CN100478009C - Chinese medicine for anti-tumor and its preparing method - Google Patents
Chinese medicine for anti-tumor and its preparing method Download PDFInfo
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Abstract
The invention relates to a Chinese medicine anticancer medication for anti liver and lung cancer and preparation thereof. Said medication is prepared comprising ginseng , toad skin, whole scorpion and pore fungi as medicinal materials, whereof part by weight is , ginseng 10.8-16.2%,toad skin 16.2-27.0%,whole scorpion 16.2-27.0%,pore fungi 32.4-54.0%. Process for said inventive medication is as follows:toad skin and whole scorpion having been through over 95% alcohol extraction, extracting solution thereof being collected and ready for use, dregs thereof being combined with other components and through water extraction , combining water extraction solution with alcohol one, distilling, spraying and drying, making into pellet, packing into capsule and to obtain said medication.
Description
One, technical field
The present invention relates to a kind of Chinese medicine anti-tumor agent with anti-hepatocarcinoma and anti-pulmonary carcinoma---flower toad capsule and preparation method thereof.
Two, background technology
According to the WHO statistics, in the whole world more than 50 hundred million populations, to die from malignant tumor person and reach 7,600,000 people in 2005, new cases are 1,090 ten thousand examples, now the trouble number is 2,460 ten thousand, and numeral is also increasing year by year.From domestic situation, malignant tumor constituent ratio in the cause of the death rises to 17.9% by 12.6% in the period of nearly 20, and the number of the infected of the annual cancer of China is 2,000,000, and the cancer mortality number surpasses 1,500,000.In the conference of the 18th world anticancer disease alliance, the research report that World Health Organization (WHO) delivers shows, whole world cancer condition will be serious day by day, new patient's number will be increased to 1,500 ten thousand by present every year 1000 ten thousand in 15 years from now on, now suffer from routine number and will increase to 3,000 ten thousand, the number dead because of cancer also will increase to 1,000 ten thousand by annual more than 700 ten thousand.Therefore, national governments, research institution and drugmaker are paid much attention to tumor research and antitumor drug research for a long time always.
Generally acknowledge that in the world curative effect antitumor drug commonly used preferably has 50～60 kinds approximately, WHO announces that antineoplastic agent commonly used is 49 kinds, and China can produce wherein about 40 kinds, reaches more than 80%.The present market situation of antitumor medication is the gesture of " standing like the legs of a tripod " substantially: antineoplastic agent accounts for 45%, and immunostimulant and immunosuppressant respectively account for about 26%, and the endocrine therapy medicine market share is then very little.There are 4 kinds to be plant alkaloid and other crude drug in the best antitumor drug and sell at 10 kinds.In addition, data shows according to the relevent statistics, be the chemical synthetic drug except that a few antineoplastic agents such as mitoxantrone, hydroxyurea and cisplatin in the antineoplastic agent of listing China nineties in last century, a series of antineoplastic agents (containing the human immunity agent) of go on the market (or still in development stage) are the composition that extracts in the natural herbal.Therefore, a few years from now on China emerges large quantities of new type natural medicine series antineoplastic medicaments probably.The natural antitumor medicine probably became the leading role of China or world's antitumor drug in 10 years from now on.
Pulmonary carcinoma (SCLC) and primary hepatocarcinoma (HCC) are the common cancers of China, and dead ranking is respectively the 1st and the 2nd.The annual newly-increased patient of pulmonary carcinoma is 1,200,000, accounts for 17.8% of cancer mortality number.The annual newly-increased patient 560,000 of hepatocarcinoma, death toll is about 120,000, accounts for 40% of whole world PLC mortality number.Chemotherapeutics is effective to control cancer cell, but very big to the damage of body, often when killing tumor cell, has destroyed normal histiocyte, has destroyed the immunocompetence of human body.
At present, the Chinese medicine that is used for antitumor drug is to be main medicine with Venenum Bufonis, is equipped with other Chinese medicine medicine, and well-known antitumor drug Venenum Bufonis is a kind of toxic herb, if single with excessive poisoning, its effective dose of bad control and the toxic dose of causing easily of Venenum Bufonis.
Three, summary of the invention
The objective of the invention is provides a kind of Chinese medicine anti-tumor agent with anti-hepatocarcinoma and anti-pulmonary carcinoma for tumor diseases such as treatment pulmonary carcinoma, hepatocarcinoma; This medicine antitumor action determined curative effect, toxic and side effects is little, characteristics such as the easy control of its effective dose and toxic dose.The present invention also provides a kind of preparation method of this kind medicine.
For achieving the above object, the technical scheme that the present invention takes is: medicine of the present invention is the pharmaceutical formulation of being made as medicine material by Radix Ginseng, Cutis Bufonis, Scorpio, Grifola frondosa.By its weight ratio be:
Radix Ginseng 10.8～16.2%, Cutis Bufonis 16.2～27.0%, Scorpio 16.2～27.0%, Grifola frondosa 32.4～54.0%.
The preparation method of the invention described above medicine may further comprise the steps, and Cutis Bufonis, Scorpio are behind 95% above ethanol extraction, extracting solution concentrates standby, and medicinal residues merge water with other drug again to be carried, and the water extract is concentrated the back merge back spray drying, granulation with the ethanol extraction concentrated solution, encapsulated, promptly.
