CN103083388B - 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 (also known as leukemia) are the malignant diseases of serious threat human life, account for first of death toll, two, although great manpower and fund have all been dropped in countries in the world, carry out capturing research, and achieve certain progress, but at present still without desirable medicine and method, clinical in early at present, in, patient with advanced cancer many employings hands art, 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 the life of patient, 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, also the curative effect had is not good enough, the cancer therapy drug of real acquisition satisfactory effect is few in number, therefore, development high-efficiency low-toxicity, quality controllable anticancer and treat leukemic medicine, there is important clinical value and wide market prospect.
Chinese medicine is a great medical treasure-house, curing difficult and complicated cases and in chronic disease, there is unique advantage, the leukemia resisting action of Fructus Gleditsiae Abnormalis, plant screening Chinese medicine from more than 100 to find, 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, effect of mass dissipating and swelling eliminating, clinical being mainly used in treats apoplexy locked mouth, remain unconscious, (China's coastal port, the Chemical Industry Press such as epilepsy abundant expectoration, version in 2005,222).Fructus Gleditsiae Abnormalis and Chinese honey locust (fruit) are all rich in saponin component, are now separated from Fructus Gleditsiae Abnormalis n-butanol portion, 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).Publication number is disclose a kind of Gleditschiasaponin extract and preparation method thereof and application in the Chinese invention patent application of CN 101537036A, there is following defect in it: extracts total saponin content in the Gleditschiasaponin extract obtained not high, drug effect is undesirable, still has the space of improvement.
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 preparation method for Fructus Gleditsiae Abnormalis total saponins, step is as follows:
(1) pre-treatment of Fructus Gleditsiae Abnormalis extraction: get dry Fructus Gleditsiae Abnormalis medical material, pulverize as coarse powder, for subsequent use;
(2) the decocting extraction process of Fructus Gleditsiae Abnormalis total saponins: get above-mentioned Fructus Gleditsiae Abnormalis coarse powder, adds deionized water 10 times amount (weight multiple), decocts 2 hours, filters; Medicinal residues add the water of 8 times amount (weight multiple) again, decoct 2 hours, filter; Medicinal residues add the water of 8 times amount (weight multiple) again, decoct 2 hours, filter; Merge three filtrates, concentrating under reduced pressure obtains the medicinal liquid that relative density is 1.10 ~ 1.15 (60 DEG C of surveys), for subsequent use;
(3) n-butanol extraction of Fructus Gleditsiae Abnormalis total saponins: get above-mentioned medicinal liquid, extracts 3 times with water saturation butanol solution, and merge n-butanol extracting liquid, reclaim under reduced pressure n-butyl alcohol to relative density is 1.15 ~ 1.20 (60 DEG C), obtains n-butanol extract;
(4) purification by macroporous resin of Fructus Gleditsiae Abnormalis total saponins is separated: get above-mentioned n-butanol extract, add the water (weight multiple) of 6 times amount, slight fever makes abundant dissolving, let cool, filter, collect filtrate, add D101 macroporous resin column (need anticipate, processing mode is conventional treatment mode) in, first be eluted to closely colourless with distilled water, then 10% ethanol (volume fraction) is used to be eluted to closely colourless, use 70% ethanol (volume fraction) eluting again, collect 70% ethanol elution, concentrating under reduced pressure obtains the thick paste that relative density is 1.20-1.25 (60 DEG C), 60 ~ 65 DEG C of drying under reduced pressure, obtain 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 obtained, is sundown powder, acrid in the mouth, salty, and have the penetrating odor of saponin, total saponin content reaches more than 80%, may be used for treating leukemia; During embody rule, fully can mix with starch and make capsule.
Method of the present invention and publication number are for compared with technique disclosed in CN 101537036A, 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, after reclaiming drying, obtain Fructus Gleditsiae Abnormalis total saponins, 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, after reclaiming drying, obtain Fructus Gleditsiae Abnormalis extract, in extract, total saponin content about 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 be obviously better than former patent process sample, its specific experiment the results are shown in embodiment two, embodiment four:
Fructus Gleditsiae Abnormalis total saponins is to the research of K562, NB4 cyto-inhibition: Fructus Gleditsiae Abnormalis total saponins 1 and 2 and the propagation of control drug amycin to K562, NB4 cell have significant inhibitory action, and along with the increase of drug level and the prolongation of action time, it strengthens gradually to the suppression ratio of tumor cell.The inhibited proliferation of Fructus Gleditsiae Abnormalis total saponins 1 pair of K562, NB4 cell is better than Fructus Gleditsiae Abnormalis total saponins 2.
Fructus Gleditsiae Abnormalis total saponins is to mice S
180sarcoma tumor-inhibiting action is studied: Fructus Gleditsiae Abnormalis total saponins 1 (this patent technique) senior middle school 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.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.Total saponins 1 couple of S that this technique is extracted
180inhibitory action be obviously better than more former patent technique total saponins 2.
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.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is further illustrated.
