihydroxyoctahydroindolizidine molecular structure, with the monose structural similitude, so the GC direct injection do not go out the peak, analyzes and must at first be prepared into the silicon ether derivant so will carry out GC.Trihydroxyoctahydroindolizidine dissolves with 100 μ l pyridines, with 100 μ l BSTFA room temperature treatment 30min, forms the TMS derivative.Carry out gas-chromatography at 0.32mm * 30m SE-30 capillary column (tool injection post).Column temperature is increased to 300 ℃ gradually from 120 ℃, and 5 ℃/min, 13.77min goes out the peak.
Trihydroxyoctahydroindolizidine MS spectroscopic data: EIMSm/e:173 (M
+), 155 (M-H
2O), 138 (M-H
2O-OH), 116,115,96,84,72,43 (parent nucleus cleaved fragment peak) (referring to Fig. 4).
3. purifying technique extraction cost of the present invention is low, the product purity height, and the trihydroxyoctahydroindolizidine extraction yield obviously is better than foreign technology.
The technical performance index of table 2 trihydroxyoctahydroindolizidine
Index system | Technical feature |
Outward appearance | The white fine needle crystal |
Fusing point | ????144~145℃ |
Thin-layer chromatography | R on silica-gel plate
fBe worth 0.44 place one red-purple spot is arranged
|
Infrared spectra (IR) | There is typical B ohlman band to absorb |
Mass spectrum (MS) | Molecular ion peak is 173 |
Gas-chromatography (GC) | TMS derivative retention time 13.77min |
Quality guaranteed period | Asepticly vacuumize packing, sealing is stored in-4 ℃ of refrigerators, 3 years quality guaranteed perioves |
Biochemical characteristic | Efficient, the single-minded inhibitor of a-mannosidase |
Molecular weight | ????173 |
Acid dissociation constant | ????Pka?7.4 |