CN101019835A - Lignum sappan and its prepn process and application - Google Patents
Lignum sappan and its prepn process and application Download PDFInfo
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- CN101019835A CN101019835A CN 200710037958 CN200710037958A CN101019835A CN 101019835 A CN101019835 A CN 101019835A CN 200710037958 CN200710037958 CN 200710037958 CN 200710037958 A CN200710037958 A CN 200710037958A CN 101019835 A CN101019835 A CN 101019835A
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Abstract
The present invention discloses one kind of lignum sappan liposome and its preparation process and application. The lignum sappan liposome has granularity of 5 nm-1 micron and contains water solution of lignum sappan water extract. The preparation process includes ultrasonic wave treatment of water solution of lignum sappan water extract and liposome until the fat layer in the supernatant disappears. The lignum sappan liposome possesses anticancer effect of inhibiting the formation of tumor cell colony and promoting tumor apoptosis. Compared with lignum sappan water extract without liposome, the lignum sappan liposome has small dosage and less toxicity.
Description
Technical field
The present invention relates to a kind of Chinese medicine preparation and its production and application, specifically, is lignum sappan that has active anticancer and its production and application.
Background technology
Cancer is one of disease that mortality rate is the highest in the global range, and doctor trained in Western medicine cooperates radiation and chemotherapy based on operative treatment, and effect is very not satisfactory.At present, worldwide, the research of cancer therapy drug turns to natural animal-plant gradually, and the screening PTS has become the focus of cancer therapy drug research and development in recent years from the natural goods Chinese herbal medicine.High curative effect, hypotoxicity, high bioavailability that while is made the new drug dosage form of carrier with liposome have also shown obvious superiority in treatment for cancer.Confirmed that at present multiple chemotherapeutics is to play antitumaous effect by cell death inducing in vivo and in vitro, tumor cell restriction that the sensitivity and the drug resistance of chemotherapeutics also is subjected to Apoptosis Mechanism simultaneously.Therefore, seeking specifically, the medicine and the Therapeutic Method of inducing apoptosis of tumour cell have become one of research focus of current oncology.Aspect treatment for cancer, the traditional Chinese medicine of China has long history.
(lignum sappan LS) is the dry duramen of leguminous plant Lignum Sappan (Caesalpinia sappan L), sweet in the mouth to Lignum Sappan, little suffering, energy promoting blood circulation by removing blood stasis, reducing swelling and alleviating pain, be clinical blood circulation promoting and blood stasis dispelling Chinese herbal medicine commonly used, contain brazilin, Brazil wood oxide and Lignum Sappan phenol, structure is as follows.Be mainly used in diseases such as treatment gastrointestinal cancer, hepatocarcinoma, cervical cancer and cancerous pain clinically.
Brazilin brazilin oxide Lignum Sappan phenol
The aqueous extract of lignum sappan main component is a brazilin, and it also is a kind of main component of histochemical stain.
Zoopery shows that Lignum Sappan has certain antitumor action.Discover that Lignum Sappan decocting liquid has the obvious suppression effect external to the inductive human B lymphocyte hypertrophy of SAC, to the inductive T lymphocytosis of PHA with induce IL one 2 activity the obvious suppression effect is arranged.Also can produce stronger inhibitory action in vivo to the activity of mice bone-marrow-derived lymphocyte, the lymphocytic propagation of T and IL 1.One of anticancer mechanism of Chinese medicine Lignum Sappan is to bring out apoptosis.But because the extremely complicated process that apoptosis is a multistage, multisystem to be participated in, Lignum Sappan inducing cell generation mechanism of apoptosis is still waiting further research.And, in order to improve the anticancer therapeutic index of Lignum Sappan, reduce the therapeutic dose of medicine and reduce the toxicity of medicine, just need a kind of good, novel medicament carrier of utilization.
Liposome is a kind of targeted drug carrier, a kind of new Types of Medicine that belong to targeting drug delivery system (targeting drug delivery system), has class cellularity (a kind of vesicle, the vesicle wall is that two-layer phospholipid molecule constitutes, water solublity and fat-soluble two types of medicines all can be wrapped in the same liposome), enter the autoimmune function that is mainly activated body in the body by reticuloendothelial system phagocytic, and change by distribution in the body of bag medicine, drug main will be liver, spleen, lung, in the histoorgans such as bone marrow, thereby accumulate the therapeutic index that improves medicine, reduce the toxicity of the therapeutic dose and the reduction medicine of medicine, liposome has shown obvious superiority in numerous disease especially treatment for cancer.
