CN103169844A - 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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- CN103169844A CN103169844A CN2013100793051A CN201310079305A CN103169844A CN 103169844 A CN103169844 A CN 103169844A CN 2013100793051 A CN2013100793051 A CN 2013100793051A CN 201310079305 A CN201310079305 A CN 201310079305A CN 103169844 A CN103169844 A CN 103169844A
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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, has heat-clearing and toxic substances removing, the 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 physiologically actives such as respiratory system and Cardiovascular Systems, 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 that the multiple recovery oral liquid of gold, softening the hard mass oral liquid, building lotus capsule, GANFULE PIAN etc. are arranged.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): disclose cell toxicant spectrum and the structure activity study of Rhizoma Paridis saponin to 10 kinds of tumor cell lines in 2057-2060; 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 Rhizoma Paridis total saponins to human lung cancer cell A549's inhibited proliferation 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 finds that Rhizoma Paridis and oxaliplatin may have the effect of certain angiogenesis inhibitor in experiment in external, body, and both use in conjunction may have synergistic function.The discovery Rhizoma Paridis total saponins such as Jia Ke can significantly suppress the growth of MGC-803 cell and meta-dose-dependence when being, and can block cell in the S phase, the rising of cell death inducing rate; Can significantly suppress EphA2, survivin protein expression, raise simultaneously the Caspase-3 protein expression, and then its mechanism of action of reaching a conclusion may and promote that with the expression of lowering EphA2 and survivin the expression of Caspase-3 is relevant.Disclose a kind of preparation method of Rhizoma Paridis total saponins in No. the 200610052223.8th, Chinese patent, also disclose a kind of pharmaceutical preparation take Rhizoma Paridis total saponins as effective ingredient and preparation method thereof in No. 200810052092.2.
The Rhizoma Curcumae Longae beginning is stated from Tang Materia Medica, be the dry rhizome of zingiberaceous plant Rhizoma Curcumae Longae Curcuma longa L., its nature and flavor acrid, bitter, warm is returned spleen, Liver Channel, has the removing blood stasis circulation of qi promoting, the 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 the 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 in 1985 first, existing a large amount of experimentatioies confirms, mechanism of action mainly comprises inducing apoptosis of tumour cell, suppress angiogenesis, the retardance cell cycle progression is arranged, 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, induce the 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 the 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 the 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).The research discovery, it can affect in a plurality of links cellular immunization, humoral immunization and the inflammatory process of autoimmune disease and the effect that analgesia is arranged, protect the liver.Peoniflorin is as accounting for the above compound of TGP90%, and discovered in recent years also has anti-tumor activity.Its mechanism of action except the immunity of enhancing body, also have the 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 such as territory and separates active component and have an anti-tumor activity external; 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.Disclose a kind of compositions with peoniflorin and lactone glucoside of Radix Paeoniae of function of increasing leukocyte in No. the 200510045840.0th, Chinese patent, be applicable to prepare the medicine of the diseases such as leukopenia, platelet and blood red globulin reduction that 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 suppression ratio preferably.
At present, the patent of the Chinese medicine composition of Rhizoma Paridis saponin and astragalus polysaccharides (CN101152408A) has been seen in report, and the Chinese medicine composition of not yet finding to have simultaneously anti-pulmonary carcinoma and hepatocarcinoma effect and being formed by Rhizoma Paridis extract, Rhizoma Curcumae Longae polysaccharide and Radix Paeoniae Alba extract.
Summary of the invention
In order to address the above problem, the object of the present invention is to provide a kind of activity stronger 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 the 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 that carries out in order:
1) Rhizoma Curcumae Longae medical material or decoction pieces are ground into fritter, then join in dehydrated alcohol and to 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 ℃, discard extracting solution, reservation 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 ℃, each 2.5 hours, merge 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 is then at 7500rmin
-1Rotating speed under centrifugalize 20min, dehydrated alcohol, acetone, ether washing precipitation are used in collecting precipitation thing and lyophilization afterwards successively, namely get Rhizoma Curcumae Longae polysaccharide crude extract;
4) above-mentioned Rhizoma Curcumae Longae polysaccharide crude extract is made 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, the chloroform and n-butanol mixed solvent and the violent jolting that add again 1/4 filtrate volume in filtrate, 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 bag filter flowing water dialysis 3 days, insoluble matter in the elimination bag filter, then add alcoholic solution to the alcoholic degree of 95% concentration to reach 85% in clear liquor, hold over night, with precipitate dehydrated alcohol and washing with acetone, the Rhizoma Curcumae Longae polysaccharide after can being made with extra care after drying;
The preparation method of described Radix Paeoniae Alba extract comprises the following step that carries 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 soaked 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 ℃, 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, be heated to filter after standing 12 hours in the place, cool place after little boiling, with precipitate evaporation drying and obtain Radix Paeoniae Alba extract at the temperature of 85 ℃.
