CN103933125B - The extracting method and products thereof of anti-tumor active substance and application in Fructus Forsythiae - Google Patents
The extracting method and products thereof of anti-tumor active substance and application in Fructus Forsythiae Download PDFInfo
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Abstract
The invention discloses a kind of method extracting anti-tumor active substance and products thereof and application from Fructus Forsythiae.A kind of method extracting anti-tumor active substance from Fructus Forsythiae of the present invention, carries out: one, the preparation of Fructus Forsythiae Aqueous extracts (FS-1) as follows; Two, ethanol precipitation initial gross separation anti-tumor effective component (FS-2); Three, macroporous adsorbent resin column chromatography method is separated Fructus Forsythiae anti-tumor effective component (FS-3); Four, thin layer chromatography is separated the anti-tumor active substance (FS-4) in Fructus Forsythiae.In addition, the present invention confirms that Fructus Forsythiae anti-tumor active substance (FS-4) can induce the apoptosis of SGC-7901gastriccarcinomacellline by experiment, further, the present invention has carried out preliminary identification to the concrete composition in Fructus Forsythiae anti-tumor active substance (FS-4) simultaneously.Extracted the Fructus Forsythiae anti-tumor active substance (FS-4) obtained by method of the present invention, have obvious anti-tumor activity effect, the treatment for tumor disease provides a kind of new technological means.
Description
Technical field
The present invention relates to extracting method of a kind of active substance and products thereof and application, the method extracting anti-tumor active substance in particular to a kind of from Fructus Forsythiae and extract the product and this product that obtain in the application of anti-tumor aspect by the method, the invention belongs to biomedicine technical field.
Background technology
Fructus Forsythiae is that Chinese Clinical commonly uses one of Chinese medicine, has another name called Hemerocallis citrina Baroni bar, Fructus Forsythiae, green grass or young crops stick up, falls to sticking up, Huang Qidan etc.Pharmacopoeia of the People's Republic of China version in 2005 Fructus Forsythiae recorded is Oleaceae (Oleaceae) Forsythia (Forsythia) plant Fructus Forsythiae (Forsythia suspensa(Thunb.) Vahl) dry fruit, the pharmacological actions such as Fructus Forsythiae has antibacterial, heart tonifying, diuresis, town to tell, the diseases such as the conventional acute anemopyretic cold of forsythia fruit for treating, carbuncle sore tumefacting virus, tuberculous lymphadenitis, urinary tract infection.
At present, gastric cancer is one of modal malignant tumor in world wide.Although its sickness rate significantly reduces in developed country, be number two in global cancer related mortality rate.This research has found the antitumor action of Fructus Forsythiae unexpectedly in the experiment of research Fructus Forsythiae extract antivirus action, thus, the present invention has carried out further improvement to the extracting method of Fructus Forsythiae anti-tumor active substance, improve extraction ratio, and this extract is carried out to the mensuration of the anti-tumor activity of system, proved that this extract has the effect of inducing in vitro human gastric cancer cell line SGC-7901 apoptosis.
Summary of the invention
Large in order to solve the chemotherapeutics side effect that existing cancer especially exists in gastric cancer clinical treatment, the problems such as easy drug resistance, the present invention enriches with china natural resources, low price, toxic and side effects is low and to be easy to universal Chinese herbal medicine Fructus Forsythiae (i.e. the dry fruit of Fructus Forsythiae) be object of study, it is separated, extracts, obtain Fructus Forsythiae anti-tumor active substance (FS-4), and it is carried out to the mensuration of anti-tumor activity, and then provide a kind of from Fructus Forsythiae separation and Extraction there is the method for the Fructus Forsythiae anti-tumor active substance of antitumor action.
In order to achieve the above object, present invention employs following technological means:
One, the preparation of Fructus Forsythiae Aqueous extracts (FS-1);
Two, ethanol precipitation initial gross separation anti-tumor effective component (FS-2);
Three, macroporous adsorbent resin column chromatography method is separated Fructus Forsythiae anti-tumor effective component (FS-3);
Four, thin layer chromatography is separated the anti-tumor active substance (FS-4) in Fructus Forsythiae;
Five, the research of Fructus Forsythiae anti-tumor active substance (FS-4) inducing in vitro human gastric cancer cell line SGC-7901 apoptosis effect;
Six, the preliminary identification of Fructus Forsythiae anti-tumor active substance (FS-4).
