CN1911258A - Extractive of parasitic loranthus, prepn. method and application thereof - Google Patents
Extractive of parasitic loranthus, prepn. method and application thereof Download PDFInfo
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Abstract
An extract of mulberry mistletoe for treating the diseases associated with cell multiplication and its preparing process are disclosed.
Description
Technical field:
The invention belongs to medical technical field, be specifically related to the extract of a kind of Herba Taxilli class plant, this extract has therapeutical effect to animal and human's cell proliferation disorders.In addition, the invention still further relates to these preparation method of extract.
Background technology:
Herba Taxilli is a Chinese medicine commonly used simply, and effects such as tool invigorating the liver and kidney, wind-damp dispelling, blood pressure lowering, the fetus nourishing of peace blood cure mainly rheumatism, arthralgia, hypertension, sciatica, lumbago, puerperal breast disease such as few.Do not see the report that it has antitumous effect.Equal several plants of Loranthaceae, for example Herba Taxilli (Taxillus chinensis), safflower loranthus parasiticus (Scurrula parasitica L.), Sichuan parasitism Chang Zuowei Chinese crude drug Herba Taxillis such as (Taxillus sutchuenensis Danser) are used as medicine.
Malignant tumor is the disease of serious harm human health and life.Epidemiological study shows, the annual newly-increased cancer patient of China has 1,600,000 people approximately, and that dies from cancer every year has 1,300,000 people approximately.Because the deterioration of aged tendency of population and environment, these numerals were also in continuous rising in the last few years.Seek the important channel that the natural antitumor active substance is the research antitumor drug from plant, plant amedica such as paclitaxel, camptothecine, vincristine are the clinical preferably antitumor drug commonly used of present effect.
Summary of the invention:
The purpose of this invention is to provide the extract of a kind of host for the Herba Taxilli of apocynaceae plant.
Another object of the present invention provides the method for these extracts of preparation the Herba Taxilli of apocynaceae plant from the host.
A further object of the present invention provides the application of this extract in treatment humans and animals cell proliferation disorders.
One aspect of the present invention relates to the Herba Taxilli extract of the host of the cell proliferation disorders that is used for the treatment of the animal and human for apocynaceae plant.
When further aspect of the present invention related to other antitumor drug coupling, the host who is used for the treatment of animal and human's cell proliferation disorders as sensitizer or attenuation agent was the Herba Taxilli extract of apocynaceae plant.
Further aspect of the present invention relates to the pharmaceutical composition of the cell proliferation disorders that is used for the treatment of the animal and human, comprises that the host is the effect of the anti-animal and human's cell proliferation disorders of combination of the Herba Taxilli extract of apocynaceae plant and other medicines, pharmaceutical carrier or excipient.Usually pharmaceutical composition of the present invention contains the Herba Taxilli extract of 0.1～99.9% weight, and drug regimen can prepare according to methods known in the art.When being used for this purpose, if desired, Herba Taxilli extract and one or more solids or liquid medicine excipient and/or adjuvant can be combined, make the appropriate format or the dosage form that can be used as human.Route of administration can be intestinal or non-intestinal, as oral, muscle, subcutaneous, nasal cavity, oral mucosa, skin, peritoneum or rectum etc.Form of administration such as tablet, capsule, drop pill, aerosol, pill, powder, solution, suspensoid, Emulsion, granule, liposome, transdermal agent, buccal tablet, suppository, lyophilized injectable powder etc. can be ordinary preparation, slow releasing preparation, controlled release preparation and various particulate delivery system.For the unit form of administration being made above-mentioned various dosage form, can be extensive use of various carrier well known in the art.In addition, as needs, also can in pharmaceutical preparation, add coloring agent, antiseptic, spice, correctives, sweeting agent or other material.
The invention still further relates to the preparation method of the Herba Taxilli extract of the cell proliferation disorders that is used for the treatment of the animal and human.
The Herba Taxilli of indication of the present invention refers to derive from parasitic belong to (the Taxillus Van Tiehg.) of the blunt fruit of Loranthaceae (Loranthaceae), parasitic any plant that belongs to (Scurrula Linn.) or Loranthus (Loranthus Jacq) of melonidum, the parasitic arbitrary plant that belongs to of melonidum preferably, the parasitic safflower loranthus parasiticus (Scurrula parasitica L.) that belongs to of melonidum most preferably, their the most basic total features are that its host is an apocynaceae plant, preferably Folium seu Cortex Nerii (Nerium indicum) and Semen Thevetiae (Thevetiaperuviana).About Loranthaceae (Loranthaceae), parasitic (Taxillus Van Tiehg.), parasitic scientific definition and the category that belongs to (Scurrula Linn.) or Loranthus (Loranthus Jacq) and Apocynaceae of melonidum of belonging to of blunt fruit, (Hou Kuanzhao compiles referring to " Chinese Higher plant section belongs to dictionary " (revised edition), Beijing: Science Press, 1984,12) reach " Chinese Plants will " the 24th volume (Beijing: Science Press, 1986).Used medical material position is generally its leaf, branch, stem, also comprises Hua Heguo.