The optimization of embodiment one Fructus Gleditsiae Abnormalis total saponins preparation method
1, the research data of decocting extraction
(1) determination of decocting condition: to Fructus Gleditsiae Abnormalis decocting technique preferred in, with total saponins yield for index, on the amount of water affecting extraction ratio, extraction time, extraction time has carried out the orthogonal test of Three factors-levels, preferably determine suitable decocting extraction conditions, the concrete test method of its orthogonal test is: accurately take Fructus Gleditsiae Abnormalis coarse powder 100g, nominal 9 parts, 9 decocting tests are carried out respectively by the decocting condition of test arrangement in table 1, be settled in 100ml measuring bottle by concentrated for each experimental liquid, the content of total saponins in each extraction sample is measured respectively by the assay method of total saponins, experimental factor and water-glass are in table 1, the arrangement of 9 orthogonal tests and the results are shown in Table 2.
Ether sedimentation method measures total saponin content: Qu Geci testing liquid 10ml (suitable crude drug amount 10g), water bath method, residue adds methanol 30ml, slight fever makes dissolving, lets cool, and filters, filtrate is under fully stirring, slowly add the ether of 3 times amount, add rear continuation stirring 10 minutes, sealing, put shady and cool place and leave standstill 24h, incline and supernatant, precipitation sucking filtration to dry, and with methanol: ether (1:2) mixed solvent 10ml washs, volatilize solvent, 60 ~ 65 DEG C of vacuum dryings, weigh, and calculate the extraction total amount of total saponins in each sample.
Table 1 experimental factor and water-glass
* amount of water is multiple first time adding the medical material amount that feeds intake, and rear secondary respectively reduces 2 times.
Table 2 decocting orthogonal test arranges and result
The result of the test of his-and-hers watches 2 carries out intuitive analysis, from extreme difference R is known C (7.3) > B (7.0) > A (1.3) is affected on total saponins yield, show that decocting time C and extraction time B is the principal element affecting total saponins extraction ratio, and C
3> C
2> C
1, B
3> B
2> B
1, therefore select C
3and B
3, amount of water is the secondary cause affecting total saponins extraction ratio, can an optional level, therefore selects A
1, therefore the suitable condition of this Fructus Gleditsiae Abnormalis decocting should be A
1b
3c
3, namely add the water of 10 times amount, 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, decocts, and filters; Extract three times, merging filtrate, be evaporated to the medicinal liquid that relative density is 1.10 ~ 1.15 (60 DEG C).
2, the Medium scale test of Fructus Gleditsiae Abnormalis total saponins
The decocting of test agent and n-butanol extraction in (1) three batch of Fructus Gleditsiae Abnormalis total saponins
In Fructus Gleditsiae Abnormalis herbal decoction, except containing except total saponins, still containing 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.Determine in the condition that decocting and n-butanol extraction are suitable for extracting in the test of Fructus Gleditsiae Abnormalis total saponins lab scale, carried out decocting and the n-butanol extraction test of test agent in three batches, its concrete grammar is: the Fructus Gleditsiae Abnormalis for subsequent use taking 20Kg, adds deionized water 10 times amount, decoct 2 hours, filter, medicinal residues add the water of 8 times amount again, the same decocting 2 times, each 2 hours, filter, merge three filtrates, be evaporated to the medicinal liquid that relative density is 1.10 ~ 1.15 (60 DEG C);
Let cool, 3 times are extracted with water saturation butanol solution, add water-saturated n-butanol 250ml by every 1000ml at every turn, merge 3 n-butanol extracting liquids, reclaim under reduced pressure to relative density is the runny plaste of 1.15 ~ 1.20 (60 DEG C), weigh, calculate n-butanol extract yield and measure the content of total saponins in each batch sample, the results are shown in Table 3.
Table 3 three batches of Fructus Gleditsiae Abnormalis total saponins n-butanol extraction results
| 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 is separated
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 lower than the technical requirement of 50%, for improving the content of total saponins in saponin extract further, again saponin extract is carried out to the technical study of macroporous adsorbent resin (D101) purifies and separates saponin extract, and assay has been carried out to total saponins in the saponin extract after separation and purification.Result shows, after the saponin extract upper prop of n-butanol extraction, first with the abundant eluting of water, then use 10% and 70% ethanol elution, can effectively make saponin extract obtain purification, in 70% alcohol washing, saponin content about reaches 80%, drying, obtains Fructus Gleditsiae Abnormalis total saponins.On the basis of lab scale purifies and separates determination 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 dissolving, let cool, filter, filtrate adds D101 macroporous resin column and (need anticipate, processing mode is conventional treatment mode) in, first be eluted to closely colourless with distilled water, then closely colourless with the same eluting that carries out of 10% ethanol, use 70% ethanol elution again, collect 70% alcohol eluen, be evaporated to the thick paste of relative density 1.20-1.25 (60 DEG C), 60-65 DEG C 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 table 4 three batches of Fructus Gleditsiae Abnormalis total saponins
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, subpackage, get product capsule, each batch of Fructus Gleditsiae Abnormalis total saponin capsules must measure and yield rate in table 5.
