CN103223144B - Traditional Chinese medicine composition for preventing liver cancer - Google Patents
Traditional Chinese medicine composition for preventing liver cancer Download PDFInfo
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Abstract
The invention discloses a traditional Chinese medicine composition for preventing liver cancer. The traditional Chinese medicine composition comprises ethanol-water co-decocted extract which is mixed by radix curcuma and white paeony root in a weight ratio of 1:(0.1-10). According to the tests, the traditional Chinese medicine composition disclosed by the invention can be used for remarkably restraining the growth of liver cancer cells including BEL-7402 and the like. According to a liver cancer mice model test, the anti-tumor rate is higher than 45% respectively, so that a mice spleen index and a thymus index can be improved; and according to a diethylnitrosamine-induced rat liver cancer test, the composition can be used for inhibiting the occurrence and development of the liver cancer more efficiently. Compared with the independently used material medicines, the traditional Chinese medicine composition has higher activity and specific effect, and has a good inhibition effect and a good defense function to the liver cancer and the occurrence of the liver cancer.
Description
Technical field
The invention belongs to technical field of Chinese medicines, be specifically related to a kind of Chinese medicine composition preventing and treating hepatocarcinoma.
Background technology
Rhizoma Curcumae Longae begins to be loaded in Tang Materia Medica, for the dry rhizome of zingiberaceous plant Rhizoma Curcumae Longae Curcuma longaL., 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.The pharmacological action of the effective ingredient such as curcumin is extensive, its antiinflammatory, antioxidation, atherosclerosis, antidepressant, antitumor etc. act on oneself and are commonly recognized, after wherein antitumor action proposed first from 1985, oneself has a large amount of experimentatioies to confirm, mechanism of action mainly comprises inducing apoptosis of tumour cell, inhibiting angiogenesis, has arresting cell cycle progression, reversing multiple medicine resistance of tumor cells etc. ten thousand.Huang Dongsheng etc. study discovery, and curcumin can strengthen the expression of caspase-8, thus start exogenous apoptosis pathway, induction human lung carcinoma cell apoptosis.Fang Yue etc. report that curcumin has the effect of the digestive system tumors such as anti-esophageal carcinoma, gastric cancer, hepatocarcinoma, colon cancer.Chinese patent CNl931353A discloses curcumin and extracts and pharmaceutical composition preparation method.
The Radix Paeoniae Alba is the peeling dry root of ranunculaceae plant Paeonia, cold nature, bitter in the mouth, acid.There is effect of nourishing blood to suppress the hyperactive liver, slow middle pain relieving, yin fluid astringing receipts antiperspirant.The active ingredient of the Radix Paeoniae Alba comprises peoniflorin, Hydroxy peoniflorin, peonin, lactone glucoside of Radix Paeoniae, benzoylpaeoniflorin etc., is referred to as Radix Paeoniae Alba total glucosides (total glucosides of peony, TGP).Research finds, it can affect the cellular immunization of autoimmune disease, humoral immunization and inflammatory process in multiple link, and the effect having analgesia, protect 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 chemotherapeutics attenuation synergistic etc.Poplar adds territory etc. and finds that Radix Paeoniae Alba alcohol extract Ultra filtration membrane active component has anti-tumor activity in vitro; Zhan Keshun etc. report that TGP combination chemotherapy nonsmall-cell lung cancer is better than chemotherapy, and can improve life in patients, improve Cellular Immunologic Function In Patients and raise peripheral hemogram.200510045840.0 disclose and a kind ofly have the peoniflorin of function of increasing leukocyte and the compositions of lactone glucoside of Radix Paeoniae, are suitable for preparing the medicine of the disease that leukopenia, platelet and hemoglobin that treatment a variety of causes causes reduce.Higher primary school Rongs etc. adopt the methods such as water extract-alcohol precipitation, membrance separation, the separation and purification of DEAE-cellulose chromatography to obtain Radix Paeoniae Alba polysaccharides BSP, all have good suppression ratio to Mice Bearing Lewis Lung Cancer and S180 sarcoma model.
