CN103083388A - Preparation method of fructus gleditsiae total saponins - Google Patents
Preparation method of fructus gleditsiae total saponins Download PDFInfo
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Abstract
The invention discloses a preparation method of fructus gleditsiae total saponins. The preparation method is characterized by comprising the following steps of: (1) taking a dried fructus gleditsiae medical material, and grinding the medical material into coarse powder; (2) taking the fructus gleditsiae coarse powder, adding deionized water, decocting and filtering out, extracting for three times, combining filtrate, and carrying vacuum concentration to obtain liquid medicine; (3) taking the liquid medicine, extracting with a water saturated n-butyl alcohol solution for 3 times, combining n-butyl alcohol extracting solutions, reducing pressure and recovering to obtain an n-butyl alcohol extract; and (4) taking the n-butyl alcohol extract, adding water for dissolution, filtering out, collecting filtrate, adding into a D101 macroporous resin column, firstly eluting with distilled water till the filtrate is near colorless, then eluting with 10% ethanol till the filtrate is near colorless, finally eluting with 70% ethanol, collecting 70% ethanol eluant, and drying under reduced pressure to obtain the fructus gleditsiae total saponins. The fructus gleditsiae total saponins obtained by the method is light brown orange powder, acrid and salty in taste and has pungent smell of saponin; the content of the total saponins is above 80%; and the fructus gleditsiae total saponins can be used for treating acute and chronic leukemia.
Description
Technical field
The present invention relates to a kind of preparation method of Fructus Gleditsiae Abnormalis total saponins.
Background technology
Cancer and leukemia (claiming again leukemia) are serious threat human life's malignant diseases, account for first of death toll, two, although great manpower and fund have all been dropped in countries in the world, captured research, and obtained certain progress, but at present still without desirable medicine and method, at present clinical in early, in, patient with advanced cancer adopts the hands art more, operation places, chemotherapy, surgical and traditional Chinese medicine and putting, chemotherapy adds the multiple different Therapeutic Method such as Chinese medicine, though these methods have certain therapeutic effect, energy partial rcsponse symptom, extend patient's life, but chemicotherapy easily causes severe trauma to human body, in the medicine of existing treatment, some poisonous side effect of medicine are larger, what have is expensive, the curative effect also had is not good enough, the cancer therapy drug of real acquisition satisfactory effect is few in number, therefore, the development high-efficiency low-toxicity, quality controllable anticancer and treat leukemic medicine, there is important clinical value and wide market prospect.
Chinese medicine is the medical treasure-house of a greatness, cure difficult and complicated cases and chronic disease aspect there is unique advantage, the leukemia resisting action of Fructus Gleditsiae Abnormalis, planting screening Chinese medicine more than 100 finds, Fructus Gleditsiae Abnormalis and Chinese honey locust (fruit) are respectively the sterile fruit of drying and the mature fruit of leguminous plant Fructus Gleditsia Gleditsia sinensis Lam., having eliminates the phlegm has one's ideas straightened out, the effect of mass dissipating and swelling eliminating, clinically be mainly used in treating the apoplexy locked mouth, remain unconscious, (Chinese Pharmacopoeia version in 2005, the Chemical Industry Press such as epilepsy abundant expectoration, version in 2005,222).Fructus Gleditsiae Abnormalis and Chinese honey locust (fruit) all are rich in saponin component, now from the Fructus Gleditsiae Abnormalis n-butanol portion, separate, identify nineteen pentacyclic triterpene type Gleditschiasaponin (J.Nat.Prod.1999,62(5): 740-745; Chem.Pham.Bull.1999,47(3): 388-393; J.Nat.Prod.1999,62(6): 877-881; Phytochemistry, 1999,52:715-722).A kind of Gleditschiasaponin extract and preparation method thereof and application are disclosed in the Chinese invention patent application that publication number is CN101537036A, there is following defect in it: in the Gleditschiasaponin extract that extraction obtains, total saponin content is not high, drug effect is undesirable, and improved space is still arranged.
Summary of the invention
For above-mentioned prior art, the invention provides a kind of preparation method of Fructus Gleditsiae Abnormalis total saponins.
The present invention is achieved by the following technical solutions:
A kind of preparation method of Fructus Gleditsiae Abnormalis total saponins, step is as follows:
(1) pre-treatment that Fructus Gleditsiae Abnormalis extracts: get dry Fructus Gleditsiae Abnormalis medical material, pulverize as coarse powder, standby;
(2) the decocting extraction process of Fructus Gleditsiae Abnormalis total saponins: get above-mentioned Fructus Gleditsiae Abnormalis coarse powder, add 10 times of amounts of deionized water (weight multiple), decoct 2 hours, filter; Medicinal residues add the water of 8 times of amounts (weight multiple) again, decoct 2 hours, filter; Medicinal residues add the water of 8 times of amounts (weight multiple) again, decoct 2 hours, filter; Merge three times filtrate, it is 1.10～1.15(60 ℃ of survey that concentrating under reduced pressure obtains relative density) medicinal liquid, standby;
(3) n-butanol extraction of Fructus Gleditsiae Abnormalis total saponins: get above-mentioned medicinal liquid, with water saturation butanol solution extraction 3 times, merge n-butanol extracting liquid, reclaim under reduced pressure n-butyl alcohol to relative density is 1.15 ~ 1.20(60 ℃), obtain n-butanol extract;
(4) purification by macroporous resin of Fructus Gleditsiae Abnormalis total saponins separates: get above-mentioned n-butanol extract, the water (weight multiple) that adds 6 times of amounts, slight fever makes abundant dissolving, let cool, filter, collect filtrate, add the D101 macroporous resin column (need anticipate, processing mode is the conventional treatment mode) in, first with distilled water, be eluted to closely colourless, then use 10% ethanol (volume fraction) to be eluted to closely colourless, use again 70% ethanol (volume fraction) eluting, collect 70% ethanol elution, it is 1.20-1.25(60 ℃ that concentrating under reduced pressure obtains relative density) thick paste, 60～65 ℃ of drying under reduced pressure, obtain the Fructus Gleditsiae Abnormalis total saponins, its total saponin content reaches more than 80%.
Utilizing the preparation method of Fructus Gleditsiae Abnormalis total saponins of the present invention to extract the Fructus Gleditsiae Abnormalis total saponins obtain, is the sundown powder, and acrid in the mouth, salty has the penetrating odor of saponin, and total saponin content reaches more than 80%, can be used for the treatment of leukemia; During concrete application, can fully mix and make capsule with starch.