Application and empirical continuous accumulation along with advanced technology, many chemical classes medicinal liposomes successfully go on the market: nineteen ninety-five, long circulation immunity liposome occurred, first DNA liposome bacterin listing in 1997, the interferon liposome of sea, Shenzhen in 2004 king Ying Telong company obtains the clinical research certification, in JIUYUE, 2004, make a breakthrough as the BLP25 proteoliposome of the pharmaceutical grade protein liposome immunne response of inducing cancer cell (be used for), by the quick tracing program of FDA (fast track programs), will and enter III phase clinical experiment as the vaccine listing.
The liposome research of China starts from the beginning of the eighties at the end of the seventies in last century.Through research and the application facet of recent two decades, obtained gratifying achievement at liposome.At present the existing a plurality of new drug dosage forms of making carrier with liposome of China have entered the clinical verification stage, liposome entrapment amycin and liposome entrapment paclitaxel are wherein arranged, and, in addition, also has more new drug in research and development recently in the application of SARS vaccine development.
List of references:
1. Han Rui, cancer therapy drug research and experiment technology [M], Beijing: Beijing Medical University, combined publication society of consonance medical university, 1998,280.
2. Si Tuzhen is strong, Wu Junzheng, cell culture [M]. Xi'an: world book publishing company, 1996,137.
3. Zhouning County is peaceful, Zhu Xiaofeng, Li Zhiming etc.Sugar apple lactone platform thing squamocin inducing leukemia HL-60 apoptosis [J], cancer.2000.19 (12) 1,098 one 1100
4. Chen Jie, Pan Qichao.The Drug resistance of apoptosis and tumor cell [J], cancer.1999.18(6)；739-741
5. Cheng Jian China, the Li Yibin chief editor, anticancer vegetable drug and proved recipe thereof [M], the 2nd edition, Jiangxi: the Jiangxi science and technology is published Du .1995:372-373.
6. appoint adhesion, silk ribbon is founded the state, Ma Junguo, etc.The research [J] of Soviet Union's art antitumaous effect.CHINA JOURNAL OF CHINESE MATERIA MEDICA, 1990,15 (5); 50-51
7. Yang Feng, Dai Guanhai, Shen Xiang, etc.Lignum Sappan is to the lymphopoietic inhibitory action of vitro human, Shanghai Journal of Immunology.1997，17(5)；212-215
8.BIOMIRA INC.Announces 3rd quarter results[EB/OL].[2004-10-28].http://www.biomira.com/news/detai lNewsRelease/204/
Summary of the invention
The object of the present invention is to provide a kind of liposome that wraps up aqueous extract of lignum sappan.
Another object of the present invention is to provide the method for this lignum sappan of preparation.
The present invention also aims to this lignum sappan is applied to prepare antitumor drug.
Technical scheme of the present invention is:
With the aqueous extract of lignum sappan liposome, make it have to a certain degree targeting, slow-releasing, can reduce drug administration dosage, alleviate drug toxicity, improve the stability of labile drug.
This experiment utilizes the liposome Lignum Sappan extract, and to select human leukemia K562 cell for use be target cell, from apoptotic angle, and by the blank experiment, the active anticancer of research lignum sappan.
Lignum sappan of the present invention, for having wrapped up the liposome of aqueous extract of lignum sappan solution, size is at 5nm-1 μ m.
Lignum sappan contains aqueous extract of lignum sappan solution 41%-55% (mass ratio).
The aqueous extract of lignum sappan solution concentration of being wrapped up is 50 μ g/ml-200mg/ml; Be preferably 10mg/ml-100mg/ml.
Preparation method is:
A. the aqueous solution with aqueous extract of lignum sappan mixes aquation with liposome;
B. supersound process is centrifugal;
C. repeating step b does not have adipose membrane sample material until the upper strata; Centrifugal again, abandon the outer unpacked aqueous extract of lignum sappan solution of supernatant weeding of grease plastid, use the normal saline washing precipitation, promptly obtained wrapping up the liposome of Lignum Sappan extract.
Used aqueous extract of lignum sappan concentration of aqueous solution is 50 μ g-200mg/ml, is preferably 10mg/ml-100mg/ml.
Used aqueous extract of lignum sappan solution, the mass ratio of contained aqueous extract of lignum sappan and liposome are 1: 2-1: 100, be preferably 1: 2-1: 30.
Aqueous extract of lignum sappan can obtain with conventional method.
