CN102872015B - Stephanotis total alkaloid extract as well as preparation method and application thereof - Google Patents
Stephanotis total alkaloid extract as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a stephanotis total alkaloid extract as well as a preparation method of the stephanotis total alkaloid extract. The preparation method of the stephanotis total alkaloid extract comprises the following steps of: grinding the dried stephanotis, heating and extracting, and decompressing and condensing the filtrate; diluting with heating water, and acidifying and filtrating to obtain a filtrate; alkalifying and filtrating to obtain the filtrate, and performing molecular weight cut off filtration on the obtained filtrate through a ceramic membrane filter; extracting with n-butyl alcohol, decompressing and condensing; spray drying the concentrated solution to obtain a dried powder extract; adding ethanol for ultrasonic extraction, mixing the extracting solution, absorbing the extracting solution by a sephadex column, eluting with 90% to 95% of ethanol, segmentally collecting, identifying through a high performance thin layer chromatography, mixing the parts containing alkaloids, freezing at a low temperature, and separating out an alkaloid crystal crude product; and adding ethanol for dissolving, standing and freezing to obtain a stephanotis total alkaloid pure product. A stephanotis total alkaloid medicinal preparation provided by the invention has the effect of adjuvant therapy on leucopenia, in particular to leucopenia of the tumor patients caused by radiotherapy and radiotherapy, and the leucopenia caused by other medicines.
Description
Technical field
The present invention relates generally to medical technical field, relates in particular to a kind of Radix Stephaniae Cepharanthae total alkaloids extract and preparation method thereof and application.
Background technology
Radix Stephaniae Cepharanthae is the tuber of menispermaceous plants headdress flower Radix Stephaniae Japonicae.Grow in fertile moistening thick grass, cloudy place, roadside, hillside or spinney, be also born on calcium carbonate tor; Gather summer and autumn, and section is dried.Aboundresources, all there is distribution China middle and south.There is heat-clearing and toxic substances removing; Wind-expelling pain-stopping; Cooling blood for hemostasis effect, ancient Chinese medicine is used for the treatment of
throatswell and ache, pyretic toxicity carbuncle, rheumatic arthralgia, stomachache
dysentery, hematemesis and epistaxis, traumatic hemorrhage etc.Bitter in the mouth is pungent, cool.
The conventional this product of modern Chinese medicine is treated all kinds of infection, the clinical remarkable result of obtaining.Therefore we carry out the researchs such as system component, drug effect, toxicity to Radix Stephaniae Cepharanthae, confirm that this product has the effect for the treatment of leukopenia, but existing research are less to the research of Radix Stephaniae Cepharanthae total alkaloids for treating leukopenia, there is no related documents and records or study report.Because the decoction of decoction is cumbersome, fall behind, dosage is large, and can not play Effect of first aid, and active ingredient is unstable, indefinite, invalid impurity is too many, be unfavorable for popularization and the clinical practice of Radix Stephaniae Cepharanthae treatment leukopenia, therefore, exploitation high standard, efficiently, long-acting, quick-acting, the treatment leukopenia Radix Stephaniae Cepharanthae effective fraction medicine of safety becomes the task of top priority.
Summary of the invention
The object of the invention is exactly the defect in order to make up prior art, and a kind of Radix Stephaniae Cepharanthae total alkaloids extract and preparation method thereof and application are provided.
