CN1108796C - Use of anticancer material and production method of said anti-cancer material - Google Patents
Use of anticancer material and production method of said anti-cancer material Download PDFInfo
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Abstract
The present invention relates to a group of specific branched chain fatty acids with obvious cancer resisting action for human beings and animals, and a production method for obtaining a plurality of specific branched chain fatty acids by chemical synthesis or fermentation or culture of bacteria containing the specific branched chain fatty acids. The fatty acids can be used for treating patients having cancer.
Description
The present invention relates to pharmaceutical sanitary field, be a kind of application and the production method thereof of material in treatment of cancer with antitumaous effect, promptly the invention relates to one group of material one idio-branched chain fatty acid with remarkable active anticancer in treatment of cancer application and contain the method for idio-branched chain fatty acid through suitability for industrialized production with specific strain.
Cancer is to threaten human health and the most serious disease of life-prolonging.The mankind mainly take operative therapy to treatment for cancer at present, chemotherapy (to call chemotherapy in the following text) and radiation therapy (to call radiotherapy in the following text).But chemotherapy and radiation all has certain toxic and side effects to human body in anticancer growth or kill cancer cell.Therefore, people are just in broad research, so that find the little and effective anticarcinogen of a kind of toxic and side effects.
The inventor in 1987 has chanced on to pollute in the culture bottle and has gone up antibacterial when cultivating K562 leukaemia, and K562 leukaemia all disappears after 48 hours.So after the antibacterial separation and purification with this pollution, be the culture medium of primary raw material, add that suitable inorganic salt cultivates with the Semen sojae atricolor.Carry out animal experiment with its culture fluid (claiming fermentation liquid again), find that it can suppress tumor growth really effectively, and normal cell is not had toxic and side effects.Over 10 years through a large amount of cancer patients, the state of an illness is alleviated after wherein having leukemia patient, carcinoma of tongue patient, rectal cancer patient, breast carcinoma patient, carcinoma of prostate patient, lung cancer patient, melanotic tumor patient, renal carcinoma patient, esophageal carcinoma patient, cancer of pancreas patient, Patients with Gastric Cancer and hepatocarcinoma patient etc. to take this fermentation liquid, or tumor is dwindled, so that disappear, many patients also live here below so far healthily.That which active substance is playing antitumaous effect on earth in the fermentation liquid? the inventor carries out the research of each side to fermentation liquid, therefrom isolates many single materials.Confirmed at last, except that genistein (Genisten), daidzein (Daidzen), soyasaponins (Saponin) from Semen sojae atricolor have some active anticancers, mainly be to play a crucial role from ten trimethyl tetradecanoic acids in the fermented product and ten dimethyl tetradecanoic acids.And then find that again except ten trimethyl tetradecanoic acids and ten dimethyl tetradecanoic acids, one group of idio-branched chain fatty acid all has cancer suppressing action, particularly carbon to surpass those branched chain fatty acids of 11, all has antitumaous effect clearly; Also find simultaneously, utilize the strain that is rich in idio-branched chain fatty acid to ferment or the product of turning out has active anticancer too.
Up to the present do not have the people that branched chain fatty acid was carried out any research as cancer-resisting substance as yet, also nobody proposed to utilize the strain that is rich in idio-branched chain fatty acid to carry out the product that industrial fermentation is produced, and can be used for treating cancer.
The objective of the invention is in order to disclose the remarkable active anticancer that this group idio-branched chain fatty acid is had, and with a series of biochemistry and morphologic experiment material, to confirm that this group idio-branched chain fatty acid can cause that the procedural dead voluntarily inhibition tumor growth mechanism of cancerous cell the more important thing is to confirming that this group idio-branched chain fatty acid can not kill and wound normal cell, to people and the equal avirulence of animal, thereby be used for human or animal cancer patient's treatment and rehabilitation.
The present invention has disclosed one group of idio-branched chain fatty acid and production method thereof that humans and animals is had remarkable active anticancer.This group branched chain fatty acid is to cause cancerous cell procedural dead (apoptosis) voluntarily to the mechanism of action of various cancerous cell, and normal human body cell, animal and human are all had no side effect.The production method of this class branched chain fatty acid involved in the present invention comprises the method for producing idio-branched chain fatty acid with chemosynthesis or biosynthesis, and described biosynthesis is meant the method that adopts the special strain fermentation that is rich in idio-branched chain fatty acid.The tunning that contains idio-branched chain fatty acid involved in the present invention not only has obvious antitumaous effect to humans and animals, but also has raising immunity, functions such as protection liver, kidney.The definition of idio-branched chain fatty acid among the present invention
The idio-branched chain fatty acid of indication of the present invention, mean with active anticancer one group saturated with undersaturated branched chain fatty acid.
The chemical constitution of saturated idio-branched chain fatty acid is:
N and m are integer independently in the formula, and (n+m) value is 0～46, and particularly m is 0 or 1, and n is 7～16.Can be expressed as again: iso-C17:1 ω 9c.The present invention also comprises said saturated and pharmaceutical salts undersaturated branched chain fatty acid, it can by with inorganic base, as reaction obtains as sodium hydroxid, have the effect of anticancer growth.
Undersaturated branched chain fatty acid of the present invention is if express with following formula, and wherein m or n are at least 2, and (CH
2)
mOr (CH
2)
nIn have (a CH at least
2-CH
2) group changes (CH=CH) group into.
The saturated branched chain fatty acid that contains X carbon among the present invention with " iso-Cx " expression, and the n=X-4 in the following formula, m=0.Be used for abbreviation with " anteiso-CX " and contain X carbon, and n=X-5, m=1.For example, 13 methyl tetradecanoic acids can be used iso-C15, and molecular formula is:
12 methyl tetradecanoic acids can be called for short with anteiso-C15, and its structural formula is as follows:
15 methyl hexadecenes are examples of undersaturated branched chain fatty acid, and its molecular formula is:
Can be expressed as again: iso-C17:1 ω 9c
The present invention also comprises said saturated and pharmaceutical salts undersaturated branched chain fatty acid, it can by with inorganic base, as reaction obtains as sodium hydroxid, have the effect of anticancer growth.
The present invention has also disclosed the medicinal lipoprotein of saturated and unsaturated idio-branched chain fatty acid, it can by with protein, comprise polypeptide and oligopeptide in conjunction with and obtain, have the effect of anticancer growth.This lipoprotein combination is general technology.
The idio-branched chain fatty acid of indication of the present invention is passable, but is not limited to, and separates obtaining from the fermented product of specific strain or culture, or by chemosynthesis, or from the nature biotechnology body, extract.Idio-branched chain fatty acid causes the procedural dead voluntarily confirmatory experiment of cancerous cell
Idio-branched chain fatty acid causes that cancerous cell procedural dead voluntarily (apoptosis) measured by three kinds of methods: 1) with the procedural morphological change of death voluntarily of observation by light microscope representative; 2) measure instrument (flowcytometry) identification just at procedural dead voluntarily cell with fluidic cell; 3) with original position cell death detection kit POD (insitu cell death detection kit, POD), on unicellular level, the dead voluntarily dna break that causes of monitoring facilities.1. morphology experiment
The procedural dead voluntarily form characteristics of cancerous cell are cell shrinkage, and chromatin is condensed into fine and close cake mass, and nucleus also can split into fragment, and cell membrane bubbles, and forms apoptotic body in a large number.
Fig. 1 represents the form of dead cancerous cell change voluntarily under the optical microscope.With 24 hours test group (Figure 1B) of iso-C15 (60 μ g/ml) handler's hepatoma carcinoma cell SNU-423, (Figure 1A) compares with untreated matched group, can find out the whole generation of cell shrinkage.With anteiso-C15 (60 μ g/ml) handler's stomach cancer cell SNU-1 after 8 hours, dye the form (Fig. 2 B) of observation of cell with HE, (Fig. 2 C) compares with untreated matched group, can find that chromatin agglutination becomes block.After people's prostate gland cancer cell DU-145 handled 8 hours with iso-C15 (60 μ ug/ml), with HE dyeing observation of cell form (Fig. 3 B), (Fig. 3 A) compared with untreated matched group, can see on the cell membrane and bubbling.2. fluidic cell metering instrument experiment
Flow cytometry FACScan and analysis software Consort 30 are adopted in this experiment, (Becton Dickinson, SanJose, CA), and with dead test kit (apoptosis detection kit, R﹠amp voluntarily; D Systems) the dead voluntarily cell number of quantitative assay is because dead voluntarily cell can combine with Annexin V and repel PI.Be resuspended in the binding buffer liquid after the cell of experiment is washed twice with refrigerative PBS, will be added in the cell through fluorescently-labeled annexinV and PI then.The cell in dead later stage and the cell membrane destroyed of non-viable non-apoptotic cell voluntarily, thereby cell DNA will combine with PI.The cell that adds dyestuff send the analysis of fluidic cell metering instrument immediately, and the cytolytic dose instrument sends the single beam laser of 488nm, accepts the signal that dyestuff annexiaV and PI are sent respectively by signal detector FL1 and FL2.So three class cells on FL1/FL2 figure, will occur: living cells, (3 district) do not gone up in two kinds of dyestuff levelling; Non-viable non-apoptotic cell and dead voluntarily later stage cell, two kinds of dyestuffs are all caught (2 district), and dead voluntarily cell is only caught annexinV (4 district).