The raw material of liposome can be selected phospholipid substance for use, as in soybean lecithin, Ovum Gallus domesticus Flavus lecithin, phospholipid dipalmitoyl phosphatidyl choline or the distearoyl phosphatidylcholine any, same cholesterol, fatsolvent mix, and evaporate fatsolvent behind the rete to be formed.Fatsolvent can use ethanol or use ether.Phospholipid substance and cholesterol mass ratio are 2: 1-3: 1.
Lignum sappan of the present invention is acted on tumor cell, observe and find, can suppress the formation of tumor cell colony, suppression ratio can reach more than 91%.In addition, observe with fluorescent staining method, lignum sappan of the present invention can impel tumor cell apoptosis to occur.
Adopt the liposome Lignum Sappan extract, advantage is, can use than low dosage to reach the effect that suppresses tumor, reduces medicine to Normocellular toxicity, improves medicine stability.
With the aqueous extract of lignum sappan of liposome and be used for the antineoplastic experiment, still the end is found.The present invention is wrapped to form nano-microcapsule with liposome and Chinese medicine aqueous extract of lignum sappan first, has carried out the antineoplastic experimentation, and has obtained believable experimental result.This method can be widely used in the research of Lignum Sappan in the Chinese medicine, and also the instrument of can be used as carries out the antineoplastic further investigation, for the traditional medicine of China has been opened up a more application prospects.
Description of drawings
Fig. 1 liposome aqueous extract of lignum sappan scans electric ytterbium observation A, B, C amplifies 15000 times liposome sem photograph; D is the liposome sem photograph of 10000 times of amplifications
The specific embodiment
The preparation of embodiment 1 aqueous extract of lignum sappan
Take by weighing Lignum Sappan 10g, measure the distilled water of 70ml with graduated cylinder, add in the beaker, the 30min that simmers in water is with eight layers of filtered through gauze, collect supernatant, add in the centrifuge tube 2500r/min, centrifugal 20min, get supernatant, and after transferring to pH=6.9 with the NaOH of 0.1N, the malleation bacteriological filtration, kept dry is stand-by.
Perhaps, the Lignum Sappan heartwood with the 0.5-1 gram is added in the distilled water of 10-20ml, sealing, and 60 ℃ of temperature are incubated 30min, and are centrifugal, get supernatant, with the filtering with microporous membrane of 0.22 μ m, transfer ph to 6.9-7.4, vacuum drying.
The preparation of embodiment 2 liposomees
0.6g soybean lecithin and 0.2g cholesterol are mixed with the 2-20mL dehydrated alcohol, dissolve in 60 ℃ of-70 ℃ of water-baths, under aseptic condition, divide and put into 10 EP pipes, cover tight EP pipe, rotate the EP pipe frequently, form layer of even rete sample material on the visible tube wall of naked eyes.At this moment, in superclean bench the EP pipe is uncapped (also can abandon supernatant) puts on the micro-thermostatic heater, 40 ℃ of volatilization ethanol.Stand-by.
The preparation (1) of embodiment 3 lignum sappans
Be dissolved in aseptic double-distilled water with making the aqueous extract of lignum sappan crystalline solid among the embodiment 1, being made into concentration is 10mg/ml solution, the aqueous extract of lignum sappan solution of getting 1ml adds aquation 15min in the EP pipe that contains the 0.3g liposome, put ultrasonic 20min in the ultrasonic device, 2000r/min, centrifugal 15min observes the effect that liposome forms.If also have more adipose membrane in the supernatant, supersound process once more, condition is the same.Adipose membrane disappears in the supernatant of centrifugal back, abandons supernatant, and is centrifugal with the normal saline washing, until the supernatant transparent clear that becomes, taking precipitate.
Ultrasonic device used in the present embodiment can use the ordinary laboratory ultrasonic washer.
The lignum sappan electron-microscope scanning figure of gained can see that as shown in Figure 1 its size is 5nm-1 μ m.
The preparation (2) of embodiment 4 lignum sappans
Be dissolved in aseptic double-distilled water with making the aqueous extract of lignum sappan crystalline solid among the embodiment 1, being made into concentration is 10mg/ml solution, and the aqueous extract of lignum sappan solution of getting 1ml adds and contains in the EP pipe of 0.2g liposome, and all the other steps are identical with embodiment 3.The lignum sappan size of gained is 5nm-1 μ m.
The preparation (3) of embodiment 5 lignum sappans
Be dissolved in aseptic double-distilled water with making the aqueous extract of lignum sappan crystalline solid among the embodiment 1, being made into concentration is 50mg/ml solution, and the aqueous extract of lignum sappan solution of getting 1ml adds and contains in the EP pipe of 0.5g liposome, and all the other steps are identical with embodiment 3.The lignum sappan size of gained is 5nm-1 μ m.