Concrete, the method that one of the present invention extracts anti-tumor active substance (FS-4) in Fructus Forsythiae is carried out in accordance with the following steps:
(1) preparation of Fructus Forsythiae Aqueous extracts
Fructus Forsythiae is after screening out dirt, and wash soaked overnight, next day, slow fire boiled, and filters, and collect filtrate, evaporation and concentration, makes Aqueous extracts, and sterilizing is labeled as FS-1, puts 4 DEG C of Refrigerator stores for subsequent use;
(2) ethanol precipitation initial gross separation anti-tumor effective component
Get Fructus Forsythiae Aqueous extracts FS-1 and be placed in plastic centrifuge tube, then gradation slowly adds the alcoholic solution that mass percent concentration is 90%-100% wherein, and constantly shake, till the volume of alcoholic solution reaches the 90%-95% of overall solution volume, centrifuge tube is placed in 4 DEG C of refrigerator 8-14h, low-temperature centrifugation, collect supernatant, supernatant through filter paper filtering, after concentrating under reduced pressure, be placed in 4 DEG C of refrigerators for subsequent use, be labeled as FS-2;
(3) macroporous adsorbent resin column chromatography method is separated Fructus Forsythiae anti-tumor effective component
Get FS-2, slowly join the macroporous adsorptive resins top that pretreatment is good, addition is 2% column volume, treat that FS-2 infiltrates resin bed, be the alcoholic solution of 30%, 60%, 90% successively with distilled water, mass percent concentration, carry out eluting with 5.00ml/min flow velocity, collect elution fraction, mass percent concentration be the ethanol elution of 90% after concentrating under reduced pressure under 100 DEG C of conditions, be labeled as FS-3;
(4) thin layer chromatography is separated Fructus Forsythiae anti-tumor active substance
According to benzene: normal hexane: methanol: the volume ratio=14-16:14-16:0.5-1.5:0.5-1.5 of glacial acetic acid prepares developing solvent, prepared on plate at thin layer at room temperature 25 DEG C by FS-3 and launch, duration of run 50-60min, dries; Then, exert a gradual, corrupting influence on thin layer by iodine fumigation and prepare plate, and observe under ultra violet lamp, namely find that FS-3 is divided into 13 bands, collect the 4th band preparing plate number from thin layer, be labeled as FS-4, be anti-tumor active substance.
In the present invention, preferably, step (1) is carried out according to following steps: Fructus Forsythiae is after screening out dirt, and with tap water cleaning 3-6 time, then wash 2-4 time with distillation, then adding distil water makes liquid level exceed Fructus Forsythiae 8-15cm, soaked overnight; Next day, slow fire boiled in 100 DEG C, filtered while hot after 1-3h with absorbent cotton, and medicinal residues repeat to extract, then filter with absorbent cotton, by twice filtrate mixing, in 100 DEG C of evaporation and concentration to 1.00mg/ml, make Aqueous extracts, through interval concora crush sterilization on the three sterilizing, each 40min, puts 4 DEG C of Refrigerator stores for subsequent use.
In the present invention, preferably, the low-temperature centrifugation described in step (2) refers in 4 DEG C, the centrifugal 15min of 4000r/min.
In the present invention, preferably, the macroporous adsorptive resins described in step (3) is X-5 type macroporous adsorptive resins.
In the present invention, preferably, the developing solvent described in step (4) is according to benzene: normal hexane: methanol: the volume ratio=15:15:1:1 preparation of glacial acetic acid, duration of run is 55min.
In the present invention, preferably, described in step (4), the collection method of the 4th band comprises the following steps:
(1) prepare upper several 4th band cut down by plate from thin layer and put into small beaker;
(2) in beaker, add dehydrated alcohol, stir, after precipitation 20-24h, collect supernatant, with fat-soluble 0.22 μm of membrane filtration, obtain the supernatant after filtering, for subsequent use, repeat step (2) 2-3 time;
(3) after the supernatant after the filtration of collecting being mixed, more centrifugal, get supernatant, with the membrane filtration of fat-soluble 0.22 μm after concentrating under reduced pressure, filtrate makes powder through lyophilization, is labeled as FS-4, is anti-tumor active substance.
Further the invention allows for the anti-tumor active substance for preparing according to above arbitrary described method and preparing the application in antitumor drug.
