CN103169844B - Traditional Chinese medicine composition having anti-lung cancer and anti-liver cancer effects - Google Patents
Traditional Chinese medicine composition having anti-lung cancer and anti-liver cancer effects Download PDFInfo
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- CN103169844B CN103169844B CN201310079305.1A CN201310079305A CN103169844B CN 103169844 B CN103169844 B CN 103169844B CN 201310079305 A CN201310079305 A CN 201310079305A CN 103169844 B CN103169844 B CN 103169844B
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Abstract
The invention discloses a traditional Chinese medicine composition having anti-lung cancer and anti-liver cancer effects. The traditional Chinese medicine composition is composed of a paris polyphylla extract, turmeric polysaccharides and a radix paeoniae alba extract in a weight ratio of 1: (0.1-5): (1-10). The in-vitro MTT (Methylthiazolyl Tetrazolium) assay activity tests proves that traditional Chinese medicine composition provided by the invention is capable of obviously inhibiting the growth of lung cancer cell lines such as LA795 and liver cancer cell lines such as Hep-B3; and lung adenocarcinoma and hepatocellular carcinoma-bearing mice model tests show that the anti-tumor rates of the traditional Chinese medicine composition both are more than 50%, and the traditional Chinese medicine composition is capable of improving the spleen index and the liver index of the mice, and obviously inhibiting pulmonary metastasis of subcutaneously transplanted tumors of the mice and causing apoptosis of tumor cells without any obvious toxic and side effects. Compared with single use of each raw material, the medicine composition is higher in anti-lung cancer and anti-liver cancer activity and clear in action, and has excellent inhibition effect on the lung cancer and the liver cancer.
Description
Technical field
The present invention relates to a kind of Chinese medicine composition with anti-pulmonary carcinoma and hepatocarcinoma effect, belong to technical field of Chinese medicines.
Background technology
Rhizoma Paridis is the dry rhizome of liliaceous plant Rhizoma Paridis Paris polyphylla Smith var.yunnanensis (Franch.) Hand.-Mazz. or Rhizoma Paridis Paris polyphylla Smith var.chinensis (Franch.) Hara, there is heat-clearing and toxic substances removing, effect of reducing swelling and alleviating pain, cool liver arresting convulsion, being used for the treatment of the diseases such as furuncle carbuncle, laryngopharynx swelling and pain, venom, traumatic pain, cool breeze tic, is the chief component medicine of the Chinese patent medicines such as YUNNAN BAIYAO, GONGXUENING JIAONANG, jidesheng sheyao tablets.Modern pharmacological research shows, Rhizoma Paridis has cytotoxic activity, antibacterial anti-inflammatory, analgesia, hemostasis, calmness, antiearly pregnancy, spermicidal, to the physiologically active such as respiratory system and Cardiovascular System, be used for the treatment of clinically the curative effect of disease such as lung system, dysfunctional uterine hemorrhage, tuberculous lymphadenitis ulcer, neurodermatitis, surgery inflammation and tumor remarkable.There is at present the listing Chinese patent medicine compound recipe that Rhizoma Paridis is used as medicine to have the multiple recovery oral liquid of gold, softening the hard mass oral liquid, building lotus capsule, GANFULE PIAN etc.In recent years, the bibliographical information of Rhizoma Paridis and antitumor effective ingredient Rhizoma Paridis total saponins thereof is increasing.CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2008,33 (16): in 2057-2060, disclose cell toxicant spectrum and the structure activity study of Rhizoma Paridis saponin to 10 kinds of tumor cell lines; The progress of Paris Linnaeus(Paris L.) medicinal plants saponins chemical composition and source of students approach thereof has been delivered in Huang Xian schools etc. at Chinese herbal medicine magazine 2009,40 (3): 483-489; Chen Zhihong etc. have investigated the inhibited proliferation of Rhizoma Paridis total saponins on human lung cancer cell A549 and the impact of cell cycle; Zhu Lili etc. suppress SGC-7901 cell proliferation and apoptosis-induced being studied to Rhizoma Paridis saponin; Lin Yunhua research find Rhizoma Paridis and oxaliplatin in vitro, in experiment, may there is the effect of certain angiogenesis inhibitor in body, both use in conjunction may have synergistic function.M-dose-dependence when the discovery Rhizoma Paridis total saponins such as Jia Ke can significantly suppress the growth of MGC-803 cell and be, can block cell in the S phase, the rising of cell death inducing rate; Can significantly suppress the expression of EphA2, survivin albumen, raise the expression of Caspase-3 albumen simultaneously, and then its mechanism of action of reaching a conclusion may and promote that with the expression of downward EphA2 and survivin the expression of Caspase-3 is relevant.In No. 200610052223.8th, Chinese patent, disclose a kind of preparation method of Rhizoma Paridis total saponins, also disclose a kind of pharmaceutical preparation taking Rhizoma Paridis total saponins as effective ingredient and preparation method thereof in No. 200810052092.2.