Each test analyzes 10 at least
4Individual cell.Untreated human leukemia K
562FL1/FL2 cell counting figure (Fig. 4 A) show that these living cells are distributed in 3 districts.And after cell was handled 24 hours with iso-C15 (30 μ g/ml), a large amount of cells is (Fig. 4 B, 4 districts, the positive and PI feminine gender of annexinV) in being in death voluntarily.Fig. 5 A, 5B, 5C show that anteiso-C15 acts on human breast cancer cell MCF-7,0,4,24 hours dynamic characteristic, after anteiso-C15 (60 μ g/ml) handles 4 hours, a large amount of cells are in dead voluntarily (Fig. 5 B, 4 districts), and after handling 24 hours, all dead (Si Wang later stage voluntarily of most cells, Fig. 5 C, 2 districts).Flow cytometry shows to untreated normal person PBL analysis result (Fig. 6 A) and to the PBL analysis result (Fig. 6 B) of handling 24 hours normal person with iso-C15 (60 μ g/ml), their FL1/FL2 figure is (3 districts much at one, living cells, dead voluntarily), it illustrates the lymphocyte not influence of iso-C15 to the normal person.3. on the individual cells level, detect the dead voluntarily experiment of programmed cell
This experiment original position cell death detection kit POD (ENZO Diagnostics, Inc.Cot No.1684817), its test philosophy is based on: cell is procedural when dead voluntarily, the cutting of genomic DNA can obtain the dna fragmentation (being mononucleosome and oligoneucleosomes) of two key small-molecular weights and the DNA of singly-bound fracture (" otch ") macromolecule, utilize archaeal dna polymerase and do not hold deoxynucleotidyl transferase (TdT), the nucleotide of labelling and the DNA key of fracture are combined in position, be called TUNEL technology (TdT mediated dUTP nick end labeling cut end labelling).TUNEL technical mark atman dead cell than labelling non-viable non-apoptotic cell sensitivity many, thereby can distinguish death voluntarily and non-viable non-apoptotic cell.This experimental procedure is undertaken by the operational flowchart that test kit shows.Laboratory sample has K
562, SNU-1, MCF7, H1688, PBL, sample is unified to be handled with iso-C15 (60 μ g/ml).Wherein the SNU-1 stomach cancer cell adds 37 ℃ of TUNEL reactant mixtures and cultivated 60 minutes down, sample through BPS give a baby a bath on the third day after its birth all over after directly analyze with fluorescence microscope.Fig. 7 B is the SNU-1 gastric carcinoma cells of handling 8 hours, with matched group Fig. 7 A relatively, can be observed several points phosphor dot of sending of dead cell voluntarily.H1688, K
562, DU145 cancerous cell and people normal circumference blood lymphocyte PBL add POD enzyme antibody (anti-fluorescein antibody) and under 37 ℃, cultivated again 30 minutes, PBS washes 3 times, developed the color 10 minutes as substrate with AEC, washing then, mounting can be observed the dead cell voluntarily that presents redness under optical microscope.Fig. 8 B is the K that handled 2 hours
562The human leukemia cancerous cell, the cell that relatively can see existing fraction with matched group Fig. 8 A begins apoptosis (cell that takes on a red color).Fig. 8 C is the K that handled 4 hours
562Cell and Fig. 8 B be apoptotic cells quantity showed increased relatively.Fig. 9 B is that the H1688 human lung carcinoma cell and the matched group Fig. 9 A that handled 8 hours compare, and can see the considerable cell that is rare redness, shows that these cells are just at apoptosis.Figure 10 B is the DU145 Human Prostate Cancer Cells of handling 8 hours, relatively can see the cell of part dead voluntarily with matched group Figure 10 A.Figure 11 B is the peripheral blood lymphocyte of handling 8 hours PBL people, and is more about the same with untreated matched group Figure 11 A, illustrates that iso-C15 does not act on normal human body cell.
Above example is that the kind of disclosed idio-branched chain fatty acid ex hoc genus anne thing is not limited to above-mentioned several, and effective cancerous cell kind is not limited to above-mentioned several cell line for convenience of explanation.ID
50ID
75And ID
90Mensuration
Sample:
Ten trimethyl tetradecanoic acids (iso-C15) are to use in the fermentation liquid of specific strain germ oligotrophy unit cell (Stenotrophomonasmaltophilia) Q-Can strain and the production of this patent method, extract with high pressure liquid chromatography (HPLC) (HPLC), sample is with NaOH solution and the dissolving of 0.5% tween 80, pH=7.5.
Cell line:
K
562The human leukemia cell; The MCF human breast cancer cell; The SNU-1 gastric carcinoma cells; The DU145 Human Prostate Cancer Cells; The SNU-423 human liver cancer cell; HCT116 people's rectum cancer cell; The H1688 human lung carcinoma cell; The normal lymphocyte of PBL people's peripheral blood.(American Type CultureCollection ATCC) buys all seven kinds of cancerous cell by American type culture collection; PBL people's normal cell is to separate from healthy people's whole blood.
Cancerous cell is cultivated by the ATCC requirement:
K
562Human leukemia cell and SNU-1 gastric carcinoma cells suspension culture are in the RPMI1640 culture medium of adding 10% heat-inactivated fetal bovine serum.
MCF human breast cancer cell and DU145 Human Prostate Cancer Cells adhere-wall culture are in the MEM culture medium of adding 10% heat-inactivated fetal bovine serum.
Thin and the H1688 human lung carcinoma cell adhere-wall culture of SNU-423 people's hepatocarcinoma is in interpolation 10% heat-inactivated fetal bovine serum RPMI1640 culture medium.
HCT116 people's rectum cancer cell adhere-wall culture is in McCoy ' the s 5a culture medium of adding 10% heat-inactivated fetal bovine serum.
People's PBL cell is kept in the RPMI1640 culture medium of 10% blood plasma that adds same individual.
Method:
Above-mentioned cell removes PBL with 10
5Outside the density of/hole, all the other are 5 * 10
4The density in/hole joins on 96 well culture plates, it is seven steps of 0～60 μ g/ml (0,0.15 μ g/ml, 3 μ g/ml, 6 μ g/ml, 15 μ g/ml, 30 μ g/ml, 60 μ g/ml) that experimental drug (diluting mother solution with culture medium) adding makes final concentration, matched group is one group and is untreated that one group of solubilizer is handled.Attached cell (SNU-423, H1688, MCF, DU145 and HCT116) after 48 hours, is removed supernatant 37 ℃ of cultivations earlier, and cell monolayer is become uniform cell suspending liquid with the EDTA-trypsinization, measures survival rate with 4% trypan blue exclusion then.Cell (the K of suspension growth
562, SNU-1 and PBL) after 37 ℃ are cultivated 48 hours, can directly from the hole, take out cell and calculate survival rate.
The ID of each cell
50, ID
75And ID
90Be to repeat twice by each group experiment to try to achieve, experiment each time is triplicate under the same conditions, ID
50, ID
75And ID
90Be defined as and kill the needed dosage of 50%, 75% and 90% cell (with untreated cellular control unit contrast), with special-purpose software CalcuSyn (Biosoft according to effect theorem establishment among the Zhou Shi father and son (Dr.T.C.Chou), Cambridge UK) calculates, and the result is as follows:
Table 1.iso-C15 is to people's cancerous cell and the external casaulty dosage cell category of normal cell casaulty dosage
ID
50(μ g/ml) ID
75(μ g/ml) ID
90(μ g/ml) MCF human breast carcinoma 10.03 ± 0.97 15.99 ± 1.28 25.49 ± 1.68K
562Human leukemia cancer 11.45 ± 1.82 22.27 ± 4.60 43.57 ± 6.71DU145 human prostata cancers 13.98 ± 2.15 40.43 ± 5.72 81.87 ± 8.85H1688 lung carcinoma cells 15.08 ± normal lymphocyte of 1.92 35.03 ± 3.59 61.37 ± 8.06HCT116 people carcinoma of the rectum, 18.49 ± 6.23 67.96 ± 8.25 108.65 ± 13.35SNU-1 people cancer of the stomach, 20.77 ± 2.47 47.43 ± 4.95 80.49 ± 10.03SNU423 people liver cancer, 24.26 ± 3.98 70.46 ± 9.36 120.77 ± 15.83PBL people＞400
Can find out that from table 1 iso-C15 all has activity to various tumor cells, wherein to people's breast carcinoma MCF cell with to people's leukemia K
562Cytosis is the strongest, and more weak to human liver cancer cell SNU423 and people's rectum cancer cell HCT116 effect, but under lethal dosage considerably beyond cancerous cell still the normal lymphocyte to the people do not have toxicity.
