Lignum Sappan extract prepares infusion liquid preparation technology and the purposes on the treatment bladder cancer thereof
Technical field
The present invention relates to a kind of preparation method of Lignum Sappan extract, be specially a kind of Lignum Sappan extract infusion liquid preparation technology and the purposes on the treatment bladder cancer thereof.
Background technology
Lignum Sappan (Caesalpinia sappan Linn.) has another name called Soviet Union's a tree, used in making timber for boats, the fragrant lumps of wood, the Soviet side.The Lignum Sappan heartwood can be used as medicine, and more than felling autumn, removes white sapwood, drying, that is and, dry duramen is elongated cylindrical or to cuing open half round post, and long 10-100 centimetre, diameter 3-13 centimetre.The surface yellowish red color to brownish red, tool cutter sheeter lines, common longitudinal crack.Matter is hard.Section is tool gloss slightly, and annual ring is obvious, the marrow of the visible burgundy that has, matter pine, band bright star, and feeble QI, it is little puckery to distinguish the flavor of.Nature and flavor are sweet, salty, flat.GUIXIN, liver, spleen channel.Has the promoting the circulation of blood blood stasis dispelling, the effect of reducing swelling and alleviating pain.Being used for amenorrhea dysmenorrhea, postpartum stagnation, breast ventral spine pain, wound swells and ache etc.
Modern medicine study finds that Lignum Sappan extract has anti-tumor activity in the animal body.Especially directly extract the brazilin that obtains from nature about Lignum Sappan extract, also have many pieces of papers and patent to deliver at the research report aspect the treatment tumor.For example the patent No. is 200610025401.8 to disclose a kind of Brazil wood chlorins compound in the application of preparation in the antitumor drug, this patent disclosure the molecular structure of Brazil wood chlorins compound, and the plant extract that contains effective ingredient is in the treatment leukemia, renal carcinoma, hepatocarcinoma, pulmonary carcinoma, rectal cancer, gastric cancer, esophageal carcinoma, and breast cancer application.In the disclosed content of its description, embodiment has only provided the brazilin that the extracts inhibition effect to various cancerous cell in experiment in vitro from Lignum Sappan, and specifically the killing effect of cancerous cell in certain one is not really furtherd investigate.
Bladder cancer is one of more common tumor of whole body, accounts for about 10% of all malignant tumor, is the modal malignant tumor of China's urinary system.Primary disease male pilosity, the ratio of men and women's sickness rate is about 4: 1, and age of onset is many more than 40 years old, and sickness rate increases with the age increase.Increase to some extent but send out a patient in recent years below 30 years old, the patient about 20 years old has finding also the time, and total sickness rate is on the rise.Among the first patient who goes to a doctor 70%-80% for the shallow property bladder cancer of table (Superficial Transitional CellCarcinoma, STCC).Show shallow property bladder cancer and be meant all tumors of bladder that comprise cancer in situ (Tis), Ta and Tl phase, no matter the height of tumor differentiation degree is not all invaded and tunica muscularis vesicae urinariae.Though transurethral resection or partial cystectomy can be excised primary tumo(u)r, because multicentricity and the multiple characteristics of STCC, relapse rate is up to 30%-85%, and recurrence in most postoperative 1 year.There is 30%-40% to make progress among the patient of recurrence and is invasive bladder cancer.
For the postoperative recurrence of prophylaxis of tumours, the mode that generally adopts is behind the excision primary lesion at present, the bladder antitumor drug perfusion therapy that continues.The main purpose of bladder instillation to treat is as follows: 1, cancer therapy drug can directly act on tumor in intravesical long period high concentration.2, can kill the tumor cell of intravesical postoperative residue, prevent the tumor cell plantation, reduce the possibility of recurrence.3, can reduce the toxic and side effects of systemic administration.4, can keep bladder, not only life is convenient, but and retention function.5, the progress that stops the malignancy of tumor degree.