In the present invention, preferably, described tumor is gastric cancer.
The Fructus Forsythiae anti-tumor active substance (FS-4) that the present invention extracts, through a series of antitumor cytolytic activity, confirms that it has the effect of inducing in vitro human gastric cancer cell line SGC-7901 apoptosis.FS-4 acts on SGC-7901 cell 36h half-inhibition concentration (IC
50) be only 3.23 μ g/ml, be better than the effect (IC of clinical commonly used drug cisplatin
50be 4.21 μ g/ml).According to " national new drug (Western medicine) preclinical study guideline compilation " regulation, Plant crude extract IC
50≤ 20 μ g/ml, and cytotoxic dose-dependent relationship, can judge that sample has lethal effect to cell in vitro, and Fructus Forsythiae anti-tumor active substance (FS-4) is a kind of strong anti-tumor active substance as can be seen here.
Accompanying drawing explanation
Fig. 1 is the apoptotic morphological change of SGC-7901 (× 40) after AO/EB Double fluorescence staining method detection of drugs effect 12h;
Note: A. matched group; B.50 μ g/ml FS-4; C.100 μ g/ml FS-4;
Fig. 2 is SGC-7901 cellular morphology under transmission electron microscope after normal cell controls group and FS-4 effect 12h;
Note: transmission electron microscope 1: normal cell × 8000; 2: experimental group cell × 8000; 3: normal cell × 20000; 4: experimental group cell × 20000;
Fig. 3 is that FCM detects FS-4 effect 12h SGC-7901 apoptosis rate result;
Note: 1: matched group; 2:12.5 μ g/ml FS-4 effect group; 3:25 μ g/ml FS-4 effect group; 4:50 μ g/mlFS-4 effect group; 5:100 μ g/ml FS-4 effect group;
Fig. 4 is blank and FS-4 mass spectrum total ion current figure.
Fig. 5 is the corresponding cutting position (right side) that thin layer after iodine fumigation exerts a gradual, corrupting influence on prepares plate (left side) and each band.
Arrow is depicted as the 4th band (FS-4) of number, wherein the 2nd, 3 band visually see less than, only just can see under uviol lamp.
Detailed description of the invention
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear with the description of specific embodiment.But these embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and do not departing under spirit and scope of the invention and can modify to technical scheme of the present invention and detail view or replace, but these amendments and replacement all fall in the scope of protection of the invention.
Embodiment 1: the preparation of Fructus Forsythiae Aqueous extracts (FS-1)
Get Fructus Forsythiae (buy from generation pharmacy, Huang sticks up, Shanxi, the place of production) 500g and, after screening out dirt, clean 5 times with tap water, with distillation washing 3 times, adding distil water makes liquid level exceed Fructus Forsythiae about 10cm, soaked overnight.Next day, slow fire boiled in 100 DEG C, filter with absorbent cotton while hot after 2h, medicinal residues repeat to extract, then filter with absorbent cotton, by twice filtrate mixing, in 100 DEG C of evaporation and concentration to 1.00mg/ml, through interval concora crush sterilization on the three sterilizing, each 40min, makes Aqueous extracts, be labeled as FS-1, put 4 DEG C of Refrigerator stores for subsequent use.
Embodiment 2: ethanol precipitation initial gross separation anti-tumor effective component (FS-2)
Get 3.5ml Fructus Forsythiae Aqueous extracts FS-1 and be placed in plastic centrifuge tube, then segmentation slowly adds the alcoholic solution that mass percent concentration is 100% wherein, and constantly shakes, and reaches till 90% of overall solution volume until volumes of aqueous ethanol.Centrifuge tube is put 4 DEG C of refrigerator 12h, in refrigerated centrifuge 4 DEG C, the centrifugal 15min of 4000r/min, collect supernatant, supernatant is through filter paper filtering, and concentrating under reduced pressure, is placed in 4 DEG C of refrigerators, for subsequent use, is labeled as FS-2.
Embodiment 3: macroporous adsorbent resin column chromatography method is separated Fructus Forsythiae anti-tumor effective component (FS-3)
Get FS-2, slowly join the good macroporous adsorbent resin of pretreatment (X-5 type) capital end, addition is 2% column volume, 8mg/ time, totally 6 times.Treat that FS-2 infiltrates resin bed, be the alcoholic solution of 30%, 60%, 90% successively with distilled water, mass percent concentration, carry out eluting with 5.00ml/min flow velocity, collect elution fraction, mass percent concentration be the alcoholic solution eluent of 90% after concentrating under reduced pressure under 100 DEG C of conditions, obtain FS-3.