Above example is that the kind of disclosed idio-branched chain fatty acid ex hoc genus anne thing is not limited to iso-C15, and effective cancerous cell kind is not limited to above-mentioned several cell line for convenience of explanation.The production method of idio-branched chain fatty acid
The present invention proposes the production method of idio-branched chain fatty acid with active anticancer.
Idio-branched chain fatty acid of the present invention can extract from natural resources, includes but not limited to contain the organism of idio-branched chain fatty acid, as the phytol of Animal fat or green plants.
Idio-branched chain fatty acid of the present invention can also be synthetic with chemistry or biological method.The Cole of branched chain fatty acid is well-known than (kolbe) electrolytic synthesis technology, and the electrolytic synthesis with iso-C15 is the example explanation below.Idio-branched chain fatty acid biological synthesis method of the present invention is to utilize the specific strain fermentation or the cultivation of being rich in idio-branched chain fatty acid in the cellular fat.1.13-the method that methyl 14 burns the electrolytic synthesis of acid
13-methyl tetradecanoic acid can be according to the Kolbe electolysis reaction principle, with isovaleric acid and dodecylic acid mono-methyl, by electrolytic synthesis in methanol solution.
Dodecanedioic acid dimethyl ester is by containing 5 times of amounts (v/w) methanol of 5% (w/v) concentrated sulphuric acid, the dodecanedioic acid esterification being prepared.This dimethyl ester after vacuum distilling is purified, is changed into monoesters with the potassium hydroxide of theoretical equivalent in absolute methanol.The dodecylic acid mono-methyl is purified by vacuum distilling.
The electrolytic coupling reaction is the isovaleric acid with monoesters and 2 times of molecular weight, in containing the absolute methanol of Feldalat NM, by two 10cm that are connected on the adjustable dc source
2Platinum electrode is kept appropriate voltage and is carried out cell reaction.When solution presents alkalescence, illustrate to react and finish.
After reaction is finished, reactant mixture is cooled to room temperature, removes by filter sedimentary by-product dodecanedioic acid dimethyl ester.Filter liquor acetic acid acidify removes methanol under reduced pressure and by vacuum topping purification 13-methyl methyl myristate.
At last, in excessive 10%NaOH aqueous solution and methanol, make the hydrolysis of 13-methyl methyl myristate by backflow.The white solid that obtains is purified by vacuum distilling, recrystallization in acetone, and vacuum drying obtains 13-methyl tetradecanoic acid white crystal, 51～52 ℃ of fusing points.
This example is for saying something, and idio-branched chain fatty acid kind and chemical synthesis process that synthetic method of the present invention relates to are not limited thereto.2. the production method that contains the fermentation liquid of idio-branched chain fatty acid
The said production method that is rich in the fermentation liquid of idio-branched chain fatty acid of the present invention comprises the steps.
Now narrate the production method of the fermentation liquid that is rich in idio-branched chain fatty acid as producing bacterial strain with germ oligotrophy unit cell (Stenotorphomonas maltophilia) Q-can bacterial strain.
This produces bacterial strain, it is germ oligotrophy unit cell (Stenotrophomonas maltophilia) Q-can bacterial strain, now be kept at American type culture collection (ATCC10801 University Boulevard, Manassas, VA 20110-2209) preserving number is ATCC 202105.ATCC identifies as follows to this strain properties:
Cellular morphology for move about, no spore, Gram-negative, aerobasilus.Colonial morphology is as follows: the ATCC#3 culture medium (cultivated on the Nutrient agar 24 hours, bacterium colony is: the I type ,-90% circle (swell, and rough surface is translucent, pale yellow cream-coloured by the about 1mm of diameter, neat in edge; The II type is that small circular (diameter＜1mm), swell, and translucent smooth, color is dark than I type bacterium colony by neat in edge.Bacterium colony ATCC#18 (T--soy agar) #44 (BHI agar), and #260 (sheep blood agar) culture medium on demonstrate identical characteristics.Two kinds of bacterium colony character are identical.
The cell fatty acid of germ oligotrophy unit cell Q-can bacterial strain is composed as follows: fatty acid (accounting for total fatty acids %)
Straight chain fatty acid *
Branched chain fatty acid * *
10：0 0.48 i11：0 3.21
14：0 3.14 i13：0 0.50
15：0 0.33 il5：0 39.34
16：0 5.52 a15：0 7.44
16：1ω9c 3.37 i15：1 1.02
16：1ω7c 12.58 il6：0 0.88
Hydroxy acid i17:0 3.68
30H-10：0 0.12 i19：0 0.33
30H-i11：0 1.51 i17：1ω9c 5.27
30H-i12：0 2.68
30H-i13：0 3.57
20H-13：0 0.29
* the colon left side is the carbon atom number, and the right side is two key numbers.
Different fatty acid before the different fatty acid of * i=, a=
Because forming, the fatty acid of antibacterial is subjected to biosynthesis condition to influence (temperature and pH etc.), so above data are one group of typical data.
At first, on the slant agar culture medium, cultivated seed 24 hours, be inoculated into then in the fluid medium in the flask, on shaking table, cultivated 24 hours.Then the culture fluid in the flask is inoculated in the seed tank, inoculative proportion is 0.1～0.5% (w/w).After seed tank fermentation 24 hours, with culture fluid move on to produce jar in fermentation 48 hours, and feed filtrated air.In general, be about 10 from seed tank to the amplification ratio of producing jar.Condition of culture is: and ventilation 1: 0.6～1.2 (quality: v/v air), min, 180～260 rev/mins of mixing speed, temperature is 28～38 ℃.After cultivate finishing, with culture fluid 100 ℃ of following autoclave sterilizations 30 minutes, then with liquid collecting, bottling, at 120 ℃ of following autoclave sterilizations.The product that obtains like this is the common oral liquid of drinking for the people.
Also can obtain other product with diverse ways, as after cultivate finishing, add a certain amount of hydrochloric acid in culture fluid, make pH drop to 3～4,100 ℃ of autoclave sterilizations 30 minutes, the cooling back was centrifugal.Main component is soybean saponin (saponin) in the supernatant that obtains, but the nutritious drink of the various tastes of furnishing.In precipitation, add 95% ethanol of equal volume and the 2N NaOH of equal volume, stir and be heated to 100 ℃, cool off centrifugal after, supernatant is collected stand-by, the IN HCl of adding equal volume in the precipitation of remainder, and heat 80 ℃ 5 minutes.Cool off centrifugal after, collect supernatant.The supernatant of twice collection is lumped together, pH is transferred to 9.0, it is exactly spissated oral liquid.Product contains especially iso-C15 (existing with sodium-salt form) of idio-branched chain fatty acid, also has Saponin, daidzin, genistein and other cancer-resisting substance in addition.We also can adopt other method.Or after cultivating end,, form the powder product directly with the culture fluid spray drying, then powder is dressed up capsule or be pressed into tablet.Or from fermentation liquid, isolate idio-branched chain fatty acid with the method for ethanol, ether and so on organic solvent and common extraction fatty acid, make the injection that main component is the branched chain fatty acid sodium salt.Or use various extraction columns, as ion handing-over post or resin column,, and then make injection, powder or the oral medicine of using for cancer patient directly from the broth extraction effective ingredient.
The typical content of fatty acid is as follows in every 500ml Q-can oral liquid:
Straight chain fatty acid * branched chain fatty acid * * 10:0 2.0-2.7mg i11:0 11.2-15.4mg12:0 2.9-4.0mg i15:0 106.0-145.8mg14:0 13.0-17.7mg i16:0 3.1-4.3mg15:0 2.8-3.8mg i17:0 12.4-17.0mg16:0 251.7-346.1mg i19:0 2.2-3.0mg17:0 2.9-4.0mg a15:0 23.4-32.1mg18:0 75.6-104.0mg i17:1 ω 9c 4.1-5.7mg20:0 5.5-7.6mg12:1 ω 8c 4.3-5.9mg carboxylic acid 16:1 ω 7c 21.0-28.9mg 3OH-i11:0 6.3-8.6mg18:1 ω 9c 488.8-672.0mg 3OH-12:0 12.0-16.5mg18:2 ω 6c 825.9-1135.6mg 3OH-i13:0 13.2-18.1mg
In said method, adopt following production culture medium.Provide composition by weight, all the other are water.Except said nutritional labeling, also add the trace element of an amount of needed by human body.