The Rhizoma Curcumae Longae beginning is loaded in Tang Materia Medica, for the dry rhizome of zingiberaceous plant Rhizoma Curcumae Longae Curcuma longa L., its nature and flavor acrid, bitter, warm, returns spleen, Liver Channel, there is removing blood stasis circulation of qi promoting, effect of inducing menstruation to relieve menalgia, can be used for the twinge of the breast side of body, obstruction of qi in the chest and cardialgia, dysmenorrhea amenorrhea mass in the abdomen, rheumatism shoulder arm pain, the treatment of the diseases such as tumbling and swelling.Rhizoma Curcumae Longae contains number of chemical composition, and main component is curcumin chemical compounds: curcumin, bisdemethoxycurcumin, demethoxycurcumin, dihydro curcumin etc.; Sesquiterpenoids: the new ketone of Rhizoma Curcumae Longae, Rhizoma Curcumae Longae keto-alcohol A, B etc.; Volatile oil (4.2%): turmerone, curcumene, curcumenol etc., contain Rhizoma Curcumae Longae polysaccharide composition in addition.Wherein the pharmacological action of curcumin effective ingredient is extensive, the effects such as its antiinflammatory, antioxidation, atherosclerosis, antidepressant, antitumor have generally been approved, wherein antitumor action after proposing for 1985 first, existing a large amount of experimentatioies confirms, mechanism of action mainly comprises inducing apoptosis of tumour cell, suppress angiogenesis, have retardance cell cycle progression, the aspects such as reversing multiple medicine resistance of tumor cells.Huang Dongsheng etc. study discovery, and curcumin can strengthen the expression of caspase-8, thereby start exogenous apoptosis pathway, induction human lung carcinoma cell apoptosis.The report curcumins such as Fang Yue have the effect of the digestive system tumors such as anti-esophageal carcinoma, gastric cancer, hepatocarcinoma, colon cancer.Chinese patent CN1931353A discloses a kind of curcumin and has extracted and Chinese medicine composition preparation method.
The Radix Paeoniae Alba is the peeling dry root of ranunculaceae plant Paeonia, cold nature, and bitter in the mouth, acid, have nourishing blood to suppress the hyperactive liver, slow middle pain relieving, yin fluid astringing to receive effect of antiperspirant.The active ingredient of the Radix Paeoniae Alba comprises peoniflorin, Hydroxy peoniflorin, peonin, lactone glucoside of Radix Paeoniae, and benzoylpaeoniflorin etc. are referred to as Radix Paeoniae Alba total glucosides (total glucosides of peony, TGP).Research discovery, it can affect in multiple links cellular immunization, humoral immunization and the inflammatory process of autoimmune disease, and the effect that has analgesia, protects the liver.Peoniflorin is as the compound accounting for more than TGP90%, and discovered in recent years also has anti-tumor activity.Its mechanism of action except the immunity of enhancing body, also have inside and outside cause tumour cell cycle stagnation, inducing apoptosis of tumour cell, to effects such as chemotherapeutics attenuation synergistics.Poplar adds the discovery Radix Paeoniae Alba alcohol extract ultrafilter membrane separation active components such as territory and has in vitro anti-tumor activity; The report TGP combination chemotherapy nonsmall-cell lung cancers such as Zhan Keshun are better than chemotherapy, and can improve life in patients, improve Cellular Immunologic Function In Patients and rising peripheral hemogram.In No. 200510045840.0th, Chinese patent, disclose a kind of compositions of peoniflorin and the lactone glucoside of Radix Paeoniae with function of increasing leukocyte, be applicable to the medicine of the diseases such as leukopenia, platelet and blood red globulin reduction that preparation treatment causes because of a variety of causes.The methods such as the employing water extract-alcohol precipitations such as the Rong of higher primary school, membrance separation, the separation and purification of DEAE-cellulose chromatography obtain Radix Paeoniae Alba polysaccharides BSP, and Mice Bearing Lewis Lung Cancer and S180 sarcoma model are all had to good suppression ratio.
At present, the patent of the Chinese medicine composition (CN101152408A) of Rhizoma Paridis saponin and astragalus polysaccharides has been seen in report, and not yet finds the Chinese medicine composition that has anti-pulmonary carcinoma and hepatocarcinoma effect simultaneously and be made up of Rhizoma Paridis extract, Rhizoma Curcumae Longae polysaccharide and Radix Paeoniae Alba extract.
Summary of the invention
In order to address the above problem, to the object of the present invention is to provide a kind of activity more by force and act on the clear and definite Chinese medicine composition with anti-pulmonary carcinoma and hepatocarcinoma effect.