The medicine of postoperative bladder instillation to treat commonly used is divided into two big classes: chemotherapeutics and immunomodulator class medicine.Chemotherapeutic mainly comprises mitomycin (MMC), epirubicin and pirarubicin etc., it is a domestic line medication at present, external multicenter prospective control study proof can both reduce (≤2 years) relapse rate in the recent period in various degree, but not obvious to the late relapse rate (〉=5 years) and the improvement of survival rate.
One of major reason of analyzing influence effect is exactly the drug resistance of tumor cell, comprises primary drug resistance and acquired drug-resistance, reduces even has eliminated effect of drugs.Now external to use more medicine be bacillus calmette-guerin vaccine (BCG), is that one of the most effective medicine of shallow property bladder cancer is shown in treatment at present and prevention.It is advantageous that and to reduce recent relapse rate, stop tumour progression simultaneously, thereby improved late result.But because the BCG toxic and side effects is big, can not behind tumor resection, use immediately, thereby influence the effect of prophylaxis of tumours cell seeding.
Although the report of Lignum Sappan extract aspect the treatment tumor arranged, also do not have at present to find about Lignum Sappan extract at the research report aspect the treatment bladder cancer.
Summary of the invention
The present invention is in order to solve Lignum Sappan extract and to provide a kind of Lignum Sappan extract to prepare infusion liquid preparation technology in the application aspect the treatment bladder cancer tumor and in the purposes aspect the treatment bladder cancer.
The present invention is realized by following technical scheme, a kind of Lignum Sappan extract infusion liquid preparation technology, step comprises, getting Lignum Sappan pulverizes, for the first time add 3-6 times of weight purified water, boil extraction and filtered to get filtrate in 45-65 minute, add 2-4 times of weight purified water then for the second time, boil extraction and filtered to get filtrate in 30-50 minute, add 1.5-2.5 times of weight purified water then for the third time, boil and extract the 25-35 branch; Merge the extracting solution that obtains for three times, concentrating under reduced pressure is to 2 times of weights of Lignum Sappan crude drug amount; Standing over night is removed precipitation; Add petroleum ether again, extraction keeps water; Add ethyl acetate again, water is removed in extraction; The decompression volatilization is ethyl acetate to the greatest extent; Weighing dry substances adds purified water, and making dry matter content is 2%, heating for dissolving, and the room temperature standing over night removes by filter precipitate; Lyophilizing, packing gets final product.
During use lyophilized powder is become infusion liquid with distilled water diluting.It is little, safe that this infusion liquid has toxic and side effects, to characteristics such as the tumor cytotoxicity effect is remarkable.Can be used as preparation treatment bladder cancer medicine.
Experimental studies results shows that this extract has significant lethal effect to the tumor of bladder cell.With multiple anticarcinogen such as clinical mitomycin commonly used, epirubicin, pirarubicin relatively, have the active anticancer height, plurality of advantages such as toxicity is little, and is safe and effective.
Through preliminary clinical observation, this medicine does not have tangible untoward reaction, and clinical efficacy is remarkable at no distant date.Particularly clinical common intractable, recurrent tumor of bladder there are better therapeutic effect, are better than antitumor drug commonly used at present.It is a kind of new antitumor drug with good DEVELOPMENT PROSPECT.
The antitumor action of extract
The experiment of 1 cell killing
Material: cell source H22 cell strain draws the Experimental Animal Center from Hebei Medical University, and the S180 cell strain draws from Chinese pharmaceutical biological product identifies institute, and two kinds of cells are preserved voluntarily by my chamber, and ascites cells is used in the abdominal cavity inoculation; Human oophoroma cell line SKOV3 is so kind as to give by Tumour Inst., Chinese Medical Academy's epi chamber; Bladder cancer cell line T24 is available from Shanghai life science institute of the Chinese Academy of Sciences; Lymphoma produces by this laboratory mice is spontaneous.Experiment medicine mitomycin for inj: cisplatin for inj (freeze-dried type): hydroxycamptothecin injection: Lignum Sappan extract (hereinafter to be referred as Br-1), provide for oneself, be mixed with desired concn with water for injection.