Soya broth:
Big by 5～10%
Yeast extract 0.02～0.5%
Or yeast powder 0.02～0.5%
Or peptone, Carnis Bovis seu Bubali cream 0.02～0.3%
CaCO
3 0.05～0.25％
K
2HPO
4 0.02～0.10％
MgSO
4 0.01～0.05％
NaCl 0.01～0.04％
Na
2MO
4 5.0～30ppm
ZnSO
4 2.5～15ppm
CoCl
2 5.0～20ppm
Except the antibacterial of being rich in branched chain fatty acid that has been identified, as oligotrophy zygosaccharomyces (Stenotrophomonas), Xanthomonas (Xanthomonas), Flavobacterium (Flavobacterium), carbon dioxide is had a liking for Cellulomonas (Capnocytophaga), alternate zygosaccharomyces (Altermonas), have a liking for cell Pseudomonas (Cytophage), Bacillus (Bacillus), golden yellow Bacillus (Chryseobacterium), steady Bacillus (Empdobacter), gold Bacillus (Aurebacterium), staphylococcus (Staphylococcus), and outside the Rhodopseudomonas (Pseudomonas), the said specific strain of the present invention also comprises other natural existence, but the present still Unidentified bacterial strain that is rich in idio-branched chain fatty acid.With the bacterial strain that is rich in idio-branched chain fatty acid, and culture medium of the present invention and production method oral liquid, capsule, tablet or the injection produced, all has anti-cancer function and to the nutrition and the protective effect of humans and animals.
Description of drawings
Fig. 1 is that the kenel of observing procedural dead human liver cancer cell SNU-423 voluntarily under the optical microscope changes.A is a matched group, and B handled 24 hours through iso-C15 (60 μ g/ml).
Fig. 2 observes procedural dead voluntarily gastric carcinoma cells SNU-1 to change with the kenel that HE dyeing shows under the optical microscope.A is a matched group, and B handled 8 hours through iso-C15 (60 μ g/ml).
Fig. 3 is that the kenel of observing procedural dead Human Prostate Cancer Cells DU-145 voluntarily under the optical microscope changes.A is a matched group, and B handled 8 hours through iso-C15 (60 μ g/ml).
Fig. 4 is human leukemia cell K
562The flow cytometry analysis chart.A is a matched group, and B handled 24 hours through iso-C15 (30 μ g/ml).
Fig. 5 is the flow cytometry analysis chart of human breast cancer cell MCF-7.A is a matched group, and B handled 4 hours through anteiso-C15 (60 μ g/ml), and C is for handling 24 hours.
Fig. 6 is the flow cytometry analysis chart of all blood lymphocytes of normal person.A is a matched group, and B handled 24 hours through iso-C15 (30 μ g/ml).
Fig. 7 observes gastric carcinoma cells SNU-1 to add the procedural death voluntarily that presents behind the TUNEL reactant under the fluorescence microscope.A is a matched group, and B handled 24 hours through iso-C15 (60 μ g/ml).
Fig. 8 observes human leukemia cell K under the optical microscope
562The people adds the procedural death voluntarily that presents behind POD reactant and the substrate.A is a matched group, and B handled 2 hours through iso-C15 (60 μ g/ml), and C is for handling 4 hours.
Fig. 9 observes human lung carcinoma cell H1688 to add the procedural death voluntarily that presents behind POD reactant and the substrate under the optical microscope.A is a matched group, and B handled 8 hours through iso-C15 (60 μ g/ml).
Figure 10 observes Human Prostate Cancer Cells DU145 to add the procedural death voluntarily that presents behind POD reactant and the substrate under the optical microscope.A is a matched group, and B handled 8 hours through iso-C15 (60 μ g/ml).
Figure 11 observes human peripheral lymphocyte PBL to add the procedural death voluntarily that presents behind POD reactant and the substrate under the optical microscope.A is a matched group, and B handled 8 hours through iso-C15 (60 μ g/ml).
Embodiment and experiment
Embodiment one
Following examples are in order more specifically to introduce the present invention, but the present invention is not limited to embodiment one.
Produce bacterial strain with the Q-can strain, with 1 ton fermentation tank is seed tank, inlet amount is 0.4 ton, the various amounts of its culture medium: Semen sojae atricolor 40kg wears into slurry (removing slag), dipotassium hydrogen phosphate 200g, calcium carbonate 200g, yeast extract 160g, magnesium sulfate 80g, sodium chloride 80g, sodium molybdate 10ppm, zinc sulfate 10ppm, cobaltous chloride 5ppm, sodium selenite 2ppm, Oleum Glycines 4kg (as defoamer) adds water to 400kg, feed 120 ℃ of sterilizations of steam 30 minutes, be cooled to 30 ℃ then and insert 3kgQ-can culture fluid (this culture fluid was cultivated gained in 24 hours 30 ℃ of following shaking table vibrations).The seed tank temperature keeps 30 ℃, 200 rev/mins of mixing speeds, ventilation 1: 1 (v/v min).Fermenting, the system inspection has not microbiological contamination after 24 hours, and the fermentation tank that changes 10 tons after definite no microbiological contamination again over to begins formal production.10 tons of fermentor cultivation based components are the same with the seed tank culture based component, and quantity is 10 times of seed tank, produce the culture medium sterilization of jar, and fermentation temperature, mixing speed, ventilation are all the same with seed tank, and continuous fermentation 48 hours, the no microbiological contamination of system inspection are fermented successfully.Be warming up to then 100 ℃ the sterilization 30 minutes, begin after cooling the bottling, install the bottle Q-can I fermentation liquid also again 118 ℃ of sterilizations of 45 minutes become semi-finished product, through quality inspection qualified after, the packing Q-can oral liquid product.Test an external active anticancer experiment
Sample:
The 13-methyl tetradecanoic acid (iso-C15) that extracts is the fermentation liquid of producing with germ oligotrophy unit cell (Stenotrophomonas maltophilia) Q-can strain and the inventive method from of the present invention, separates with high-pressure liquid phase instrument (HPLC).
The iso-C15 of chemosynthesis, (St.Louis, MO USA) buy by Sigma Chemical company.
Other idio-branched chain fatty acid comprises:
10-methyl hendecanoic acid (iso-C12)
11-methyl dodecylic acid (iso-C13)
12-methyl tridecanoic acid (iso-C14)
11-methyl tridecanoic acid (anteiso-C14)
12-methyl tetradecanoic acid (anteiso-C15)
14-methyl pentadecanoic acid (iso-C16)
13-methyl pentadecanoic acid (anteiso-C16)
15-methyl hexadecanoic acid (iso-C17)
16-methylheptadecanoic acid (iso-C18)
15-methylheptadecanoic acid (anteiso-C18)
17-methyl octadecanoid acid (iso-C19)
18-methyl nonadecylic acid (iso-C20)
(St.Louis, MO USA) buy above sample by Sigma Chemical company.
Cell line;
Human leukemia cell line K562 and people's adenocarcinoma of stomach cell line SGC-7901.
Method:
Detect cytotoxic activity with the MTT test.K562 and SGC7901 cell culture are selected cell exponential phase of growth for use in the RPMI1640 culture fluid that adds 15% heat-killed hyclone.The density that the every hole of 96 hole microtest plates adds cell is 2 * 10
4/ 100 μ l, add the culture medium that contains the variable concentrations sample at each experimental port, making the final concentration that adds 13 methyl tetradecanoic acids (sample that comprises synthetic and natural extract) experimental port is five gradients, and the final concentration of other sample is 30 μ g/ml, and 6 control wells add the culture medium that does not contain sample.Culture plate is put 37 ℃ of CO2 gas incubator, 5%CO
2, saturated humidity cultivated 24 hours.Remove supernatant with the method for turning over soon, in each hole, add the MTT of 20 μ l 50mg/ml, cultivated again 4 hours, add DMSO100 μ g/ml, shaken cultivation plate 10 minutes, 570nm surveys absorbance A in enzyme-linked immunosorbent assay instrument (BioTek EL 311S type)
570:
Suppression ratio (%)=1-(experimental port average A
570Value/control wells average A
570Value)
The result:
The iso-C15* of table 2. synthetic is to the suppression ratio (%) of growth of cancer cells
Cell 90 μ g/ml 60 μ g/ml 30 μ g/ml 15 μ g/ml 7.5 μ
g/ml
K
562 85.3 83.1 71.6 50.1 26.2
SGC7901 68.4 63.1 50.5 27.5 -
* sample 10% dissolve with ethanol
The iso-C15* of table 3. natural extract is to the suppression ratio (%) of growth of cancer cells
Cell 90 μ g/ml 60 μ g/ml 30 μ g/ml 15 μ g/ml 7.5 μ
g/ml
K
562 87.2 83.7 72.2 51.2 27.1
SGC7901 68.8 62.1 51.2 28.1 -
* sample 10% dissolve with ethanol
Several idio-branched chain fatty acids of table 4.