The present invention is achieved by the following technical solutions:
A kind of Radix Stephaniae Cepharanthae total alkaloids extract, it contains the isoquinoline alkaloid with following chemical constitution:
(1) cepharanthine molecular formula: C
37h
38n
2o
6
Molecular weight: 606.71
(2) isotetrandrine molecular formula: C
38h
42n
2o
6
Molecular weight: 622.75
(3) cycleanine molecular formula: C
38h
42n
2o
6
Molecular weight: 622.75
(4) berbamine molecular formula: C
37h
40n
2o
6
Molecular weight: 608.72
A preparation method for Radix Stephaniae Cepharanthae total alkaloids extract, comprises the following steps:
(1) Radix Stephaniae Cepharanthae dry product is ground into coarse powder, adding concentration is 70～100% ethanol heating extraction, and extracting solution is crossed 100～200 mesh sieves, and filtrate decompression concentrates to obtain concentrated solution;
(2) the concentrated solution heating water dilution of being prepared by step (1), adds hydrochloric acid and adjusts pH to 2～5 acidify, stirs, and leaves standstill, and filters to get filtrate;
(3) prepared by step (2) to filtrate hydro-oxidation sodium and adjust pH to 9～12 alkalization, stir, leave standstill, filter to get filtrate, gained filtrate is carried out to molecular retention filtration through purpose ceramic-film filter, and ceramic membrane filter aperture is 0.1 μ m～0.3 μ m, and gained filtrate is fine straining liquid;
(4) the fine straining liquid in step (3) is passed through to processed good ion exchange resin column, impurity is removed in washing, then uses 80～95% ethanol elutions, collects eluent, concentrating under reduced pressure; Or by the fine straining liquid n-butanol extraction of step (3), collect extract, concentrating under reduced pressure;
(5) the concentrated solution spraying of step (4) is dried to obtain to powder extract;
(6) powder extract in step (5) is added to ethanol ultrasonic extraction, merge extractive liquid,, extracting solution passes through sephadex column, with 90～95% ethanol elution, Fractional Collections, differentiate through high performance thin-layer chromatography, merge and contain alkaloid position, freezing, separates out alkaloid crystal crude product;
(7) the alkaloid crystal crude product of step (6) is added to dissolve with ethanol, leave standstill, freezing, obtain Radix Stephaniae Cepharanthae total alkaloids sterling, dry, pulverize, obtain Radix Stephaniae Cepharanthae total alkaloids.
The application of described Radix Stephaniae Cepharanthae total alkaloids extract in preparation treatment leukopenia disease drug.
Advantage of the present invention is: described Radix Stephaniae Cepharanthae total alkaloids pharmaceutical preparation, and for leukopenia, the leukopenia that especially tumour patient Radiotherapy chemotherapy causes, and leukopenia due to other drug has the effect of auxiliary treatment.
Detailed description of the invention
Embodiment 1
Radix Stephaniae Cepharanthae total alkaloids extracting method:
The Radix Stephaniae Cepharanthae of getting recipe quantity adds 6 times of amounts of ethanol and carries out the extraction of adverse current dynamic heat, each 3 hours, collect extracting solution, filter, filtrate decompression is concentrated into (temperature 70 C, vacuum number-0.08Npa) every 1ml is equivalent to primary crude drug 5g, with hot water dissolving, adds hcl acidifying, stir, leave standstill, filter to obtain filtrate, cold preservation leaves standstill 48 hours, filter, add alkali alkalization, stir, leave standstill, filter to obtain filtrate, gained medicinal liquid is carried out to molecular retention filtration through purpose ceramic-film filter, and ceramic membrane filter aperture is 0.1 μ m～0.3 μ m, and gained filtrate is fine straining liquid; By fine straining liquid, by processed good ionic resin post, impurity is removed in washing, then uses 85% ethanol elution, collects eluent, concentrating under reduced pressure; Spraying is dried to obtain powder extract again.Powder extract adds ethanol ultrasonic extraction secondary, merge extractive liquid,, and extracting solution passes through sephadex column, collection of biological alkali position, freezing, separates out alkaloid crystal crude product; Alkaloid crystal crude product adds dissolve with ethanol, leaves standstill, freezing, obtains Radix Stephaniae Cepharanthae total alkaloids sterling, dry, pulverize, and obtains Radix Stephaniae Cepharanthae total alkaloids;
Adjusting pH to 2～5 is acidify, adjusts pH to 9～12 for alkalization, lower same.