The results are shown in following table
The various medicines of table 1 are to the lethal effect of T24 cell
The various medicines of table 2 are to the lethal effect of SKOV3 cell
The various medicines of table 3 are to the lethal effect of H22 cell
The various medicines of table 4 are to the lethal effect of S180 cell
The various medicines of table 5 are to the lethal effect of lymphoma cell
The experiment of 2 apoptosis
Material bladder cancer cell line T24 is available from Shanghai life science institute of the Chinese Academy of Sciences, 1640 culture medium available from U.S. Gibco company hyclone available from Hangzhou Sijiqing Biological Engineering Material Co., Ltd..Br-1 extracts preparation by this laboratory from the Chinese crude drug Lignum Sappan.The apoptosis test kit is available from U.S. company BD.The fluidic cell detector, U.S. B-D company produces, model FACSCalibur.The method cell culture is in the PRMI1640 culture medium that contains 10% hyclone, in 5%CO
2The cell of logarithmic growth is chosen in the cultivation of going down to posterity in 37 ℃ of incubators, add medicinal liquid after concentration reach 0,30 μ g/ml respectively, 60 μ g/ml, positive control drug are mitomycin, content is 60 μ g/ml.Digest collecting cell behind drug effect 6h, PBS washes twice, and nylon mesh is filtered, and adds apoptosis reagent A nnexin V-FITC, and Propidium Iodide goes up machine testing apoptosis situation behind the lucifuge 15min.
As a result, cellular control unit early apoptosis rate is 0.15%, and late period, apoptosis rate was 0.21%, and total apoptosis rate is 0.36, belongs to the normal apoptotic of cell, and dead cell ratio is 13.34%; The apoptosis rate in early stage, late period of 30 μ g/ml dosage group cells is respectively 0.36% and 0.99%, and total apoptosis rate is 1.35, than matched group tangible increase is arranged, and the mortality rate of cell also is increased to 55% simultaneously, and target cell is shown the obvious suppression effect; The apoptosis rate in late period of 60 μ g/ml dosage group cells reaches 36.46, shows tangible promotion apoptotic effect, and the mortality rate of cell also rises to 63.05%, shows that medicine has significant apoptosis-induced and lethal effect to target cell.Total apoptosis rate of positive controls cell is 1.27%, shows certain facilitation.(seeing Fig. 1,2,3,4)
3 SABC
The material cell strain: the SK-OV-3 cell is the human ovarian cancer source, is so kind as to give by Tumour Inst., Chinese Medical Academy's epi chamber.Main agents: 1640 culture medium available from U.S. Gibco company hyclone available from Hangzhou Sijiqing Biological Engineering Material Co., Ltd..Br-1 extracts preparation by this laboratory from the Chinese crude drug Lignum Sappan.On the coverslip of method cell inoculation in six orifice plates, when treating its well-grown, add medicinal liquid, concentration is respectively 0,10 μ g/ml, 20 μ g/ml, and 40 μ g/ml, 80 μ g/ml, positive control drug are cisplatin, concentration is 60 μ g/ml.Behind the drug effect 24h, PBS slowly cleans, and 4% paraformaldehyde is fixed, and adds caspase3 behind the distilled water flushing respectively, caspase9, and the corresponding dilution one of survivin resists, and 4 ℃ are spent the night.Take out room temperature next day and place 10min, PBS washes 5min, 3 times; Drip two and resist 37 ℃ of 30min; PBS washes 5min, 3 times; The DAB colour developing is breathed out the Rui Shi haematoxylin and is redyed nucleus, the acidic alcohol differentiation, and mirror control down, tap water returns blue 10min; Dehydration, transparent: 70%, 80%, 90%, 100% ethanol (1), (2) each 5min, dimethylbenzene (1), (2) each 10min, the neutral gum mounting, high power lens is observed down.