Its technical process is: Cutis Bufonis, Scorpio are through 95% above ethanol extraction 3 times, promptly use soak with ethanol, 80～85 ℃ of heat are extracted, time was respectively 2.5 hours, 1 hour, 1 hour, the weight ratio of used ethanol and Cutis Bufonis and Scorpio is respectively: for the first time be 10 times of amounts, the second time are 6 times of amounts, be 6 times of amounts for the third time, merge the ethanol extract after the immersion three times, reclaim ethanol then, the medicinal liquid stand for standby use after concentrating; Residue and other drug merge, water is carried 3 times, be that decocting boils, temperature is 95～100 ℃, time was respectively 2 hours, 1.5 hours, 1 hour, the weight ratio of institute's water and residue and other drug gross weight is respectively: for the first time be 10 times of amounts, be for the second time 8 times of amounts, be 6 times of amounts for the third time, extract three times water extract merging, concentrated (being concentrated into relative density is 1.08); With water extract and the merging of ethanol concentrated medicament, filtration, spray drying, granulation, encapsulated again, promptly.We are called No. 1, colored toad capsule.
The spray drying condition: it is 20ml/min that spray dryer is adjusted to the medicinal liquid admission velocity, 165 ℃ of inlet temperature, and 90 ℃ of leaving air temps promptly get dry medicated powder in the collecting tank.
Medicine of the present invention also can be the endo-medicine preparation of being made as follows by following medicaments in part by weight raw material:
Radix Ginseng 12.5～18.8%, Cutis Bufonis 12.5～18.8%, Scorpio 12.5～18.8%, Grifola frondosa 25.0～37.7%, Rhizoma Pinelliae 18.8～25.0%.
The preparation method of the invention described above medicine may further comprise the steps: Cutis Bufonis and Scorpio are through 95% ethanol extraction, and medicinal residues and concentrated solution are standby; After the Rhizoma Pinelliae decocting boiled 2 hours, pour Radix Ginseng, Grifola frondosa and medicinal residues again into, water is carried 3 times, add water inventory and be controlled at 10 times of medical material amounts for the first time, for the second time be 8 times of amounts, be 6 times of amounts for the third time, the water extract that extracts three times merges, filtration, concentrated (being concentrated into relative density is 1.08), again with the merging of alcohol extraction concentrated medicament, spray drying, granulation, encapsulated, promptly.We are called No. 2, colored toad capsule.(methods such as ethanol extraction, water are carried, spray drying are the same)
The present invention is directed to hepatocarcinoma patient and have the characteristics of deficiency in origin and excess in superficiality, with the Cutis Bufonis is monarch drug, Radix Ginseng is that ministerial drug, Scorpio are adjuvant drug, Grifola frondosa is a messenger drug, set up colored toad capsule side,, sesame toad diffusing with ancient prescription toad sand from the medicine composition looses and compares, Radix Ginseng has powerful strengthening vital QI to eliminate pathogenic factors effect, with Scorpio, Grifola frondosa serves as that assistant makes, strengthened the antitumor action of Cutis Bufonis, Grifola frondosa is good for the stomach, regulates the flow of vital energy, diuretic, has increased the detoxifcation diuretic function of Cutis Bufonis again, specially control mid and late liver cancer and cirrhotic ascites, pulmonary carcinoma is equally also had good efficacy.
The present invention used the extremely strong Cutis Bufonis of antitumaous effect and with it compatibility enhance immunity such as Radix Ginseng, Grifola frondosa, Scorpio, suppress the Chinese medicine of tumor after, not only can kill and wound cancerous cell, and to improving patient's life qualities such as pulmonary carcinoma, hepatocarcinoma, improving the general situation of body and prolonging life cycle all has good effect.
Well-known Venenum Bufonis is a kind of toxic herb, if list causes poisoning easily with Venenum Bufonis is excessive, and the present invention uses Cutis Bufonis to replace Venenum Bufonis, then can play antitumor action, is unlikely to again as bad control effective dose of Venenum Bufonis and toxic dose.Except that the Venenum Bufonis that contains trace, also contain the peptide constituents----anticancer peptide of antitumor action, so its tumor-inhibiting action is stronger, but is unlikely to cause poisoning in the Cutis Bufonis.Because monomeric antitumor research is one of the focus in tumor research field in recent years, numerous medicine scholars have explored Chinese medicine monomer composition-----cinobufacin to hepatocarcinoma, pulmonary carcinoma, esophageal carcinoma, leukemic inhibitory action, and these researchs have far reaching significance to developing new cancer treatment drugs.Toadpoison Medicine (Bufalin) is one of a kind of toxicity aglucon that extracts in the Chinese medicine Venenum Bufonis, belongs to the inhibitor of cell topoisomerase II, is the powerhouse of antitumor action in the bufotalin.Toadpoison Medicine is at the existing report of the research of anti-tumor aspects such as neoplastic hematologic disorder, carcinoma of prostate, gastric cancer, the research of mechanism aspect has related to tumor cell differentiation, apoptosis induced and Expression of Related Genes, aspects such as signal transduction and reverse multidrug resistance, but be limited to the more of in vitro study, contain the Venenum Bufonis composition that comprises cinobufacin, Toadpoison Medicine in the Cutis Bufonis, and containing the antitumor action peptide constituents, this respect is existing both at home and abroad to be studied and report.For this reason, we are principal agent with the Cutis Bufonis in the past to Venenum Bufonis, Cutis Bufonis antitumor research basis, add that Radix Ginseng, Grifola frondosa, Scorpio etc. have drafted colored toad capsule formula, and confirm that through experimentation its antitumous effect is good.Particularly put, have the symptom of nausea and vomiting more serious after the chemotherapy at the part tumor patient in addition, invented again and add the prescription of the Rhizoma Pinelliae with the potentiation preventing or arresting vomiting.