Embodiment 4: thin layer chromatography is separated Fructus Forsythiae anti-tumor active substance (FS-4)
(1) activation of lamellae: gradually heated up in an oven by lamellae, maintains 105 DEG C ~ 110 DEG C activation 30min.It is stand-by that Preparative TLC plate after activation is placed on preservation in exsiccator;
(2) point sample: first with pencil apart from 1cm place, lamellae one end gently standardized horizontal line as start line, then draw FS-3 with the microcap of 100 microlitres, in start line, careful point sample, makes applied sample amount reach 2mg/ plate, the hot blast drying of the excellent Preparative TLC plate hair dryer of point, for subsequent use;
(3) launch: first press benzene: normal hexane: methanol: the volume ratio=15:15:1:1 of glacial acetic acid configures developing solvent.In expansion cylinder, add the developing solvent prepared, make it highly be no more than 1cm, excellent for some lamellae is carefully put into expansion cylinder, point sample one end down, is immersed in developing solvent, is built lid.Room temperature 25 DEG C, duration of run 55min, dries;
(4) develop the color
Iodine vapor develops the color: the lamellae evaporation of solvent after launching is placed on one and is placed with 1 ~ 2min in the closed glass container of a small amount of solid particle iodine, then from iodine vapor container, takes out lamellae, draws with needle point the band that component manifests;
(5) collect:
The lamellae obtained through upper several step (1)-(4) by polylith is first rule under uviol lamp, cuts after color fade:
Being cut down by the 4th band several on lamellae collects in small beaker, add the ethanol solution of its volume 5 times, stir, supernatant is collected after precipitation 24h, with fat-soluble 0.22 μm of membrane filtration supernatant, above step repeat 3 times (concrete operations: 1. use syringe sucking-off supernatant from beaker A, then with fat-soluble 0.22 membrane filtration in beaker B; 2. the anhydrous ethanol solvent adding 5 times of volumes again in beaker A repeats above-mentioned steps; 3. blast drier evaporate to dryness can be used at any time in beaker B.); After supernatant is mixed, more centrifugal, get supernatant, be finally evaporated to about 10ml, then with the membrane filtration of 0.22 μm, filtrate makes powder through cryodesiccated method by extracting the medicine obtained, and be labeled as FS-4 ,-20 DEG C save backup.
Embodiment 5: Fructus Forsythiae anti-tumor active substance (FS-4) inducing in vitro human gastric cancer cell line SGC-7901 apoptosis effect
Adopt the external inhibitory action to SGC-7901gastriccarcinomacellline growth, propagation of MTT colorimetric determination Fructus Forsythiae anti-tumor active substance.FS-4 act on SGC-7901gastriccarcinomacellline 6,12,24, the half-inhibition concentration (IC of 36h
50) be respectively 87.38 μ g/ml, 56.10 μ g/ml, 34.45 μ g/ml, 3.23 μ g/ml.
Observed the FS-4 effect SGC-7901gastriccarcinomacellline of variable concentrations by AO-EB Double fluorescence staining method after, there is a large amount of apoptotic cells (shown in Fig. 1).
After FS-4 effect SGC-7901gastriccarcinomacellline observed by transmission electron microscope (goldstandard), the ultrastructure of apoptotic cell.Display karyoplasmic ratio reduces, and kernel disappears, and in endochylema, cavity is obvious, the intensive marginalisation of chromatin, cell surface fine hair depigmentation, forms plasma membrane blade sample projection, and has apoptotic body to be formed (shown in Fig. 2).
After FS-4 acts on SGC-7901 cell, cell DNA can be made to rupture, form " scalariform " DNA.
The two dye of flow cytometer Annexin V-PI detects apoptotic situation (shown in Fig. 3).After normal cell controls group and 12.5 μ g/ml, 25 μ g/ml, 50 μ g/ml, 100 μ g/ml FS-4 treatment S GC-7901 cell 12h, apoptosis rate is respectively 4.15%, 8.04%, 29.46%, 44.66%, 58.53%, FS-4 each effect group and matched group difference all has significance (P < 0.01).