The Medium scale result of table 5 three batches of Fructus Gleditsiae Abnormalis total saponin capsules
Note: 1. this product often criticizes that theoretical capsule must measure is 10000.
Embodiment two Fructus Gleditsiae Abnormalis total saponins is to the research of K562, NB4 cyto-inhibition
1 experiment material and instrument
The preparation of 1.1 Fructus Gleditsiae Abnormalis test specimen liquid:
Fructus Gleditsiae Abnormalis total saponins 1 is 70% ethanol elution thing (present invention process sample) after upper macroporous resin column, sample lot number: 20111013.By development, seminar provides.Method is as follows:
(1) pre-treatment of Fructus Gleditsiae Abnormalis extraction: get dry Fructus Gleditsiae Abnormalis medical material, pulverize as coarse powder, for subsequent use;
(2) the decocting extraction process of Fructus Gleditsiae Abnormalis total saponins: get above-mentioned Fructus Gleditsiae Abnormalis coarse powder, adds deionized water 10 times amount (weight multiple), decocts 2 hours, filters; Medicinal residues add the water of 8 times amount (weight multiple) again, decoct 2 hours, filter; Medicinal residues add the water of 8 times amount (weight multiple) again, decoct 2 hours, filter; Merge three filtrates, concentrating under reduced pressure obtains the medicinal liquid that relative density is 1.10 ~ 1.15 (60 DEG C of surveys), for subsequent use;
(3) n-butanol extraction of Fructus Gleditsiae Abnormalis total saponins: get above-mentioned medicinal liquid, extracts 3 times with water saturation butanol solution, and merge n-butanol extracting liquid, reclaim under reduced pressure to relative density is that the runny plaste of 1.15 ~ 1.20 (60 DEG C) obtains n-butanol extract;
(4) purification by macroporous resin of Fructus Gleditsiae Abnormalis total saponins is separated: get above-mentioned n-butanol extract, add the water (weight multiple) of 6 times amount, slight fever makes abundant dissolving, let cool, filter, collect filtrate, add in the D101 macroporous resin column of having anticipated, first be eluted to closely colourless with distilled water, then 10% ethanol (volume fraction) is used to be eluted to closely colourless, use 70% ethanol (volume fraction) eluting again, collect 70% ethanol elution, concentrating under reduced pressure obtains the thick paste that relative density is 1.20-1.25 (60 DEG C), 60 ~ 65 DEG C of drying under reduced pressure, obtain Fructus Gleditsiae Abnormalis total saponins.
Fructus Gleditsiae Abnormalis total saponins 2 for patent publication No. be in CN 101537036A embodiment 1 prepare product, sample lot number: 20120316.By development, seminar provides.
Accurately take Fructus Gleditsiae Abnormalis total saponins respectively appropriate, be made into every milliliter of medicinal liquid containing 50mg with water.Degerming with 0.22 μm of syringe needle frit, be the test specimen of this external leukemia cell.
The preparation of 1.2 control drug injection amycin: injection amycin is test positive control drug, produced by Actavis Italy S.p.A, 10mg/ props up, lot number 0QL0073, normal saline dilution during test.
1.3 tumor cell line
K562: human chronic polymorpho nuclear leukemia cells strain, suspension culture, adjusts cell number 4 × 10 during test
5/ ml.
NB4: people's acute promyelocytic leukemia, suspension culture, adjusts cell number 4 × 10 during test
5/ ml.
1.4 test reagents: RPMI-1640 dry powder (U.S. Gibco Products); New-born calf serum; Tetramethyl ribavirin is blue; Dimethyl sulfoxide; 0.25% trypsin.
1.5 instruments: SW-CJ-2FD superclean bench, BDS200-PH inverted microscope, IL-161CI CO2 gas incubator, xMark microplate reader.
2 test methods and result
The cultivation of 2.1 tumor cells: K562, NB4 cell RPMI1640 complete culture solution, at 37 DEG C, 5%CO
2incubator is cultivated.Take the logarithm trophophase cell for experiment.
2.2 mtt assay measure: the tumor cell getting exponential phase of growth is centrifugal, counting, prepares cell suspension, is inoculated in 96 orifice plates, every hole 100 μ l.Add the medicinal liquid to be measured of variable concentrations respectively.And set up positive control and the negative control group of variable concentrations.Every concentration is four multiple holes.Put 37 DEG C, 5%CO
2in incubator, cultivate 24 hours and 72 hours respectively.Then every hole adds MTT (5mg/ml) 10 μ l, continue cultivation to take out after 4 hours, centrifugal, suck supernatant, every hole adds DMSO 150 μ l, again culture plate is shaken even about 5min, basis of microscopic observation colored particles surveys OD value in 20min microplate reader after disappearing under 490nm wavelength condition.Calculate growth of tumour cell suppression ratio.Separately ask its IC to improve Kou Shi computing method
50value, refers 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 the propagation of amycin to K562, NB4 cell have significant inhibitory action, and along with the increase of drug level and the prolongation of action time, the suppression ratio of medicine to cell also increases.