Medicament of the present invention proves through toxicity test and clinical trial, have good antitumor action and end the cancer pain effect, for various alimentary tract cancer esophageal carcinoma, carcinoma of gastric cardia, gastric cancer, hepatocarcinoma, colorectal cancer and pulmonary carcinoma, nasopharyngeal carcinoma etc. significant curative effect is arranged all, especially hepatocarcinoma, cirrhotic ascites, pulmonary carcinoma are had specially good effect; And have no side effect or toxic and side effects minimum.That preparation method provided by the invention has is simple to operate, technology is reasonable, stable and controllable for quality, be suitable for suitability for industrialized production, characteristics such as the ingredient loss is few.
Four, the specific embodiment
The present invention will be described in detail below in conjunction with embodiment:
Embodiment 1 (No. 1, flower toad capsule):
Radix Ginseng 4～6kg, Cutis Bufonis 6～10kg, Scorpio 6～10kg, Grifola frondosa 12～20kg
Cutis Bufonis, Scorpio are through 95% above ethanol extraction 3 times, promptly use soak with ethanol, 80～85 ℃ of heat are extracted, time was respectively 2.5 hours, 1 hour, 1 hour, the weight ratio of used ethanol and Cutis Bufonis and Scorpio is respectively: for the first time be 10 times of amounts, the second time are 6 times of amounts, be 6 times of amounts for the third time, merge the ethanol extract after the immersion three times, reclaim ethanol then, the medicinal liquid stand for standby use after concentrating; Residue and other drug merge, water is carried 3 times, be that decocting boils, 95～100 ℃, time were respectively 2 hours, 1.5 hours, 1 hour, the weight ratio of institute's water and residue and other drug gross weight is respectively: for the first time be 10 times of amounts, the second time are 8 times of amounts, be 6 times of amounts for the third time, the water extract that extracts three times merges, and concentrates (being concentrated into relative density is 1.08); With water extract and the merging of ethanol concentrated medicament, filtration, spray drying, granulation, encapsulated again, promptly.
Usage and consumption: oral, 2～3 times on the 1st, each 4～6.
Embodiment 2 (No. 2, flower toad capsule):
Radix Ginseng 4～6kg, Cutis Bufonis 4～6kg, Scorpio 4～6kg, Grifola frondosa 8～12kg, Rhizoma Pinelliae 6～8kg
Cutis Bufonis (is respectively 8 times of amounts, 6 times of amounts, 6 times of amounts) behind 95% above ethanol extraction 3 times, merge, reclaim ethanol liquid, and residue is standby; After Rhizoma Pinelliae being decocted first 1 hour in addition (water yield when assurance is boiled medicine), merge water with above-mentioned residue and other drug again and carry (being respectively 8 times of amounts, 6 times of amounts, 6 times of amounts) 3 times, ethanol extract merging with water extract and after reclaiming, concentrated, dry, granulation, encapsulated, promptly get colored toad capsule preparations No. 2.
Usage and consumption: oral, 2～3 times on the 1st, each 4～6.
Colored toad capsule preparations of the present invention has good antitumor action and ends the cancer pain effect, for various alimentary tract cancer esophageal carcinoma, carcinoma of gastric cardia, gastric cancer, hepatocarcinoma, colorectal cancer and pulmonary carcinoma, nasopharyngeal carcinoma etc. significant curative effect is arranged all, especially hepatocarcinoma, cirrhotic ascites, pulmonary carcinoma are had specially good effect.
Routine patient's clinical observation surplus 10, effective percentage 100%, improvement rate 90%, and tried the patient and be the Advanced Carcinoma Patient that hospital no longer accepts for medical treatment, the diet difficulty, the health extremely feeble, part cancer patient's many places diffusion promptly begins QI movement being in harmonious way, QI and blood and accent with a couple of days behind this preparation, reach treating the poisonous disease with poisonous drugs, removing obstruction in the collateral to relieve pain is controlled its mark, blocking-up tumor-blood-vessel growth, human body immunity improving power are improved the life quality of tumor patient greatly with Zhi Qiben, and part patient fully recovers, none death of all patients in treating at present shows unprecedented therapeutic effect.
According to modern study, antitumor drug and method mainly contain following a few class both at home and abroad:
1. topoisomerase enzyme inhibitor
2. tubulin activity inhibitor
3. tumor neogenetic blood vessels generates (TA) inhibitor
4. gene therapy
Main method comprises: 1. cytokine gene therapy: by external/intracorporal method transducer cell factor gene, regulate immunoreation; 2. drug sensitive gene therapy: i.e. suicide gene therapy; 3. multidrug resistance gene therapy: forward multidrug resistance gene to marrow hemopoietic stem cells, in conjunction with the high-dose chemotherapy antitumor; 4. gene substitution or additional: the antioncogene of the oncogene of replacement mutation or additional disappearance; 5. antisense nucleic acid technique: be used to suppress the expression of oncogene; 6. oncogene engineering tumor vaccine: promptly utilize gene recombination technology etc. but gene therapy still must be used with traditional antitumor drug, both have synergism.