Method of the present invention is that in CN101537036A, disclosed technique is compared with publication number, and improvements are as follows:
(1) comparison of preparation method aspect:
Preparation method of the present invention: raw material adopts water extraction, n-butanol extraction and macroporous resin D101 purification, eluant is water, 10% ethanol and 70% ethanol, collect 70% ethanol elution thing, obtain the Fructus Gleditsiae Abnormalis total saponins after reclaiming drying, its total saponin content reaches more than 80%.
Former patent technique: raw material is through water or 85% ethanol extraction, macroporous resin column AB-8 on extract, eluant is water, 20 ethanol, 40% ethanol, 60% ethanol, collects 40% and 60% ethanol elution thing, obtain Fructus Gleditsiae Abnormalis extract after reclaiming drying, in extract, total saponin content approximately reaches 65%.
(2) comparison of drug effect aspect:
Result of the test shows, the sample after purifies and separates of the present invention is to K562, NB4 and S
180inhibitory action obviously be better than former patent technique sample, its concrete experimental result is shown in embodiment bis-, embodiment tetra-:
The research of Fructus Gleditsiae Abnormalis total saponins to K562, NB4 cyto-inhibition: Fructus Gleditsiae Abnormalis total saponins 1 and 2 and the control drug amycin propagation of K562, NB4 cell is had to significant inhibitory action, and, along with the increase of drug level and the prolongation of action time, its suppression ratio to tumor cell strengthens gradually.The inhibited proliferation of 1 couple of K562 of Fructus Gleditsiae Abnormalis total saponins, NB4 cell is better than Fructus Gleditsiae Abnormalis total saponins 2.
The Fructus Gleditsiae Abnormalis total saponins is to mice S
180the research of sarcoma tumor-inhibiting action: Fructus Gleditsiae Abnormalis total saponins 1(this patent technique) senior middle school's dosage group is to S
180the growth of sarcoma has obvious inhibitory action, and its suppression ratio is respectively 39.5% and 34.2%, and low dose group has no obvious inhibitory action.The former patent technique of Fructus Gleditsiae Abnormalis total saponins 2() high dose group is to S
180the growth of sarcoma has obvious inhibitory action, and its suppression ratio is 31.2%, and middle low dose group has no obvious inhibitory action.1 couple of S of total saponins that this technique is extracted
180inhibitory action obviously be better than more former patent technique total saponins 2.
The accompanying drawing explanation
Fig. 1: the blank interferogram of echinocystic acid and oleanolic acid in working sample, wherein, Fig. 1: Fig. 1-1 neat solvent methanol peak; Fig. 1-2: reference substance; Fig. 1-3: test sample.
Fig. 2-1: the standard curve of echinocystic acid.
Fig. 2-2: the standard curve of oleanolic acid.
The specific embodiment
Below in conjunction with embodiment, the present invention is further illustrated.
The optimization of embodiment mono-Fructus Gleditsiae Abnormalis total saponins preparation method
1, the research data that decocting extracts
(1) determining of decocting condition: to Fructus Gleditsiae Abnormalis decocting technique preferably in, take the total saponins yield as index, on affecting the amount of water of extraction ratio, extraction time, extraction time has been carried out the orthogonal test of Three factors-levels, preferably determined suitable decocting extraction conditions, the concrete test method of its orthogonal test is: accurately take Fructus Gleditsiae Abnormalis coarse powder 100g, 9 parts of nominals, carry out 9 decocting tests by the decocting condition of test arrangement in table 1 respectively, each experimental liquid is concentrated and is settled in the 100ml measuring bottle, assay method by total saponins is measured respectively the content that respectively extracts total saponins in sample, experimental factor and water-glass are in Table 1, the arrangement of 9 orthogonal tests and the results are shown in Table 2.
The ether sedimentation method is measured total saponin content: get the suitable crude drug amount of the 10ml(of testing liquid 10g each time), water bath method, residue adds methanol 30ml, slight fever makes to dissolve, and lets cool, and filters, filtrate is under fully stirring, the ether that slowly adds 3 times of amounts, add rear continuation and stir 10 minutes, sealing, put the standing 24h in shady and cool place, incline and supernatant, the precipitation sucking filtration is extremely dry, and uses methanol: ether (1:2) mixed solvent 10ml washing, volatilize solvent, 60～65 ℃ of vacuum dryings, weigh, and calculates the extraction total amount of total saponins in each sample.
Table 1 experimental factor and water-glass
* amount of water is for to add the multiple of the medical material amount that feeds intake, rear secondary respectively to reduce 2 times for the first time.
Table 2 decocting orthogonal test arranges and result
The result of the test of his-and-hers watches 2 is carried out intuitive analysis, from the known C(7.3 that affects on the total saponins yield of extreme difference R)>B(7.0)>A(1.3), show that decocting time C and extraction time B are the principal elements that affects the total saponins extraction ratio, and C
3>C
2>C
1, B
3>B
2>B
1therefore, select C
3and B
3, amount of water is for affecting the secondary cause of total saponins extraction ratio, can an optional level, therefore select A
1, so the suitable condition of this Fructus Gleditsiae Abnormalis decocting should be A
1b
3c
3, add the water of 10 times of amounts, decoct 3 times, be 2 hours at every turn.
(2) the decocting extraction process of Fructus Gleditsiae Abnormalis total saponins: get dry Fructus Gleditsiae Abnormalis medical material, pulverize as coarse powder, add deionized water, decoct, filter; Extract three times, merging filtrate, being evaporated to relative density is 1.10～1.15(60 ℃) medicinal liquid.
2, the Medium scale of Fructus Gleditsiae Abnormalis total saponins test
Decocting and the n-butanol extraction of test agent in (1) the three batch of Fructus Gleditsiae Abnormalis total saponins
In the Fructus Gleditsiae Abnormalis herbal decoction, except containing total saponins, still contain other water soluble ingredients such as polysaccharide, for removing the water-soluble component of the more polysaccharide of content, improve the content of total saponins in saponin extract, therefore adopt n-butanol extraction to carry out preliminary purification to it.In the test of Fructus Gleditsiae Abnormalis total saponins lab scale, determine on the condition of decocting and the suitable extraction of n-butanol extraction, the decocting of test agent and n-butanol extraction test in having carried out three batches, its concrete grammar is: take the standby Fructus Gleditsiae Abnormalis of 20Kg, add 10 times of amounts of deionized water, decoct 2 hours, filter, medicinal residues add the water of 8 times of amounts again, the same decocting 2 times, each 2 hours, filter, merge filtrate three times, being evaporated to relative density is 1.10～1.15(60 ℃) medicinal liquid;
Let cool, with water saturation butanol solution extraction 3 times, add water-saturated n-butanol 250ml by every 1000ml at every turn, merge 3 times n-butanol extracting liquid, reclaim under reduced pressure to relative density is 1.15 ~ 1.20(60 ℃) runny plaste, weigh, calculate the n-butanol extract yield and measure the content of total saponins in each batch sample, the results are shown in Table 3.