The inhibitory action (24h) of table 6 Fructus Gleditsiae Abnormalis total saponins 1 pair of K562, NB4 cell proliferation
Compare with cell controls group, △ P<0.05; N=4
The inhibitory action (24h) of table 7 Fructus Gleditsiae Abnormalis total saponins 2 pairs of K562, NB4 cell proliferation
Compare with cell controls group, △ P<0.05; N=4
Table 8 amycin is to the inhibitory action (24h) of K562, NB4 cell proliferation
Compare with cell controls group, △ P<0.05; N=4
The inhibitory action (72h) of table 9 Fructus Gleditsiae Abnormalis total saponins 1 pair of K562, NB4 cell proliferation
Compare with cell controls group, △ P<0.05; N=4
The inhibitory action (72h) of table 10 Fructus Gleditsiae Abnormalis total saponins 2 pairs of K562, NB4 cell proliferation
Compare with cell controls group, △ P<0.05; N=4
Table 11 amycin is to the inhibitory action (72h) of K562, NB4 cell proliferation
Compare with cell controls group, △ P<0.05; N=4
3 brief summaries
3.1 Fructus Gleditsiae Abnormalis total saponins 1 and 2 and the propagation of control drug amycin to K562, NB4 cell have significant inhibitory action, and along with the increase of drug level and the prolongation of action time, it strengthens gradually to the suppression ratio of tumor cell.
Inhibited proliferation comparatively Fructus Gleditsiae Abnormalis total saponins the last 2 of 3.2 Fructus Gleditsiae Abnormalis total saponins, 1 pair of K562, NB4 cell.
Embodiment three Fructus Gleditsiae Abnormalis total saponins is to the research of HL-60 cyto-inhibition
1 experiment material and instrument
The preparation of 1.1 Fructus Gleditsiae Abnormalis test specimen liquid: accurately take Fructus Gleditsiae Abnormalis total saponins (with embodiment two) respectively appropriate, is made into every milliliter of medicinal liquid containing 50mg with water.Degerming with 0.22 μm of syringe needle frit, be the test specimen of this external leukemia cell.
1.2 cisplatin for inj (CDDP): be test positive control drug, Shandong Qilu Pharmaceutical Factory produce, 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: HL-60, suspension culture, adjusts cell number 1.5 × 105/ml during test.
2 test methods and result
HL-60 cell centrifugation, the counting of exponential phase of growth are got in the cultivation of 2.1 tumor cells, the cell suspension of preparation 1.5 × 105 cell/ml, be inoculated in 96 orifice plates, every hole 0.1ml, put in 37 DEG C of 5%CO2 incubators and cultivate after 24 hours, add the medicinal liquid to be measured of variable concentrations respectively, 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 cultivation respectively centrifugal after 24 hours and 72 hours, incline liquid in hole, twice is washed with PH7.41/15MPBS, then the MTT liquid adding 0.2ml continues cultivation 4 hours, liquid in centrifugal hole of inclining again, every hole adds dimethyl sulfoxide 0.2ml vibration and dissolves its crystallization, surveys OD value by microplate reader under 570nm wavelength condition, and be calculated as follows the suppression ratio of medicine to cell, separately ask its IC with the simple and direct composite accounting of Sun Rui unit
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 each dosage group of table 12 Fructus Gleditsiae Abnormalis total saponins is to the inhibitory action (24h) of HL-60 cell
The each dosage group of table 13 Fructus Gleditsiae Abnormalis total saponins is to the inhibitory action (72h) of HL-60 cell
In table 12,13, data show that Fructus Gleditsiae Abnormalis total saponins has obvious inhibitory action to HL-60 cell, and its inhibition strength obviously strengthens with the increasing of drug dose.According to Fructus Gleditsiae Abnormalis total saponins, the medicament contact IC of 24,72 hours is calculated respectively to the suppression ratio that HL-60 produces
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 people in loop.
3 discuss and brief summary
3.1 Fructus Gleditsiae Abnormalis total saponins have obvious inhibitory action and dosage, time effect dependency to HL-60 cell.
3.2 drug doses testing each group are determined according to trial test result, and when the neighbouring two groups of dose ratios of trial test display 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 saponin extract effect 24, the 72 hours IC to HL-60 cell
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 Fructus Gleditsiae Abnormalis total saponins has more stable extracorporeal anti-tumor effect.
Embodiment four Fructus Gleditsiae Abnormalis total saponins is to mice S
180sarcoma tumor-inhibiting action is studied
1 material
Trial drug: Fructus Gleditsiae Abnormalis total saponins 1, Fructus Gleditsiae Abnormalis total saponins 2, with embodiment two.
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
180sarcoma mice, draws from pharmacological room of medicine institute of Shandong Academy of Medical Sciences.