*To K
562The suppression ratio of cell growth
Sample iso-C12 iso-C13 iso-C14 iso-C16 iso-C17 iso-C18
Suppression ratio 70.69 71.03 72.15 71.58 70.79 68.39
Sample Iso-C19 iso-C20 anteiso-anteiso-anteiso-anteiso-
C15 C14 C16 C18
Suppression ratio 69.15 62.58 73.10 72.59 70.68 71.73
* the concentration of idio-branched chain fatty acid is 30 μ g/ml, with NaOH dissolving, whole pH value of solution=7.5
Above example is for convenience of explanation, the kind of disclosed idio-branched chain fatty acid ex hoc genus anne thing, and effectively the cancerous cell kind is not limited thereto.Test two couples of mice LD
50Mensuration
Material:
13-methyl tetradecanoic acid (chemosynthesis) is dissolved in 0.35% tween 80, PH=7.5 after forming sodium salt with the NaOH reaction; The ICR mice, body weight 20.5～22.5g male and female half and half.
Method:
Dosage is from 10～800mg/kg, 2 mices of each dosage level, and other establishes the solvent control group, concrete dosage group is 10mg/kg, 20mg/kg, 40mg/kg, 80mg/kg, 160mg/kg, 800mg/kg, one by one by above-mentioned dosage group, every mice lumbar injection every day three times was observed 7.
Conclusion:
13-methyl tetradecanoic acid can not surveyed median lethal dose(LD 50) LD still there not to be dead mouse under the 800mg/kg high dose
50Test three pairs of human breast carcinoma MCF7 nude mouses and transplant the experiment of cancer therapeutical effect
Material:
13-methyl tetradecanoic acid (synthetic) and NaOH reaction forms and is dissolved in behind the sodium salt in 0.35% the tween 80, and pH is 7.5; SPF level Babl/c nude mouse, the female Mus of body weight 18～22g
Method:
MCF7 tumor subcutaneous transplantation exists, aseptic condition is operation down, get eugonic MCF7 tumor tissue several, shred mix the back with the trocar in nude mouse right side breast fat pad place's transplanted tumor piece of tissue (being about about 0.18 * 0.18 * 0.2mm), the transplanted tumor inoculation selected tumor to grow to 14mm approximately after seven days
3The nude mice of size, random packet, every group 6, divide matched group and treatment group, every day first and second day lumbar injection 60mg/kg 13-methyl tetradecanoic acid once, since the 4th day every other day the injection once, still be 60mg/kg at every turn, totally 14 times, and the normal saline of the same dosage of the each lumbar injection of matched group.Drug withdrawal is dissected after one day and is claimed tumor heavy and mice body weight, and calculates inhibition rate of tumor growth by following formula.
And carry out t and check
Experimental condition: the SPF level, 25 ± 2 ℃ of temperature, humidity 50 ± 10% matched group tumors are heavy
Conclusion:
As shown in table 5,13-methyl tetradecanoic acid interval lumbar injection 14 times has than the obvious suppression effect human breast carcinoma.
Table 5.13-methyl tetradecanoic acid is to the heavy suppression ratio P of human breast carcinoma KCF-Babl/c Nude Mice therapeutical effect rank dosed administration side number of animals body weight tumor value
Formula begins/begins at last/contrast ip 6/6 20.1/17.7 1.09 ± 0.28-test 60mg/kg ip 6/6 20.4/19.6 0.19 ± 0.13 82.2＜0.05 at last
More than the experiment illustration is that the kind of disclosed idio-branched chain fatty acid ex hoc genus anne thing is not limited to above-mentioned several, and effective cancerous cell kind is not limited to above-mentioned several cell line for convenience of explanation.Anticancer and the health protection effect that contains the fermentation liquid of idio-branched chain fatty acid contains the fermentation liquid of idio-branched chain fatty acid
The said fermentation liquid of the present invention is culture medium and the production method that adopts specific strain and the present invention's design, produces.This fermentation liquid contains the various idio-branched chain fatty acids with remarkable active anticancer, and from the abundant nutrition of soya broth and opsonigenous substance.In order fully to prove the anticancer and health care of fermentation liquid,, special following experiment and the result thereof of introducing.The fermentation liquid of the experiment employing of introducing below as an example, is " a Q-can oral liquid ".This oral liquid is with having a liking for Fructus Hordei Germinatus oligotrophy unit cell (Stenotrphomonas maltophilia) Q-can bacterial strain as producing bacterial strain, and adopts the production method of above-mentioned soya broth and the present invention's design to produce.Similarly zoopery and clinical trial were also carried out the capsule form product, and its result is consistent with the result of Q-can oral liquid product.
The typical content of fatty acid is as follows in every 500ml Q-can oral liquid:
The acute toxicity testing of straight chain fatty acid * branched chain fatty acid * * 10:0 2.0-2.7mg i11:0 11.2-15.4mg12:0 2.9-4.0mg i15:0 106.0-145.8mg14:0 13.0-17.7mg i16:0 3.1-4.3mg15:0 2.8-3.8mg i17:0 12.4-17.0mg16:0 251.7-346.1mg i19:0 2.2-3.0mg17:0 2.9-4.0mg a15:0 23.4-32.1mg18:0 75.6-104.0mg i17:1 ω 9c 4.1-5.7mg20:0 5.5-7.6mg12:1 ω 8c 4.3-5.9mg carboxylic acid 16:1 ω 7c 21.0-28.9mg 3OH-i11:0 6.3-8.6mg18:1 ω 9c 488.8-672.0mg 3OH-12:0 12.0-16.5mg18:2 ω 6c 825.9-1135.6mg 3OH-i13:0 13.2-18.1mgQ-can oral liquid
Material:
The Q-can oral liquid; The ICR mice, body weight 20.5-22.5g
Method and result:
Get empty stomach body weight 20.5-22.5g ICR mice 20 (male and female close half) because preliminary experiment can't be measured LD50, the oral liquid measure of Q-can that can bear with maximum is 3 times of concentrated solutions, 1ml/ only irritates stomach, irritates altogether in 24 hours four times (10:00a.m., 4:00p.m., 10:00p.m. and next day 6:00a.m.).The result observes all and is tried mice and irritate stomach movable minimizing after 5 minutes each, just recovers after 1 hour greatly, has 3 mices to occur suffering from diarrhoea in 1～2 day behind medicine, but continuous 7 light none tried dead mouse.To be tried Mus after finishing the course of treatment and put to death the dissection extremely of living, perusal does not see that internal organs are unusual.Above-mentioned limiting test shows, even Q-can oral liquid non-toxic reaction still when acute heavy dose of administration.Press body surface area and convert, this dosage is equivalent to body weight 70kg per day for adults and takes Q-can oral liquid 4,652ml.Q-can oral liquid sub-acute toxicity test
Material:
The Q-can oral liquid, kunming mice body weight 22～24g.
Method and result:
24 mices (male and female half and half) are divided into matched group and experimental group at random, gavage normal saline and Q-can oral liquid respectively, and dosage is 0.8ml/, gavages 21 days continuously.Extracted 2 mices at random out by each group on the 22nd day and live extremely, get internal organs and do to check under the paraffin section mirror.Remaining every group of 10 mices were observed seven days more continuously.As a result, perusal and mirror are checked down and are not found that all internal organs have any pathological changes, observe none death in seven days after the remaining mice drug withdrawal.Above-mentioned experimental result explanation gavages 21 days Q-can oral liquids continuously and can not cause toxic reaction, can not make mice pathological changes occur.Q-can oral liquid long term toxicity test
Material:
The Q-can oral liquid; 80 Spraque-Dawley rat (male and female half and half), body weight 60 ± 0.75g.