In order to achieve the above object, the Chinese medicine composition with anti-pulmonary carcinoma and hepatocarcinoma effect provided by the invention by Rhizoma Paridis extract, Rhizoma Curcumae Longae polysaccharide and Radix Paeoniae Alba extract with 1: the weight ratio of 0.1-5: 1-10 forms; Wherein Rhizoma Paridis extract is commercial goods, and specification is 5: 1,10: 1 or 20: 1;
The preparation method of described Rhizoma Curcumae Longae polysaccharide comprises the following step carrying out in order:
1) Rhizoma Curcumae Longae medical material or decoction pieces are ground into fritter, then join in dehydrated alcohol and soak 1 hour, the amount ratio of medical material and dehydrated alcohol is 1g: 9.5mL, then heating and refluxing extraction 1 hour at the temperature of 90 DEG C, discard extracting solution, retain reflux residue;
2) above-mentioned reflux residue is added to the water, the amount ratio of reflux residue and water is 1g: 9-10mL, and then heating and refluxing extraction 2 times at the temperature of 90 DEG C, each 2.5 hours, merges 2 times extracting solution;
3) said extracted liquid is joined in the alcoholic solution of 95% concentration to alcoholic degree and reach 75%, hold over night, then at 7500rmin
-1rotating speed under centrifugalize 20min, collecting precipitation thing lyophilization, use dehydrated alcohol, acetone, ether washing precipitation afterwards successively, obtains Rhizoma Curcumae Longae polysaccharide crude extract;
4) above-mentioned Rhizoma Curcumae Longae polysaccharide crude extract is made to sample solution with water dissolution, then adding consumption is that chloroform and the n-butanol mixed solvent of sample solution volume 1/4 extracts, violent jolting 20-30min, centrifugalize, in filtrate, add again chloroform and n-butanol mixed solvent the violent jolting of 1/4 filtrate volume, repeat aforesaid operations, until the denatured protein of water layer and solvent layer intersection no longer occurs, extract is evaporated to 1/4 of original volume;
5) extract after above-mentioned concentrating is placed in to bag filter flowing water dialysis 3 days, insoluble matter in elimination bag filter, then in clear liquor, add alcoholic solution to the alcoholic degree of 95% concentration to reach 85%, hold over night, by dehydrated alcohol and washing with acetone for precipitate, after being dried, can obtain the Rhizoma Curcumae Longae polysaccharide after refining;
The preparation method of described Radix Paeoniae Alba extract comprises the following step carrying out in order:
1) white Peony Root or decoction pieces are ground into coarse powder, then join in the alcoholic solution of 65% concentration and soak 1 hour, the ethanol amount ratio of the Radix Paeoniae Alba and 65% concentration is 1g: 8-10mL, then heating and refluxing extraction 3 times at the temperature of 95 DEG C, the 1st time 2 hours, 2nd, 3 times each 1 hour, merge 3 times extracting solution;
2) be evaporated to 1/2 of original volume after said extracted liquid is filtered, then add the water of 1.5 times of volumes, filter after being heated to leave standstill 12 hours in cool place place after micro-boiling, by precipitate evaporation drying and obtain Radix Paeoniae Alba extract at the temperature of 85 DEG C.
In the preparation method of above-mentioned Rhizoma Curcumae Longae polysaccharide, between described step 2} and step 3}, also comprise a decolouring step, method is by step 2) to be evaporated to relative density at 25 DEG C after the extracting liquid filtering that obtains be 1.0, then adopt macroporous weakly basic anion exchange resin D301-G at 35 DEG C, under the condition of pH=5.5, decolour.
In the preparation method of above-mentioned Rhizoma Curcumae Longae polysaccharide, described step 4) in chloroform and n-butanol mixed solvent the volume ratio of chloroform and n-butyl alcohol be 4.5: 1.
The amount ratio of described Rhizoma Paridis extract, Rhizoma Curcumae Longae polysaccharide and Radix Paeoniae Alba extract is 1: 2.5: 5.
The Chinese medicine composition with anti-pulmonary carcinoma and hepatocarcinoma effect provided by the invention is to be mixed by commercially available Rhizoma Paridis extract, Rhizoma Curcumae Longae polysaccharide and Radix Paeoniae Alba extract.Prove through external mtt assay activity test, this Chinese medicine composition can significantly suppress the growth of the hepatoma cell strains such as lung cancer cell line and Hep-B3 such as LA795; To adenocarcinoma of lung and hepatocarcinoma bearing mouse model, test shows, tumour inhibiting rate all more than 50%, improves mouse spleen index and liver index respectively, and the higher and effect of the activity of anti-pulmonary carcinoma and hepatocarcinoma clearly, has good inhibitory action to pulmonary carcinoma, hepatocarcinoma.In addition, described Chinese medicine composition preparation process is simple, and can be prepared into various different dosage forms.
Detailed description of the invention
Embodiment mono-:
(1) get 1g and cross the commercially available Rhizoma Paridis extract that specification is 10: 1 after 80 mesh sieves (bright biological development corporation, Ltd. of Baoji side produces) powder.