Not yet find so far to have prevent and treat hepatocarcinoma and be seen in report by the Chinese medicine composition that extract forms of decocting altogether of Rhizoma Curcumae Longae and the Radix Paeoniae Alba.
Summary of the invention
The object of the present invention is to provide a kind of Chinese medicine composition preventing and treating hepatocarcinoma.
Technical scheme of the present invention is summarized as follows:
Prevent and treat a Chinese medicine composition for hepatocarcinoma, make by following method:
(1) ratio being 1:0.1 ~ 10 in quality takes raw material Rhizoma Curcumae Longae and the Radix Paeoniae Alba, and the volumetric concentration adding raw materials quality 7 ~ 9 times is the ethanol water of 60% ~ 90%, soaks 0.5 ~ 1.5 hour; heating and refluxing extraction; each extraction 1 ~ 3 hour, extracts 1 ~ 3 time, merge extractive liquid;
(2) extracting solution is through being evaporated to 1/10 ~ 1/8 of extracting liquid volume, dry, obtains a kind of Chinese medicine composition preventing and treating hepatocarcinoma.
Described step (1) is preferably: the ratio being 1:5 in quality takes raw material Rhizoma Curcumae Longae and the Radix Paeoniae Alba, and the volumetric concentration adding raw materials quality 8 times is the ethanol water of 65%, soaks 1 hour, heating and refluxing extraction, each extraction 2 hours, extracts 2 times, merge extractive liquid.
Described step (2) is preferably: extracting solution is through being evaporated to 1/9 of extracting liquid volume, dry, obtains a kind of Chinese medicine composition preventing and treating hepatocarcinoma.
Advantage of the present invention:
Prove through test, Chinese medicine composition of the present invention significantly can suppress the growth of the hepatoma cell strains such as BEL-7402; To hepatocarcinoma bearing mouse model test display, tumour inhibiting rate, respectively all more than 45%, improves mouse spleen index and thymus index; To Hepatoma Induced By Diethylnitrosamine test display, compositions more effectively can suppress generation and the development of hepatocarcinoma.With apply separately compared with crude drug, the higher and effect of the Chinese medicine composition activity of composition for preventing and controlling hepatocarcinoma of the present invention clearly, has good inhibitory action and defense function to the generation of hepatocarcinoma and hepatocarcinoma.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is further illustrated.
The following examples understand the present invention better to enable those skilled in the art to, but do not impose any restrictions the present invention.
Embodiment 1
Prevent and treat a Chinese medicine composition for hepatocarcinoma, make by following method:
(1) ratio being 1:5 in quality takes raw material Rhizoma Curcumae Longae and the Radix Paeoniae Alba, and the volumetric concentration adding raw materials quality 8 times is the ethanol water of 65%, soaks 1 hour, heating and refluxing extraction, and each extraction 2 hours, extracts 2 times, merge extractive liquid;
(2) extracting solution is through being evaporated to 1/9 of extracting liquid volume, dry, obtains a kind of Chinese medicine composition preventing and treating hepatocarcinoma.
Embodiment 2
Preventing and treating a Chinese medicine composition for hepatocarcinoma, is make by following method:
(1) ratio being 1:0.1 in quality takes raw material Rhizoma Curcumae Longae and the Radix Paeoniae Alba, and the volumetric concentration adding raw materials quality 7 times is the ethanol water of 90%, soaks 0.5 hour, heating and refluxing extraction, and each extraction 1 hour, extracts 1 time, merge extractive liquid;
(2) extracting solution is through being evaporated to 1/8 of extracting liquid volume, dry, obtains a kind of Chinese medicine composition preventing and treating hepatocarcinoma.