Embodiment 2
Radix Stephaniae Cepharanthae total alkaloids extracting method:
The Radix Stephaniae Cepharanthae of getting recipe quantity adds 8 times of amounts of ethanol and carries out the extraction of adverse current dynamic heat, each 2 hours, collect extracting solution, filter, filtrate decompression is concentrated into (temperature 70 C, vacuum number-0.08Npa) every 1ml is equivalent to primary crude drug 4g, with hot water dissolving, adds hcl acidifying, stir, leave standstill, filter to obtain filtrate, cold preservation leaves standstill 48 hours, filter, add alkali alkalization, stir, leave standstill, filter to obtain filtrate, gained medicinal liquid is carried out to molecular retention filtration through purpose ceramic-film filter, and ceramic membrane filter aperture is 0.1 μ m～0.2 μ m, and gained filtrate is fine straining liquid; By fine straining liquid, by processed good ionic resin post, impurity is removed in washing, then uses 90% ethanol elution, collects eluent, concentrating under reduced pressure; Spraying is dried to obtain powder extract again.Powder extract adds ethanol ultrasonic extraction secondary, merge extractive liquid,, and extracting solution passes through sephadex column, collection of biological alkali position, freezing, separates out alkaloid crystal crude product; Alkaloid crystal crude product adds dissolve with ethanol, leaves standstill, freezing, obtains Radix Stephaniae Cepharanthae total alkaloids sterling, dry, pulverize, and obtains Radix Stephaniae Cepharanthae total alkaloids;
Embodiment 3
Radix Stephaniae Cepharanthae total alkaloids extracting method:
The Radix Stephaniae Cepharanthae of getting recipe quantity adds 5 times of amounts of ethanol and carries out the extraction of adverse current dynamic heat, each 4 hours, collect extracting solution, filter, filtrate decompression is concentrated into (temperature 70 C, vacuum number-0.08Npa) every 1ml is equivalent to primary crude drug 5g, with hot water dissolving, adds sulfuric acid acidation, stir, leave standstill, filter to obtain filtrate, cold preservation leaves standstill 48 hours, filter, add alkali alkalization, stir, leave standstill, filter to obtain filtrate, gained medicinal liquid is carried out to molecular retention filtration through purpose ceramic-film filter, and ceramic membrane filter aperture is 0.2 μ m～0.3 μ m, and gained filtrate is fine straining liquid; By fine straining liquid, by processed good ionic resin post, impurity is removed in washing, then uses ethanol elution, collects eluent, concentrating under reduced pressure; Spraying is dried to obtain powder extract again.Powder extract adds ethanol ultrasonic extraction secondary, merge extractive liquid,, and extracting solution passes through sephadex column, collection of biological alkali position, freezing, separates out alkaloid crystal crude product; Alkaloid crystal crude product adds dissolve with ethanol, leaves standstill, freezing, obtains Radix Stephaniae Cepharanthae total alkaloids sterling, dry, pulverize, and obtains Radix Stephaniae Cepharanthae total alkaloids;
Embodiment 4
Radix Stephaniae Cepharanthae total alkaloids extracting method:
The Radix Stephaniae Cepharanthae of getting recipe quantity adds 6 times of amounts of ethanol and carries out the extraction of adverse current dynamic heat, each 3 hours, collect extracting solution, filter, filtrate decompression is concentrated into (temperature 70 C, vacuum number-0.08Npa) every 1ml is equivalent to primary crude drug 5g, with hot water dissolving, adds sulfuric acid acidation, stir, leave standstill, filter to obtain filtrate, cold preservation leaves standstill 48 hours, filter, add alkali alkalization, stir, leave standstill, centrifugal filtering obtains filtrate, gained medicinal liquid is carried out to molecular retention filtration through purpose ceramic-film