5. telomerase inhibitor
Telomerase (telomerease) is the archaeal dna polymerase that a kind of RNA relies on, can be template with RNA own, at the repetitive sequence of end of chromosome synthetic six poly-Deoxydization nucleotide TTAGGG, the end of chromosome during with the compensation cell division shortens, and solves " end duplicates problem ".Telomerase is present in most normal human cells, but in tumor cell wide expression, point out it may be essential by the malignant cell infinite multiplication.Suppress the vigor of telomerase, might stop the tumor cell malignant proliferation.
6. interventional therapy etc.;
Wherein tumor neogenetic blood vessels generation (TA) inhibitor is the relatively more active class of present antitumor research.The growth of the research proof primary tumo(u)r and transfer depend on new vessels and generate.Tumor not only can be obtained nutrition and oxygen but also can carry transitional cell to the host continuously by tumor vessel from the host by tumor vessel, and formed at other position continued growths and the induction of vascular of body, caused neoplasm metastasis.The vascular system of tumor has become brand-new, an antineoplaston target spot likely.People just are being devoted to R and D can destroy or suppress angiogenesis, the medicine that stops growth of tumor and transfer effectively, this class medicine is called the TA inhibitor, and the TA inhibitor for treating has many advantages: when 1. tumor takes place, vascularization is activated, so have good specificity; 2. vascular endothelial cell is exposed in the blood flow, and medicine can directly play a role, so dosage is little, curative effect is high, untoward reaction is little; 3. the endothelial cell gene expression is relatively stable, is difficult for producing drug resistance.
Clinical data shows that Bufo siccus class medical material is the animal medicinal material that is commonly used to treat cancers such as hepatocarcinoma, and for " deficiency in origin and excess in superficiality " symptom at liver cancer patient, need to cooperate with Radix Ginseng, etc. the Chinese medicine of QI invigorating tonify deficiency, prove effective repeatedly.By the clinical practice of more than ten years, we find with Cutis Bufonis under the situation of medical materials such as compatibility Radix Ginseng, Grifola frondosa, Scorpio, patients such as hepatocarcinoma, pulmonary carcinoma to be had better therapeutic effect as essential drugs.Zoopery also illustrates, flower toad capsule up to 51.8% and 70.1%, all is higher than 44.6% tumour inhibiting rate of simple usefulness " toad sand looses " to the tumor control rate of S180 and H22 tumor-bearing mice.In view of this seminar in the last few years to the clinical and experimentation of hepatocarcinoma, think that colored toad capsule has possessed higher development and has been worth, and the used medical material overwhelming majority is all Shandong special product medical material, can bring into play the advantage of the local high-quality genuine medicinal materials that produced better, better is people's health service.
" toad sand looses " is to derive from treatment " tympanites " ancient prescription in " deep red capsule brief points ", is essential drugs with Cutis Bufonis, Fructus Amomi.Folk prescription, the proved recipe for the treatment of hepatocarcinoma, liver cirrhosis with Cutis Bufonis that for many years just spreading among the people; The hepatopathy center doctor of Tai'an hospital looses treating hepatocirrhosis, hepatocarcinoma for many years with toad sand, receives effect preferably; Document announcement sesame toad diffusing (Ganoderma, Cutis Bufonis etc.) treatment hepatocarcinoma, hepatic ascites, the result is satisfactory.We have the characteristics of deficiency in origin and excess in superficiality at hepatocarcinoma patient on this basis, with the Cutis Bufonis is monarch drug, Radix Ginseng is that ministerial drug, Scorpio are adjuvant drug, Grifola frondosa is a messenger drug, set up colored toad capsule side,, sesame toad diffusing with toad sand from the medicine composition looses and compares, Radix Ginseng has powerful strengthening vital QI to eliminate pathogenic factors effect, with Scorpio, Grifola frondosa serves as that assistant makes, strengthened the antitumor action of Cutis Bufonis, Grifola frondosa is good for the stomach, regulates the flow of vital energy, diuretic, has increased the detoxifcation diuretic function of Cutis Bufonis again, specially control mid and late liver cancer and cirrhotic ascites, pulmonary carcinoma is also had good efficacy.
Side's Chinese medicine benefit warm in nature is and slightly poisonous, and full side has the merit of tonifying Qi and lifting yang ， Xiao Disorderization knot, blood circulation promoting and blood stasis dispelling, detoxifcation row water.