4.2 the result is as schematically shown in Figure 5,
Fig. 5 A1-A6 analyzes explanation:
1. cellular control unit expression of apoptosis protein feminine gender only has extremely discrete Cytoplasm to have shallow table painted;
2.25 μ g/ml group and matched group no significant difference;
The weak positive expression of 45% cell 3.50 μ g/ml group is had an appointment, cytoplasm is painted;
4.100 μ g/ml group has the cell positive more than 90% to express the Cytoplasm engrain;
5.200 μ g/ml organizes about 90% cell strong positive expression, the equal engrain of Cytoplasm and nucleus;
6. the cisplatin group compares no significant difference with matched group, shows that the apoptosis of pair cell does not have obvious influence.
The explanation of Fig. 5 B1-B6:
1. drug level compares no significant difference with matched group when 50 μ g/ml are following, shows the variation that can not cause apoptosis protein Casepase3 under this concentration;
2. drug level reaches 100 μ g/ml when above, and Cytoplasm dyeing obviously increases, and prompting medicine under this concentration can produce a large amount of apoptotic proteins by inducing cell, thereby causes cell generation apoptosis;
3. cisplatin group cell and matched group compare no significant difference, show that medicine pair cell apoptosis does not produce tangible influence
Fig. 5 C1-C6 explanation:
Cellular control unit nuclear and Cytoplasm all have significantly painted, illustrate Apoptosis Inhibitor Protein Survivin in normal cell, express stronger, the apoptosis generation inhibitory action of pair cell.
2. the expression of Apoptosis Inhibitor Protein Survivin all is suppressed in each dosage group cell of administration, shows that the apoptosis of medicine pair cell has facilitation.But there is not evident difference between each dosage group.
Infusion liquid of the present invention is to the therapeutical effect of the first shallow property of the table tumor of bladder of sending out
Intend the shallow property of systematic observation table tumor of bladder patient 60 examples, the observation period is limited to 24 months.Method: postoperative continous pouring 8 times; Since the 3rd week, pour into continous pouring 8 times weekly 1 time; Next perfusion in every month is 1 time, continous pouring 10 times.Patient's medicining condition, untoward reaction and clinical efficacy in the itemized record observation period, respectively at postoperative March, June, JIUYUE, December, row cystoscopy 18 months, 24 months the time, and make itemized record, carry out curative effect relatively with existing clinical chemistry commonly used and biotherapeutics.
6.2 therapeutical effect to the recurrent tumor of bladder
Plan adopts the PGF perfusion therapy to using conventional chemotherapy medicine and the out of contior postoperative recurrence patient of biotherapeutics.Primary part observation PGF is to the therapeutical effect of recurrent tumor of bladder.Therapeutic Method is the same, and observing quantity is 60 examples.
PGF prevention superficial bladder cancer clinical observation statistical table
Description of drawings
Fig. 1 matched group: apoptosis rate is 0.36%
Figure 22 0 μ g/ml dosage group, apoptosis rate is 1.35%
Figure 38 0 μ g/ml dosage group, apoptosis rate is 36.76%
Fig. 4 positive controls, apoptosis rate are 1.27%
Fig. 5 A (1-6) is the influence of Br-1 to Caspase9
Fig. 5 B (1-6) is influence to Caspase3 for Br-1
Fig. 5 C (1-6) is the influence of Br-1 to Survivin
Fig. 6 is a thin-layer chromatogram
1 row-HAEMATOXYLIN standard substance, 2 row-PGF samples, 3 row-acetic acid ethyl ester extracts, 4 row-n-butyl alcohol extracts, 5 row-acetic acid ethyl ester extracts through 4, the 6 row-acetic acid ethyl ester extracts of the fraction behind the column chromatography through the fraction 6 of 5, the 7 row-acetic acid ethyl ester extracts of the fraction behind the column chromatography behind column chromatography
The liquid chromatogram of Fig. 7 PGF water extract
Fig. 8 a acetic acid ethyl ester extract liquid chromatogram
Fig. 8 b n-butyl alcohol extract liquid chromatogram
Fig. 9 column chromatography separating ethyl acetate extract
The liquid chromatogram of the fraction 5 of Figure 10 b acetic acid ethyl ester extract behind column chromatography
The liquid chromatogram of the fraction 6 of Figure 10 c acetic acid ethyl ester extract behind column chromatography