Method and result:
80 white mouse are divided into 4 groups at random, heavy dose of group (20ml/kg Q-can oral liquid), middle dosage group (10ml/kgQ-can oral liquid), small dose group (5ml/kg Q-can oral liquid) and matched group (with the normal saline of volume).Gastric infusion, once a day, continuous three months.In experimentation, observe the action of white mouse, appetite, gastrointestinal reaction and body weight change are measured routine blood test, platelet, electrocardiogram, liver function and renal function.Administration was put to death dissection with experimental mouse after 3 months, and main organs is made perusal and pathologic finding, comprised the heart, liver,spleen,kidney, stomach, jejunum and brain.
Generally speaking, it is all right to be tried white mouse, no abnormal behavior, and no gastrointestinal reaction, appetite is good.The body weight gain curve of experimental group similar substantially to matched group (P＞0.05), Electrocardioscopy is normal, and hematology's (comprising routine blood test and platelet) experimental group and matched group do not have the difference (P＞0.05) on the statistics.Liver function (comprising ALT and TTT) and renal function (comprising BUN and Cr) no significant difference (P＞0.05).Though the creatinine of experimental group slightly raises, but still in range of normal value.The main organs check pathological section shows that the cellularity of experimental group does not relatively have obviously different with tectology and matched group.In a word, Q-can oral liquid successive administration toxic reaction can not occur, so can use by safety clothes.The Q-can oral liquid protect the liver function
Material:
The Q-can oral liquid; 60 ICR mices.
Method and result:
60 ICR mices are divided into six groups at random, 10 every group, comprise 3 experimental grouies and 3 matched groups, weighed in 8 hours after the fasting.
The 1st, 2,3 groups (experimental group) drunk to mice with 75%, 50% and 33% Q-can oral liquid replacement clear water respectively, and in the 7th day lumbar injection thioacetamide (TAA) 80mg/kg.
The 4th group (positive controls) at the 5th day and the 6th day, every day lumbar injection diisopropylamine ascorbate 80mg/kg each once, and in the 7th day lumbar injection TAA 80mg/kg.
The 5th group (negative control group) was in the 7th day lumbar injection TAA 80mg/kg.
The 6th group (blank group) was in the 7th day intraperitoneal injection of saline 0.1mg/kg.
Contaminate at TAA and mice to be plucked eyeball in back 1.4 hours and get blood, separation of serum also detects the serum transaminase level of respectively organizing mice with 721 spectrophotometric OD values.At last, will be tried Mus and take off cervical vertebra and put to death, and separate and also extract liver, and obtain the percentage ratio of liver percentage of liveweight, the result carries out variance analysis:
Table 6.Q-can oral liquid brings out the influence of mice serum SGPT rising and hepatomegaly to TAA
Group medicine Mus OD value LW/BW P*
Number (X ± and SD) (%, X ± SD)
1 75％Q-can+TAA 10 0.705±0.107 5.825±0.590 ＜0.001
2 50％Q-can+TAA 10 0.764±0.078 6.181±0.710 ＜0.001
3 33％Q-can+TAA 10 0.847±0.073 6.323±0.942 ＜0.001
4 diisopropylamine ascorbate+TAA 10 0.728 ± 0.091 6.180 ± 0.514＜0.001
5 TAA 10 0.935±0.032 7.380±0.338
6 normal saline 10 0.318 ± 0.014 6.234 ± 0.358＜0.001
* compare with the 5th group.The heavy BW=body weight of LW=liver
Above result shows that the Q-can oral liquid all has the comparison significant inhibitory effect to serum transaminase rising and the hepatomegaly that TAA brings out, and its intensity is similar to ip diisopropylamine ascorbate (80mg/kg), even more stronger.Strengthen the chemotherapeutics effect
Material:
The Q-can oral liquid, the strain of rat liver cancer HAC tumor, male mice in kunming, body weight 20-25g, Cyclophosphamide for injection (CP).
Method and result:
Get 40 of mices and be divided into 5 groups at random.Three experimental grouies are fed 36%, 60% and 100% Q-can oral liquid respectively, and matched group and positive controls are fed drinking water.In experiment the 8th day, under the sterile working, give whole mouse peritoneal injection cancerous cell HAC suspensions (10
7/ ml).Behind inoculation HAC cell 1,3,5 days, three experimental grouies and each intraperitoneal injection of cyclophosphamide 50mg/kg of positive controls mice were once.Behind the inoculation HAC the 9th day, recover normal diet before the experiment.Observe and write down the death time of each Mus, calculate average survival period and increase in life span.Its increase in life span (ILS%) is defined as:
ILS%=(experimental group survival period-matched group survival period)/matched group survival period
Table 7.Q-can oral liquid is to the potentiation of CP treatment hepatocarcinoma
Group medicine survival period 〉=17 days prolongation natural law ILS (%) P*
The Mus number
1 10.63±1.03 0
2 36％Q-can+CP 12.38±2.39 1 1.75±2.05 16.46 ＜0.10
3 60％Q-can+CP 14.44±3.54 2 3.94±2.98 35.84 ＜0.02
4 100％Q-can+CP 16.56±3.96 6 5.94±2.96 55.75 ＜0.01
5 CP 13.06±3.03 1 2.44±2.58 22.86 ＜0.05
* compare with the 1st group (matched group).
Above result shows that chemotherapeutics CP and Q-can oral liquid merge to be used, and its antitumaous effect is enhanced.The survival period of no medicine negative control group mice with tumor only 10.63 ± 1.03 days, simple ring phosphamide positive controls is 13.06 ± 3.03 days, increase in life span 22.86%.And merge to use CP and Q-can oral liquid (dosage 60% and 100%), no matter calculate, all above the antitumaous effect of simple CP with the average life rate elongation or with the Mus number that survival period prolongs 60% (17 days).So, merging use 60% and 100%Q-can oral liquid, the increase in life span that CP is produced improves 56.78% and 143.86% more respectively, and its difference has the remarkable meaning of statistics.The Q-can oral liquid is to the test report of Mice Bearing Lewis Lung Cancer effect
Material:
Q-can oral liquid (concentrating) contains idio-branched chain fatty acid 3.6mg for every milliliter; F1 mice (C57/B1 and DBA/2 first filial generation mice) body weight 18～22g, female; Lawis lung cancer tumor kind.
Method and result:
10 oral Q-can oral liquids of mices elder generation of treatment group 10 days, the idio-branched chain fatty acid dosage that take in every day is 36mg/kg, inoculate Lewis lung cancer then and continued oral administration again 18 days, dissect simultaneously with other group, just began administration behind 10 mouse inoculation Lewis lung cancer of positive controls in second day, lumbar injection CTX, continuous 8 days of dosage 30mg/kg, 12 mices of matched group also are intraperitoneal injection of saline 8 days.
Result of the test, though the Q-can group is lower by 21% than positive controls tumour inhibiting rate, from the survival rate of mice, 100% survival of Q-can group, and positive controls has only 70%.Positive controls is to use intraperitoneal injection, and dosage is 30mg/kg, and the Q-can test group is oral Q-can oral liquid every day, and contained idio-branched chain fatty acid dosage only has 36mg/kg in the oral dose.If oral dose strengthens, might improve tumour inhibiting rate.
Table 8.Q-can oral liquid is to the heavy tumour inhibiting rate P of heavily administration number of animals of the therapeutical effect group agent body weight tumor value of Mice Bearing Lewis Lung Cancer
Mode begins/begins at last/and last X ± SD (g) % physiological saline-ip 12/11 21.2/22.5 1.90 ± 0.96-CTX 30mg/kg ip 10/7 21.2/20.3 0.71 ± 0.36 62.2＜0.01Q-can 36mg/kg po, 10/10 20.9/21.9 1.11 ± 0.46 41.6＜0.05Q-can oral liquid is to the therapeutic action of people's cancer of the stomach SGC-7901 Transplanted Into Nude Mice cancer
Material:
Female Balb/c nude mouse, 6 ages in week, body weight 18-22g, whole experimental session is fed under specific-pathogen free (SPF) condition.Concentrate in every milliliter of the Q-can oral liquid and contain idio-branched chain fatty acid 3.6mg.Ametycin (MMC) is bought.
Method and result:
Set up people's gastric cancer SGC～7901 transplanted tumoies, cut into the fragment about diameter 2mm during experiment, it is subcutaneous to be inoculated in the right axillary fossa of nude mouse.After inoculation, be divided into 5 groups at random on the 5th day.Normal control group and positive controls are distinguished intraperitoneal injection of saline and mitomycin (20mg/kg) once a day, experimental group divides 18mg/kg, 36mg/kg and three kinds of dosage of 72mg/kg (idio-branched chain fatty acid content) in beginning oral administration one-time continuous 14 days every day on the same day.Inoculate back 20 days and stop experiment, the disconnected neck of mice is put to death.Take out tumor and heavy to the tumor of this experimental group and matched group, calculate suppression ratio, experiment repeats twice.