Western Blot measures different time and acts on SGC-7901 cell, and after variable concentrations FS-4 acts on SGC-7901 cell, all can activate the expression of apoptosis-related protein (Caspase-3, Caspase-9, Bcl-2, Bax).
The Fructus Forsythiae anti-tumor active substance (FS-4) that the present invention extracts, through a series of antitumor cytolytic activity, confirms that it has the effect of inducing in vitro human gastric cancer cell line SGC-7901 apoptosis.FS-4 acts on SGC-7901 cell 36h half-inhibition concentration (IC
50) be only 3.23 μ g/ml, be better than the effect (IC of clinical commonly used drug cisplatin
50be 4.21 μ g/ml).According to " national new drug (Western medicine) preclinical study guideline compilation " regulation, Plant crude extract IC
50≤ 20 μ g/ml, and cytotoxic dose-dependent relationship, can judge that sample has lethal effect to cell in vitro, and Fructus Forsythiae anti-tumor active substance (FS-4) is a kind of strong anti-tumor active substance as can be seen here.
The preliminary identification of embodiment 6:FS-4 composition
WATERS company Acquity Ultra Performance Liquid Chromatography instrument series connection level Four bar time of-flight mass spectrometer (UPLC/Q-TOF MS), ACQUITY UPLC BEH C18(2.1 × 50mm, 1.7 μm) chromatographic column, be furnished with PDA detector and MassLynx4.1 work station.
1, sample solution preparation
Solvent is 80% methanol aqueous solution.Take FS-410mg and be dissolved in 10ml80% methanol aqueous solution, make the stock solution of 1mg/ml, 4 DEG C of storages, to be measured.
2, chromatographic separation condition
Mobile phase A is 0.1% formic acid high purity water solution, and Mobile phase B is acetonitrile; Flow velocity is 0.35ml/min.Gradient elution is separated, and Initial Gradient keeps 2% Mobile phase B (acetonitrile) 0.5min, and in 10min, acetonitrile linearly rises to 100%, and maintain 1.5min, in 3.5min, ethane nitrile content falls back 2% afterwards, refers to following table 1.Balance chromatographic column 1min.Each sample size is 5 μ L; Column temperature 35 DEG C; Automatic sampler environment temperature maintains 4 DEG C.
Table 1 Fructus Forsythiae FS-4 condition of gradient elution
3, Mass Spectrometry Conditions
Ionization source: electric spray ion source (ESI); Negative ion mode detects.Testing conditions is: capillary voltage 2700V, taper hole voltage 35V, extraction aperture voltage 3V, ion source temperature 100 DEG C, desolventizing gas velocity is 650L/h, EFI fog (desolventizing gas) temperature 300 DEG C, blowback air flow velocity is 50L/h, and impact energy 6.0, MCP detector voltage is 2350V.Do desolventizing gas with high pure nitrogen, high-purity argon gas does collision gas.Use full scan pattern, mass scan range is that 50-1000amu collects data with Centroid pattern.
4, the component analysis of Fructus Forsythiae anti-tumor active ingredient (FS-4)
This experiment adopts Acquity Ultra Performance Liquid Chromatography instrument series connection level Four bar time of-flight mass spectrometer (UPLC/Q-TOF MS) chemical composition to Fructus Forsythiae anti-tumor activity composition (FS-4) to analyze.Utilize flight time mass spectrum can obtain the feature of accurate mass number, by Masslynx4.1 software, retrieval fragment ion is elementary composition, analytical pyrolysis mode, adopt the mass number that records and the less principle (error is less than 10 × 10-6) of calculated mass number relative error, and comprehensively analyze in conjunction with retention time etc.Result infers the possible structure 14 chemical compositions altogether, contrast through molecular weight, find wherein 6 kinds be known substance: i.e. betulic acid (Betulinicacid), oleanolic acid (Oleanolic acid), ursolic acid (Ursolic acid), caffeic acid (Caffeic acid), palmitic acid (Palmitic acid), stearic acid (Stearic acid), wherein only stearic acid content is higher; All the other 8 kinds of materials not from known substance to going out, may be some novel substances.Its retrieval molecular formula of gained, result for retrieval, mass spectrum total ion current figure and moieties second order ms data are shown in Fig. 4, table 2.