The liquid chromatogram of the fraction 7 of Figure 10 d acetic acid ethyl ester extract behind column chromatography
Figure 11 column chromatography is separated n-butyl alcohol extract
The liquid chromatogram of the fraction 3 of Figure 12 a n-butyl alcohol extract behind column chromatography
The liquid chromatogram of the fraction 4 of Figure 12 b n-butyl alcohol extract behind column chromatography
The liquid chromatogram of the fraction 6 of Figure 12 c n-butyl alcohol extract behind column chromatography
The proton nmr spectra of Figure 13 fraction 4
The carbon-13 nmr spectra of Figure 14 fraction 4
The hydrocarbon relevant spectrum of Figure 15 fraction 4
The hydrocarbon relevant spectrum of Figure 15 fraction 4
The uv-visible absorption spectra of Figure 16 fraction 4
The infrared spectrum of Figure 17 fraction 4
The infrared standard spectrum of Figure 18 brazilin
Figure 19 acetic acid ethyl ester extract is through the structural formula of column chromatography tails 4
The specific embodiment
Embodiment 1, a kind of irrigation of bladder liquid preparing process, step comprises, getting Lignum Sappan pulverizes, add for the first time 5 times of weight purified water, boil extraction and filtered to get filtrate in 60 minutes, add 3 times of weight purified water for the second time then, boil extraction and filtered to get filtrate, add for the third time then 2 times of weight purified water in 40 minutes, boil and extract the extracting solution that merging in 30 fens obtains for three times, concentrating under reduced pressure is to 2 times of weights of crude drug amount; Standing over night is removed precipitation; The petroleum ether that adds the long-pending amount of monoploid again, extraction keeps water; The ethyl acetate that adds the long-pending amount of triploid again, water is removed in extraction; The decompression volatilization is ethyl acetate to the greatest extent; Weighing dry substances adds purified water, and making dry matter content is 2%, heating for dissolving.The room temperature standing over night removes by filter precipitate; Lyophilizing, packing gets final product.
Below be the qualification result to effective ingredient in the extracting solution: PGF represents described extracting solution in the following content.
As can be seen from Figure 6: HAEMATOXYLIN mark content is very little in the PGF sample; At least contain four components in the PGF sample; And the ratio of component PGF sample component of ethyl acetate and n-butyl alcohol extract is relative less, illustrates with extraction to have played certain separation purification effect; Do not contain component 2 in the n-butyl alcohol extract, illustrate that ethyl acetate has played enrichment to component 2.
High performance liquid chromatography is separated PGF, efficient liquid phase chromatographic analysis result: PGF sample, acetic acid ethyl ester extract and n-butyl alcohol extract are carried out liquid-phase chromatographic analysis, result such as Fig. 7, shown in Figure 8.As can be seen from Figure 7, have 9 components in the PGF water extract at least, what wherein content was bigger has four, and its appearance time is respectively at 9.8min, 12.1min, 15.8min and 19.0min.
As can be seen from Figure 8, acetic acid ethyl ester extract is mainly 9min and 12min component, the 9min component disappears substantially in the n-butyl alcohol extract, be mainly 12min, 15min and 19min component, can illustrate also that with can be behind the ethyl acetate extraction result of this and Thin-layer separation is in full accord with the enrichment of 9min component.
3. column chromatography is separated the chemical constituent among the PGF, column chromatography separating ethyl acetate part: ethyl acetate is partly carried out column chromatography separate, the result as shown in Figure 9.And detect the purity of each fraction with high performance liquid chromatography, the result as shown in figure 10,
As can be seen from Figure 10, the purity of fraction 4 is higher, can reach more than 90%; Fraction 6 is consistent with the main constituent of fraction 7, and wherein the purity of fraction 7 is higher, can reach more than 90%; Fraction 5 purity are relatively poor, are mainly 14min and 17min component, also need further separation and purification.