Three batches of Fructus Gleditsiae Abnormalis total saponins n-butanol extraction results of table 3
| Sequence number | Throw cream amount (Kg) | N-butyl alcohol thing (Kg) | Yield * (%) | Total saponin content (%) |
| 1 | 20.0 | 2.78 | 13.9 | 45.6 |
| 2 | 20.0 | 2.68 | 13.4 | 46.3 |
| 3 | 20.0 | 2.55 | 12.8 | 46.8 |
* yield is in the medical material 20.0Kg that feeds intake.
3, the purification by macroporous resin of Fructus Gleditsiae Abnormalis extract separates
In saponin extract after decocting extraction and n-butanol extraction, the content of total saponins is about 45%, not yet reach declare new Chinese medicine effective site content should be lower than 50% specification requirement, for further improving the content of total saponins in saponin extract, again saponin extract has been carried out the technical study of macroporous adsorbent resin (D101) purifies and separates saponin extract, and total saponins in the saponin extract after separation and purification has been carried out to assay.Result shows, after the saponin extract upper prop of n-butanol extraction, and the abundant eluting of first water, then use 10% and 70% ethanol elution, and can effectively make saponin extract obtain purification, in 70% pure washing, saponin content approximately reaches 80%, and drying obtains the Fructus Gleditsiae Abnormalis total saponins.In the lab scale purifies and separates, determine on the basis of separation condition, carried out the purifies and separates test of three batches of Fructus Gleditsiae Abnormalis total saponins, its concrete test method is as follows.
Method for purifying and separating: claim n-butanol extract 150g, add 6 times of water gagings, slight fever makes to dissolve, let cool, filter, filtrate adds the D101 macroporous resin column (need anticipate, processing mode is the conventional treatment mode) in, first with distilled water, be eluted to closely colourless, then closely colourless with the same eluting that carries out of 10% ethanol, use again 70% ethanol elution, collect 70% alcohol eluen, be evaporated to relative density 1.20-1.25(60 ℃) thick paste, 60-65 ℃ of drying under reduced pressure, weigh and measure the content of total saponins in three batch samples, in three batches of pilot scale sample separation total saponins must measure and saponin content in Table 4.
The purifies and separates result of the test of three batches of Fructus Gleditsiae Abnormalis total saponins of table 4
4, the preparation (preparing 10000 capsules) of test agent in three batches
Take the Fructus Gleditsiae Abnormalis total saponins 1.5Kg after above-mentioned purification, add starch 1.0Kg, fully mix, in incapsulating by every 0.25g, packing, the capsule that gets product, each batch of Fructus Gleditsiae Abnormalis total saponin capsules must measure and yield rate in Table 5.
The Medium scale result of three batches of Fructus Gleditsiae Abnormalis total saponin capsules of table 5
Annotate: 1. must to measure be 10000 to every batch of theoretical capsule of this product.
The research of embodiment bis-Fructus Gleditsiae Abnormalis total saponins to K562, NB4 cyto-inhibition
1 experiment material and instrument
1.1 the preparation of Fructus Gleditsiae Abnormalis test specimen liquid:
Fructus Gleditsiae Abnormalis total saponins 1 is 70% ethanol elution thing after upper macroporous resin column (technique sample of the present invention), sample lot number: 20111013.By development, seminar provides.Method is as follows:
(1) pre-treatment that Fructus Gleditsiae Abnormalis extracts: get dry Fructus Gleditsiae Abnormalis medical material, pulverize as coarse powder, standby;
(2) the decocting extraction process of Fructus Gleditsiae Abnormalis total saponins: get above-mentioned Fructus Gleditsiae Abnormalis coarse powder, add 10 times of amounts of deionized water (weight multiple), decoct 2 hours, filter; Medicinal residues add the water of 8 times of amounts (weight multiple) again, decoct 2 hours, filter; Medicinal residues add the water of 8 times of amounts (weight multiple) again, decoct 2 hours, filter; Merge three times filtrate, it is 1.10～1.15(60 ℃ of survey that concentrating under reduced pressure obtains relative density) medicinal liquid, standby;
(3) n-butanol extraction of Fructus Gleditsiae Abnormalis total saponins: get above-mentioned medicinal liquid, with water saturation butanol solution extraction 3 times, merge n-butanol extracting liquid, reclaim under reduced pressure to relative density is 1.15 ~ 1.20(60 ℃) runny plaste obtain n-butanol extract;
(4) purification by macroporous resin of Fructus Gleditsiae Abnormalis total saponins separates: get above-mentioned n-butanol extract, the water (weight multiple) that adds 6 times of amounts, slight fever makes abundant dissolving, let cool, filter, collect filtrate, add in the D101 macroporous resin column of having anticipated, first with distilled water, be eluted to closely colourlessly, then use 10% ethanol (volume fraction) to be eluted to closely colourless, then use 70% ethanol (volume fraction) eluting, collect 70% ethanol elution, it is 1.20-1.25(60 ℃ that concentrating under reduced pressure obtains relative density) thick paste, 60～65 ℃ of drying under reduced pressure, obtain the Fructus Gleditsiae Abnormalis total saponins.
The product that Fructus Gleditsiae Abnormalis total saponins 2 is embodiment 1 preparation in CN101537036A for patent publication No., sample lot number: 20120316.By development, seminar provides.
Accurately take respectively the Fructus Gleditsiae Abnormalis total saponins appropriate, water is made into every milliliter of medicinal liquid containing 50mg.With 0.22 μ m syringe needle filter filtration sterilization, it is the test specimen of this external leukemia cell.
1.2 the preparation of control drug injection amycin: the injection amycin, for the test positive control drug, is produced by Actavis Italy S.p.A, and 10mg/ props up, and lot number 0QL0073 uses normal saline dilution during test.
1.3 tumor cell line
K562: the human chronic polymorpho nuclear leukemia cells strain, suspension culture, adjust cell number 4 * 10 during test
5/ ml.
NB4: people's acute promyelocytic leukemia, suspension culture, adjust cell number 4 * 10 during test
5/ ml.