The present invention studies have shown that, contains antitumor effective ingredient such as bufogenin, Toadpoison Medicine, cinobufacin in the Cutis Bufonis, and experiment and clinical proof have tangible antitumous effect for many years.And contain multiple small-molecule peptide material in Cutis Bufonis and the Scorpio, it also is unique anticancer intelligent factor, has obvious inhibition new vessels nucleus formation, these small-molecule peptides can enter in the blood of human body, selectively the attack tumor tissues at first cuts off the nutrient source approach that corpus carcinosus is depended on for existence---newborn blood capillary, allows cancerous cell lose nutrition supply and can't survive, grows, breed, until apoptosis, withered as the fallen leaves in autumn.This intelligent targeting fundamentally cuts off the fission breeding of cancerous cell, causes the apoptosis of cancerous cell, has reached orientation and has put down tumoricidal effect.Simultaneously owing to contain multiple saponin active ingredient in the Chinese medicine Radix Ginseng, the effect in QI invigorating Yiyang is arranged not only, and ginsenoside Rg1 wherein and Rg3 have antitumor action, the ginseng polysaccharide also has certain anti-tumor activity and immunological enhancement.One of best antitumor medicinal fungi that Grifola frondosa is so far to be found, tumour inhibiting rate is only second to Agaricus blazei Murrill, far above mushroom medicines such as Polyporus, Lentinus Edodes, Coriolous Dersicolor (Fr.) Quel, Ganoderma applanatum (Pers. Ex Wallr) Pat., Ganodermas, tumour inhibiting rate is more than 80% in the Japanology grifolan body, its external inhibitory rate 100% of domestic research.Above medicine active anticancer component accumulates in around the tumor, form focus topical remedy high concentration region, the part active ingredient enters inside tumor, directly kills tumor cell, make its necrosis, softening, absorption, reach the effect that " gathering is assaulted fortified position " directly kills tumor cell.Simultaneously, preparation of the present invention has stronger immunostimulant ability, can improve cancer patient's autoimmunity, brings into play autonomous anti-cancer ability, thereby effectively controls the transfer and the recurrence of tumor, plays anticancer effect altogether.
In a word, we have the body resistance strengthening and constitution consolidating, the effect of note of the ancient Chinese knot that disappears, detoxifcation row water, so can control mid and late liver cancer, pulmonary carcinoma, curative effect is reliable.
And summing up clinical experience through the inventor proves: Rhizoma Pinelliae has good preventing or arresting vomiting effect, can obviously resist because of chemotherapeutics to body do not accommodate that toxicity causes feel sick, vomiting, also can eliminate or alleviate individual patient to taking uncomfortable and the feeling sick of causing of Cutis Bufonis, vomiting.Need be decocted first more than 2 hours but in this preparation, add Rhizoma Pinelliae, to give full play to its stopping nausea and vomiting by lowering the adverse flow of QI and antitumor action.Report existing several pieces about the experimentation and the clinical application of Rhizoma Pinelliae preventing or arresting vomiting, document sees reference.
Medicine of the present invention is used for oncotherapy, ends cancer pain etc. and have good effect, instant effect, wide spectrum, the characteristics of multiple effect such as good analgesic effect.
Medicine of the present invention proves through toxicity test, and no obvious toxic-side effects is safe in utilization.
That preparation method of the present invention has is simple to operate, technology is reasonable, stable and controllable for quality, be suitable for suitability for industrialized production, characteristics such as the ingredient loss is few.
Colored toad capsule preparations with the foregoing description 1, carry out following zoopery:
Antitumor animal experiment and mechanism of action
1 materials and methods
The colored toad capsule preparations of 1.1 medicine: embodiment 1.
1.2 animal model is set up and grouping
Medicine FT-207: Qilu Pharmaceutical Factory, batch number: 509004LG; PINGXIAO PIAN is produced by Jintaiyang Pharmaceutical Co., Ltd., Ningxia, batch number: 20051102;
Propidium iodide (PI), Sigma company product, cat:p4170.The RNaseA enzyme is a Sigma company product.
Instrument mainly contains: BCM-1000A type biologic cleanliness workbench, SuZhou Antai Air Tech Co., Ltd. of Su Jing group.The automatic pure water distillator of SZ-III type, roc Science and Technology Ltd. is praised in Shanghai.LDZ4-0.8 type centrifuge, Beijing Medical Centrifugal Machine Factory.FACSCalibur type flow cytometer, U.S. company BD (BectonDickinson).Japan's blood counting instrument.
1.2 experimental technique
1.2.1 animal Kunming mouse male and female half and half, this Leco Corp. provides by Shanghai, animal credit number SCXK (Shanghai) 2003-0003.
1.2.2 tumor strain H22, S180 are provided by Shandong Academy of Medical Sciences.
1.2.3 the experiment compound method disinfection salt solution (PBS) of reagent: NaH2PO42H2O 0.6g, Na2HPO412H2O 5.8g, NaCl 16.4g adds tri-distilled water to 2000ml, puts into disinfection with high pressure steam pot sterilization back and uses; Propidium iodide (PI): the preparation of PBS liquid, concentration is 50ug/ml; The RNaseA enzyme: the preparation of PBS liquid, concentration is 1ug/ml.
1.2.4 the dislocation of tumor source mouse carotid is put to death in the modelling of lotus tumor, be fixed on the Mus plate, routine disinfection, in superclean bench, extract tumor liquid out from the abdominal cavity, normal saline is diluted to 1 * 107ml-1, and other respectively organize mice all in the above-mentioned tumor cell suspension 0.2ml of right armpit subcutaneous injection, to set up solid tumor models except that blank; Make the above-mentioned tumor cell suspension 0.2ml of need lumbar injection of increase in life span bearing mouse model, to set up the ascites tumor model.