In the preparation method of above-mentioned Rhizoma Curcumae Longae polysaccharide, also comprise a decolouring step between described step 2} and step 3}, method is with step 2) to be evaporated to 25 ℃ of lower relative densities after the extracting liquid filtering that obtains be 1.0, then adopt macroporous weakly basic anion exchange resin D301-G at 35 ℃, decolour under the condition of pH=5.5.
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.
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 the lung cancer cell lines such as LA795 and Hep-B3; Adenocarcinoma of lung and the test of hepatocarcinoma bearing mouse model are shown, tumour inhibiting rate improves mouse spleen index and liver index respectively all more than 50%, and the activity of anti-pulmonary carcinoma and hepatocarcinoma is higher and effect is clear and definite, and pulmonary carcinoma, hepatocarcinoma are had good inhibitory action.In addition, described Chinese medicine composition preparation process is simple, and can be prepared into various different dosage forms.
The specific embodiment
Embodiment one:
(1) getting 1g, to cross specification after 80 mesh sieves be commercially available Rhizoma Paridis extract (the bright biological development corporation, Ltd. in the Baoji side produces) powder of 10: 1.
(2) preparation of Rhizoma Curcumae Longae polysaccharide
1) 300g Rhizoma Curcumae Longae pulverizing medicinal materials is become fritter, then join in dehydrated alcohol and to 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 ℃, discard extracting solution, reservation 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 ℃, each 2.5 hours, merge 2 times extracting solution;
3) be 1.0 with being evaporated to 25 ℃ of lower relative densities after extracting liquid filtering obtained above, then adopt macroporous weakly basic anion exchange resin D301-G at 35 ℃, decolour under the condition of pH=5.5;
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 is then at 7500rmin
-1Rotating speed under centrifugalize 20min, dehydrated alcohol, acetone, ether washing precipitation are used in collecting precipitation thing and lyophilization afterwards successively, namely get Rhizoma Curcumae Longae polysaccharide crude extract;
5) above-mentioned Rhizoma Curcumae Longae polysaccharide crude extract is made 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, the chloroform and n-butanol mixed solvent and the violent jolting that add again 1/4 filtrate volume in filtrate, 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 bag filter flowing water dialysis 3 days, insoluble matter in the elimination bag filter, then add alcoholic solution to the alcoholic degree of 95% concentration to reach 85% in clear liquor, hold over night, with precipitate dehydrated alcohol and washing with acetone, the Rhizoma Curcumae Longae polysaccharide 20.1g after can being made with extra care after drying.
(3) preparation of Radix Paeoniae Alba extract
1) the 100g white Peony Root is ground into coarse powder, then join in the alcoholic solution of 65% concentration and soaked 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 ℃, 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, be heated to filter after standing 12 hours in the place, cool place after little boiling, with precipitate evaporation drying and obtain Radix Paeoniae Alba extract 13.5g at the temperature of 85 ℃.
(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 namely can be made into the Chinese medicine composition with anti-pulmonary carcinoma and hepatocarcinoma effect provided by the invention.
Embodiment two:
(1) getting 10g, to cross specification after 80 mesh sieves be the commercially available Rhizoma Paridis extract powder of 5: 1.