1.4 test reagent: RPMI-1640 dry powder (U.S. Gibco company product); New-born calf serum; Tetramethyl ribavirin indigo plant; Dimethyl sulfoxide; 0.25% trypsin.
1.5 instrument: SW-CJ-2FD superclean bench, BDS200-PH inverted microscope, IL-161CI CO2 gas incubator, xMark microplate reader.
2 test methods and result
2.1 the cultivation of tumor cell: K562, NB4 cell RPMI1640 complete culture solution, at 37 ℃, 5%CO
2incubator is cultivated.Take the logarithm the trophophase cell for experiment.
2.2MTT method is measured: fetching counts that the tumor cell of trophophase is centrifugal, counting, prepares cell suspension, is inoculated in 96 orifice plates every hole 100 μ l.The medicinal liquid to be measured that adds respectively variable concentrations.And set up positive control and the negative control group of variable concentrations.Every concentration is four multiple holes.Put 37 ℃, 5%CO
2in incubator, cultivate respectively 24 hours and 72 hours.Then every hole adds MTT(5mg/ml) 10 μ l, continue to cultivate after 4 hours and take out, centrifugal, suck supernatant, every hole adds DMSO150 μ l, again culture plate is shaken to even 5min left and right, after micro-Microscopic observation colored particles disappears, at 20min, by microplate reader, survey the OD value under 490nm wavelength condition.Calculate the growth of tumour cell suppression ratio.Separately with improvement Kou Shi computing method, ask its IC
50value, refer to table 6～table 11.
Suppression ratio (%)=(negative control OD value-test hole OD value)/negative control OD value * 100%
2.3 result of the test
Result of the test shows, Fructus Gleditsiae Abnormalis total saponins 1 and 2 and amycin the propagation of K562, NB4 cell is had to significant inhibitory action, and, along with the increase of drug level and the prolongation of action time, medicine also increases the suppression ratio of cell.
The inhibitory action (24h) of 1 couple of K562 of table 6 Fructus Gleditsiae Abnormalis total saponins, NB4 cell proliferation
Compare △ P<0.05 with the cell matched group; N=4
The inhibitory action (24h) of 2 couples of K562 of table 7 Fructus Gleditsiae Abnormalis total saponins, NB4 cell proliferation
Compare △ P<0.05 with the cell matched group; N=4
The inhibitory action (24h) of table 8 amycin to K562, NB4 cell proliferation
Compare △ P<0.05 with the cell matched group; N=4
The inhibitory action (72h) of 1 couple of K562 of table 9 Fructus Gleditsiae Abnormalis total saponins, NB4 cell proliferation
Compare △ P<0.05 with the cell matched group; N=4
The inhibitory action (72h) of 2 couples of K562 of table 10 Fructus Gleditsiae Abnormalis total saponins, NB4 cell proliferation
Compare △ P<0.05 with the cell matched group; N=4
The inhibitory action (72h) of table 11 amycin to K562, NB4 cell proliferation
Compare △ P<0.05 with the cell matched group; N=4
3 brief summaries
3.1 Fructus Gleditsiae Abnormalis total saponins 1 and 2 and the control drug amycin propagation of K562, NB4 cell is had to significant inhibitory action, and, along with the increase of drug level and the prolongation of action time, its suppression ratio to tumor cell strengthens gradually.
3.2 the inhibited proliferation of 1 couple of K562 of Fructus Gleditsiae Abnormalis total saponins, NB4 cell is than Fructus Gleditsiae Abnormalis total saponins the last 2.
The research of embodiment tri-Fructus Gleditsiae Abnormalis total saponins to the HL-60 cyto-inhibition
1 experiment material and instrument
1.1 the preparation of Fructus Gleditsiae Abnormalis test specimen liquid: accurately take respectively Fructus Gleditsiae Abnormalis total saponins (with embodiment bis-) appropriate, water is made into every milliliter of medicinal liquid containing 50mg.With 0.22 μ m syringe needle filter filtration sterilization, it is the test specimen of this external leukemia cell.
1.2 cisplatin for inj (CDDP): be the test positive control drug, the production of Shandong Qilu Pharmaceutical Factory, 20ml/ props up, lot number 200212024, during test with normal saline dilution to desired concn.
MTT: purchased from Sino-American Biotechology company, lot number 20100712.
DG3022A type enzyme-linked immunosorbent assay instrument: East China Electronics Co., Ltd pipe factory produces.
Subculture in vitro separately cell: HL-60: the HL-60, suspension culture, adjust cell number 1.5 * 105/ml during test.
2 test methods and result
2.1 the cultivation fetching of tumor cell is counted HL-60 cell centrifugation, the counting of trophophase, the cell suspension of 1.5 * 105 cell/ml of preparation, be inoculated in 96 orifice plates, every hole 0.1ml, put in 37 ℃ of 5%CO2 incubators and cultivate after 24 hours, the medicinal liquid to be measured that adds respectively variable concentrations, concentration is respectively 1.10 * 104,1.10 * 103,1.10 * 102,1.10 * 101,1.10,1.10 * 10-1 μ g/ml, and establish the positive control of variable concentrations, concentration is respectively 102,101,1,10-1,10-2,10-3ug/ml and negative control group.Every concentration is four multiple holes.Continue respectively to cultivate after 24 hours and 72 hours centrifugal, liquid in hole inclines, with PH7.41/15MPBS, wash twice, then add the MTT liquid of 0.2ml to continue to cultivate 4 hours, liquid in the centrifugal hole of inclining again, every hole adds dimethyl sulfoxide 0.2ml vibration and dissolves its crystallization, by microplate reader, surveys the OD value under 570nm wavelength condition, and be calculated as follows the suppression ratio of medicine to cell, separately with the simple and direct composite accounting of Sun Rui unit, ask its IC
50value, result is as shown in table 12, table 13.
Suppression ratio (%)=(negative control OD value-test hole OD value)/negative control OD value * 100%
The inhibitory action (24h) of each dosage group of table 12 Fructus Gleditsiae Abnormalis total saponins to the HL-60 cell
The inhibitory action (72h) of each dosage group of table 13 Fructus Gleditsiae Abnormalis total saponins to the HL-60 cell
In table 12,13, data show that the Fructus Gleditsiae Abnormalis total saponins has obvious inhibitory action to the HL-60 cell, and its inhibition strength obviously strengthens with the increasing of drug dose.Suppression ratio HL-60 produced according to the Fructus Gleditsiae Abnormalis total saponins has calculated respectively the medicament contact IC of 24,72 hours
50be 15.3 μ g/ml and 10.6 μ g/ml, show that this product has significant external tumor-inhibiting action to HL-60 human promyelocytic leukemia cell.
