CN103463602A - Medicine for treating cutaneous squamous cell carcinoma and preparation method of medicine for treating cutaneous squamous cell carcinoma - Google Patents
Medicine for treating cutaneous squamous cell carcinoma and preparation method of medicine for treating cutaneous squamous cell carcinoma Download PDFInfo
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Abstract
The invention discloses a medicine for treating cutaneous squamous cell carcinoma and a preparation method of the medicine. The medicine comprises the following materials in parts by weight: 4-6 parts of roots of Chinese pulsatilla, 2-4 parts of curcuma zedoary and 1-3 parts of ginger. The preparation method of the medicine comprises the following steps: adding 70% ethanol into medicinal materials for extracting for three times, wherein the addition amount of ethanol is 12 times that of the medicinal materials and the extraction is carried out for 2 hours each time, combining filter liquor and concentrating to be fluid extract with the ratio of 1:1.3, placing into a thermotank with the temperature of 65-75 DEG C to dry into hard brittle pieces, and thus obtaining the medicine by storing at a low temperature and in a dry place. The medicine for treating cutaneous squamous cell carcinoma can obviously inhibit the proliferation of cutaneous squamous cell carcinoma A431 cells, meanwhile has effects on inducing the apoptosis of A431 cells, and also can prevent the cells from staying at a G0/G1 period so as to prevent the DNA synthesis of an S period and inhibit the proliferation of tumor cells.
Description
Technical field
The present invention relates to technical field of Chinese medicines, specifically a kind of medicine that is used for the treatment of cutaneous squamous cell carcinoma also relates to the preparation method that this is used for the treatment of the medicine of cutaneous squamous cell carcinoma simultaneously.
Background technology
Cutaneous squamous cell carcinoma (squamous cell carcinoma, SCC) is a kind of malignant tumor that originates from epidermis or appendages keratinocyte.Its sickness rate is only second to basal cell carcinoma in skin non-black element cell carcinoma, accounts for 20% of whole non-black element cell carcinomas.The Skin Squamous Cell Carcinoma morbidity is the trend risen year by year in recent years, and PD is fast, destructive large, and lymphatic metastasis can occur, and late period, visceral metastases can occur, and cutaneous squamous cell carcinoma has become the main cutaneous tumor that threatens the human life.
The method of existing treatment Skin Squamous Cell Carcinoma mainly contains following several: 1, operative treatment is the most frequently used art formula, while selecting operation, except considering lesions position, size, the depth, also will consider beauty treatment reparation, the impact of reduce on appearance.But operation is had relatively high expectations to professional technique, length consuming time, and somewhat expensive.2 radiotherapies: radiotherapy is to utilize that to have ionization density large and the characteristics of certain range are arranged, and the high-energy ray irradiated tumor cell destroys that its DNA double chain structure makes it apoptosis and/or its biofilm structure makes it degeneration necrosis, thereby reaches therapeutic purposes.Radiotherapy is mainly application x-ray, radium and electron beam etc.The skin carcinoma that privileged sites are located as face ear nose etc., radiotherapy is more superior than operation, does not affect beauty treatment.But radiation therapy session is long, and existence makes temporary transient red and swollen, the pain of skin, even ulcerated potential danger; Also can cause pigmentation, surrounding tissue is as impaired as the sweat gland function, alopecia areata, cicatrix deterioration etc.3 Drug therapys: have at present the medicines such as each imiquimod ointment, 5-fluorouracil ointment, injection of interferon, Bec-5 ointment, Bleomycin A5, cisplatin to carry out chemotherapy, but common the side effect such as red swelling of the skin, pain, desquamation, sclerosis incrustation are arranged.4 cryotherapys: the advantage of freezing maximum is that cicatrix is poor, in the physiological function that keeps the disease damage position and beauty treatment, has unique advantage, and easy, safety, instant effect, few intercurrent disease, spends low.Though itself can not bring pain freezing, the tissue after thawing can produce pain, swelling and the untoward reaction such as ooze out.5 photodynamic therapy (PDT): photodynamic therapy (photo dynamic therapy; PDT) be a kind of use in conjunction photosensitizer and respective sources; destroy the brand-new treatment technology of pathological tissues by the photodynamics reaction selectivity; the optical dynamic treatment of tumor targeting can effectively be killed and wounded the local tumor cell by force fast; and on normal cell without impact, protected that appearance is injury-free, the vitals function is unaffected.Laser therapy can be applied separately, also can with the surgical operation coupling, consuming time few, but need be completed by the specialist, therefore costly.