1.2.5 animal grouping with medication with this method with Kunming mouse right fore axil subcutaneous vaccination H22 hepatocarcinoma entity tumor and S180 solid tumor, and lumbar injection H22 hepatic ascites oncocyte.Inoculate back first day, animal weighed be divided into 10 groups at random, every group 10, all after inoculation the 2nd day the beginning gastric infusion, observe mice diet and tumor growth situation, drug withdrawal after continuous 8 days is weighed, is put to death animal and strip tumor, thymus, spleen in drug withdrawal and weigh tumour inhibiting rate and organ index of each group of statistics respectively next day.Lumbar injection H22 hepatic ascites tumor group, drug withdrawal behind successive administration 9d, the life span of observing mice, and calculate the tumor-bearing mice increase in life span.
1.3 observation index and assay method
Tumour inhibiting rate: mice was weighed in 24 hours after the last administration, disconnected neck is put to death, and strips the tumor piece and weighs
(body weight deducts the heavy difference of tumor for the mice body weight)
1.4 compound recipe flower toad capsule is to the influence in transplanted tumor mouse cell cycle
Mechanical Method prepares tumor tissue cell's suspension: strip fresh tumor tissues, with PBS with its rinsing clean after, be immersed in the plate that contains PBS, with eye scissors tumor tissues is shredded, grind, tissue homogenate filters with 200 order stainless (steel) wires, and cell suspension is in PBS liquid, the centrifugal 5min of 1000rpm abandons supernatant; After adding PBS made cell resuspended in precipitate, centrifugal again 5min abandoned supernatant, if cell still has a large amount of erythrocyte to repeat above step.Add PBS liquid suspension cell at last, adjusting cell number is 1 * 106, and obtained cell suspension 100ul adds the 1mg/mlRNA enzyme, places 30min in 37 ℃ of water-baths, uses 500ul4 ℃ of lucifuge dyeing of PI (propidium iodide) 10min then.Last machine is with obtaining cell with cellquest software, the flow cytometry analysis cell cycle.
1.5 the flow cytometer list dyes the detection apoptosis rate
Fresh tumor tissues is handled the same, and obtained cell suspension 100ul adds 1mg/mlRNA enzyme 20ul, and 37 ℃ of water-bath 30min add 4 ℃ of lucifuge dyeing of PI 1ml 10min again, and flow cytometer obtains cell, with the hypodiploid peak of Modifit software analysis apoptotic cell.
1.6 routine blood test detects
Mice is plucked eyeball get blood, separation of serum is done routine blood test and is detected.
1.7 statistical procedures
Experimental data is carried out statistical analysis with the SPSS11.5 statistical software, and mean represents with X+SD, relatively the checking with t of two means.
2 results
2.1 flower toad capsule is to the inhibitory action of tumor-bearing mice S180 tumor cell
High, medium and low 3 the dosage groups of flower toad capsule are the highest with the high dose group tumour inhibiting rate, middle dosage group is taken second place, the low dose group tumour inhibiting rate is minimum, but the tumour inhibiting rate that all is higher than positive control Chinese drug-treated group PINGXIAO PIAN, and positive control chemotherapeutic group FT-207 is bigger because of toxic and side effects, the mortality of mice height is not done the tumour inhibiting rate statistics.Pharmacodynamic study shows: flower toad capsule all has obvious inhibitory action to mice S180 sarcoma, reaches as high as 51.8%, and is good dose-effect relationship, and comparing with the positive control PINGXIAO PIAN all has significant difference, P＜0.01.
Table 1 compound recipe flower toad capsule is to the influence of tumor-bearing mice S180 tumor bulk-growth
| Group | Number of animals (beginning) | Number of animals (end) | Tumor body weight (g) X ± SD | Tumour inhibiting rate (%) |
| The normal saline contrast | 10 | 10 | 1.37±0.34 | |
| FT-207 | 10 | 1 | 0.106 | |
| The positive control PINGXIAO PIAN | 10 | 9 | 1.08±0.49 | 21.2 |
| Flower toad capsule 0.15g/ kg | 10 | 10 | 0.84±0.43 | 33.3＊ |
| Flower toad capsule 0.3g/kg | 10 | 10 | 0.72±0.33 | 42.8＊ |
| Flower toad capsule 0.6g/kg | 10 | 10 | 0.66±0.27 | 51.8＊ |
* compare P＜0.01 with matched group
2.2 flower toad capsule is to the inhibitory action of mice H22 hepatocarcinoma tumor cell
High, medium and low 3 the dosage groups of flower toad capsule are the highest with the high dose group tumour inhibiting rate, middle dosage group is taken second place, the low dose group tumour inhibiting rate is minimum, but the tumour inhibiting rate that all is higher than positive control Chinese drug-treated group PINGXIAO PIAN, and positive control chemotherapeutic group FT-207 is bigger because of toxic and side effects, the mortality of mice height is not done the tumour inhibiting rate statistics.Pharmacodynamic study shows: flower toad capsule all has obvious inhibitory action to mice H22 hepatoma carcinoma cell, reaches as high as 60.4%, and is good dose-effect relationship, and comparing with the positive control PINGXIAO PIAN all has significant difference, P＜0.01.