Embodiment 3
Preventing and treating a Chinese medicine composition for hepatocarcinoma, is make by following method:
(1) ratio being 1:5 in quality takes raw material Rhizoma Curcumae Longae and the Radix Paeoniae Alba, and the volumetric concentration adding raw materials quality 8 times is the ethanol water of 80%, soaks 1 hour, heating and refluxing extraction, and each extraction 2 hours, extracts 2 times, merge extractive liquid;
(2) extracting solution is through being evaporated to 1/9 of extracting liquid volume, dry, obtains a kind of Chinese medicine composition preventing and treating hepatocarcinoma.
Embodiment 4
Preventing and treating a Chinese medicine composition for hepatocarcinoma, is make by following method:
(1) ratio being 1:10 in quality takes raw material Rhizoma Curcumae Longae and the Radix Paeoniae Alba, and the volumetric concentration adding raw materials quality 9 times is the ethanol water of 60%, soaks 1.5 hours, heating and refluxing extraction, and each extraction 3 hours, extracts 3 times, merge extractive liquid;
(2) extracting solution is through being evaporated to 1/10 of extracting liquid volume, dry, obtains a kind of Chinese medicine composition preventing and treating hepatocarcinoma.
The test of pesticide effectiveness of Chinese medicine composition of the present invention is as follows:
1. anti tumor activity in vitro test
1.1 cell strains and cultivation
BEL-7402 hepatoma cell strain, is incubated at containing 10% inactivated fetal bovine serum, 100U/mL penicillin, in the RPMn640 culture medium of 100ug/mL streptomycin, cultivates under 37 ° of C, 5%CO2 incubators and saturated humidity condition, within 3-4 days, goes down to posterity once.
Hep-3B hepatoma cell strain, is incubated at containing 10% inactivated fetal bovine serum, 100U/mL penicillin, in the DMEM culture medium of 100 μ g/mL streptomycins, in 37 DEG C, cultivates under 5%CO2 incubator and saturated humidity condition, within 3 ~ 4 days, goes down to posterity once.
SMMC-7721 hepatoma cell strain, is incubated at containing 10% inactivated fetal bovine serum, 100U/mL penicillin, in the DMEM culture medium of 100 μ g/mL streptomycins, in 37 DEG C, cultivates under 5%CO2 incubator and saturated humidity condition, within 3 ~ 4 days, goes down to posterity once.
Antitumor Activity of Drugs measures
The preparation of test medicine: raw material Rhizoma Curcumae Longae, the Radix Paeoniae Alba are individually extracted by with embodiment 1 same procedure, obtains Rhizoma Curcumae Longae extract and Radix Paeoniae Alba extract.Pulverized respectively by a kind of Chinese medicine composition preventing and treating hepatocarcinoma that two kinds of extracts and each embodiment obtain, the mixing that adds water shakes up, and supersound process makes each extract dissolve all completely.
Mtt assay: be mixed with the cell suspension that concentration is 3 × 104/mL by after the trypsinization of exponential phase cell, is inoculated in 96 hole ELISA Plate, and every hole adds 200 μ L.Change after 24h and containing serum free culture system liquid, cell synchronization is grown, every hole 200 μ L.Within 3rd day, add by reagent in hole, namely add the independent solution of extract of Rhizoma Curcumae Longae (being called for short Rhizoma Curcumae Longae group) respectively, composition solution prepared by the independent solution of extract of the Radix Paeoniae Alba (being called for short Radix Paeoniae Alba group) and the present invention's 4 embodiments; Separately establish matched group: 0.1% dimethyl sulfoxide (DMSO), every hole adds 200 μ L.Often organize by reagent and establish 0.01 respectively, 0.1,1,10,100,1000 μ g/mL, six concentration, each concentration establishes 8 parallel holes, and cultivate 24h at 37 DEG C after, every hole adds serum-free without phenol red culture fluid freshly prepared 0.5mg/mLMTTl00 μ L, continue to cultivate 4h, then, every hole adds 100 μ LDMSO and dissolves MTT formazan granule, after microoscillator vibration mixing, in microplate reader, measure optical density value (OD), experiment in triplicate, is averaged.With solvent control process tumor cell for matched group, suppression ratio is calculated by OD value, formula is: inhibitory rate of cell growth=(matched group OD value-experimental group OD value)/matched group ODX100%, and be abscissa with drug level, OD value is vertical coordinate, draw cell growth curve, and try to achieve IC50 (half suppression ratio), IC50=inhibitory rate of cell growth is the drug level of 50%.The results are shown in Table 1.