2 experimental techniques and result
After mice buys 24h, give respectively by the mice S of normal saline dilution
180ascites fluid, containing oncocyte 5 × 10
6/ ml inoculates in left oxter, after inoculation 24h, random packet administration, be grouped into NS matched group 24 (each 12 of ♀ ♂), 20mg/kg cyclophosphamide SC group 10 (each 5 of ♀ ♂), total saponins and total extract various dose group, often organize 12 (being each 6 of ♀ ♂), daily once, successive administration 9 days, after last administration 24h, de-neck puts to death mice, peel off tumor body, GB303 electronic balance claims tumor body weight, t inspection respectively organizes tumor weight, 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.
Table 14 total saponins 1 and total saponins 2 couples of mice S
180sarcoma tumor-inhibiting action
| Group | n | Tumor weight | Tumour inhibiting rate % |
| NS matched group | 24 | 2.05±1.02 | —— |
| Cyclophosphamide 20mg/kg | 10 | 1.07±0.26 | 47.8 |
| Total saponins 1 high dose group | 12 | 1.24±0.30 | 39.5 |
| Dosage group in total saponins 1 | 12 | 1.35±0.71 | 34.2 |
| Total saponins 1 low dose group | 12 | 1.75±0.76 | 14.6 |
| Total saponins 2 high dose group | 12 | 1.41±0.34 | 31.2 |
| Dosage group in total saponins 2 | 12 | 1.57±0.66 | 23.4 |
| Total saponins 2 low dose group | 12 | 1.94±0.84 | 5.37 |
3 results and discussion
3.1 experimental results show, Fructus Gleditsiae Abnormalis total saponins 1 senior middle school 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 results show, 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.
3.3 above-mentioned experimental results show, the total saponins that this technique is extracted is to S
180inhibitory action be obviously better than former patent technique.
The studies on acute toxicity of embodiment five Fructus Gleditsiae Abnormalis saponin extract and total saponins
1. material
1.1 by test product: Fructus Gleditsiae Abnormalis extract is the n-butanol extract of n-butanol extraction before upper macroporous resin in embodiment two; Fructus Gleditsiae Abnormalis total saponins is with embodiment two.
1.2 animals, environment and feedstuff:
Animal: KM mice, 18-22g, ♀ ♂ half and half, Shandong University's Experimental Animal Center provides, the quality certification number: SCXK(Shandong) 20090001.
Environment: Medicine Institute, Shandong Province animal experimental center, credit number: SYXK (Shandong) 20050052.
Feedstuff: management of laboratory animal center, Shandong Province provides, the quality certification number: SCXK (Shandong) 20040014.
1.3 instruments: PL303 type electronic balance, mettler tolido product.
2. method and result
2.1 preliminary experiment: get 18 ~ 22g (in group, weight differences is less than 4g) mice, ♀ ♂ half and half, water 12h is can't help in fasting, often organize 6, carry out the preliminary experiment of 0%, 100% fatal dose (Dmin, Dmax) of Fructus Gleditsiae Abnormalis total saponins and Fructus Gleditsiae Abnormalis extract respectively, draw approximate 0%, 100% fatal dose of Fructus Gleditsiae Abnormalis total saponins and Fructus Gleditsiae Abnormalis extract.According to preliminary result, carry out the LD of Fructus Gleditsiae Abnormalis total saponins and Fructus Gleditsiae Abnormalis extract respectively
50measure.18-22g KM mice, ♀ ♂ half and half, often organizes 10, and each take Dmax as maximum dosage-feeding by test product, is coefficient determination different dosing dosage with 0.8.Before administration, water 12h is can't help in fasting, 0.2mL/10g gavage, Continuous Observation 14d after gavage, normal diet, records 14 days mouse death rates, calculates the different LD by test product
50interval with the credibility of 95.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
Each group of LD is calculated according to Bliss method
50interval with 95% credibility, 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 non-upper prop
50for 2.72g/kg.
Embodiment six HPLC measures the content of echinocystic acid and oleanolic acid in Fructus Gleditsiae Abnormalis total saponins
Object: the content measuring oleanolic acid and echinocystic acid in Fructus Gleditsiae Abnormalis total saponins, sets up the HPLC assay method of 2 kinds of compositions in said preparation.