The preparation (4) of embodiment 6 lignum sappans
Make the aqueous extract of lignum sappan crystalline solid among the embodiment 1 and be dissolved in aseptic double-distilled water, being made into concentration is 100mg/ml solution, and the aqueous extract of lignum sappan solution of getting 1ml adds and contains in the EP pipe of 0.5g liposome, and all the other steps are identical with embodiment 3.The lignum sappan size of gained is 5nm-1 μ m.
The influence that embodiment 7 lignum sappans form K562 leukaemia's colony
K562 leukaemia is available from Shanghai cell institute of the Chinese Academy of Sciences.Culture medium is for containing 10%FCS RPMI1640 complete medium (containing each 100U/ml of 10% calf serum, penicillin and streptomycin, 1% glutamine, pH7.2～7.4).Human leukemia K562 cell inserts in the 50ml Tissue Culture Flask, puts 37 ℃, saturated humidity, 5%CO
2Cultivate in the incubator.Every 48h goes down to posterity once.The propagation of taking the logarithm phase cell is used for test.
Adopt the agar colony forming method.The take the logarithm K562 cell of trophophase, counting.With 1X10
3Ml
-1Tumor cell joins in the 24 porocyte culture plates (culture medium that contains 20%FCS, RPMI-1640 and 0.3% agar), and experimental group adds not commensurability lignum sappan respectively, and (the final content of Lignum Sappan extract is respectively 32 μ gml
-1, 320 μ gml
-1, 640 μ gml
-1, 960 μ gml
-1, 1280 μ gml
-1) or wrap up blank liposome (back is represented with Liposome) the 50 μ l of dual distilled water, not dosing of matched group, every group of 4 holes, 37 ℃, 5%CO
2, continuous culture 5 days under the saturated humidity condition, the colony number of forming with 〉=50 K562 cells of inverted microscope counting.Calculate the colony suppression ratio by following formula: colony suppression ratio (%)=(1-administration group colony number/matched group colony number) * 100%.The result is as shown in table 1.
Behind the lignum sappan effect K562 leukaemia 5 days, colony forming cell obviously arrives inhibition, and the amount of liposome is significance negative correlation (r=-0.981) in colony-forming efficiency and the adding system.
Table 1: the influence that lignum sappan forms K562 leukaemia's colony
| Lignum sappan dosage (μ gml -1, by aqueous extract of lignum sappan) | n | Colony number of cell (X ± S) | Suppression ratio (%) |
| Control | 3 | 197.5±58.5 | 0.00 |
| 32 | 3 | 137.5±20.6 | 30.38 |
| 320 | 3 | 117.5±17.0 | 40.51 |
| 640 | 3 | 87.5±9.5 | 55.70 |
| 960 | 3 | 72.5±5 | 63.29 |
| 1280 | 3 | 17.5±12.5★ | 91.14 |
| Liposome | 3 | 191.5±55.1* | 0.009 |
Compare P＜0.05 with matched group, ★ P＜0.01*P＞0.05
Embodiment 8 fluorescent staining methods are calculated apoptosis rate, observe lignum sappan to the apoptotic influence of K562
In K562 leukaemia's system of logarithmic proliferation phase, add not commensurability lignum sappan effect after 1-3 days, fluorescence microscope detects apoptotic cell.
The take the logarithm K562 cell of trophophase, counting.With 1X10
5Ml
-1(the final content of Lignum Sappan extract is respectively 320 μ gml for tumor cell, 20%FCS RPMI-1640 and not commensurability lignum sappan
-1, 640 μ gml
-1, 960 μ gml
-1, 1280 μ gml
-1), or wrap up blank liposome (back is represented with Liposome) the 50 μ l of dual distilled water, add respectively in the 96 porocyte culture plates (Costar), put 37 ℃, saturated humidity, 5%CO
2Leave standstill cultivation in the incubator, not dosing of matched group.When 24,48,72 hours of cultivating, get the piping and druming of test group and matched group K562 cell suspension evenly respectively.Respectively getting 30 μ l cell suspension is added on the clean slide, add 10 μ l acridine orange (AO, 10 μ g/ml) add coverslip behind the mixing, (250～300nm) times observation of cell forms of wavelength are also carried out apoptotic cell counting (cell membrane foaming, karyopycnosis, nuclear skew, karyorrhexis, crescent form and apoptotic body forms) to place fluorescence microscope, every slide is observed 200 cells, observe 3 slides, be calculated as follows apoptosis rate: apoptosis rate (%)=(apoptosis cell/200) * 100% for every group
Statistical procedures: data with
Two sample means t check and correlation analysis relatively adopted in expression.