3 discuss and brief summary
3.1 the Fructus Gleditsiae Abnormalis total saponins has obvious inhibitory action and dosage, time effect dependency to the HL-60 cell.
3.2 the drug dose of each group of this test is definite according to the trial test result, trial test shows that when neighbouring two groups of dose ratios are 1:3, immeasurable heterodyne is different, therefore dose ratio is increased to 1:10.With reference to the simple and direct composite accounting of Sun Rui unit, tentatively obtain 24,72 hours IC to the HL-60 cell of saponin extract effect
50be respectively 15.3 μ g/ml and 10.6 μ g/ml.All meet Ministry of Public Health Clinical Researches of New Drugs guideline and require (LC
50<30 μ g/ml), illustrate that the Fructus Gleditsiae Abnormalis total saponins has more stable extracorporeal anti-tumor effect.
Embodiment tetra-Fructus Gleditsiae Abnormalis total saponins are to mice S
180the research of sarcoma tumor-inhibiting action
1 material
Trial drug: Fructus Gleditsiae Abnormalis total saponins 1, Fructus Gleditsiae Abnormalis total saponins 2, with embodiment bis-.
Neosar, Shanghai No.12 Pharmaceutical Factory's product.
Experimental animal: Kunming kind 18-22g mice, Shandong University's Experimental Animal Center, the quality certification number: SCXK (Shandong) 20090001, ♀ ♂ dual-purpose; Plant Mus S
180the sarcoma mice, draw from pharmacological room of medicine institute of Shandong Academy of Medical Sciences.
2 experimental techniques and result
After mice is bought 24h, give respectively the mice S by normal saline dilution
180ascites fluid, containing oncocyte 5 * 10
6/ ml inoculates in left oxter, after inoculation 24h, the random packet administration, be grouped into 24 of NS matched groups (each 12 of ♀ ♂), 10 of 20mg/kg cyclophosphamide SC groups (each 5 of ♀ ♂), total saponins and total extract various dose group, every group 12 (being each 6 of ♀ ♂), be administered once every day, successive administration 9 days, after last administration 24h, de-neck is put to death mice, peel off the tumor body, the GB303 electronic balance claims the tumor body weight, the tumor weight is respectively organized in the t check, and by (NS matched group tumor weight-administration group tumor weight)/NS matched group tumor weight, calculate the tumour inhibiting rate of each medicine group, result is as shown in table 14.
2 couples of mice S of table 14 total saponins 1 and total saponins
180the sarcoma tumor-inhibiting action
| Group | n | The tumor weight | Tumour inhibiting rate % |
| The NS matched group | 24 | 2.05±1.02 | —— |
| Cyclophosphamide 20mg/ |
10 | 1.07±0.26 | 47.8 |
| Total saponins 1 |
12 | 1.24±0.30 | 39.5 |
| Dosage group in total saponins 1 | 12 | 1.35±0.71 | 34.2 |
| Total saponins 1 |
12 | 1.75±0.76 | 14.6 |
| |
12 | 1.41±0.34 | 31.2 |
| Dosage group in |
12 | 1.57±0.66 | 23.4 |
| |
12 | 1.94±0.84 | 5.37 |
3 results and discussion
3.1 experimental result shows, Fructus Gleditsiae Abnormalis total saponins 1 senior middle school's dosage group is to S
180the growth of sarcoma has obvious inhibitory action, and its suppression ratio is respectively 39.5% and 34.2%, and low dose group has no obvious inhibitory action.
3.2 experimental result shows, Fructus Gleditsiae Abnormalis total saponins 2 high dose group are to S
180the growth of sarcoma has obvious inhibitory action, and its suppression ratio is 31.2%, and middle low dose group has no obvious inhibitory action.
3.3 above-mentioned experimental result shows, the total saponins that this technique is extracted is to S
180inhibitory action obviously be better than former patent technique.
The studies on acute toxicity of embodiment five Fructus Gleditsiae Abnormalis saponin extracts and total saponins
1. material
1.1 be subject to test product: the n-butanol extract that Fructus Gleditsiae Abnormalis extract is the front n-butanol extraction of upper macroporous resin in embodiment bis-; The Fructus Gleditsiae Abnormalis total saponins is with embodiment bis-.
1.2 animal, environment and feedstuff:
Animal: the KM mice, 18-22g, ♀ ♂ half and half, does Shandong University's Experimental Animal Center provide, the quality certification number: the SCXK(Shandong)?
Environment: zoopery center, Medicine Institute, Shandong Province, credit number: the SYXK(Shandong) 20050052.
Feedstuff: management of laboratory animal center, Shandong Province provides, the quality certification number: the SCXK(Shandong) 20040014.
1.3 instrument: PL303 type electronic balance, mettler tolido product.
2. method and result
2.1 preliminary experiment: get the different 4g of being less than of 18 ~ 22g(group endosome method of double differences) mice, ♀ ♂ half and half, water 12h is can't help in fasting, every group 6, carry out respectively the preliminary experiment of 0%, 100% fatal dose (Dmin, Dmax) of Fructus Gleditsiae Abnormalis total saponins and Fructus Gleditsiae Abnormalis extract, draw approximate 0%, 100% fatal dose of Fructus Gleditsiae Abnormalis total saponins and Fructus Gleditsiae Abnormalis extract.According to the preliminary experiment result, carry out respectively the LD of Fructus Gleditsiae Abnormalis total saponins and Fructus Gleditsiae Abnormalis extract
50measure.18-22g KM mice, ♀ ♂ half and half, 10 every group, each is subject to test product to take Dmax as maximum dosage-feeding, take 0.8 as parameter identification different dosing dosage.Before administration, water 12h is can't help in fasting, the 0.2mL/10g gavage, and Continuous Observation 14d after gavage, normal diet, record 14 days mouse death rates, calculates the different LD that are subject to test product
50with 95 credibility interval.Result is as shown in table 15～17.
Table 15 Fructus Gleditsiae Abnormalis total saponins LD
50measure
Table 16 Fructus Gleditsiae Abnormalis extract LD
50measure
Calculate and respectively organize LD according to the Bliss method
50with 95% credibility interval, result is as table 3:
Table 17 Fructus Gleditsiae Abnormalis total saponins and Fructus Gleditsiae Abnormalis extract LD
50with 95% credibility interval
3 brief summaries
Record the Ld of Fructus Gleditsiae Abnormalis total saponins
50for 1.21g/kg, the Fructus Gleditsiae Abnormalis extract LD before upper prop not
50for 2.72g/kg.