In recent years, along with the develop rapidly of life sciences, the signal conduction in malignant cell, the adjusting of cell cycle, the apoptotic process such as induce is illustrated substantially.Using the key enzyme of some signal transduction pathways relevant to the tumor cell differentiation and proliferation as the target spot of drug screening, and the exploitation selectively acting has become the important directions of current antitumor drug research and development in efficient, the low toxicity of specific target spot, the new type anticancer medicine of high specificity.In recent years, quantity research shows greatly, and Chinese medicine is having its exclusive advantage aspect antineoplastic vascular formation, inducing apoptosis of tumour cell, the differentiation of promotion tumor cell and raising immunologic function.
Summary of the invention
A kind of propagation that can significantly suppress skin scale cancer A431 cell that the object of the invention is to overcome above-mentioned shortcoming and provide, it significantly induces the apoptotic effect of A431 in addition simultaneously, and can hinder cell processed and rest on the G0/G1 phase, make the DNA biosynthesis block of S phase, the medicine that is used for the treatment of cutaneous squamous cell carcinoma of the propagation of inhibition tumor cell.
Another object of the present invention is to provide this to be used for the treatment of the preparation method of the medicine of cutaneous squamous cell carcinoma.
A kind of medicine that is used for the treatment of cutaneous squamous cell carcinoma of the present invention, its parts by weight of raw materials proportioning is:
Radix Pulsatillae 4-6 part, Rhizoma Curcumae 2-4 part, Rhizoma Zingiberis Recens 1-3 part.
Above-mentioned a kind of medicine that is used for the treatment of cutaneous squamous cell carcinoma, its parts by weight of raw materials proportion optimization:
5 parts of the Radix Pulsatillaes, 3 parts of Rhizoma Curcumae, 2 parts, Rhizoma Zingiberis Recens.
A kind of preparation method that is used for the treatment of the medicine of cutaneous squamous cell carcinoma of the present invention, comprise the steps:
(1) add the ethanol extraction three times of 12 times of medical material weight amounts 70%, each 2h;
(2) merging filtrate, be condensed into 1 to 1.3 fluid extract, puts in 65-75 ℃ of calorstat and dry to becoming hard crisp, and cold drying is preserved and be get final product.
The present invention compared with prior art, has obvious beneficial effect, and as can be known from the above technical solutions: the theory of Chinese medical science analysis: the skin scale cancer is many because shining upon, and the burning hot skin that burns for a long time, cause the accumulation of heat stasis, take the Radix Pulsatillae as monarch drug, heat-clearing and toxic substances removing; Take Rhizoma Curcumae as ministerial drug, blood circulation promoting and blood stasis dispelling; The Rhizoma Zingiberis Recens of take makes as helping, and priming enters institute, plays a role.This method anemoside B4 extraction ratio is not less than 54.72, and dry cream rate is not less than 28.78, and comprehensive scores is not less than 41.75.
Evidence medicine of the present invention has propagation and the cell death inducing effect that suppresses fell scale cancer A431 cell, and can block cell in the G0/G1 phase, and stop the synthetic of S phase DNA and copy, thus the propagation of inhibition tumor cell.
The specific embodiment of the present invention is provided in detail by following examples.