filter, and ceramic membrane filter aperture is 0.1 μ m～0.3 μ m, and gained filtrate is fine straining liquid; By fine straining liquid n-butanol extraction, combining extraction liquid, concentrating under reduced pressure; Spraying is dried to obtain powder extract again.Powder extract adds ethanol ultrasonic extraction secondary, merge extractive liquid,, and extracting solution passes through sephadex column, collection of biological alkali position, freezing, separates out alkaloid crystal crude product; Alkaloid crystal crude product adds dissolve with ethanol, leaves standstill, freezing, obtains Radix Stephaniae Cepharanthae total alkaloids sterling, dry, pulverize, and obtains Radix Stephaniae Cepharanthae total alkaloids;
Embodiment 5
Radix Stephaniae Cepharanthae total alkaloids extracting method:
The Radix Stephaniae Cepharanthae of getting recipe quantity adds 6 times of amounts of ethanol and carries out the extraction of adverse current dynamic heat, each 3 hours, collect extracting solution, filter, filtrate decompression is concentrated into (temperature 70 C, vacuum number-0.08Npa) every 1ml is equivalent to primary crude drug 5g, with hot water dissolving, adds hcl acidifying, stir, leave standstill, filter to obtain filtrate, cold preservation leaves standstill 48 hours, filter, add alkali alkalization, stir, leave standstill, filter to obtain filtrate, gained medicinal liquid is carried out to molecular retention filtration through purpose ceramic-film filter, and ceramic membrane filter aperture is 0.1 μ m～0.3 μ m, and gained filtrate is fine straining liquid; By fine straining liquid n-butanol extraction, combining extraction liquid, concentrating under reduced pressure; Spraying is dried to obtain powder extract again.Powder extract adds ethanol ultrasonic extraction secondary, merge extractive liquid,, and extracting solution passes through sephadex column, collection of biological alkali position, freezing, separates out alkaloid crystal crude product; Alkaloid crystal crude product adds dissolve with ethanol, leaves standstill, freezing, obtains Radix Stephaniae Cepharanthae total alkaloids sterling, dry, pulverize, and obtains Radix Stephaniae Cepharanthae total alkaloids;
Embodiment 6
Radix Stephaniae Cepharanthae total alkaloids extracting method:
The Radix Stephaniae Cepharanthae of getting recipe quantity adds 8 times of amounts of 85% ethanol and carries out the extraction of adverse current dynamic heat, each 3 hours, collect extracting solution, filter, filtrate decompression is concentrated into (temperature 70 C, vacuum number-0.08Npa) every 1ml is equivalent to primary crude drug 4g, with hot water dissolving, adds hcl acidifying, stir, leave standstill, filter to obtain filtrate, cold preservation leaves standstill 48 hours, filter, add alkali alkalization, stir, leave standstill, filter to obtain filtrate, gained medicinal liquid is carried out to molecular retention filtration through purpose ceramic-film filter, and ceramic membrane filter aperture is 0.1 μ m～0.3 μ m, and gained filtrate is fine straining liquid; By fine straining liquid, by processed good ionic resin post, impurity is removed in washing, then uses ethanol elution, collects eluent, concentrating under reduced pressure; Spraying is dried to obtain powder extract again.Powder extract adds ethanol ultrasonic extraction secondary, merge extractive liquid,, and extracting solution passes through sephadex column, collection of biological alkali position, freezing, separates out alkaloid crystal crude product; Alkaloid crystal crude product adds dissolve with ethanol, leaves standstill, freezing, obtains Radix Stephaniae Cepharanthae total alkaloids sterling, dry, pulverize, and obtains Radix Stephaniae Cepharanthae total alkaloids;
Embodiment 7
Radix Stephaniae Cepharanthae total alkaloids extracting method:
The Radix Stephaniae Cepharanthae of getting recipe quantity adds 8 times of amounts of 75% ethanol and carries out the extraction of adverse current dynamic heat, each 4 hours, collect extracting solution, filter, filtrate decompression is concentrated into (temperature 70 C, vacuum number-0.08Npa) every 1ml is equivalent to primary crude drug 5g, with hot water dissolving, adds hcl acidifying, stir, leave standstill, filter to obtain filtrate, cold preservation leaves standstill 48 hours, filter, add alkali alkalization, stir, leave standstill, filter to obtain filtrate, gained medicinal liquid is carried out to molecular retention filtration through purpose ceramic-film filter, and ceramic membrane filter aperture is 0.1 μ m～0.3 μ m, and gained filtrate is fine straining liquid; By fine straining liquid, by processed good ionic resin post, impurity is removed in washing, then uses 95% ethanol elution, collects eluent, concentrating under reduced pressure; Spraying is dried to obtain powder extract again.Powder extract adds ethanol ultrasonic extraction secondary, merge extractive liquid,, and extracting solution passes through sephadex column, collection of biological alkali position, freezing, separates out alkaloid crystal crude product; Alkaloid crystal crude product adds dissolve with ethanol, leaves standstill, freezing, obtains Radix Stephaniae Cepharanthae total alkaloids sterling, dry, pulverize, and obtains Radix Stephaniae Cepharanthae total alkaloids;
Contrast test 1:
120 of Kunming mouses, male and female half and half, are divided into 6 groups: Normal group, model control group, the high, medium and low dosage group of Radix Stephaniae Cepharanthae total alkaloids, batilol group.Above 6 treated animals, except normal control treated animal, all the other 5 treated animals are all in first 1 day use of administration
60once, dosage is 4Gy to Co-radiation gamma, after 24 hours, is divided into immediately 5 groups again, only removes Normal group and model control group gavage distilled water 0.4ml/, and all the other each groups are by various dose gastric infusion, every day 1 time, continuous 7 days.Last administration 24 hours, pulls out eyeball of mouse and gets blood, detects total white blood cells (WBC), erythrocyte sum (RBC), platelet count (PLT) in blood, the results are shown in Table 1.
Table 1 Radix Stephaniae Cepharanthae total alkaloids pair
60the impact (X ± SD) that Co-gamma-radiation damage mouse blood is learned
Note: model group and Normal group comparison,
△ △p < 0.01
Medication therapy groups and model group comparison, * P < 0.05, * * P < 0.01
The demonstration of table 1 result, the each dosage of Radix Stephaniae Cepharanthae total alkaloids and model control group relatively have extremely significantly row or significant difference, i.e. Radix Stephaniae Cepharanthae total alkaloids pair
60due to Co-gamma-radiation, external blood leukocytes reduces obvious protective effect, and big or middle dosage group curative effect is obviously better than positive control drug batilol group.
Contrast test 2:
120 of Kunming mouses, male and female half and half, are divided into 6 groups at random, i.e. Normal group, cyclophosphamide model control group (Cy), the high, medium and low dosage group of Radix Stephaniae Cepharanthae total alkaloids (Cy+), Cy+ batilol group.Except Normal group, all the other are respectively organized the equal intraperitoneal injection of cyclophosphamide of mice (Cy) 150mg/kg and cause leukopenia model.Cy started to give distilled water by 20ml/kg respectively by normal group and Cy model control group the same day with injection.All the other each group gives respectively different pharmaceutical by various dose, every day 1 time, gastric infusion, continuous 10 days.After last administration, after 24 hours, pluck eyeball and get blood, and detect total white blood cells WBC in blood, the results are shown in Table 2.