(2) preparation of Rhizoma Curcumae Longae polysaccharide
1) 300g Rhizoma Curcumae Longae pulverizing medicinal materials is become to fritter, then join in dehydrated alcohol and soak 1 hour, the amount ratio of medical material and dehydrated alcohol is 1g: 9.5mL, and then heating and refluxing extraction 1 hour at the temperature of 90 DEG C, discards extracting solution, retains reflux residue;
2) above-mentioned reflux residue is added to the water, the amount ratio of reflux residue and water is 1g: 9mL, and then heating and refluxing extraction 2 times at the temperature of 90 DEG C, each 2.5 hours, merges 2 times extracting solution;
3) be 1.0 by being evaporated to relative density at 25 DEG C after extracting liquid filtering obtained above, then adopt macroporous weakly basic anion exchange resin D301-G at 35 DEG C, under the condition of pH=5.5, decolour;
4) extracting solution after above-mentioned decolouring is joined in the alcoholic solution of 95% concentration to alcoholic degree and reach 75%, hold over night, then at 7500rmin
-1rotating speed under centrifugalize 20min, collecting precipitation thing lyophilization, use dehydrated alcohol, acetone, ether washing precipitation afterwards successively, obtains Rhizoma Curcumae Longae polysaccharide crude extract;
5) above-mentioned Rhizoma Curcumae Longae polysaccharide crude extract is made to sample solution with water dissolution, then adding consumption is chloroform and the n-butanol mixed solvent (V/V=4.5: 1) extract of sample solution volume 1/4, violent jolting 20-30min, centrifugalize, in filtrate, add again chloroform and n-butanol mixed solvent the violent jolting of 1/4 filtrate volume, repeat aforesaid operations, until the denatured protein of water layer and solvent layer intersection no longer occurs, extract is evaporated to 1/4 of original volume;
6) extract after above-mentioned concentrating is placed in to bag filter flowing water dialysis 3 days, insoluble matter in elimination bag filter, then in clear liquor, add alcoholic solution to the alcoholic degree of 95% concentration to reach 85%, hold over night, by dehydrated alcohol and washing with acetone for precipitate, after being dried, can obtain the Rhizoma Curcumae Longae polysaccharide 20.1g after refining.
(3) preparation of Radix Paeoniae Alba extract
1) 100g white Peony Root is ground into coarse powder, then join in the alcoholic solution of 65% concentration and soak 1 hour, the ethanol amount ratio of the Radix Paeoniae Alba and 65% concentration is 1g: 9mL, then heating and refluxing extraction 3 times at the temperature of 95 DEG C, the 1st time 2 hours, 2nd, 3 times each 1 hour, merge 3 times extracting solution;
2) be evaporated to 1/2 of original volume after said extracted liquid is filtered, then add the water of 1.5 times of volumes, filter after being heated to leave standstill 12 hours in cool place place after micro-boiling, by precipitate evaporation drying and obtain Radix Paeoniae Alba extract 13.5g at the temperature of 85 DEG C.
(4) preparation of Chinese medicine composition
After being mixed by the weight ratio of 1: 2.5: 5, above-mentioned Rhizoma Paridis extract, Rhizoma Curcumae Longae polysaccharide and Radix Paeoniae Alba extract can be made into the Chinese medicine composition with anti-pulmonary carcinoma and hepatocarcinoma effect provided by the invention.
Embodiment bis-:
(1) get 10g and cross the commercially available Rhizoma Paridis extract powder that specification is 5: 1 after 80 mesh sieves.
(2) preparation of Rhizoma Curcumae Longae polysaccharide
1) 300g Rhizoma Curcumae Longae pulverizing medicinal materials is become to fritter, then join in dehydrated alcohol and soak 1 hour, the amount ratio of medical material and dehydrated alcohol is 1g: 9.5mL, and then heating and refluxing extraction 1 hour at the temperature of 90 DEG C, discards extracting solution, retains reflux residue;
2) above-mentioned reflux residue is added to the water, the amount ratio of reflux residue and water is 1g: 10mL, and then heating and refluxing extraction 2 times at the temperature of 90 DEG C, each 2.5 hours, merges 2 times extracting solution;
3) be 1.0 by being evaporated to relative density at 25 DEG C after extracting liquid filtering obtained above, then adopt macroporous weakly basic anion exchange resin D301-G at 35 DEG C, under the condition of pH=5.5, decolour;
4) extracting solution after above-mentioned decolouring is joined in the alcoholic solution of 95% concentration to alcoholic degree and reach 75%, hold over night, then at 7500rmin
-1rotating speed under centrifugalize 20min, collecting precipitation thing lyophilization, use dehydrated alcohol, acetone, ether washing precipitation afterwards successively, obtains Rhizoma Curcumae Longae polysaccharide crude extract;
5) above-mentioned Rhizoma Curcumae Longae polysaccharide crude extract is made to sample solution with water dissolution, then adding consumption is chloroform and the n-butanol mixed solvent (V/V=4.5: 1) extract of sample solution volume 1/4, violent jolting 20-30min, centrifugalize, in filtrate, add again chloroform and n-butanol mixed solvent the violent jolting of 1/4 filtrate volume, repeat aforesaid operations, until the denatured protein of water layer and solvent layer intersection no longer occurs, extract is evaporated to 1/4 of original volume;
6) extract after above-mentioned concentrating is placed in to bag filter flowing water dialysis 3 days, insoluble matter in elimination bag filter, then in clear liquor, add alcoholic solution to the alcoholic degree of 95% concentration to reach 85%, hold over night, by dehydrated alcohol and washing with acetone for precipitate, after being dried, can obtain the Rhizoma Curcumae Longae polysaccharide 20.6g after refining.