The specific embodiment
Describe the present invention below in conjunction with example, further explain and illustrate technical scheme characteristics of the present invention.
embodiment 1:
A kind of preparation method that is used for the treatment of the medicine of cutaneous squamous cell carcinoma, comprise the steps:
(1) 5 parts of the Radix Pulsatillaes, 3 parts of Rhizoma Curcumae, 2 parts, Rhizoma Zingiberis Recens, add the ethanol extraction three times of 12 times of medical material weight amounts 70%, each 2h;
(2) merging filtrate, be condensed into 1 to 1.3 fluid extract, puts in 65-75 ℃ of calorstat and dry to becoming hard crisp, and cold drying is preserved and be get final product.
embodiment 2:
A kind of preparation method that is used for the treatment of the medicine of cutaneous squamous cell carcinoma, comprise the steps:
(1) 4 parts of the Radix Pulsatillaes, 4 parts of Rhizoma Curcumae, 1 part, Rhizoma Zingiberis Recens, add the ethanol extraction three times of 12 times of medical material weight amounts 70%, each 2h;
(2) merging filtrate, be condensed into 1 to 1.3 fluid extract, puts in 65-75 ℃ of calorstat and dry to becoming hard crisp, and cold drying is preserved and be get final product.
embodiment 3:
A kind of preparation method that is used for the treatment of the medicine of cutaneous squamous cell carcinoma, comprise the steps:
(1) 6 parts of the Radix Pulsatillaes, 2 parts of Rhizoma Curcumae, 2 parts, Rhizoma Zingiberis Recens, add the ethanol extraction three times of 12 times of medical material weight amounts 70%, each 2h;
(2) merging filtrate, be condensed into 1 to 1.3 fluid extract, puts in 65-75 ℃ of calorstat and dry to becoming hard crisp, and cold drying is preserved and be get final product.
test example:
1 effect experiment that makes medicine by the following examples further proves beneficial effect of the present invention:
1 material
1.1 medicine embodiment 1 makes medicine, every ml is equivalent to crude drug 2g, usings anemoside B4 content and extractum yield as quality control standard (anemoside B4 extraction ratio 89.10, dry cream rate 38.4, comprehensive scores 63.75), and cryopreservation is standby.
1.2 the DMEM of the high sugar of reagent, hyclone are purchased from Hyclone company, MTT is purchased from Amresco company (201105).
1.3 cell line A431 cell is purchased from Wuhan University's Chinese Typical Representative culture collection center.
2 methods
2.1 cell culture A431 cell culture in containing in the DMEM in high glucose culture medium of 10% hyclone, penicillin (100U/L) and streptomycin (100U/L), is placed in the incubator of 5%CO2,37 ℃ and saturated humidity.
2.2 mtt assay is measured embodiment 1 and made medicine to the take the logarithm cell of trophophase of the inhibited proliferation of A431 cell, the adjustment cell density is 1.25 * 105/ml, and 160 μ l/ holes are inoculated in 96 orifice plates.After cultivating 24h, every hole adds the medicine of 40 μ l variable concentrations, and drug level is established 5 gradients, is respectively 1/400,1/200,1/100,1/50,1/25 of former medicine.Establish blank zeroing group (adding 200 μ l serum-free mediums), negative control group (not adding medicine), positive controls (5-Fu10 μ g/ml) simultaneously.Because embodiment 1 makes medicine, color is arranged, add variable concentrations color matched group.After cultivating respectively 24h, 48h, 72h, supernatant discarded, add 200 μ l serum-free mediums and 20 μ lMTT(5mg/ml), after being placed in CO2 gas incubator cultivation 4h, abandon supernatant, add 150 μ lDMSO, after vibration 10min, measure absorbance (OD value) in enzyme-linked immunosorbent assay instrument 490nm place.The experiment triplicate.