The content of echinocystic acid and oleanolic acid in embodiment six HPLC mensuration Fructus Gleditsiae Abnormalis total saponins
Purpose: measure the content of oleanolic acid and echinocystic acid in the Fructus Gleditsiae Abnormalis total saponins, set up the HPLC assay method of 2 kinds of compositions in said preparation.
The Fructus Gleditsiae Abnormalis total saponins is Chinese medicine 5 kind new medicines of developing, and Pharmacodynamic test of active extract shows, the Fructus Gleditsiae Abnormalis total saponins is antileukemie main active component, and leukemia is had to good therapeutic effect.And the main aglycon that echinocystic acid and oleanolic acid are saponin in total saponins, for controlling the inherent quality of this product, formulate the strong quantitative quality control standard of controllability, related documents with reference to echinocystic acid and content of oleanolic acid mensuration, echinocystic acid in the Fructus Gleditsiae Abnormalis total saponins and oleanolic acid have been carried out to the research of content assaying method, assay by methodological system thinking and three batch samples, set up the HPLC of echinocystic acid and oleanolic acid in this preparation containing the survey method, formulated the content limit of 2 kinds of compositions, now be reported as follows:
1 instrument and reagent
High performance liquid chromatograph (Waters 2695Separations Module, Waters 2996Photodiode Array Detector).Chromatographic column is the Diamonsil of Di Ma company
tM(diamond) C18,5 μ m, 150 * 4.6r, ultra-pure water (Millipore company demineralizer), the mobile phase acetonitrile is chromatographically pure, other reagent are analytical pure.Echinocystic acid reference substance (Chinese pharmaceutical biological product is checked institute, lot number 110756-2011010), oleanolic acid reference substance (Chinese pharmaceutical biological product is checked institute, lot number 0709-9803), for assay.
2 methods and result
2.1 chromatographic condition: with octadecylsilane chemically bonded silica, be filler, acetonitrile-water (90:10) is mobile phase, flow velocity 1.0mlmin
-1, the detection wavelength is 210nm, 25 ℃ of column temperatures, and sample size 10uL, number of theoretical plate calculates and should be not less than 2500 by echinocystic acid and oleanolic acid peak.Echinocystic acid, oleanolic acid separate good under these conditions.
2.2 the preparation of echinocystic acid and oleanolic acid reference substance solution: get the echinocystic acid reference substance in right amount in the 2mL measuring bottle, accurately weighed is 2.60mg; Even up pier fruit acid reference substance in right amount in the 2mL measuring bottle, accurately weighed is 2.12mg.Respectively add methanol to make in right amount to dissolve and be diluted to scale, shake up, as two reference substance storing solutions, (its concentration is respectively 1.30mgmL
-1, 1.06mgmL
-1); The accurate absorption in each 1mL to 2mL measuring bottle of above-mentioned two storing solutions, shake up again, product mixed solution in contrast, and the concentration of its echinocystic acid and oleanolic acid reference substance is respectively 0.65mgmL
-1, 0.53mgmL
-1.
2.3 the preparation of need testing solution: get the about 0.15g of Fructus Gleditsiae Abnormalis total saponins, accurately weighed, add kieselguhr 3g, mix thoroughly, put in apparatus,Soxhlet's, add methanol 100mL, reflux 3 hours, let cool, evaporate to dryness, residue adds dilute hydrochloric acid 80mL to be made to dissolve, quantitatively be transferred in round-bottomed flask, heating in water bath hydrolysis 2h, let cool, quantitatively be transferred in separatory funnel, with chloroform extraction 3 times (50, 50, 30mL), the combined chloroform extracting solution, wash with water 2 times, each 60mL, discard water liquid, the chloroform solution evaporate to dryness, residue is with dissolve with methanol and be settled in the 10mL measuring bottle, shake up, as need testing solution.
2.4 methodological study
2.4.1 measure the selection of wavelength: precision is drawn reference substance solution and each 10 μ L of need testing solution respectively, and the injection liquid chromatography detects in 200～400nm scope, draws chromatogram.Result shows, echinocystic acid and oleanolic acid have maximum absorption band at 205nm and 200nm place respectively, are reduce disturbance, guarantees baseline stability, with reference to the pertinent literature of the assay of 2 kinds of compositions, selects 210nm as measuring wavelength.
2.4.2 blank interference test: precision is drawn each 10 μ L of need testing solution, reference substance solution and neat solvent methanol solution respectively, and the injection liquid chromatography, detect at the 210nm place.Result shows, in the test sample chromatograph, on the position identical with the oleanolic acid chromatographic retention with the reference substance echinocystic acid, obvious absworption peak is arranged, and solvent methanol solution has no obvious absorption peaks on correspondence position, shows the obviously interference of this mensuration nothing, sees Fig. 1.
2.4.3 linear relationship is investigated: it is appropriate that precision takes the echinocystic acid reference substance respectively, and being made into concentration is 1.335mgmL
-1, 0.6675mgmL
-1,, 0.33375mgmL
-1, 0.166875mgmL
-1, 0.0834375mgmL
-1the reference substance solution of five variable concentrations, accurate above-mentioned each reference substance solution 10 μ L that draw, the injection liquid chromatography, measure the peak area integrated value, the results are shown in Table 18, the peak area integrated value (A) of take is vertical coordinate, concentration (mgmL
-1) be abscissa, the drawing standard curve, echinocystic acid is at 0.0834375mgmL as a result
-1～1.335mgmL
-1in scope, with peak area integrated value (A), be the good linear relation, see Fig. 2-1.The data obtained is carried out to regression analysis, obtain regression equation y=789999x ﹢ 135181, correlation coefficient r=0.9999.
Table 18 echinocystic acid linear relationship is investigated result
It is appropriate that precision takes the oleanolic acid reference substance respectively, and being made into concentration is 1.07mgmL
-1, 0.535mgmL
-1, 0.2675mgmL
-1, 0.13375mgmL
-1, 0.066875mgmL
-1the reference substance solution of five variable concentrations, accurate above-mentioned each reference substance solution 10 μ L that draw, the injection liquid chromatography, measure the peak area integrated value, the results are shown in Table 19, the peak area integrated value (A) of take is vertical coordinate, concentration (mgmL
-1) be abscissa, the drawing standard curve, oleanolic acid is at 0.066875mgmL as a result
-1～1.07mgmL
-1in scope, with peak area integrated value (A), be the good linear relation, see Fig. 2-2.The data obtained is carried out to regression analysis, obtain regression equation y=854782x ﹢ 74164, correlation coefficient r=1.