(3) preparation of Radix Paeoniae Alba extract
1) 100g white Peony Root is ground into coarse powder, then join in the alcoholic solution of 65% concentration and soak 1 hour, the ethanol amount ratio of the Radix Paeoniae Alba and 65% concentration is 1g: 8mL, then heating and refluxing extraction 3 times at the temperature of 95 DEG C, the 1st time 2 hours, 2nd, 3 times each 1 hour, merge 3 times extracting solution;
2) be evaporated to 1/2 of original volume after said extracted liquid is filtered, then add the water of 1.5 times of volumes, filter after being heated to leave standstill 12 hours in cool place place after micro-boiling, by precipitate evaporation drying and obtain Radix Paeoniae Alba extract 13.5g at the temperature of 85 DEG C.
(4) preparation of Chinese medicine composition
After being mixed by the weight ratio of 1: 0.1: 1, above-mentioned Rhizoma Paridis extract, Rhizoma Curcumae Longae polysaccharide and Radix Paeoniae Alba extract can be made into the Chinese medicine composition with anti-pulmonary carcinoma and hepatocarcinoma effect provided by the invention.
Embodiment tri-:
(1) get 10g and cross the commercially available Rhizoma Paridis extract powder that specification is 20: 1 after 80 mesh sieves.
(2) preparation of Rhizoma Curcumae Longae polysaccharide
1) 300g Rhizoma Curcumae Longae pulverizing medicinal materials is become to fritter, then join in dehydrated alcohol and soak 1 hour, the amount ratio of medical material and dehydrated alcohol is 1g: 9.5mL, and then heating and refluxing extraction 1 hour at the temperature of 90 DEG C, discards extracting solution, retains reflux residue;
2) above-mentioned reflux residue is added to the water, the amount ratio of reflux residue and water is 1g: 10mL, and then heating and refluxing extraction 2 times at the temperature of 90 DEG C, each 2.5 hours, merges 2 times extracting solution;
3) be 1.0 by being evaporated to relative density at 25 DEG C after extracting liquid filtering obtained above, then adopt macroporous weakly basic anion exchange resin D301-G at 35 DEG C, under the condition of pH=5.5, decolour;
4) extracting solution after above-mentioned decolouring is joined in the alcoholic solution of 95% concentration to alcoholic degree and reach 75%, hold over night, then at 7500rmin
-1rotating speed under centrifugalize 20min, collecting precipitation thing lyophilization, use dehydrated alcohol, acetone, ether washing precipitation afterwards successively, obtains Rhizoma Curcumae Longae polysaccharide crude extract;
5) above-mentioned Rhizoma Curcumae Longae polysaccharide crude extract is made to sample solution with water dissolution, then adding consumption is chloroform and the n-butanol mixed solvent (V/V=4.5: 1) extract of sample solution volume 1/4, violent jolting 20-30min, centrifugalize, in filtrate, add again chloroform and n-butanol mixed solvent the violent jolting of 1/4 filtrate volume, repeat aforesaid operations, until the denatured protein of water layer and solvent layer intersection no longer occurs, by extract concentrating under reduced pressure;
6) extract after above-mentioned concentrating is placed in to bag filter flowing water dialysis 3 days, insoluble matter in elimination bag filter, then in clear liquor, add alcoholic solution to the alcoholic degree of 95% concentration to reach 85%, hold over night, by dehydrated alcohol and washing with acetone for precipitate, after being dried, can obtain the Rhizoma Curcumae Longae polysaccharide 20.6g after refining.
(3) extracting method of described Radix Paeoniae Alba extract comprises the following step carrying out in order:
1) the white Peony Root 100g of coarse powder will be ground into, adopt (W/W) 65% ethanol of 1L Radix Paeoniae Alba coarse powder dry weight, soak after 1 hour at 95 DEG C of temperature reflux, extract, 2 hours, and carry out again reflux, extract, 2 times by the 1st reflux, extract, operating condition, each 1 hour, merge 3 times extracting solution;
2) after extracting liquid filtering through being evaporated to 1/2 of original volume, add the water of 1.5 times of volumes, filter after being heated to leave standstill 12 hours in cool place place after micro-boiling, precipitate is evaporation drying at 85 DEG C, obtains Radix Paeoniae Alba extract.
(4) preparation of Chinese medicine composition
After being mixed by the weight ratio of 1: 5: 10, above-mentioned Rhizoma Paridis extract, Rhizoma Curcumae Longae polysaccharide and Radix Paeoniae Alba extract can be made into the Chinese medicine composition with anti-pulmonary carcinoma and hepatocarcinoma effect provided by the invention.
In order to verify the drug effect of the Chinese medicine composition with anti-pulmonary carcinoma and hepatocarcinoma effect provided by the invention, the inventor has carried out following test:
One. anti-tumor activity test
1. anti tumor activity in vitro test
1.1 cell strains and cultivation
Mouse pulmonary adenocarcinoma cell line LA 795 strain, is incubated at containing in the RPMn640 culture medium of 10% inactivated fetal bovine serum, 100U/mL penicillin and 100 μ g/mL streptomycins, in 37 DEG C, and 5%CO
2under incubator and saturated humidity condition, cultivate, within 3-4 days, go down to posterity once.