Can reference: Jiang Bo, open inferior strict, the Zhou Dianyuan chief editor. molecular biology common experimental method [M]. Beijing: the medical officer people publishes, February in 1996 the 1st edition: 101; 176-177
Results suggest, the lignum sappan of each concentration all can impel the leukaemia apoptosis to occur, and after liposome, aqueous extract of lignum sappan content all has active anticancer between 32 μ g-1.28mg/ml, tumor cell is had inhibitory action; The time of its apoptosis number and cultivation presents positive correlation.Simultaneously each concentration point and matched group are checked through t, have diversity (p＜0.05-0.001).See Table 2.
Table 2: lignum sappan promotes the time-effect relationship of K562 apoptosis of leukemia
| Lignum sappan dosage (μ gml -1, by Lignum Sappan extract) | n | Apoptotic cell (the 24h 48h 72h of X ± S) | The r value | ||
| Control | 3 | 4.33±2.08 | 7.67±1.52 | 8.67±1.52 | 0.9548 |
| 320 | 3 | 15.00±1.00● | 19.00±1.00 | 20.67±2.08● | 0.973 |
| 640 | 3 | 19.00±2.64★ | 23.67±1.15 | 23.67±2.08★ | 0.9367 |
| 960 | 3 | 20.00±2.64● | 25.67±2.18 | 27.00±1.00★ | 0.9818 |
| 1280 | 3 | 24.67±2.08● | 29.00±2.00★ | 33.00±1.73● | 0.9997 |
| Liposome | 3 | 3.07±0.667* | 4.67±1.667* | 7.08±2.33* | 0.9932 |
Compare with matched group, check through t: P＜0.05,1, ★ P＜0.01, ● P＜0.001 * P＞0.05
Claims (8)
1. a lignum sappan is characterized in that, parcel aqueous extract of lignum sappan solution in this liposome, and size is at 5nm-1 μ m.
2. the lignum sappan of claim 1 is characterized in that, described aqueous extract of lignum sappan solution accounts for the 41%-55% of lignum sappan gross mass.
3. claim 1 or 2 lignum sappan is characterized in that described aqueous extract of lignum sappan solution concentration is 50 μ g/ml-200mg/ml.
4. the lignum sappan of claim 3 is characterized in that, described aqueous extract of lignum sappan solution concentration is 10mg/ml-100mg/ml.
5. the preparation method of a lignum sappan may further comprise the steps:
A. aqueous extract of lignum sappan solution is mixed aquation with liposome;
B. use ultrasonic Treatment, centrifugal then,
C. repeating step b does not have adipose membrane sample material until the upper strata, the outer unpacked aqueous extract of lignum sappan solution of weeding of grease plastid, wash centrifugal, taking precipitate.
6. the lignum sappan preparation method of claim 2 is characterized in that, the mass ratio of employed aqueous extract of lignum sappan and liposome is 1: 2-1: 100.
7. the lignum sappan preparation method of claim 6 is characterized in that, the mass ratio of described aqueous extract of lignum sappan and liposome is 1: 2-1: 30.
8. each lignum sappan of claim 1-4 is in the application of preparation in the antitumor drug.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101904892A (en) * | 2010-05-22 | 2010-12-08 | 山西省肿瘤医院 | Preparation process for Sappan Wood extract perfusate and application thereof in treating bladder cancer |
| CN101683380B (en) * | 2009-05-11 | 2011-08-24 | 山西省肿瘤医院 | Method for preparing irrigating solution with anti-tumor function |
| CN101411739B (en) * | 2008-10-27 | 2011-10-26 | 山西省肿瘤医院 | Method for preparing Caesalpinia sappan water extract |
-
2007
- 2007-03-09 CN CNB2007100379588A patent/CN100502837C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101411739B (en) * | 2008-10-27 | 2011-10-26 | 山西省肿瘤医院 | Method for preparing Caesalpinia sappan water extract |
| CN101683380B (en) * | 2009-05-11 | 2011-08-24 | 山西省肿瘤医院 | Method for preparing irrigating solution with anti-tumor function |
| CN101904892A (en) * | 2010-05-22 | 2010-12-08 | 山西省肿瘤医院 | Preparation process for Sappan Wood extract perfusate and application thereof in treating bladder cancer |
| CN101904892B (en) * | 2010-05-22 | 2012-06-06 | 山西省肿瘤医院 | Preparation process for Sappan Wood extract perfusate and application thereof in treating bladder cancer |
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