Table 2 Radix Stephaniae Cepharanthae total alkaloids causes on cyclophosphamide the impact (X ± SD) that murine interleukin reduces
| Group | Number of animals (only) | Dosage (mg/kg) | WBC（×10 9/L） |
| Normal group | 20 | -- | 8.02±1.78 |
| Cy model group | 20 | -- | 0.66±0.11 △△ |
| Cy+ high dose group | 20 | 0.60 | 1.03±0.88 |
| Dosage group in Cy+ | 20 | 0.30 | 4.62±2.91** |
| Cy+ low dose group | 20 | 0.15 | 1.59±1.74** |
| Cy batilol group | 20 | 100 | 2.67±1.44** |
Note: model group and Normal group comparison,
△ △p < 0.01
Medication therapy groups and model group comparison, * P < 0.05, * * P < 0.01
The demonstration of table 2 result, the each dosage of Radix Stephaniae Cepharanthae total alkaloids and model control group relatively have utmost point significance or significant difference, and Radix Stephaniae Cepharanthae total alkaloids reduces and has obvious protective effect caused by cyclophosphamide peripheral blood leucocyte.
Can prove that by above pharmacological evaluation Radix Stephaniae Cepharanthae total alkaloids of the present invention has obvious therapeutical effect to bone marrow injury and leukopenia.
Claims (2)
1. a preparation method for Radix Stephaniae Cepharanthae total alkaloids extract, is characterized in that, concrete steps are as follows:
(1) Radix Stephaniae Cepharanthae dry product is ground into coarse powder, adding concentration is 70～100% ethanol heating extraction, and extracting solution is crossed 100～200 mesh sieves, and filtrate decompression concentrates to obtain concentrated solution;
(2) the concentrated solution heating water dilution of being prepared by step (1), adds hydrochloric acid and adjusts pH to 2～5 acidify, stirs, and leaves standstill, and filters to get filtrate;
(3) prepared by step (2) to filtrate hydro-oxidation sodium and adjust pH to 9～12 alkalization, stir, leave standstill, filter to get filtrate, gained filtrate is carried out to molecular retention filtration through purpose ceramic-film filter, and ceramic membrane filter aperture is 0.1 μ m～0.3 μ m, and gained filtrate is fine straining liquid;
(4) the fine straining liquid in step (3) is passed through to ion exchange resin column, impurity is removed in washing, then uses 80～95% ethanol elutions, collects eluent, concentrating under reduced pressure; Or by the fine straining liquid n-butanol extraction of step (3), collect extract, concentrating under reduced pressure;
(5) the concentrated solution spraying of step (4) is dried to obtain to powder extract;
(6) powder extract in step (5) is added to ethanol ultrasonic extraction, merge extractive liquid,, extracting solution passes through sephadex column, with 90～95% ethanol elution, Fractional Collections, differentiate through high performance thin-layer chromatography, merge and contain alkaloid position, freezing, separates out alkaloid crystal crude product;
(7) the alkaloid crystal crude product of step (6) is added to dissolve with ethanol, leave standstill, freezing, obtain Radix Stephaniae Cepharanthae total alkaloids sterling, dry, pulverize, obtain Radix Stephaniae Cepharanthae total alkaloids.
2. the application of Radix Stephaniae Cepharanthae total alkaloids extract claimed in claim 1 in preparation treatment leukopenia disease drug.
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| CN103405436A (en) * | 2013-07-25 | 2013-11-27 | 合肥七星医药科技有限公司 | Cycleanine drug for treating leucopenia and aplastic anemia and preparation thereof |
| CN103432134B (en) * | 2013-07-25 | 2016-01-20 | 合肥七星医药科技有限公司 | The isotetrandrine medicine of leukocyte increasing and treatment aplastic anemia and preparation thereof |
| CN105853424A (en) * | 2016-03-30 | 2016-08-17 | 安徽九方药物研究院有限公司 | Alkaloid composition and application thereof |
| CN109111458A (en) * | 2018-07-06 | 2019-01-01 | 湖州展舒生物科技有限公司 | The preparation method of cycleanine |
| CN112142667B (en) * | 2019-06-28 | 2021-12-07 | 沈阳药科大学 | Alkaloid compound and preparation method and application thereof |
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