Human lung adenocarcinoma A549 cell strain, is incubated at containing in the DMEM culture medium of 10% inactivated fetal bovine serum, 100U/mL penicillin and 100 μ g/mL streptomycins, in 37 DEG C, and 5%CO
2under incubator and saturated humidity condition, cultivate, within 3～4 days, go down to posterity once.
BEL-7402 hepatoma cell strain, is incubated at containing in the RPMn640 culture medium of 10% inactivated fetal bovine serum, 100U/mL penicillin and 100 μ g/mL streptomycins, in 37 DEG C, and 5%CO
2under incubator and saturated humidity condition, cultivate, within 3-4 days, go down to posterity once.
Hep-B3 hepatoma cell strain, is incubated at containing in the DMEM culture medium of 10% inactivated fetal bovine serum, 100U/mL penicillin and 100 μ g/mL streptomycins, in 37 DEG C, and 5%CO
2under incubator and saturated humidity condition, cultivate, within 3～4 days, go down to posterity once.
1.2 Antitumor Activity of Drugs are measured
Mtt assay: be 3 × 10 with being mixed with concentration after trypsinization by exponential phase cell
4the cell suspension of individual/mL, is inoculated in 96 hole ELISA Plate, and every hole adds 200 μ L.After 24h, change not containing serum free culture system liquid, make cell synchronization growth, every hole 200 μ L.Within the 3rd day, be subject to reagent to adding in hole, add respectively Rhizoma Paridis extract aqueous solution, Rhizoma Curcumae Longae polysaccharide solution, Radix Paeoniae Alba extract aqueous solution and this Chinese medicine composition aqueous solution; Separately establish matched group: 0.1% dimethyl sulfoxide (DMSO), every hole adds 200 μ L.Be subject to every group of reagent to establish respectively 0.01,0.1,1,10,100,1,000 six of μ g/mL concentration, each concentration is established 8 parallel holes, at 37 DEG C, cultivates after 24h, and every hole adds serum-free without the freshly prepared 0.5mg/mL MTT100 of phenol red culture fluid μ L, continue to cultivate 4h, then, every hole adds 100 μ L DMSO and dissolves MTT formazan granule, after mixing with microoscillator vibration, in microplate reader, measure optical density value (OD), experiment in triplicate, is averaged.Process tumor cell as matched group taking solvent control, calculate suppression ratio by OD value, formula is: inhibitory rate of cell growth=(matched group OD value one experimental group OD value)/matched group OD × 100%, and taking drug level as abscissa, OD value is vertical coordinate, draw cell growth curve, and try to achieve IC
50(half suppression ratio), IC
50=inhibitory rate of cell growth is 50% drug level.The results are shown in Table 1.
2. anti-tumor in vivo activity test
Respectively 100 T739 mices (female, 18-20g) are given to Chinese medicine composition prepared by above-described embodiment and carry out the test of pesticide effectiveness in mice LA795 adenocarcinoma of lung metastasis model body.
Respectively 100 kunming mices (female, 18-20g) are given to Chinese medicine composition prepared by above-described embodiment and carry out the test of pesticide effectiveness in mice HepA liver cancer model and H22 liver cancer model body.
2.1 animal model
2.1.1HepA liver cancer model is got the full tumor-bearing mice of ascites after inoculation HepA hepatoma carcinoma cell, with normal saline washing abdominal part to carry out disinfection, in superclean bench, extract 5mL milky ascites as tumor source, then add 5mL normal saline and be mixed with the cell suspension of 1: 1 ratio, extract 0.1mL be inoculated in a mice oxter with asepsis injector, inoculate altogether 100.Whole operating under aseptic condition carried out.
2.1.2H22 liver cancer model is got the full tumor-bearing mice of ascites after inoculation H22 hepatoma carcinoma cell, with normal saline washing abdominal part to carry out disinfection, in superclean bench, extract 5mL milky ascites as tumor source, then add 5mL normal saline and be mixed with the cell suspension of 1: 1 ratio, extract 0.1mL be inoculated in a mice oxter with asepsis injector, inoculate altogether 100.Whole operating under aseptic condition carried out.
2.1.3LA795 adenocarcinoma of lung metastasis model is got the 6-8 days good T739 mice with tumor of tumor growth after inoculation LA795 lung adenocarcinoma cell, break cervical vertebra and put to death mice, under aseptic condition, peel off tumor, select freshly to shred and be placed in glass grinding device without downright bad tumor tissue, adding appropriate normal saline grinds gently and is prepared into tumor cell suspension, after filtering, cell sieve adds normal saline dilution, counting, being deployed into cell concentration is the cell suspension of 1,000 ten thousand/milliliter, be inoculated in healthy mice right fore axillary fossa subcutaneous, every 0.2ml.