Table 2 flower toad capsule is to the influence of H22 Mouse Liver growth of cancer cells
| Group | Number of animals (beginning) | Number of animals (end) | Tumor body weight (g) X ± SD | Tumour inhibiting rate (%) |
| The normal saline contrast | 10 | 9 | 1.44±0.4 | |
| FT-207 | 10 | 2 | 0.06 | 95.2. |
| The positive control PINGXIAO PIAN | 10 | 9 | 1.04±0.49 | 27.8 |
| Flower toad capsule 0.15g/kg | 10 | 10 | 0.76±0.38 | 47.2 |
| Flower toad capsule 0.3g/kg | 10 | 10 | 0.58±0.36 | 59.7 |
| Flower toad capsule 0.6g/kg | 10 | 10 | 0.43±0.29 | 70.1 |
* compare P＜0.01 with matched group
2.3 flower toad capsule is to the influence of lotus H22 hepatic ascites cell mouse life span
Flower toad capsule height, in, low 3 dosage groups are the highest to lotus H22 ehrlich ascites carcinoma mice increase in life span with high dose group, middle dosage group is taken second place, the low dose group rate elongation is minimum, but all be higher than positive control Chinese drug-treated group PINGXIAO PIAN to lotus H22 ehrlich ascites carcinoma mice increase in life span, pharmacodynamic study shows: flower toad capsule all has certain inhibitory action to mice H22 ascites cells, can prolong lotus H22 ehrlich ascites carcinoma mice life span, reach as high as 29.9%, and be proportionate with dosage, compare high dose group with the positive control PINGXIAO PIAN significant difference is arranged, P＜0.01, in, low dose group is compared there was no significant difference with the positive control PINGXIAO PIAN.P＞0.05。
Table 3 flower toad capsule is to the result of lotus H22 ehrlich ascites carcinoma mice increase in life span
| Group | Life span | Rate elongation (%) |
| The normal saline contrast | 15.2 | |
| The positive control PINGXIAO PIAN | 18.0 | 18.4 |
| Flower toad capsule 0.15g/kg | 18.2 | 19.7 |
| Flower toad capsule 0.3g/kg | 18.7 | 23.0 |
| Flower toad capsule 0.6g/kg | 19.8 | 30.2＊ |
* compare P＜0.01 with matched group
2.4 flower toad capsule is to the influence of lotus S180 mice and H22 liver cancer mouse immune organ
Behind the continuous gastric infusion 9d of mice, thymus is got in execution and spleen is weighed, and calculates spleen index and thymus index.The difference of comparing with matched group that there are no significant.
Table 4 compound recipe flower toad capsule is to mouse immune organ weight's influence
| Group | Index and spleen index | Thymus index |
| The normal saline contrast | 6.23±0.34 | 4.70±0.29 |
| The positive control PINGXIAO PIAN | 6.14±0.49 | 3.99±0.38 |
| Flower toad capsule 0.15g/kg | 5.71±0.43 | 3.36±0.44 |
| Flower toad capsule 0.3g/kg | 5.68±0.33 | 4.28±0.58 |
| Flower toad capsule 0.6g/kg | 6.00±0.52 | 3.94±0.49 |
By statistics, compare P＞0.05 with matched group
2.5 flow cytometer detects the cell cycle and the apoptosis rate of H22 rat liver cancer tissue
Flower toad capsule raises with dosage, makes tumor cell increase G in the ratio of S phase
0-G
1The phase ratio descends, G
2-M phase ratio changes little, Chinese medicine contrast PINGXIAO PIAN, and chemotherapeutic FT-207 toxicity is bigger, experimental results show that colored toad capsule is to make G to the influence in H22 rat liver cancer histiocyte cycle
0-G
1The phase cell reduces, and cell is arrested in the S phase, and induces the part apoptosis of tumor cells.See Table 5
The cell cycle of table 5H22 rat liver cancer tissue changes and apoptosis rate
| Group | G 0-G 1 | S | G 2-M | Apoptosis rate % |
| The normal saline contrast | 93.38 | 6.34 | 0.28 | 1.3 |
| FT-207 | ||||
| The positive control PINGXIAO PIAN | 52.54 | 46.66 | 0.80 | 12.31＊ |
| Flower toad capsule 0.15g/kg | 67.44 | 31.71 | 0.85 | 16.13＊ |
| Flower toad capsule 0.3g/kg | 62.98 | 36.95 | 0.07 | 20.99＊ ＊ |
| Flower toad capsule 0.6g/kg | 57.78 | 42.22 | 0.02 | 24.32＊ ＊ |
* compare P＜0.05 with matched group; * compares with matched group, P＜0.01
2.6 compound recipe flower toad capsule is to the influence of mouse blood system
Behind the continuous gastric infusion 9d of mice, get and win eyeball after mice is weighed and get blood and carry out blood cell analysis, the RGB of each tumor-bearing mice group all reduces to some extent than blank group as a result, and PLT is all but the increase to some extent of all blank group, but there was no significant differences all; There is not significant change between each group of WBC and HGB, all not statistically significant.