Table 19 oleanolic acid linear relationship is investigated result
2.4.4 precision test: the accurate need testing solution 10 μ L that draw, continuous sample introduction 5 times, measure the peak area integrated value, measures the meansigma methods of echinocystic acid 5 times as a result
rSD=0.44%; The meansigma methods of oleanolic acid
rSD=0.75%.Show that instrument precision is good, in Table 20.
Table 20 Precision test result
2.4.5 stability test: the accurate need testing solution 10 μ L that draw, every the 2h sample introduction, to measure 1 time, continuous sample introduction is measured 5 times, and results peaks area integral value (A) is substantially constant in minute 8h, measures the meansigma methods of echinocystic acid 5 times
rSD=0.46%; The meansigma methods of oleanolic acid
rSD=0.38%.Show that test sample 8h internal stability is good, in Table 21.
Table 21 stability test result
2.4.6 replica test: precision takes same batch sample 0.15g, get altogether 6 parts, accurately weighed, press preparation and the content assaying method of Fructus Gleditsiae Abnormalis total saponins need testing solution, prepare echinocystic acid and oleanolic acid in each need testing solution working sample, calculate the content in each sample.The average content of 6 test determination echinocystic acid as a result
rSD=0.69%; The average content of oleanolic acid
rSD=1.29%.Show this experimental technique favorable reproducibility, in Table 22.
Table 22 replica test result
2.4.7 application of sample recovery test: precision takes 5 parts, repeatability sample, every part of 0.075g, add respectively echinocystic acid and oleanolic acid reference substance appropriate, press preparation and the content assaying method of Fructus Gleditsiae Abnormalis total saponin extracts need testing solution, make each recovery test liquid and measure echinocystic acid and the oleanolic acid in each response rate sample, calculating the response rate of each sample, the average recovery rate that result records 5 test echinocystic acid is 97.8%, coefficient of variation RSD is 1.36%, in Table 23; The average recovery rate of oleanolic acid is 98.84%, and coefficient of variation RSD is 0.86%, in Table 24.
Table 23 echinocystic acid application of sample recovery test result
Table 24 oleanolic acid application of sample recovery test result
2.5 assay
2.5.1 the assay of echinocystic acid and oleanolic acid in three batch samples: accurate respectively 10 μ L of reference substance and three batches of need testing solutions that draw respectively, the injection liquid chromatography, measure and calculate the content of echinocystic acid and oleanolic acid in each batch sample, the results are shown in Table 25.
The assay result of echinocystic acid and oleanolic acid in three batches of Fructus Gleditsiae Abnormalis total saponins of table 25
2.5.2 the assay of echinocystic acid and oleanolic acid in the three batches of Chinese medicine III medical materials: get each about 1g of Fructus Gleditsiae Abnormalis medicinal powder, accurately weighed, press preparation and the content assaying method of Fructus Gleditsiae Abnormalis total saponin extracts need testing solution, make each medical material experimental liquid and measure echinocystic acid and the oleanolic acid in each medical material, calculate the content in each batch of medical material, the results are shown in Table 26.
The assay result of echinocystic acid and oleanolic acid in three batches of Fructus Gleditsiae Abnormalis medical materials of table 26
3 brief summaries and discussion
(1) echinocystic acid in the Fructus Gleditsiae Abnormalis total saponins and oleanolic acid have been carried out to the research of content assaying method, assay by methodological system thinking and three batch samples, set up the HPLC content assaying method of echinocystic acid and oleanolic acid in the preparation, recorded echinocystic acid at 0.0834375mgmL
-1～1.335mgmL
-1in scope, peak area (A) and concentration (mgmL
-1) be good linear relationship, regression equation y=789999x ﹢ 135181, correlation coefficient r=0.9999; Oleanolic acid is at 0.066875mgmL
-1～1.07mgmL
-1in scope, peak area (A) and concentration (mgmL
-1) be good linear relationship, regression equation y=854782x ﹢ 74164, correlation coefficient r=1.0.
In (2) three batches of Fructus Gleditsiae Abnormalis total saponins, the echinocystic acid total content is respectively 213.2mgg
-1, 225.0mgg
-1, 219.3mgg
-1average average recovery is 98.22%, and coefficient of variation RSD is 1.35%; The oleanolic acid total content is respectively 120.0mgg
-1, 127.8mgg
-1, 123.5mgg
-1, average average recovery 98.84%, coefficient of variation RSD is 0.86%.
Echinocystic acid total content difference 26.70mgg in (3) three batches of Fructus Gleditsiae Abnormalis medical materials
-1, 27.47mgg
-1, 27.24mgg
-1, the oleanolic acid total content is respectively 17.32mgg
-1, 16.18mgg
-1, 16.69mgg
-1.
(4) this mensuration methanol extraction, measure the main aglycon echinocystic acid of Fructus Gleditsiae Abnormalis saponin and the content of oleanolic acid after hydrochloric acid hydrolysis.This law has highly sensitive, favorable reproducibility, and the characteristics such as specificity is strong, be to control the ideal of this preparation inherent quality containing the survey method.
Attached: the quality standard of Fructus Gleditsiae Abnormalis total saponins (draft)
The Fructus Gleditsiae Abnormalis total saponins
Zhuyazao?Zongzaogan
[prescription] Fructus Gleditsiae Abnormalis 1000g.
[method for making] gets the Fructus Gleditsiae Abnormalis coarse powder, add 10 times of amounts of deionized water, decoct 2 hours, filter, medicinal residues add the water of 8 times of amounts again, the same decocting 2 times, each 2 hours, filter, merge three times filtrate, be evaporated to relative density 1.10～1.15(60 ℃ survey) medicinal liquid, with water saturation n-butanol extraction 3 times, merge n-butanol extracting liquid, reclaim under reduced pressure to relative density is 1.15 ~ 1.20(60 ℃), add the appropriate slight fever of water and make abundant dissolving, let cool, filter, collect filtrate, add in processed good D101 macroporous resin column, first with distilled water, be eluted to closely colourless, then closely colourless with the same eluting that carries out of 10% ethanol, use again 70% ethanol elution, collect 70% alcohol eluen, be evaporated to relative density 1.20-1.25(60 ℃) thick paste, 60-65 ℃ of drying under reduced pressure, obtain.