2.2 animal grouping and administrations
2.2.1HepA liver cancer model starts administration, every day 1 time in inoculating next day: (1) model group, gavages normal saline, every 0.2mL; (2) chemotherapy group: press 20mg/kgbw intraperitoneal injection of cyclophosphamide; (3) Rhizoma Paridis high dose group: gavage by 400mg/kgbw; (4) Rhizoma Paridis low dose group: gavage by 200mg/kgbw; (5) Rhizoma Curcumae Longae polysaccharide high dose group: gavage by 400mg/kgbw; (6) Rhizoma Curcumae Longae polysaccharide low dose group: gavage by 200mg/kgbw; (7) Radix Paeoniae Alba high dose group: gavage by 400mg/kgbw; (8) Radix Paeoniae Alba low dose group: gavage by 200mg/kgbw; (9) Chinese medicine composition high dose group: gavage by 400mg/kgbw; (10) Chinese medicine composition low dose group: gavage by 200mg/kgbw; Two weeks, finishes rear cervical region dislocation next day in administration and puts to death mice, dissects and gets its tumor tissue and weigh, and is calculated as follows tumour inhibiting rate; Win spleen, thymus is also weighed, and is calculated as follows spleen index and thymus index simultaneously.The results are shown in Table 2, table 3.
Tumour inhibiting rate (%)=(the average tumor weight of the average tumor weight/normal saline of 1-administration group group) × 100%
Spleen index=(spleen weight/body weight) * 1000
Thymus index=(thymic weight/body weight) * 1000
2.2.2H22 liver cancer model starts administration, every day 1 time in inoculating next day: (1) model group, gavages normal saline, every 0.2mL; (2) chemotherapy group: press 20mg/kgbw intraperitoneal injection of cyclophosphamide; (3) Rhizoma Paridis high dose group: gavage by 400mg/kgbw; (4) Rhizoma Paridis low dose group: gavage by 200mg/kgbw; (5) Rhizoma Curcumae Longae polysaccharide high dose group: gavage by 400mg/kgbw; (6) Rhizoma Curcumae Longae polysaccharide low dose group: gavage by 200mg/kgbw; (7) Radix Paeoniae Alba high dose group: gavage by 400mg/kgbw; (8) Radix Paeoniae Alba low dose group: gavage by 200mg/kgbw; (9) Chinese medicine composition high dose group: gavage by 400mg/kgbw; (10) Chinese medicine composition low dose group: gavage by 200mg/kgbw; Two weeks, finishes rear cervical region dislocation next day in administration and puts to death mice, dissects and gets its tumor tissue and weigh, and calculates tumour inhibiting rate by formula above; Win spleen, thymus is also weighed simultaneously, calculates spleen index and thymus index by formula above.The results are shown in Table 4, table 5.
2.2.3LA795 adenocarcinoma of lung metastasis model starts administration, every day 1 time in inoculating next day: (1) model group, gavages normal saline, every 0.2mL; (2) chemotherapy group: press 20mg/kgbw intraperitoneal injection of cyclophosphamide; (3) Rhizoma Paridis high dose group: gavage by 400mg/kgbw; (4) Rhizoma Paridis low dose group: gavage by 200mg/kgbw; (5) Rhizoma Curcumae Longae polysaccharide high dose group: gavage by 400mg/kgbw; (6) Rhizoma Curcumae Longae polysaccharide low dose group: gavage by 200mg/kgbw; (7) Radix Paeoniae Alba high dose group: gavage by 400mg/kgbw; (8) Radix Paeoniae Alba low dose group: gavage by 200mg/kgbw; (9) Chinese medicine composition high dose group: gavage by 400mg/kgbw; (10) Chinese medicine composition low dose group: gavage by 200mg/kgbw.Two weeks, finishes rear cervical region dislocation next day in administration and puts to death mice, dissects and gets its tumor tissue and weigh, and calculates tumour inhibiting rate by formula above; Win liver, spleen, thymus, kidney, the heart, brain, lung, stomach, ileum, colon, wherein liver, spleen is weighed respectively, calculates spleen index and thymus index by formula above.The tissue taking off is stored in 10% neutral formalin, and does paraffin section, carry out conventional H E dyeing, wherein tumor tissue also does TUNEL dyeing, observation of cell apoptosis.The results are shown in Table 6-table 8.
Two, anti-tumor activity test result
This Chinese medicine composition of table 1 Anticancer Activity in vitro result of the test
This Chinese medicine composition of table 2 is to HepA hepatocarcinoma tumor-bearing mice tumour inhibiting rate result of the test
This Chinese medicine composition of table 3 is to the comparison of HepA hepatocarcinoma tumor-bearing mice immune organ weight index
Note: compare * P < 0.05, * * P < 0.01 with model group
This Chinese medicine composition of table 4 is to H22 hepatocarcinoma tumor-bearing mice tumour inhibiting rate result of the test
This Chinese medicine composition of table 5 is to the comparison of H22 hepatocarcinoma tumor-bearing mice immune organ weight index
Note: compare * P < 0.05, * * P < 0.01 with model group
This Chinese medicine composition of table 6 shifts mice tumour inhibiting rate result of the test to LA795 adenocarcinoma of lung
This Chinese medicine composition of table 7 shifts the comparison of mouse immune organ weight index to LA795 adenocarcinoma of lung
Note: compare * P < 0.05, * * P < 0.01 with model group
Table 8 LA795 adenocarcinoma of lung shifts mice and respectively organizes HE dyeing and tumor tissue TUNEL coloration result
Three, conclusion
Through evidence, the Chinese medicine composition preparation method with anti-pulmonary carcinoma and hepatocarcinoma effect provided by the invention is simple, prove through external mtt assay activity test, this Chinese medicine composition can significantly suppress the growth of the hepatoma cell strains such as lung cancer cell line and Hep-B3 such as LA795; Adenocarcinoma of lung and the test of hepatocarcinoma bearing mouse model are shown, tumour inhibiting rate after compatibility significantly improves (more than 50%), mouse spleen index and liver index increase to some extent, and the lung that can obviously suppress mouse subcutaneous transplanting tumor shifts and cause the apoptosis of tumor cell, and without obvious toxic-side effects.Compared with independent use, the anti-pulmonary carcinoma of this Chinese medicine composition, liver cancer activity are higher and effect is clear and definite, and pulmonary carcinoma, hepatocarcinoma are had to good inhibitory action.