Table 6 compound recipe flower toad capsule is to the influence of mouse blood system
| Group | RGB/×10 12·L -1 | WBC/×10 9·L -1 | PLT/×10 9·L -1 | HGB/g·L -1 |
| Blank group | 8.51 | 5.26 | 996.2 | 128.1 |
| The positive control PINGXIAO PIAN | 7.93 | 5.33 | 1069.7 | 123.2 |
| Flower toad capsule 0.15g/kg | 7.67 | 4.74 | 1230.7 | 121.9 |
| Flower toad capsule 0.3g/kg | 7.52 | 5.03 | 1119.5 | 113.8 |
| Flower toad capsule 0.6g/kg | 7.64 | 6.69 | 1026.4 | 118.5 |
By statistics, compare P＞0.05 with matched group
Brief summary:
Pharmacodynamic study shows: flower toad capsule all has obvious inhibitory action to mice S180 sarcoma, H22 hepatocarcinoma, and is good dose-effect relationship.The result shows that colored toad capsule is respectively 33.3%, 42.8% and 51.8% to the suppression ratio of mice S180 sarcoma; To the suppression ratio of mice H22 hepatocarcinoma up to 70.4%; High dose group makes the tumor-bearing mice increase in life span improve 30%.Flow cytometer detects and shows: compound recipe flower toad capsule stops tumour cell cycle in the S phase with the raising of dosage, and can obviously induce the H22 hepatoma cell apoptosis, and can improve immune function of mice, atrophy of thymus gland due to effective inhibition chemotherapeutic, its mechanism of action may with the retardance cell cycle, cell death inducing is relevant.In addition, the mice routine blood test detects and shows that colored toad capsule gastric infusion does not have obvious influence to the mouse blood system, proves that flower toad capsule does not have obvious toxic and side effects.
List of references:
1, Xu Shuyun etc.: pharmacological experimental methodology [M], the 2nd edition, Beijing: People's Health Publisher, 1991:722～723.
2, the king bathes life: herbal pharmacology and application [M], the 1st edition, Beijing: People's Health Publisher, 1983:446,1102.
3, Jiangsu Polytechnical College is compiled: Chinese medicine voluminous dictionary [M], Shanghai, Science and Technology of Shanghai publishing house, 1977:2714.
4, Xiao Peigen chief editor: newly organized Chinese medicinal herbal the 4th volume [M], Beijing, Chemical Industry Press, 2002:270.
5, Yu Rongli, Zhang Guiling, Qin Xusheng: Grifola frondosa progress.Shanghai Agricultural journal [J], 2005,21 (3): 101～105.
Claims (4)
1. antineoplastic Chinese medicine medicine is characterized in that: be by Radix Ginseng, and Cutis Bufonis, Scorpio, Grifola frondosa is made as medicine material, by its weight ratio is:,
Radix Ginseng 10.8～16.2%, Cutis Bufonis 16.2～27.0%, Scorpio 16.2～27.0%, Grifola frondosa 32.4～54.0%.
2. antineoplastic Chinese medicine medicine is characterized in that: be by Radix Ginseng, and Cutis Bufonis, Scorpio, Grifola frondosa, Rhizoma Pinelliae is made as medicine material, and medicine material by its weight ratio is:
Radix Ginseng 12.5～18.8%, Cutis Bufonis 12.5～18.8%, Scorpio 12.5～18.8%, Grifola frondosa 25.0～37.7%, Rhizoma Pinelliae 18.8～25.0%.
3, the preparation method of the described antineoplastic Chinese medicine of a kind of claim 1 medicine, it is characterized in that: may further comprise the steps, Cutis Bufonis, Scorpio are behind 95% above ethanol extraction, extracting solution concentrates standby, medicinal residues merge water with other drug again and carry, the water extract is concentrated the back merge back spray drying, granulation with the ethanol extraction concentrated solution, encapsulated
Its technical process is: Cutis Bufonis, Scorpio are through 95% above ethanol extraction 3 times, promptly use soak with ethanol, 80～85 ℃ of heat are extracted, time was respectively 2.5 hours, 1 hour, 1 hour, the weight ratio of used ethanol and Cutis Bufonis and Scorpio is respectively: for the first time be 10 times of amounts, the second time are 6 times of amounts, be 6 times of amounts for the third time, merge the ethanol extract after the immersion three times, reclaim ethanol then, the medicinal liquid stand for standby use after concentrating; Residue and other drug merge, water is carried 3 times, be that decocting boils, temperature is 95～100 ℃, time was respectively 2 hours, 1.5 hours, 1 hour, the weight ratio of institute's water and residue and other drug gross weight is respectively: for the first time is 10 times of amounts, is for the second time 8 times of amounts, is 6 times of amounts for the third time, extract three times water extract merging, and concentrated; With water extract and the merging of ethanol concentrated medicament, filtration, spray drying, granulation, encapsulated again, promptly.
4. the preparation method of the described antineoplastic Chinese medicine of claim 2 medicine, it is characterized in that: may further comprise the steps, Cutis Bufonis and Scorpio are through 95% ethanol extraction, and medicinal residues and concentrated solution are standby; After the Rhizoma Pinelliae decocting boiled 2 hours, pour Radix Ginseng, Grifola frondosa and medicinal residues again into, water is carried 3 times, add water inventory and be controlled at 10 times of medical material amounts for the first time, for the second time be 8 times of amounts, be 6 times of amounts for the third time, the water extract that extracts three times merges, filtration, concentrated, again with the merging of alcohol extraction concentrated medicament, spray drying, granulation, encapsulated, promptly.
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| 灰树花多糖的抗肿瘤作用及对巨噬细胞、自然杀伤细胞的影响. 劳华均等.上海农业学报，第13卷第1期. 1997 |
| 灰树花多糖的抗肿瘤作用及对巨噬细胞、自然杀伤细胞的影响. 劳华均等.上海农业学报，第13卷第1期. 1997 * |
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