The blank zeroing group of inhibitory rate of cell growth (%)=1-(experimental group OD-color matched group OD/ negative control group OD-OD) * 100%
2.3 flow cytometer detects and analyzes the cell of collecting exponential phase, cell density with 6.25 * 105/ml is inoculated in Tissue Culture Flask, after cultivating 48h, treat cell cover with bottle at the bottom of about 50%-60%, random packet, each group gives corresponding intervention factor, and negative control group adds the equivalent serum-free medium.After cultivating 48h, collect medication group and negative control group and respectively organize cell conditioned medium, with after trypsin digestion cell, centrifugal together with supernatant, 1000r/min, 3min.
The adjustment cell density is 1.0 * 106/ml, and careful supernatant discarded adds PBS and cleans 2 times, adds 1ml ice ethanol, and-20 ℃ are fixedly spent the night.The negative control pipe adds 100 μ l cell suspension, and the every pipe of sample tube adds 2.5 μ l Annexin V/FITC, 2.5 μ lPI, adds 100 μ l cell suspension, mixes rear room temperature lucifuge and hatches 30min.The employing flow cytometer detects.
2.4 statistical method adopts SPSS13.0 software to carry out statistical analysis, all measurement datas are used
mean, relatively adopt one factor analysis of variance between many groups, all checks are two-sided test, and using α=0.05 as inspection level.P<0.05 o'clock is thought statistical significance.
3 results
3.1 mtt assay is measured embodiment 1 and is made the inhibited proliferation of medicine to the A431 cell
In concentration, be 1/400,1/200,1/100,1/50,1/25 act on A431 cell 24h, 48h, 72h after, with negative control group relatively, each concentration group all has inhibited proliferation to the A431 cell, and, along with the prolongation with action time of increasing of drug level, its inhibited proliferation to the A431 cell more obviously.In Table 1.
Table 1 embodiment 1 makes the inhibited proliferation (n=5) of medicine to the A431 cell
With negative control group, compare, ▲ ▲ P<0.01
3.2 morphological observation
Inverted microscope is observed the metamorphosis of variable concentrations drug effect after A431 cell 48h.The growth of negative control group cell attachment, form is polygon or irregular shape, and nuclear membrane is clear, and refractivity is strong, and Growth of Cells is good.Fusion phenomenon appears in 1/100 group of part cell, and pyknosis appears in 1/25 group of cell, dissolves the cell mortality.Along with drug level is higher, cell detachment is more obvious
3.3 the two transfect cells of Annexin V/PI detect apoptosis rate
According to the flow cytometer testing result, its left upper quadrant UL represents the cell debris that mechanical injuries cause; Right upper quadrant UR represents non-viable apoptotic cell or dead cell; Left lower quadrant LL represents negative normal cell; Right lower quadrant LR represents viable apoptotic cell.The flow cytometer testing result shows, with negative control group, compares, and compound Chinese medicinal preparation group G0/G1 phase cell increases than negative control group, S phase, G2/M phase Leukopenia.Wherein the compound Chinese medicinal preparation high dose group G0/G1 phase increases than negative control group, and the S phase reduces than negative control group, has significant difference (P<0.05).With negative control group, compare, compound Chinese medicinal preparation group apoptosis rate, apparently higher than negative control group, has significant difference (P<0.05).
Embodiment 1 makes the impact of medicine on A431 Cellular cycle and apoptosis rate
Compare ▲ P<0.05 with negative control group
4 conclusions
Skin carcinoma is the same with other malignant tumor, is also the process of a multi-step, multifactor participation.And wherein one of important reason is exactly that cell cycle and apoptosis regulation are unbalance.
Cell cycle by DNA the presynthetic phase (G1 phase), DNA synthesis stage (s phase), DNA post-synthetic phase (G2 phase), mitotic phase (M phase) form.In cell generation cycle, G1 phase, s phase, G2 phase, M phase are played very important effect separately.In addition, cell can also stop mitosis from G1 phase cell cycle away from keyboard, enters G0 phase (resting stage).Accepting external signal at G0 phase cell stimulates, and determines that cell enters the S phase and carries out DNA replication dna and complete division malignant proliferation occurring or entering the stage of latency G0 phase.If stop cell to enter the S phase by the G0 phase, the infinite multiplication of tumor cell can be controlled.