Table 1 anti tumor activity in vitro result of the test
2. anti-tumor in vivo activity test
2.1HepA liver cancer model is tested
2.1.1 animal model
Kunming mice (female, 18-20g) 110 is divided into 11 groups at random, often organizes 10 (each group name claims to see 2.1.2), the pharmaceutical composition giving embodiment 1 preparation carries out the test of pesticide effectiveness in mice HepA liver cancer model body.Modeling method is as follows:
Get the tumor-bearing mice that ascites after inoculation HepA hepatoma carcinoma cell is full, sterilization abdominal part, in superclean bench, extract milky ascites is tumor source, brine, ascites 5.5mL, the ratio of ascites and normal saline is 1:1, normal saline 5.5mL, be made into cell suspension, extract 0.lmL with asepsis injector, be inoculated in 110 mice oxters respectively.Whole operation is aseptically carried out.
2.1.2 animal grouping and administration
Inoculate and start administration, every day 1 time next day: (1) model group, gavages normal saline, every 0.2mL; (2) chemotherapy group: by 20mg/kgbw intraperitoneal injection of cyclophosphamide; (3) Rhizoma Curcumae Longae high dose group: gavage by 12.5g/kgbw; (4) dosage group in Rhizoma Curcumae Longae: gavage by 2.5g/kgbw; (5) Rhizoma Curcumae Longae low dose group: gavage by 0.5g/kgbw; (6) Radix Paeoniae Alba high dose group: gavage by 12.5g/kgbw; (7) dosage group in the Radix Paeoniae Alba: gavage by 2.5g/kgbw; (8) Radix Paeoniae Alba low dose group: gavage by 0.5g/kgbw; (9) pharmaceutical composition (embodiment 1) high dose: gavage by 12.5g/kgbw; (10) dosage group in pharmaceutical composition (embodiment 1): gavage (11) pharmaceutical composition (embodiment 1) low dose group by 2.5g/kgbw: gavage by 0.5g/kgbw.Each group of successive administration two weeks, terminate rear next day cervical dislocation in administration and put to death mice, solution takes its tumor tissue, wins liver, spleen, and thymus is also weighed.
Survey tumour inhibiting rate: (i) tumour inhibiting rate (%)=(the average tumor weight of 1-administration group average tumor weight/normal saline group) × 100%, pluck liver, spleen, thymus is also weighed.Liver refers to=(liver weight/body weight) * 1000, and spleen refers to=(spleen weight/body weight) * 1000, thymus index=(thymic weight/body weight) * 1000.The results are shown in Table 2 and table 3.
Table 2 compositions is to HepA hepatocarcinoma tumor-bearing mice tumour inhibiting rate result of the test
Table 3 compositions compares HepA hepatocarcinoma tumor-bearing mice immune organ weight index
Note: compare with model group that there were significant differences, * P < 0.05
Experimental result shows, the compositions of embodiment 2, embodiment 3 and embodiment 4 is on HepA hepatocarcinoma tumor-bearing mice tumour inhibiting rate, similar to embodiment 1 on the impact of HepA hepatocarcinoma tumor-bearing mice immune organ.