The result of twice experiment (experiment I and experiment II) is as follows
Table 9.Q-can oral liquid is to the therapeutical effect of people's gastric cancer SGC-7901 nude mouse oxter transplanted tumor
Experiment I
The heavy suppression ratio P of group dosed administration administration number of mice body weight tumor
The other mode time begins/begins at last/last X ± SDg
NS - ip Qdx14 12/12 21.9/23.3 0.17±0.45 -
MMC 20mg/kg ip Qdx14 6/6 22.4/22.0 0.33±0.24 71.49 ＜0.01
Q-can 18mg/kg Po Qdx14 6/6 22.0/22.5 0.80±0.42 31.19 ＞0.05
Q-can 36mg/kg Po Qdx14 6/6 21.9/22.0 0.60±0.45 48.23 ＜0.05
Q-can 72mg/kg po Qdx14 6/6 21.6/21.7 0.57±0.35 51.28 ＜0.05
Experiment II
The heavy suppression ratio P of group dosed administration administration number of mice body weight tumor
The mode time begins/begins at last/last X ± SDg
MMC 20mg/kg ip Qdx14 6/6 21.1/21.1 0.30±0.33 73.51 ＜0.01
Q-can?18mg/kg po Qdx14 6/6 21.9/22.3 0.90±0.59 21.58 ＞0.05
Q-can?36mg/kg po Qdx14 6/6 22.0/21.6 0.66±0.49 42.47 ＜0.05
Q-can?72mg/kg po Qdx14 6/6 22.3/20.3 0.51±0.37 55.45 ＜0.01
The The above results explanation, behind tumor inoculation, with 18mg/kg, 36mg/kg and three kinds of dosage of 72mg/kg (idio-branched chain fatty acid content) oral Q-can oral liquid every day once, can produce inhibitory action to people's gastric cancer SGC-7901 transplanted tumor in continuous 14 days, and with oral dosage increase, tumour inhibiting rate improves.Q-can oral liquid auxiliary for treating cancer 170 routine observation of curative effect
This clinical trial is carried out in China five tame hospitals, chemotherapy also relatively with this product treatment cancer 130 examples and simple chemotherapy 131 examples, what have statistical significance is, improves the quality of living, appetite stimulator is eliminated weak weight increase; Improve immunologic function; Alleviate the program that hemogram that chemotherapy causes reduces, and be lower than normal person certain therapeutical effect is arranged controlling proleukocyte and hemoglobin, also have certain protect the liver, the kidney effect.Radiotherapy is also relatively routine with radiation alone 32 with this product 34 examples, and the level that improves lgA has statistical significance.Grouping: various cancer random stratified groupings, be divided into two and organize greatly, wherein chemotherapy group is also used this product (treatment group) 136, simple chemotherapy (matched group) 131 examples; Radiotherapy is also used this product (treatment group) 34 examples, radiation alone (matched group) 32 examples.The sick kind: chemotherapy group is based on stomach, liver, esophagus, large intestine, lung, breast carcinoma etc., and combination radiotherapy group is based on nasopharyngeal carcinoma, laryngeal carcinoma.Sex: chemotherapy is controlled male 94 examples of group, women 42 examples, and matched group man 84 examples, women 47 examples, radiotherapy in the treatment group man 25 examples, women 9 examples, matched group man 26 examples, women 6 examples, two groups all have comparability (P＞0.1).Age: chemotherapeutic treatment group 17-83 year, average 53.1 years old, matched group 16-80 year, average 53.2 years old; In radiotherapy in the treatment group 30-73 year, average 49.7, in matched group 31-72 year, average 50.1 years old, two groups all had comparability (P＞0.1).Therapeutic Method: chemotherapeutic treatment group.Every day and with Q-can oral liquid 2 each 80ml chemotherapy matched groups and with other " rising in vain " medicine, 60 days courses of treatment.
Have 9 examples to obey Q-can oral liquid 160ml every day in the radiotherapy in the treatment group, everyone only obeys 100ml, 60 days courses of treatment 25 examples.
Observed result:
Table 10.Q-can oral liquid is to the chemotherapy group influence of quality of life
Before controlling, group example number controls back P value
(X ± SD) divides that (X ± SD) divides between self group
Treatment organizes 136 76.3 ± 14.69 83.90 ± 15.36＜0.01
Matched group 131 79.96 ± 10.96 76.22 ± 12.80＜0.01＜0.01
Table 11.Q-can oral liquid has the improvement effect to the chemotherapy group symptom
Symptom group example number alleviates example and stablizes example and increase the weight of routine P value
(％) (％) (％)
The loss of appetite treatment organizes 77 49 (68.6) 18 (25.00) 5 (6.94)＜0.01
Matched group 81 18 (22.22) 33 (40.74) 30 (37.04)
Weak treatment organizes 90 56 (62.22) 28 (31.11) 6 (16.67)＜0.01
Matched group 70 13 (18.57) 20 (41.43) 28 (40.00)
Matched group 18 5 (27.78) 12 (66.67) 1 (5.55)
Table 12.Q-can oral liquid influences chemotherapy group body weight *
Group example number increases the stable routine routine P value that descends of example
(％) (％) (％)
Treatment organizes 136 63 (46.32) 33 (24.27) 40 (29.41)
＜0.01
Before average specific was controlled after matched group 131 2 (15.27) 35 (26.72) 76 (58.0) * controlled 〉=and 0.5 kilogram of person is for increasing, and≤0.5 kilogram of person is decline, and the person of falling between is for stable.
Table 13.Q-can oral liquid has the effect that improves cellular immune function to chemotherapy group
Before controlling, index group example number controls back P value
(% of X ± SD) (X ± SD) between % self group
The LTT treatment organizes 65 55.95 ± 8.02 56.28 ± 8.55＞0.05＜0.01
Matched group 75 55.85 ± 8.87 49.41 ± 12.21＜0.01
CD
3Treatment organizes 30 43.53 ± 4.55 43.47 ± 5.10＞0.05＜0.01
Matched group 30 45.47 ± 3.56 38.57 ± 4.50＜0.01
CD
2Treatment organizes 30 44.07 ± 4.60 43.10 ± 5.13＞0.05＜0.01
Matched group 30 42.60 ± 5.20 38.27 ± 5.62＜0.01
The NK treatment organizes 30 9.60 ± 5.11 12.00 ± 4.23＜0.05＜0.01
Cell matched group 30 12.96 ± 4.31 10.80 ± 4.00＜0.05
Table 14.Q-can oral liquid has the effect that improves humoral immune function to chemotherapy group
Before controlling, index group example number controls back P value
(% of X ± SD) (X ± SD) between % self group
The IgC treatment organizes 71 10.90 ± 4.69 11.92 ± 5.06＜0.05＜0.01
Matched group 75 11.90 ± 4.38 11.05 ± 4.99＜0.05
The IgA treatment organizes 72 1.63 ± 0.67 1.73 ± 1.32＞0.05＜0.01
Matched group 75 1.65 ± 0.76 1.38 ± 0.76＜0.01
The IgM treatment organizes 71 1.24 ± 0.59 1.55 ± 1.05＜0.05＜0.01
Matched group 75 1.53 ± 0.78 1.30 ± 0.73＜0.01
Table 15.Q-can oral liquid has raising immunologic function (IgA) effect to combination radiotherapy group
Before controlling, index group example number controls back P value
(% of X ± SD) (X ± SD) between % self group
The IgA treatment organizes 31 1.74 ± 1.31 20.39 ± 0.90＜0.05＜0.01
G/L matched group 34 2.07 ± 10.3 1.73 ± 0.80＞0.05
The effect that table 16.Q-can oral liquid has prevention routine blood test and platelet to descend to chemotherapy group
Before controlling, project group example number controls back P value
(% of X ± SD) (X ± SD) between % self group
The WBC treatment organizes 30 4.745 ± 1.21 5.451 ± 0.86＜0.01＜0.01
(* 10
9/ L) matched group 30 5.29 ± 0.85 4.450 ± 0.80＜0.01
The granulocyte treatment organizes 30 3.20 ± 0.82 3.66 ± 0.69＜0.01＜0.01
(* 10
9) matched group 30 3.72 ± 0.58 3.09 ± 0.45＜0.01
The platelet treatment organizes 30 140.30 ± 4.88 160.03 ± 4.36＜0.01＜0.01
(* 10
9) matched group 30 157.33 ± 3.52 145.53 ± 5.33＜0.01
The Hb treatment organizes 30 94.63 ± 18.00 96.89 ± 16.08＞0.05＜0.01
(g/L) matched group 30 103.67 ± 13.24 99.20 ± 11.63＜0.01
Table 17.Q-can oral liquid can reduce SGDT and ALP value to chemotherapy group, and the effect of protection liver is arranged
Group example number SGPT (mmol/L) the P routine number ALP of value (U/L) P value
Control before controlling between self organizes after controlling before controlling between the group of back self
(X±SD) (X±SD)
Treatment organizes 89 460.06 ± 330.11 ±＞0.05＜0.05 88 72.34 ± 70.42 ±＞0.05
753.4 245.01 57.81 48.71 ＞0.05
Matched group 84 261.47 ± 284.00 ±＞0.05 83 65.26 ± 77.64 ±＜0.05