Claims (3)
1. a Chinese medicine composition with anti-pulmonary carcinoma and hepatocarcinoma effect, is characterized in that: the described Chinese medicine composition with anti-pulmonary carcinoma and hepatocarcinoma effect by Rhizoma Paridis extract, Rhizoma Curcumae Longae polysaccharide and Radix Paeoniae Alba extract with 1: the weight ratio of 0.1-5: 1-10 forms; Wherein Rhizoma Paridis extract is commercial goods, and specification is 5: 1,10: 1 or 20: 1;
The preparation method of described Rhizoma Curcumae Longae polysaccharide comprises the following step carrying out in order:
1) Rhizoma Curcumae Longae medical material or decoction pieces are ground into fritter, then join in dehydrated alcohol and soak 1 hour, the amount ratio of medical material and dehydrated alcohol is 1g: 9.5mL, then heating and refluxing extraction 1 hour at the temperature of 90 DEG C, discard extracting solution, retain reflux residue;
2) above-mentioned reflux residue is added to the water, the amount ratio of reflux residue and water is 1g: 9-10mL, and then heating and refluxing extraction 2 times at the temperature of 90 DEG C, each 2.5 hours, merges 2 times extracting solution;
3) said extracted liquid is joined in the alcoholic solution of 95% concentration to alcoholic degree and reach 75%, hold over night, then at 7500rmin
-1rotating speed under centrifugalize 20min, collecting precipitation thing lyophilization, use dehydrated alcohol, acetone, ether washing precipitation afterwards successively, obtains Rhizoma Curcumae Longae polysaccharide crude extract;
4) above-mentioned Rhizoma Curcumae Longae polysaccharide crude extract is made to sample solution with water dissolution, then adding consumption is that chloroform and the n-butanol mixed solvent of sample solution volume 1/4 extracts, violent jolting 20-30min, centrifugalize, in filtrate, add again chloroform and n-butanol mixed solvent the violent jolting of 1/4 filtrate volume, repeat aforesaid operations, until the denatured protein of water layer and solvent layer intersection no longer occurs, extract is evaporated to 1/4 of original volume;
5) extract after above-mentioned concentrating is placed in to bag filter flowing water dialysis 3 days, insoluble matter in elimination bag filter, then in clear liquor, add alcoholic solution to the alcoholic degree of 95% concentration to reach 85%, hold over night, by dehydrated alcohol and washing with acetone for precipitate, after being dried, can obtain the Rhizoma Curcumae Longae polysaccharide after refining;
The preparation method of described Radix Paeoniae Alba extract comprises the following step carrying out in order:
1) white Peony Root or decoction pieces are ground into coarse powder, then join in the alcoholic solution of 65% concentration and soak 1 hour, the ethanol amount ratio of the Radix Paeoniae Alba and 65% concentration is 1g: 8-10mL, then heating and refluxing extraction 3 times at the temperature of 95 DEG C, the 1st time 2 hours, 2nd, 3 times each 1 hour, merge 3 times extracting solution;
2) be evaporated to 1/2 of original volume after said extracted liquid is filtered, then add the water of 1.5 times of volumes, filter after being heated to leave standstill 12 hours in cool place place after micro-boiling, by precipitate evaporation drying and obtain Radix Paeoniae Alba extract at the temperature of 85 DEG C;
In the preparation method of above-mentioned Rhizoma Curcumae Longae polysaccharide, described step 2) and step 3) between also comprise one decolouring step, method is by step 2) to be evaporated to relative density at 25 DEG C after the extracting liquid filtering that obtains be 1.0, then adopt macroporous weakly basic anion exchange resin D301-G at 35 DEG C, under the condition of pH=5.5, decolour.
2. the Chinese medicine composition with anti-pulmonary carcinoma and hepatocarcinoma effect according to claim 1, it is characterized in that: in the preparation method of above-mentioned Rhizoma Curcumae Longae polysaccharide, described step 4) in chloroform and n-butanol mixed solvent the volume ratio of chloroform and n-butyl alcohol be 4.5: 1.
3. the Chinese medicine composition with anti-pulmonary carcinoma and hepatocarcinoma effect according to claim 1, is characterized in that: the amount ratio of described Rhizoma Paridis extract, Rhizoma Curcumae Longae polysaccharide and Radix Paeoniae Alba extract is 1: 2.5: 5.
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