2.2H22 liver cancer model is tested
2.2.1 animal grouping and administration
Administration in advance is respectively organized 1 week, every day 1 time: (1) model group, gavages normal saline, every 0.2mL with kunming mice (female, 18-20g) 110 (be divided into 11 groups at random, often organize 10, each group name claims to see 2.1.2); (2) chemotherapy group: by 20mg/kgbw intraperitoneal injection of cyclophosphamide; (3) Rhizoma Curcumae Longae high dose group: gavage by 12.5g/kgbw; (4) dosage group in Rhizoma Curcumae Longae: gavage by 2.5g/kgbw; (5) Rhizoma Curcumae Longae low dose group: gavage by 0.5g/kgbw; (6) Radix Paeoniae Alba high dose group: gavage by 12.5g/kgbw; (7) dosage group in the Radix Paeoniae Alba: gavage by 2.5g/kgbw; (8) Radix Paeoniae Alba low dose group: gavage by 0.5g/kgbw; (9) pharmaceutical composition (embodiment 1) high dose: gavage by 12.5g/kgbw; (10) dosage group in pharmaceutical composition (embodiment 1): gavage (11) pharmaceutical composition (embodiment 1) low dose group by 2.5g/kgbw: by 0.5g/kgbw
2.2.2 animal model
Get the tumor-bearing mice that ascites after inoculation H22 hepatoma carcinoma cell is full, sterilization abdominal part, in superclean bench, extract milky ascites is tumor source, brine, ascites 5.5mL, the ratio of ascites and normal saline is 1:1, normal saline 5.5mL, be made into cell suspension, extract 0.lmL with asepsis injector, be inoculated in 110 the mice oxters giving medicine in advance respectively.Whole operation is aseptically carried out.
2.2.3 administration again after modeling
After modeling, each group is continued to gavage the medicine identical with described in 2.2.1, continuous two weeks, and terminate rear cervical dislocation execution next day mice in last administration, solution takes its tumor tissue, wins liver, spleen, and thymus is also weighed.
Survey tumour inhibiting rate: (i) tumour inhibiting rate (%)=(the average tumor weight of 1-administration group average tumor weight/normal saline group) × 100%, pluck liver, spleen, thymus is also weighed.Liver refers to=(liver weight/body weight) * 1000, and spleen refers to=(spleen weight/body weight) * 1000, and thymus index=(thymic weight/body weight) * 1000, the results are shown in Table 4 and table 5.
Table 4 compositions is to H22 hepatocarcinoma tumor-bearing mice tumour inhibiting rate result of the test
Table 5 compositions compares H22 hepatocarcinoma tumor-bearing mice immune organ weight index
Note: compare with model group that there were significant differences, * P < 0.05
Experimental result shows, the compositions of embodiment 2, embodiment 3 and embodiment 4 is on H22 hepatocarcinoma tumor-bearing mice tumour inhibiting rate, similar to embodiment 1 on the impact of H22 hepatocarcinoma tumor-bearing mice immune organ.
2.3 Hepatoma Induced By Diethylnitrosamine experiments
2.3.1 animal grouping and administration
120 rats are divided into 11 groups at random, and except B group 20, all the other respectively organize 10, deliver medicine to each group of rat respectively according to agents and dosage, continuous 5 weeks: (1) normal group (A group): freely drink water, eat food; (2) model group (B group): freely drink water, eat food; (3) Rhizoma Curcumae Longae high dose group (C group): gavage by 12.5g/kgbw; (4) dosage group (D group) in Rhizoma Curcumae Longae: gavage by 2.59/kgbw; (5) Rhizoma Curcumae Longae low dose group (E group): gavage by 0.5g/kgbw; (6) Radix Paeoniae Alba high dose group (F group): gavage by 12.5g/kgbw; (7) dosage group (G group) in the Radix Paeoniae Alba: gavage by 2.5g/kgbw; (8) Radix Paeoniae Alba low dose group (H group): gavage by 0.5g/kgbw; (9) pharmaceutical composition (prepared by embodiment 1) high dose group (I group): gavage by 12.5g/kgbw; (10) dosage group (J group) in pharmaceutical composition (prepared by embodiment 1): gavage by 2.5g/kgbw.(11) pharmaceutical composition (prepared by embodiment 1) low dose group (K group): gavage by 0.59/kgbw.