(2) preparation of Rhizoma Curcumae Longae polysaccharide
1) 300g Rhizoma Curcumae Longae pulverizing medicinal materials is become fritter, then join in dehydrated alcohol and to 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 ℃, discard extracting solution, reservation 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 ℃, each 2.5 hours, merge 2 times extracting solution;
3) be 1.0 with being evaporated to 25 ℃ of lower relative densities after extracting liquid filtering obtained above, then adopt macroporous weakly basic anion exchange resin D301-G at 35 ℃, decolour under the condition of pH=5.5;
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 is then at 7500rmin
-1Rotating speed under centrifugalize 20min, dehydrated alcohol, acetone, ether washing precipitation are used in collecting precipitation thing and lyophilization afterwards successively, namely get Rhizoma Curcumae Longae polysaccharide crude extract;
5) above-mentioned Rhizoma Curcumae Longae polysaccharide crude extract is made 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, the chloroform and n-butanol mixed solvent and the violent jolting that add again 1/4 filtrate volume in filtrate, 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 bag filter flowing water dialysis 3 days, insoluble matter in the elimination bag filter, then add alcoholic solution to the alcoholic degree of 95% concentration to reach 85% in clear liquor, hold over night, with precipitate dehydrated alcohol and washing with acetone, the Rhizoma Curcumae Longae polysaccharide 20.6g after can being made with extra care after drying.
(3) preparation of Radix Paeoniae Alba extract
1) the 100g white Peony Root is ground into coarse powder, then join in the alcoholic solution of 65% concentration and soaked 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 ℃, 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, be heated to filter after standing 12 hours in the place, cool place after little boiling, with precipitate evaporation drying and obtain Radix Paeoniae Alba extract 13.5g at the temperature of 85 ℃.
(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 namely can be made into the Chinese medicine composition with anti-pulmonary carcinoma and hepatocarcinoma effect provided by the invention.
Embodiment three:
(1) getting 10g, to cross specification after 80 mesh sieves be the commercially available Rhizoma Paridis extract powder of 20: 1.
(2) preparation of Rhizoma Curcumae Longae polysaccharide
1) 300g Rhizoma Curcumae Longae pulverizing medicinal materials is become fritter, then join in dehydrated alcohol and to 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 ℃, discard extracting solution, reservation 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 ℃, each 2.5 hours, merge 2 times extracting solution;
3) be 1.0 with being evaporated to 25 ℃ of lower relative densities after extracting liquid filtering obtained above, then adopt macroporous weakly basic anion exchange resin D301-G at 35 ℃, decolour under the condition of pH=5.5;
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 is then at 7500rmin
-1Rotating speed under centrifugalize 20min, dehydrated alcohol, acetone, ether washing precipitation are used in collecting precipitation thing and lyophilization afterwards successively, namely get Rhizoma Curcumae Longae polysaccharide crude extract;
5) above-mentioned Rhizoma Curcumae Longae polysaccharide crude extract is made 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, the chloroform and n-butanol mixed solvent and the violent jolting that add again 1/4 filtrate volume in filtrate, repeat aforesaid operations, until the denatured protein of water layer and solvent layer intersection no longer occurs, with the extract concentrating under reduced pressure;
6) extract after above-mentioned concentrating is placed in bag filter flowing water dialysis 3 days, insoluble matter in the elimination bag filter, then add alcoholic solution to the alcoholic degree of 95% concentration to reach 85% in clear liquor, hold over night, with precipitate dehydrated alcohol and washing with acetone, the Rhizoma Curcumae Longae polysaccharide 20.6g after can being made with extra care after drying.
(3) extracting method of described Radix Paeoniae Alba extract comprises the following step that carries out in order:
1) will be ground into the white Peony Root 100g of coarse powder, adopt (W/W) 65% ethanol of 1L Radix Paeoniae Alba coarse powder dry weight, soak after 1 hour in 95 ℃ of lower reflux, extract, of temperature 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) extracting liquid filtering by being evaporated to 1/2 of original volume, adds the water of 1.5 times of volumes, is heated to filter after standing 12 hours in the place, cool place after little boiling, and precipitate obtains Radix Paeoniae Alba extract 85 ℃ of lower evaporation dryings.
(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 namely 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 with Chinese medicine composition of 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 strain and cultivation
The mouse pulmonary adenocarcinoma cell line LA 795 strain is incubated in the RPMn640 culture medium that contains 10% inactivated fetal bovine serum, 100U/mL penicillin and 100 μ g/mL streptomycins, in 37 ℃, and 5%CO
2Cultivate under incubator and saturated humidity condition, went down to posterity once in 3-4 days.
Human lung adenocarcinoma A549 cell strain is incubated in the DMEM culture medium that contains 10% inactivated fetal bovine serum, 100U/mL penicillin and 100 μ g/mL streptomycins, in 37 ℃, and 5%CO
2Cultivate under incubator and saturated humidity condition, went down to posterity once in 3～4 days.