Fructus Gleditsiae Abnormalis total saponins is Chinese medicine 5 kind new medicine developed, and Pharmacodynamic test of active extract shows, Fructus Gleditsiae Abnormalis total saponins is antileukemie main active component, has good therapeutic effect to leukemia.And echinocystic acid and oleanolic acid are the main aglycon of saponin in total saponins, for controlling the inherent quality of this product, formulate the quantitative quality control standard that controllability is strong, with reference to the related documents that echinocystic acid and content of oleanolic acid measure, the research of content assaying method has been carried out to the echinocystic acid in Fructus Gleditsiae Abnormalis total saponins and oleanolic acid, by the assay of methodological system thinking and three batch samples, establish the HPLC of echinocystic acid and oleanolic acid in this preparation containing survey method, formulate the content limit of 2 kinds of compositions, be now reported as follows:
1 instrument and reagent
High performance liquid chromatograph (Waters 2695 Separations Module, Waters 2996 Photodiode Array Detector).Chromatographic column is Di Ma company Diamonsil
tM(diamond) C18,5 μm, 150 × 4.6r, ultra-pure water (Millipore company demineralizer), mobile phase acetonitrile is chromatographically pure, and other reagent are analytical pure.Echinocystic acid reference substance (Chinese pharmaceutical biological product inspection institute, lot number 110756-2011010), oleanolic acid reference substance (Chinese pharmaceutical biological product inspection institute, lot number 0709-9803), for assay.
2 methods and result
2.1 chromatographic conditions: be filler with octadecylsilane chemically bonded silica, acetonitrile-water (90:10) is mobile phase, flow velocity 1.0mlmin
-1, determined wavelength is 210nm, column temperature 25 DEG C, sample size 10uL, and number of theoretical plate calculates should be not less than 2500 by echinocystic acid and oleanolic acid peak.Echinocystic acid, oleanolic acid are separated good under these conditions.
The preparation of 2.2 echinocystic acid and oleanolic acid reference substance solution: get echinocystic acid reference substance in right amount in 2mL measuring bottle, accurately weighed is 2.60mg; Get oleanolic acid reference substance in right amount in 2mL measuring bottle, accurately weighed is 2.12mg.Respectively add methanol make dissolving in right amount and be diluted to scale, shake up, as two reference substance storing solutions, (its concentration is respectively 1.30mgmL
-1, 1.06mgmL
-1); Again in each 1mL to the 2mL measuring bottle of above-mentioned two storing solution of accurate absorption, shake up, in contrast product mixed solution, the concentration of its echinocystic acid and oleanolic acid reference substance is respectively 0.65mgmL
-1, 0.53mgmL
-1.
The preparation of 2.3 need testing solutions: get Fructus Gleditsiae Abnormalis total saponins and be about 0.15g, 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 makes dissolving, 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), combined chloroform extracting solution, wash 2 times with water, each 60mL, discard water liquid, chloroform solution evaporate to dryness, residue dissolve with methanol is also settled in 10mL measuring bottle, shake up, as need testing solution.
2.4 methodological study
2.4.1 the selection of wavelength is measured: accurate absorption reference substance solution and each 10 μ L of need testing solution respectively, injection liquid chromatography, detects within the scope of 200 ~ 400nm, draws chromatogram.Result shows, echinocystic acid and oleanolic acid have maximum absorption band at 205nm and 200nm place respectively, for reducing interference, ensures baseline stability, with reference to the pertinent literature of the assay of 2 kinds of compositions, selects 210nm as mensuration wavelength.
2.4.2 blank interference test: each 10 μ L of accurate absorption need testing solution, reference substance solution and neat solvent methanol solution respectively, injection liquid chromatography, detects at 210nm place.Result shows, in test sample chromatograph, on the position identical with oleanolic acid chromatographic retention with reference substance echinocystic acid, have obvious absworption peak, and solvent methanol solution is on correspondence position, has no obvious absorption peaks, shows that this mensuration is without obviously interference, sees Fig. 1.
2.4.3 linear relationship is investigated: precision takes echinocystic acid reference substance in right amount 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, accurately draw above-mentioned each reference substance solution 10 μ L, injection liquid chromatography, measure integrating peak areas value, the results are shown in Table 18, with integrating peak areas value (A) for vertical coordinate, concentration (mgmL
-1) be abscissa, drawing standard curve, result echinocystic acid is at 0.0834375mgmL
-1~ 1.335mgmL
-1in scope, with integrating peak areas value (A) in good linear relation, see Fig. 2-1.Regression analysis is carried out to the data obtained, obtains regression equation y=789999x ﹢ 135181, correlation coefficient r=0.9999.
Result investigated by table 18 echinocystic acid linear relationship
Precision takes oleanolic acid reference substance in right amount 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, accurately draw above-mentioned each reference substance solution 10 μ L, injection liquid chromatography, measure integrating peak areas value, the results are shown in Table 19, with integrating peak areas value (A) for vertical coordinate, concentration (mgmL
-1) be abscissa, drawing standard curve, result oleanolic acid is at 0.066875mgmL
-1~ 1.07mgmL
-1in scope, with integrating peak areas value (A) in good linear relation, see Fig. 2-2.Regression analysis is carried out to the data obtained, obtains regression equation y=854782x ﹢ 74164, correlation coefficient r=1.