191.23 217.30 37.32 64.03
Table 18.Q-can oral liquid has protective effect to the drug-induced renal function injury of chemotherapy group
Group hematuria element (mmol/L) the P routine number serum creatinine of value (mmol/L) P value
Nitrogen is controlled between the group of back self before controlling and controlling between the group of back self
Before controlling (X ± SD) (X ± and SD) (X ± SD)
(X±
SD)
Treatment group 5.13 ± 4.95 ±＞0.05＜0.01 110 97.15 ± 97.99 ±＞0.05＜0.01
2.95 1.33 30.64 23.46
Matched group 4.26 ± 5.04 ±＜0.01 90 89.28 ± 107.08 ±＜0.01
1.03 1.42 22.13 41.27
Table 19.Q-can oral liquid all has the rising effect to chemotherapy group total serum protein and albumin
Before controlling, project group example number controls back P value
(X ± SD) (between X ± SD) self organizes
The total protein treatment organizes 101 65.31 ± 10.01 67.47 ± 5.99＞0.05＜0.01
(g/L) matched group 103 65.64 ± 6.53 64.20 ± 6.07＜0.05
The albumin treatment organizes 107 38.78 ± 5.65 39.13 ± 5.26＞0.05＜0.05
(g/L) matched group 102 39.44 ± 4.74 38.18 ± 5.24＜0.05
With the spray-dried made capsule of the Q-can strain fermentating liquid of indication of the present invention, clinically to the effect of 116 routine cancer patients observations and the effect basically identical of Q-can oral liquid.So no longer repeat.Q-can oral liquid treatment 35 routine U.S. carcinoma of prostate patients' clinical observation
Detect 36 carcinoma of prostate patients (in age from 37-80 year, the mean age is 66 years old) in the tame hospitals of U.S. two, take Q-can concentrated oral liquid (every bottle of 250ml contains idio-branched chain fatty acid 300mg) 8 week～18 weeks (average 14 weeks).Patient does not accept radiotherapy, chemotherapy or hormone therapy during treating, take 250ml every day, the Q-can concentrated oral liquid.35 routine patients draw blood and measure the variation of PSA (Prostate Specific Antigen) index, and its result is as follows:
Table 20.Q-can oral liquid is to the influence of carcinoma of prostate patient P SA index
(mg/ml) X ± SD took back (mg/ml) X ± SD P value before case load was taken
35 10.22±10.72 7.45±6.06 ＜0.01
Above clinical observation shows that the Q-can oral liquid has the effect that reduces its PSA to the carcinoma of prostate patient.Full name of the used abbreviation of the present patent application: HPLC=High Peformance Ligqid Chromatography high performance liquid chromatography LD
50The indirect dUTP otch of=50% lethal dose median lethal dose(LD 50) PBL=peripherel blood lymphocytes peripheral blood lymphocyte TUNEL=TdT-mediated dUTP nick end labeling TdT-end labelling POD=peroxidase peroxidase FBS=fetal bovine serum hyclone PBS=phosphate-biffered saline phosphate-buffered saline CTX=Cytoxin cytotoxin CP=cyclophosphamide cyclophosphamide
Claims (4)
1. a class has the material of antitumaous effect, it is characterized in that this material is saturated and corresponding unsaturated side chain fatty acid, its pharmaceutical salts and axunge albumen, be made into oral liquid, powder, capsule, tablet, injection product, be used for cancer patient anti-cancer, anticancer, strengthen the radiotherapy chemotherapy effect, improve immune function of human body and health care.Wherein saturated branched chain fatty acid molecular formula is:
N and m are integers independently in the formula, and n+m is 0～46; M or n are at least 2 in the above-mentioned molecular formula of unsaturated side chain fatty acid, at (CH
2)
mOr (CH
2)
nIn have (a CH at least
2-CH
2) group changes (CH=CH) group into.
2. the material with antitumaous effect according to claim 1 is characterized in that this type of saturated and unsaturated side chain fatty acid is to obtain with the electrochemical method synthetic method.
3. the material with antitumaous effect according to claim 1 is characterized in that this type of saturated and unsaturated side chain fatty acid is obtained by extracting in the natural material.
4. the material with antitumaous effect according to claim 1 is characterized in that this type of saturated and unsaturated side chain fatty acid is through the fermentation culture medium that contains more than one above-mentioned substances at least that fermentation culture obtained by the bacterial isolates that is rich in branched chain fatty acid.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN98120509A CN1108796C (en) | 1998-10-16 | 1998-10-16 | Use of anticancer material and production method of said anti-cancer material |
| CA002286750A CA2286750C (en) | 1998-10-16 | 1999-10-18 | Application and production process of a group of anticancer compounds |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN98120509A CN1108796C (en) | 1998-10-16 | 1998-10-16 | Use of anticancer material and production method of said anti-cancer material |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1251296A CN1251296A (en) | 2000-04-26 |
| CN1108796C true CN1108796C (en) | 2003-05-21 |
Family
ID=5226780
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN98120509A Expired - Lifetime CN1108796C (en) | 1998-10-16 | 1998-10-16 | Use of anticancer material and production method of said anti-cancer material |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN1108796C (en) |
| CA (1) | CA2286750C (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110771694A (en) * | 2019-10-31 | 2020-02-11 | 江南大学 | Grease composition |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001076588A1 (en) * | 2000-04-06 | 2001-10-18 | Coastside Research | Method to inhibit lipoxygenase and cancer cell proliferation |
| AU2002322828A1 (en) * | 2001-08-03 | 2003-02-24 | Zhenhua Yang | A group of novel anti-cancer compounds with specific structure |
| BR112013026032A2 (en) * | 2011-04-12 | 2016-12-20 | Nestec Sa | nutritional compositions including branched chain fatty acids for wound healing |
| FR3007418B1 (en) * | 2013-06-21 | 2015-07-17 | Interoleagineuse D Assistance Et De Dev S I A Soc | LIPIDIC COMPOSITION BASED ON TRIACYLGLYCEROL |
| CN105982883A (en) * | 2015-02-17 | 2016-10-05 | 复旦大学 | Application of palmitic acid to preparation of drug for treating colon cancer |
| CN109997918A (en) * | 2018-01-05 | 2019-07-12 | 福建杨振华851生物科技股份有限公司 | One kind is containing newborn fermented soybean compound protein product and preparation method thereof |
| CN109022309B (en) * | 2018-07-27 | 2023-04-18 | 福建杨振华851生物科技股份有限公司 | Stenotrophomonas maltophilia capable of producing free fatty acid and application thereof |
| CN112089711A (en) * | 2020-09-28 | 2020-12-18 | 石河子大学 | Application of free fatty acid in preparation of medicine for treating prostatic cancer |
| KR102395453B1 (en) * | 2020-11-11 | 2022-05-09 | 제주대학교 산학협력단 | Composition for inhibiting breast cancer metastasis comprising odd long chain fatty acid as effective component |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4985466A (en) * | 1988-06-14 | 1991-01-15 | Dai-Ichi Kogyo Seiyaku Co., Ltd. | Method for treating tumors susceptible to treatment with reduced wool alcohol |
-
1998
- 1998-10-16 CN CN98120509A patent/CN1108796C/en not_active Expired - Lifetime
-
1999
- 1999-10-18 CA CA002286750A patent/CA2286750C/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4985466A (en) * | 1988-06-14 | 1991-01-15 | Dai-Ichi Kogyo Seiyaku Co., Ltd. | Method for treating tumors susceptible to treatment with reduced wool alcohol |
Non-Patent Citations (1)
| Title |
|---|
| BIOCHEM,第53卷 1953-01-01 HANSEN 等"THE BRANCH-HAINED FATTY ACID OF MUTTON FAT" * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110771694A (en) * | 2019-10-31 | 2020-02-11 | 江南大学 | Grease composition |
Also Published As
| Publication number | Publication date |
|---|---|
| CA2286750A1 (en) | 2000-04-16 |
| CN1251296A (en) | 2000-04-26 |
| CA2286750C (en) | 2003-10-14 |
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