[character] this product is the sundown powder, and acrid in the mouth, salty has the penetrating odor of saponin.
The about 0.2g of this product is got in [discriminating] (1), adds ethanol 8ml, and reflux 5 minutes, let cool, and filters, and gets filtrate 0.5ml, puts in little porcelain dish, and evaporate to dryness, let cool, and adds 3 of acetic anhydride, stirs evenly, and along the ware wall, adds 2, sulphuric acid, and solution fades in reddish violet.
(2) get the about 0.2g of this product, add water 10ml, boil 10 minutes, filter, the strong jolting of filtrate, produce lasting foam (persistent period is more than 15 minutes).
(3) get the about 0.5g of this product, add ethyl acetate-methanol (2:8, V/V) mixed liquor 15ml, reflux, extract, 30 minutes, let cool, and filters, and gets filtrate as need testing solution.Separately get Fructus Gleditsiae Abnormalis control medicinal material 1.0g, add above mixed solution 15ml, be made in the same way of Fructus Gleditsiae Abnormalis medical material contrast liquid.Draw respectively each 5 μ l of above-mentioned two kinds of solution, on the silica gel G plate that point is binding agent in same 0.3% sodium carboxymethyl cellulose, n-butyl alcohol-methanol-the ammonia (10:2:5) of take is developing solvent, launches, and takes out, dry, spray is with 5% phosphomolybdic acid ethanol, and 105 ℃ to be heated to the speckle colour developing clear, in the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, the blue spot of aobvious same color.
[inspection] moisture: according to aquametry (appendix LX H of Chinese Pharmacopoeia version in 2010) first method, the water content of Fructus Gleditsiae Abnormalis total saponin extracts must not cross 6.0%.
[assay]
1, the mensuration of total saponins: get Fructus Gleditsiae Abnormalis total saponins 2g, accurately weighed, put in the 250ml tool plug triangular flask of constant weight, add methanol 30ml, slight fever makes to dissolve, filter, with 10ml methanol gradation washing nozzle, washing liquid is incorporated in filtrate, under fully stirring, the ether that slowly adds 3 times of amounts, add rear continuation and stir 3-5 minute, sealing, put the standing 24h in shady and cool place, with the filter paper of constant weight, filter, and use methanol: ether (1:2) mixed solvent 10ml washing precipitation and filter, washing liquid filters, filter paper volatilizes solvent, put together 60～65 ℃ of vacuum dryings with triangular flask, weigh, in calculation sample, total saponins must measure.
This saponin extract must not be less than 75.0% containing total saponins.
2. oleanolic acid, echinocystic acid are measured according to high performance liquid chromatography (appendix VI D of Chinese Pharmacopoeia version in 2010)
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filler, and acetonitrile-water (90:10) is mobile phase, flow velocity 1.0mlmin-1, the detection wavelength is 210nm, 25 ℃ of column temperatures, sample size 10uL, number of theoretical plate calculates and should be not less than 2500 by echinocystic acid and oleanolic acid peak.
Echinocystic acid is got in the preparation of reference substance solution and the oleanolic acid reference substance is appropriate, adds methanol and makes the solution of every 1ml containing 0.60mg/ml and 0.50mg/ml, product solution in contrast.
The about 0.15g of Fructus Gleditsiae Abnormalis total saponins is got in the preparation of need testing solution, accurately weighed, add kieselguhr 3g, mix thoroughly, put in apparatus,Soxhlet's, add methanol 100mL, backflow 3h, let cool, evaporate to dryness, residue adds dilute hydrochloric acid 80mL to be made to dissolve, quantitatively be transferred in round-bottomed flask, heating in water bath hydrolysis 2h, let cool, quantitatively be transferred in separatory funnel, with chloroform extraction 3 times (50, 50, 30mL), the combined chloroform extracting solution, wash with water 2 times, each 60mL, discard water liquid, the chloroform solution evaporate to dryness, residue is with dissolve with methanol and be settled in the 10mL measuring bottle, shake up, as need testing solution.
Algoscopy is accurate reference substance solution and each 10 μ l of need testing solution of drawing respectively, and the injection liquid chromatography, measure, and obtains.
This product must not be less than 180.0mg/g containing echinocystic acid, containing oleanolic acid, must not be less than 100.0mg/g.
[storage] sealing, put shady and cool dry place and preserve.
[function with cure mainly] this product is the leukemia new drug, is applicable to the treatment of leukemia.
[attention] serious gastric ulcer person and anemia of pregnant woman, spitting of blood, haematemesis person are cautious use of.
[preparation] Fructus Gleditsiae Abnormalis total saponin capsules.
List of references:
[1] Chinese Pharmacopoeia Commission. the Pharmacopoeia of the People's Republic of China. Beijing: Chemical Industry Press, 2005:44.
Claims (1)
1. the preparation method of a Fructus Gleditsiae Abnormalis total saponins, it is characterized in that: step is as follows:
(1) pre-treatment that Fructus Gleditsiae Abnormalis extracts: get dry Fructus Gleditsiae Abnormalis medical material, pulverize as coarse powder, standby;
(2) the decocting extraction process of Fructus Gleditsiae Abnormalis total saponins: get above-mentioned Fructus Gleditsiae Abnormalis coarse powder, add 10 times of amounts of deionized water, decoct 2 hours, filter; Medicinal residues add the water of 8 times of amounts again, decoct 2 hours, filter; Medicinal residues add the water of 8 times of amounts again, decoct 2 hours, filter; Merge three times filtrate, concentrating under reduced pressure obtains the medicinal liquid that relative density is 1.10～1.15, standby;
(3) n-butanol extraction of Fructus Gleditsiae Abnormalis total saponins: get above-mentioned medicinal liquid, with water saturation butanol solution extraction 3 times, merge n-butanol extracting liquid, reclaim under reduced pressure n-butyl alcohol to relative density is 1.15～1.20, obtains n-butanol extract;
(4) purification by macroporous resin of Fructus Gleditsiae Abnormalis total saponins separates: get above-mentioned n-butanol extract, add the water of 6 times of amounts, fully dissolve, filter, collect filtrate, add in the D101 macroporous resin column, first be eluted to closely colourless with distilled water, then use 10% ethanol elution to closely colourless, use 70% ethanol elution again, collect 70% ethanol elution, concentrating under reduced pressure obtains the thick paste that relative density is 1.20-1.25,60～65 ℃ of drying under reduced pressure, obtain the Fructus Gleditsiae Abnormalis total saponins.
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