Result investigated by table 19 oleanolic acid linear relationship
2.4.4 precision test: accurate absorption need testing solution 10 μ L, continuous sample introduction 5 times, measures integrating peak areas 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: accurate absorption need testing solution 10 μ L, measure 1 time every 2h sample introduction, continuous sample introduction measures 5 times, and results peaks area integral value (A) is substantially constant in minute 8h, measures the meansigma methods of echinocystic acid for 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 6 parts altogether, accurately weighed, by preparation and the content assaying method of Fructus Gleditsiae Abnormalis total saponins need testing solution, prepare each need testing solution and echinocystic acid in working sample and oleanolic acid, 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 echinocystic acid respectively and oleanolic acid reference substance is appropriate, by preparation and the content assaying method of Fructus Gleditsiae Abnormalis total saponin extracts need testing solution, obtain each recovery test liquid and the echinocystic acid measured in each response rate sample and oleanolic acid, calculate 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%, 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: accurately respectively draw reference substance and three crowdes of each 10 μ L of need testing solution, injection liquid chromatography, measures and calculates 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 table 25 three batches of Fructus Gleditsiae Abnormalis total saponins
2.5.2 the assay of echinocystic acid and oleanolic acid in three batches of Chinese medicine No. III medical material: get each about 1g of Fructus Gleditsiae Abnormalis medicinal powder, accurately weighed, by preparation and the content assaying method of Fructus Gleditsiae Abnormalis total saponin extracts need testing solution, obtain each medical material experimental liquid and the echinocystic acid measured in each medical material and oleanolic acid, 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 table 26 three batches of Fructus Gleditsiae Abnormalis medical materials
3 brief summaries and discussion
(1) research of content assaying method has been carried out to the echinocystic acid in Fructus Gleditsiae Abnormalis total saponins and oleanolic acid, by the assay of methodological system thinking and three batch samples, establish the HPLC content assaying method of echinocystic acid and oleanolic acid in preparation, record echinocystic acid at 0.0834375mgmL
-1~ 1.335mgmL
-1in scope, peak area (A) and concentration (mgmL
-1) in 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) in good linear relationship, regression equation y=854782x ﹢ 74164, correlation coefficient r=1.0.
In (2) three batches of Fructus Gleditsiae Abnormalis total saponins, echinocystic acid total content is respectively 213.2mgg
-1, 225.0mgg
-1, 219.3mgg
-1mean sample recovery rate is 98.22%, coefficient of variation RSD is 1.35%; Oleanolic acid total content is respectively 120.0mgg
-1, 127.8mgg
-1, 123.5mgg
-1, mean sample recovery rate 98.84%, coefficient of variation RSD is 0.86%.
Echinocystic acid total content 26.70mgg respectively in (3) three batches of Fructus Gleditsiae Abnormalis medical materials
-1, 27.47mgg
-1, 27.24mgg
-1, oleanolic acid total content is respectively 17.32mgg
-1, 16.18mgg
-1, 16.69mgg
-1.
(4) this mensuration methanol extraction, measures the main aglycon echinocystic acid of Fructus Gleditsiae Abnormalis saponin and the content of oleanolic acid after hydrochloric acid hydrolysis.The features such as this law has highly sensitive, favorable reproducibility, and specificity is strong control the ideal of this preparation inherent quality containing survey method.
Attached: the quality standard (draft) of Fructus Gleditsiae Abnormalis total saponins
Fructus Gleditsiae Abnormalis total saponins
Zhuyazao Zongzaogan
[prescription] Fructus Gleditsiae Abnormalis 1000g.
[method for making] gets Fructus Gleditsiae Abnormalis coarse powder, add deionized water 10 times amount, decoct 2 hours, filter, medicinal residues add the water of 8 times amount again, the same decocting 2 times, each 2 hours, filter, merge three filtrates, be evaporated to the medicinal liquid of relative density 1.10 ~ 1.15 (60 DEG C of surveys), 3 times are extracted with water-saturated n-butanol, merge n-butanol extracting liquid, reclaim under reduced pressure to relative density is 1.15 ~ 1.20 (60 DEG C), the appropriate slight fever that adds water makes abundant dissolving, let cool, filter, collect filtrate, add in processed good D101 macroporous resin column, first be eluted to closely colourless with distilled water, then closely colourless with the same eluting that carries out of 10% ethanol, use 70% ethanol elution again, collect 70% alcohol eluen, be evaporated to the thick paste of relative density 1.20-1.25 (60 DEG C), 60-65 DEG C of drying under reduced pressure, obtain.
[character] this product is sundown powder, acrid in the mouth, salty, has the penetrating odor of saponin.
[discriminating] (1) is got this product and is about 0.2g, adds ethanol 8ml, and reflux 5 minutes, lets cool, and filter, get filtrate 0.5ml, put in little porcelain dish, evaporate to dryness, lets cool, and adds acetic anhydride 3, stirs evenly, and add 2, sulphuric acid along ware wall, solution fades in reddish violet.
(2) get this product and be about 0.2g, add water 10ml, boils 10 minutes, and filter, the strong jolting of filtrate, namely produces lasting foam (more than 15 minutes persistent period).