2.3.2 modeling and again administration
Administration, after 5 weeks, starts modeling.A treated animal is freely drunk water, is eaten food, and diethylnitrosamine (DEN) is mixed with 1% aqueous solution by B treated animal, weekly every Mus timing gavage, and weekly dose is pressed 70mg/kg and calculated, continuous gavage 15 weeks.C to H treated animal, except giving except DEN by above-mentioned dosage method, continues each group of Chinese medicine given described in 2.3.1, continuous gavage 15 weeks simultaneously.
2.3.3 detection method and index
Above-mentioned each treated animal was put to death the 15th weekend, get blood separation of serum and the liver function indexs such as automatic biochemistry analyzer detection alanine aminotransferase (ALT), aspartate amino transferase (AST), gamma glutamyl transpeptidase (γ-GT), hepatic tissue fixes 24h in 10% formalin.Row dehydration after fixing, transparent, waxdip, routine paraffin wax embedding, preparation section, adopts HE staining in the histological change of light Microscopic observation liver.The results are shown in Table 6 and table 7.
Table 6 is group Liver Function Testing index results contrast respectively
Note: compare with model group that there were significant differences,
Δp < 0.05,
Δ Δp < 0.01
Table 7 respectively group liver tissues of rats pathological examination compares
Test compositions prepared by embodiment 2, embodiment 3 and embodiment 4 by method identical above, experimental result shows, embodiment 2, embodiment 3 also have above-mentioned similar effect with the compositions of embodiment 4.
3. conclusion
The Chinese medicine composition preparation method that anti-hepatocarcinoma provided by the invention occurs is simple, and prove through external MTT test, this pharmaceutical composition significantly can suppress the hepatoma cell strain growths such as BEL-7402; Hepatocarcinoma bearing mouse model test display, after compatibility, tumour inhibiting rate significantly improves (more than 45%), and mouse spleen index and thymus index also increase to some extent; To Hepatoma Induced By Diethylnitrosamine test display, compositions more effectively can suppress generation and the development of hepatocarcinoma.With apply separately compared with crude drug, the higher and effect of this pharmaceutical composition activity clearly, has good inhibitory action and defense function to the treatment of hepatocarcinoma and the generation that prevents liver cancer.
Claims (3)
1. prevent and treat a Chinese medicine composition for hepatocarcinoma, it is characterized in that making by following method:
(1) ratio being 1:0.1 ~ 10 in quality takes raw material Rhizoma Curcumae Longae and the Radix Paeoniae Alba, and the volumetric concentration adding raw materials quality 7 ~ 9 times is the ethanol water of 60% ~ 90%, soaks 0.5 ~ 1.5 hour; heating and refluxing extraction; each extraction 1 ~ 3 hour, extracts 1 ~ 3 time, merge extractive liquid;
(2) extracting solution is through being evaporated to 1/10 ~ 1/8 of extracting liquid volume, dry, obtains a kind of Chinese medicine composition preventing and treating hepatocarcinoma.
2. a kind of Chinese medicine composition preventing and treating hepatocarcinoma according to claim 1, it is characterized in that described step (1) is: the ratio being 1:5 in quality takes raw material Rhizoma Curcumae Longae and the Radix Paeoniae Alba, the volumetric concentration adding raw materials quality 8 times is the ethanol water of 65%, soak 1 hour, heating and refluxing extraction, each extraction 2 hours, extracts 2 times, merge extractive liquid.
3. a kind of Chinese medicine composition preventing and treating hepatocarcinoma according to claim 1, is characterized in that described step (2) is: extracting solution is through being evaporated to 1/9 of extracting liquid volume, dry, obtains a kind of Chinese medicine composition preventing and treating hepatocarcinoma.
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| 姜黄素抗消化系肿瘤作用研究;方悦等;《浙江中西医结合杂志》;20121231;第22卷(第8期);651-652 * |
| 白芍总苷对HepG2细胞增殖的抑制作用;王世宏等;《安徽医科大学学报》;20061031;第41卷(第5期);547-549 * |
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