The BEL-7402 hepatoma cell strain is incubated in the RPMn640 culture medium that contains 10% inactivated fetal bovine serum, 100U/mL penicillin and 100 μ g/mL streptomycins, in 37 ℃, and 5%CO
2Cultivate under incubator and saturated humidity condition, went down to posterity once in 3-4 days.
The Hep-B3 hepatoma cell strain is incubated in the DMEM culture medium that contains 10% inactivated fetal bovine serum, 100U/mL penicillin and 100 μ g/mL streptomycins, in 37 ℃, and 5%CO
2Cultivate under incubator and saturated humidity condition, went down to posterity once in 3～4 days.
1.2 Antitumor Activity of Drugs is measured
Mtt assay: it is 3 * 10 that the exponential phase cell is mixed with concentration after with trypsinization
4The cell suspension of individual/mL is inoculated in 96 hole ELISA Plate, and every hole adds 200 μ L.Change after 24h and do not contain serum free culture system liquid, make the cell synchronization growth, every hole 200 μ L.Added in the hole in the 3rd day to be subjected to reagent, namely add respectively the 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 add 200 μ L.Be subjected 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, and after cultivating 24h under 37 ℃, 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 dissolving MTT formazan granules, after microoscillator vibration mixing, measure optical density value (OD) on microplate reader, the experiment triplicate is averaged.Process tumor cell as matched group take solvent control, calculate suppression ratio by the OD value, formula is: inhibitory rate of cell growth=(matched group OD value one experimental group OD value)/matched group OD * 100%, and take drug level as abscissa, the OD value is vertical coordinate, draws cell growth curve, and tries 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
(female, the Chinese medicine composition that 18-20g) gives above-described embodiment preparation carries out the test of pesticide effectiveness in mice LA795 adenocarcinoma of lung metastasis model body with 100 T739 mices respectively.
(female, the Chinese medicine composition that 18-20g) gives above-described embodiment preparation carries out the test of pesticide effectiveness in mice HepA liver cancer model and H22 liver cancer model body with 100 kunming mices respectively.
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, wash abdominal part to carry out disinfection with normal saline, extract 5mL milky ascites as the tumor source in superclean bench, then add the 5mL normal saline and be mixed with the cell suspension of 1: 1 ratio, extract 0.1mL and 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, wash abdominal part to carry out disinfection with normal saline, extract 5mL milky ascites as the tumor source in superclean bench, then add the 5mL normal saline and be mixed with the cell suspension of 1: 1 ratio, extract 0.1mL and be inoculated in a mice oxter with asepsis injector, inoculate altogether 100.Whole operating under aseptic condition carried out.
2.1.3LA795 the adenocarcinoma of lung metastasis model is got 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, peel off tumor under aseptic condition, select fresh tumor tissue without necrosis to shred and be placed in the glass grinding device, adding appropriate normal saline grinds gently and is prepared into tumor cell suspension, after filtering, the 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 administration
2.2.1HepA liver cancer model begins administration, every day 1 time in inoculating next day: (1) model group gavages normal saline, every 0.2mL; (2) chemotherapy group: press the 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, next day, mice was put to death in the cervical region dislocation after administration finishes, and dissected and got its tumor tissue and weigh, and was calculated as follows tumour inhibiting rate; Win simultaneously spleen, thymus is also weighed, and is calculated as follows spleen index and thymus index.The results are shown in Table 2, table 3.
Tumour inhibiting rate (%)=(the average tumor of the average tumor weight/normal saline of 1-administration group group is heavy) * 100%
Spleen index=(spleen weight/body weight) * 1000
Thymus index=(thymic weight/body weight) * 1000
2.2.2H22 liver cancer model begins administration, every day 1 time in inoculating next day: (1) model group gavages normal saline, every 0.2mL; (2) chemotherapy group: press the 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, next day, mice was put to death in the cervical region dislocation after administration finishes, and dissected and got its tumor tissue and weigh, and calculated tumour inhibiting rate by top formula; Win simultaneously spleen, thymus is also weighed, and calculates spleen index and thymus index by top formula.The results are shown in Table 4, table 5.