(3) get this product and be about 0.5g, add ethyl acetate-methanol (2:8, V/V) mixed liquor 15ml, reflux, extract, 30 minutes, lets cool, and filters, 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 each 5 μ l of above-mentioned two kinds of solution respectively, point is on the silica gel G plate that same 0.3% sodium carboxymethyl cellulose is binding agent, with n-butyl alcohol-methanol-ammonia (10:2:5) for developing solvent, launch, take out, dry, spray is with 5% phosphomolybdic acid ethanol, and 105 DEG C to be heated to spot development clear, in test sample chromatograph, on the position corresponding to control medicinal material chromatograph, the blue spot of aobvious same color.
[inspection] moisture: according to aquametry (Chinese Pharmacopoeia version in 2010 annex LX H) 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 dissolving, filter, with 10ml methanol gradation washing nozzle, washing liquid is incorporated in filtrate, under fully stirring, slowly add the ether of 3 times amount, add rear continuation and stir 3-5 minute, sealing, put shady and cool place and leave standstill 24h, filter with the filter paper of constant weight, and with methanol: the 10ml washing precipitation of ether (1:2) mixed solvent and filter, washing liquid filters, filter paper volatilizes solvent, with triangular flask with putting 60 ~ 65 DEG C of vacuum dryings, 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 measure according to high performance liquid chromatography (Chinese Pharmacopoeia version in 2010 annex VI D)
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filler, acetonitrile-water (90:10) is mobile phase, flow velocity 1.0mlmin-1, determined wavelength is 210nm, column temperature 25 DEG C, sample size 10uL, number of theoretical plate calculates 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 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 preparation of need testing solution is got Fructus Gleditsiae Abnormalis total saponins and is about 0.15g, 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 makes dissolving, 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), combined chloroform extracting solution, wash 2 times with water, each 60mL, discard water liquid, chloroform solution evaporate to dryness, residue dissolve with methanol is also settled in 10mL measuring bottle, shake up, as need testing solution.
Algoscopy is accurate respectively draws reference substance solution and each 10 μ l of need testing solution, injection liquid chromatography, measures, to obtain final product.
This product must not be less than 180.0mg/g containing echinocystic acid, must not be less than 100.0mg/g containing oleanolic acid.
[storage] seals, and puts shady and cool dry place and preserves.
[function with cure mainly] this product is 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. a preparation method for Fructus Gleditsiae Abnormalis total saponins, is characterized in that: step is as follows:
(1) pre-treatment of Fructus Gleditsiae Abnormalis extraction: get dry Fructus Gleditsiae Abnormalis medical material, pulverize as coarse powder, for subsequent use;
(2) the decocting extraction process of Fructus Gleditsiae Abnormalis total saponins: get above-mentioned Fructus Gleditsiae Abnormalis coarse powder, adds deionized water 10 times amount, decocts 2 hours, filters; Medicinal residues add the water of 8 times amount again, decoct 2 hours, filter; Medicinal residues add the water of 8 times amount again, decoct 2 hours, filter; Merge three filtrates, concentrating under reduced pressure, 60 DEG C time, record the medicinal liquid that relative density is 1.10 ~ 1.15, for subsequent use;
(3) n-butanol extraction of Fructus Gleditsiae Abnormalis total saponins: get above-mentioned medicinal liquid, extracts 3 times with water saturation butanol solution, and merge n-butanol extracting liquid, reclaim under reduced pressure n-butyl alcohol, 60 DEG C time, record relative density is 1.15 ~ 1.20, obtains n-butanol extract;
(4) purification by macroporous resin of Fructus Gleditsiae Abnormalis total saponins is separated: get above-mentioned n-butanol extract, add the water of 6 times amount, slight fever makes abundant dissolving, let cool rear filtration, collect filtrate, add in D101 macroporous resin column, first be eluted to closely colourless with distilled water, then use 10% ethanol elution to closely colourless, then use 70% ethanol elution, collect 70% ethanol elution, concentrating under reduced pressure, 60 DEG C time, record the thick paste that relative density is 1.20-1.25,60 ~ 65 DEG C of drying under reduced pressure, obtain Fructus Gleditsiae Abnormalis total saponins; Described Fructus Gleditsiae Abnormalis total saponins is buff powder, acrid in the mouth, salty, and have the penetrating odor of saponin, its total saponin content more than 80%, water content is no more than 6.0%, is no less than 180.0mg/g containing echinocystic acid, is no less than 100.0mg/g containing oleanolic acid; Described macroporous resin column need be anticipated, and processing mode is conventional treatment mode.
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| CN1429834A (en) * | 2002-12-31 | 2003-07-16 | 中国药科大学 | Fructus gleditsiae total saponin, its preparation method and use in preparation of medicine |
| EP1803351A1 (en) * | 2005-12-28 | 2007-07-04 | Ideasupply.com Argentina, S.A. | Extract of the seedless pod of gleditsia amorphoides and its use as an agricultural adjuvant |
| CN101537036A (en) * | 2009-03-30 | 2009-09-23 | 山东省中医药研究院 | Soap pod saponin extract as well as preparation method and application thereof |
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