Apoptosis is a kind of spontaneous programmed cell death process that body cell occurs under normal physiological or pathological state.Apoptosis, as a kind of major way of cell death, has considerable status in neoplastic process.
The Flow cytometry cell cycle each the time distribute mutually and apoptosis rate, be the biological study of tumor molecule, an important technology in the antitumor drug Mechanism Study.In recent years, increasing scholar applies modern Protocols in Molecular Biology Chinese medicine antineoplastic mechanism is furtherd investigate.In this experiment, embodiment 1 makes medicine has obvious inhibited proliferation to the A431 cell, and, along with the increase of drug level and the prolongation of action time, its suppression ratio is more obvious.Further by flow cytometer, detect the variation that embodiment 1 makes Cellular cycle and apoptosis rate after drug effect, result shows, with matched group relatively, G0/G1 phase cytosis, S phase Leukopenia.Prompting embodiment 1 makes medicine and stops tumor cell in the G0/G1 phase, and inhibition tumor cell enters the S phase.And, along with the increasing of drug level, the apoptosis rate of tumor cell also increases.
In sum, embodiment 1 makes the propagation that medicine can significantly suppress skin scale cancer A431 cell, and simultaneously it significantly induces the apoptotic effect of A431 in addition, and can hinder cell processed and rest on the G0/G1 phase, makes the DNA biosynthesis block of S phase, the propagation of inhibition tumor cell.
The above, it is only preferred embodiment of the present invention, not the present invention is done to any pro forma restriction, any technical solution of the present invention content that do not break away from, any simple modification, equivalent variations and the modification above embodiment done according to technical spirit of the present invention, all still belong in the scope of technical solution of the present invention.
Claims (3)
1. a medicine that is used for the treatment of cutaneous squamous cell carcinoma, its parts by weight of raw materials proportioning is:
Radix Pulsatillae 4-6 part, Rhizoma Curcumae 2-4 part, Rhizoma Zingiberis Recens 1-3 part.
2. a kind of medicine that is used for the treatment of cutaneous squamous cell carcinoma as claimed in claim 1, its parts by weight of raw materials proportioning:
5 parts of the Radix Pulsatillaes, 3 parts of Rhizoma Curcumae, 2 parts, Rhizoma Zingiberis Recens.
3. as claim 1 or described a kind of preparation method that is used for the treatment of the medicine of cutaneous squamous cell carcinoma, comprise the steps:
(1) add the ethanol extraction three times of 12 times of medical material weight amounts 70%, each 2h;
(2) merging filtrate, be condensed into 1 to 1.3 fluid extract, puts in 65-75 ℃ of calorstat and dry to becoming hard crisp, and cold drying is preserved and be get final product.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112451674A (en) * | 2020-12-29 | 2021-03-09 | 西安交通大学 | Skin squamous cell carcinoma treatment preparation |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1961954A (en) * | 2005-11-08 | 2007-05-16 | 上海中药制药技术有限公司 | Externally applied transdermal Chinese medicinal formulation for treating skin cancer |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1961954A (en) * | 2005-11-08 | 2007-05-16 | 上海中药制药技术有限公司 | Externally applied transdermal Chinese medicinal formulation for treating skin cancer |
Non-Patent Citations (3)
| Title |
|---|
| 佚名: "生姜能防皮肤癌", 《生物学通报》 * |
| 潘新社等: "白头翁生物活性研究现状及展望", 《西北农业学报》 * |
| 钟华等: "不同莪术提取物对小鼠皮肤癌的药效研究", 《时珍国医国药》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112451674A (en) * | 2020-12-29 | 2021-03-09 | 西安交通大学 | Skin squamous cell carcinoma treatment preparation |
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