The mass spectrometric data of the known compound that table 2UPLC is separated
Claims (6)
1. from Fructus Forsythiae, extract a method for anti-tumor active substance, it is characterized in that comprising the following steps:
(1) preparation of Fructus Forsythiae Aqueous extracts
Fructus Forsythiae is after screening out dirt, and wash soaked overnight, next day, slow fire boiled, and filters, and collect filtrate, evaporation and concentration, makes Aqueous extracts, and sterilizing is labeled as FS-1, puts 4 DEG C of Refrigerator stores for subsequent use;
(2) ethanol precipitation initial gross separation anti-tumor effective component
Get Fructus Forsythiae Aqueous extracts FS-1 and be placed in plastic centrifuge tube, then gradation slowly adds the alcoholic solution that mass percent concentration is 90%-100% wherein, and constantly shake, till the volume of alcoholic solution reaches the 90%-95% of overall solution volume, centrifuge tube is placed in 4 DEG C of refrigerator 8-14h, low-temperature centrifugation, collect supernatant, supernatant through filter paper filtering, after concentrating under reduced pressure, be placed in 4 DEG C of refrigerators for subsequent use, be labeled as FS-2;
(3) macroporous adsorbent resin column chromatography method is separated Fructus Forsythiae anti-tumor effective component
Get FS-2, slowly join the macroporous adsorptive resins top that pretreatment is good, addition is 2% column volume, treat that FS-2 infiltrates resin bed, be the alcoholic solution of 30%, 60%, 90% successively with distilled water, mass percent concentration, carry out eluting with 5.00ml/min flow velocity, collect elution fraction, mass percent concentration be the ethanol elution of 90% after concentrating under reduced pressure under 100 DEG C of conditions, be labeled as FS-3;
(4) thin layer chromatography is separated Fructus Forsythiae anti-tumor active substance
According to benzene: normal hexane: methanol: the volume ratio=14-16:14-16:0.5-1.5:0.5-1.5 of glacial acetic acid prepares developing solvent, prepared on plate at thin layer at room temperature 25 DEG C by FS-3 and launch, duration of run 50-60min, dries; Then, exert a gradual, corrupting influence on thin layer by iodine fumigation and prepare plate, and observe under ultra violet lamp, namely find that FS-3 is divided into 13 bands, collect the 4th band preparing plate number from thin layer, be labeled as FS-4, be anti-tumor active substance.
2. in accordance with the method for claim 1, it is characterized in that step (1) is carried out according to following steps: Fructus Forsythiae, after screening out dirt, with tap water cleaning 3-6 time, then washes 2-4 time with distillation, then adding distil water makes liquid level exceed Fructus Forsythiae 8-15cm, soaked overnight; Next day, slow fire boiled in 100 DEG C, filtered while hot after 1-3h with absorbent cotton, and medicinal residues repeat to extract, then filter with absorbent cotton, by twice filtrate mixing, in 100 DEG C of evaporation and concentration to 1.00mg/ml, make Aqueous extracts, through interval concora crush sterilization on the three sterilizing, each 40min, puts 4 DEG C of Refrigerator stores for subsequent use.
3. in accordance with the method for claim 1, it is characterized in that the low-temperature centrifugation described in step (2) refers in 4 DEG C, the centrifugal 15min of 4000r/min.
4. in accordance with the method for claim 1, it is characterized in that the macroporous adsorptive resins described in step (3) is X-5 type macroporous adsorptive resins.
5. in accordance with the method for claim 1, it is characterized in that the developing solvent described in step (4) is according to benzene: normal hexane: methanol: the volume ratio=15:15:1:1 preparation of glacial acetic acid, duration of run is 55min.
6. in accordance with the method for claim 1, it is characterized in that the collection method of the 4th band described in step (4) comprises the following steps:
(1) prepare upper several 4th band cut down by plate from thin layer and put into small beaker;
(2) in beaker, add dehydrated alcohol, stir, after precipitation 20-24h, collect supernatant, with fat-soluble 0.22 μm of membrane filtration, obtain the supernatant after filtering, for subsequent use, repeat step (2) 2-3 time;
(3) after the supernatant after the filtration of collecting being mixed, more centrifugal, get supernatant, with the membrane filtration of fat-soluble 0.22 μm after concentrating under reduced pressure, filtrate makes powder through lyophilization, is labeled as FS-4, is anti-tumor active substance.
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