2.2.3LA795 the adenocarcinoma of lung metastasis model begins administration, every day 1 time in inoculating next day: (1) model group gavages normal saline, every 0.2mL; (2) chemotherapy group: press the 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, next day, mice was put to death in the cervical region dislocation after administration finishes, and dissected and got its tumor tissue and weigh, and calculated tumour inhibiting rate by top formula; Win liver, spleen, thymus, kidney, the heart, brain, lung, stomach, ileum, colon, liver wherein, spleen is weighed respectively, calculates spleen index and thymus index by top formula.The tissue that takes off is stored in 10% neutral formalin, and does paraffin section, carry out conventional H E dyeing, wherein tumor tissue is also done 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 to HepA hepatocarcinoma tumor-bearing mice immune organ weight index relatively
Annotate: 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 to H22 hepatocarcinoma tumor-bearing mice immune organ weight index relatively
Annotate: 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 the LA795 adenocarcinoma of lung
This Chinese medicine composition of table 7 shifts mouse immune organ weight index relatively to the LA795 adenocarcinoma of lung
Annotate: 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, 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 the lung cancer cell lines such as LA795 and Hep-B3; 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 the mouse subcutaneous transplanting tumor shifts and cause the apoptosis of tumor cell, and without obvious toxic-side effects.Compare with independent use, the anti-pulmonary carcinoma of this Chinese medicine composition, liver cancer activity is higher and the effect is clear and definite, and pulmonary carcinoma, hepatocarcinoma are had good inhibitory action.
Claims (3)
1. Chinese medicine composition with anti-pulmonary carcinoma and hepatocarcinoma effect is characterized in that: 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 the 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 that carries out in order:
1) Rhizoma Curcumae Longae medical material or decoction pieces are ground into fritter, then join in dehydrated alcohol and to 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 ℃, discard extracting solution, reservation 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 ℃, each 2.5 hours, merge 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 is then at 7500rmin
-1Rotating speed under centrifugalize 20min, dehydrated alcohol, acetone, ether washing precipitation are used in collecting precipitation thing and lyophilization afterwards successively, namely get Rhizoma Curcumae Longae polysaccharide crude extract;
4) above-mentioned Rhizoma Curcumae Longae polysaccharide crude extract is made 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, the chloroform and n-butanol mixed solvent and the violent jolting that add again 1/4 filtrate volume in filtrate, 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 bag filter flowing water dialysis 3 days, insoluble matter in the elimination bag filter, then add alcoholic solution to the alcoholic degree of 95% concentration to reach 85% in clear liquor, hold over night, with precipitate dehydrated alcohol and washing with acetone, the Rhizoma Curcumae Longae polysaccharide after can being made with extra care after drying;
The preparation method of described Radix Paeoniae Alba extract comprises the following step that carries 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 soaked 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 ℃, 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, be heated to filter after standing 12 hours in the place, cool place after little boiling, with precipitate evaporation drying and obtain Radix Paeoniae Alba extract at the temperature of 85 ℃.
In the preparation method of above-mentioned Rhizoma Curcumae Longae polysaccharide, described step 2) and step 3) between also comprise one the decolouring step, method is with step 2) to be evaporated to 25 ℃ of lower relative densities after the extracting liquid filtering that obtains be 1.0, then adopt macroporous weakly basic anion exchange resin D301-G at 35 ℃, decolour under the condition of pH=5.5.
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, it 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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| CN103933472A (en) * | 2014-04-04 | 2014-07-23 | 天津大学 | Traditional Chinese medicine composition for resisting liver cancer and lung cancer |
| CN104623215A (en) * | 2015-01-30 | 2015-05-20 | 天津大学 | Anti-tumor medicine composition |
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| CN101400359A (en) * | 2006-03-17 | 2009-04-01 | 草药科学新加坡私人有限公司 | Extracts and methods comprising curcuma species |
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| CN101400359A (en) * | 2006-03-17 | 2009-04-01 | 草药科学新加坡私人有限公司 | Extracts and methods comprising curcuma species |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103933472A (en) * | 2014-04-04 | 2014-07-23 | 天津大学 | Traditional Chinese medicine composition for resisting liver cancer and lung cancer |
| CN103933472B (en) * | 2014-04-04 | 2016-10-05 | 天津大学 | A kind of Traditional Chinese medicinal composition for treating lung cancer and liver cancer |
| CN104623215A (en) * | 2015-01-30 | 2015-05-20 | 天津大学 | Anti-tumor medicine composition |
| CN104623215B (en) * | 2015-01-30 | 2018-05-04 | 天津大学 | A kind of antitumor medicine composition |
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