Column chromatography is separated the n-butyl alcohol part, and the result as shown in figure 11.And detecting the purity of each fraction with high performance liquid chromatography, the result is as shown in figure 12.
As can be seen from Figure 12, n-butyl alcohol is relatively poor to extract purity of each fraction behind column chromatography, needs further separation condition to be selected.
The structure of 4 chemical compounds is identified, ins all sorts of ways the fraction 4 of ethyl acetate behind column chromatography carried out the structure evaluation, and the result is shown in Figure 13-17.
Fraction 4 gets red crystalline powder after boiling off solvent.
1H NMR (300MHz, solvent: D
2O) show two methene proton δ 2.78 (1H, d, J=16.2Hz, H-7), 2.98 (1H, d, J=16.2Hz, H-7 '); Two even oxygen methene proton δ 3.61 (1H, d, J=11.7Hz, H-6), 3.91 (1H, d, J=11.7Hz, H-6 '); Methine protons δ 3.7 (1H, S, H-11b); And five fragrant proton: δ 6.32 (1H, S, H-4); Belong to the AB coupling δ 6.54 (1H, d, J=8.4Hz, H-2), 7.27 (1H, d, J=8.4Hz, H-1); Be positioned at the phenyl ring para-position δ 6.69 (1H, S, H-8), 6.73 (1H, S, H-11).
13C NMR (300MHz, solvent: D
2O) show 16
13The C signal, δ ppm:41.63 (C-7), 49.93 (C-12), 70.23 (C-6), 77.94 (C-6a), 104.22 (C-4), 110.60 (C-2), 112.92 (C-11), 113.96 (C-8), 115.59 (C-1a), 132.39 (C-7a), 132.74 (C-1), 137.38 (C-11a), 143.82 (c-10), 144.08 (C-9), 154.46 (C-3), 156.24 (C-4a).By above information and document
[3]Unanimity, and infrared absorption spectroscopy is consistent with standard spectrogram (Figure 18), and authenticating compound 3 is a brazilin, and structure is as shown in figure 19.
Embodiment 2, a kind of irrigation of bladder liquid preparing process, step comprises, getting Lignum Sappan pulverizes, add for the first time 3 times of amount purified water, boil extraction and filtered to get filtrate in 65 minutes, add 2 times of amount purified water then for the second time, boil extraction and filtered to get filtrate, add for the third time then 1.5 times of amount purified water in 50 minutes, boil and extracted 35 fens; Merge the extracting solution that obtains for three times, concentrating under reduced pressure is 2 doubly heavy to Lignum Sappan crude drug amount; Standing over night is removed precipitation; The petroleum ether that adds doubling dose again, extraction keeps water; Add four times of ethyl acetate again, water is removed in extraction; The decompression volatilization is ethyl acetate to the greatest extent; Weighing dry substances adds purified water, and making dry matter content is 2%, heating for dissolving, and the room temperature standing over night removes by filter precipitate; Lyophilizing, packing gets final product.Structure and composition qualification result are with embodiment 1.
Embodiment 3, a kind of irrigation of bladder liquid preparing process, step comprises, getting Lignum Sappan pulverizes, add for the first time 6 times of amount purified water, boil to extract and filtered to get filtrate in 45 minutes, add for the second time 4 times of amount purified water then, boil extraction and filtered to get filtrate, add for the third time then 2.5 times of amount purified water in 30 minutes, boil and extracted 25 fens; Merge the extracting solution that obtains for three times, concentrating under reduced pressure is 2 doubly heavy to Lignum Sappan crude drug amount; Standing over night is removed precipitation; Add 1.5 times of amount petroleum ether again, extraction keeps water; The ethyl acetate that adds 2 times of amounts again, water is removed in extraction; The decompression volatilization is ethyl acetate to the greatest extent; Weighing dry substances adds purified water, and making dry matter content is 2%, heating for dissolving, and the room temperature standing over night removes by filter precipitate; Lyophilizing, packing gets final product.Structure and composition qualification result are with embodiment 1.