This host for the preparation method of extract of the Herba Taxilli on the apocynaceae plant is:
(1) the harvesting host is clean airing of Herba Taxilli medical material or the pulverizing of oven dry back on the apocynaceae plant, with polarity or non-polar solven heating or extract at room temperature, the preferred solvent that extracts is 60～80% ethanol or other alcohols solvent of same concentration, preferably extracts temperature in 60～80 ℃ of scopes; Extract 1～10 time, preferably extracting pass is 3～5 times, every all over extraction time heating extraction be general 1～4 hour or longer, the extract at room temperature time is general every all over 1 day or longer.Also medical material elder generation can be extracted with organic solvent (as petroleum ether, benzene, toluene or ethane etc.), filter, discard filtrate and keep filtering residue with liposoluble substances such as removal pigments, filtering residue reuse said method extracts.
(2) it is about 50～70% that filtrate being adjusted to contains alcohol, puts 4 ℃ of refrigerator overnight or longer time, so that pigment and oils and fats are separated out; Remove by filter pigment and oils and fats; Also but the reuse petroleum ether extraction is further removed pigment and oils and fats.Filtrate is reclaimed solvent, concentrate extractum.
(3) extractum mixes pasty state with Silon or silica white, grind to form fine powder behind the dry removal moisture, cross polyamide column (preferred polyamide granules size is 100～200 orders) purification, water, 10%～90% gradient alcohol liquid eluting, collect eluent, reclaim the concentrated extractum that obtains of solvent, this extractum gets required extract through the further dry moisture of removing.
Also can only be condensed into denseer aqueous solution when (4) concentrating in the step (2), concentrated solution at this moment also can directly be crossed the polyamide column purification.
Through component analysis, mainly contain Flavonoid substances (containing flavone more than 50%) in 30～90% pure eluates, chemical constituents such as cardiotonic glycoside and alkaloid; Contain polysaccharide (containing sugar more than 56%) in water and the 10% pure eluate, chemical constituents such as glycoprotein and polypeptide.
The composition characteristics of the extract that the present invention obtains is wherein to contain the cardiotonic glycoside constituents, and the Herba Taxilli extract that parasitizes other host (for example mulberry, Arillus Longan, cassia tree etc.) does not contain the cardiotonic glycoside composition.The contained cardiotonic glycoside composition of this type of cardiotonic glycoside composition and host's apocynaceae plant with, find that the cardiotonic glycoside that extracts is different with the cardiotonic glycoside molecular weight of material that extracts because adopt Agilent company 1100 types high performance liquid chromatography-ion trap mass spectrometry coupling instrument (LC-MSD Trap) check and analysis from the safflower loranthus parasiticus leaf from its host's sweetscented oleander leaf.
The Herba Taxilli extract that parasitizes on the apocynaceae plant with method for preparing is found to be a kind of broad-spectrum high-efficiency low-toxicity antineoplastic component through the inside and outside pharmacodynamic experiment.External have intensive cytotoxic activity, IC to kinds of tumor cells (for example leukemia, lymphoma, hepatocarcinoma, pulmonary carcinoma, cervical cancer, nasopharyngeal carcinoma etc.)
50Generally at 0.1～1 μ g/ml; Even but to the normal human cell at the also acellular cytotoxic activity of the concentration of 50 μ g/ml.Suppress the growth of kinds of tumors in the body, and the antitumor drug (for example amycin) of other clinical use is had powerful sensitization and Attenuation.
Description of drawings:
Fig. 1 variable concentrations safflower loranthus parasiticus extract is to the suppression ratio of NB4 cell strain.
Fig. 2 variable concentrations safflower loranthus parasiticus extract is to the suppression ratio of Jurtet cell strain.
Fig. 3 variable concentrations safflower loranthus parasiticus extract is to the suppression ratio of U266 cell strain.
Fig. 4 variable concentrations safflower loranthus parasiticus extract is to the suppression ratio of HL-60 cell strain.
Fig. 5 variable concentrations safflower loranthus parasiticus extract is to the suppression ratio of K562 cell strain.
Fig. 6 variable concentrations safflower loranthus parasiticus extract is to the suppression ratio of NCI-H446 cell strain.
Fig. 7 variable concentrations safflower loranthus parasiticus extract is to the suppression ratio of slow grain primary cell.
Fig. 8 variable concentrations safflower loranthus parasiticus extract is to the suppression ratio of Hela cell strain.
Fig. 9 variable concentrations safflower loranthus parasiticus extract is to the suppression ratio of CNE cell strain.
Figure 10 variable concentrations safflower loranthus parasiticus extract is to the suppression ratio of 293T cell strain.
Behind Figure 11 variable concentrations safflower loranthus parasiticus extract-treated 293T cell strain absorbance value (OD value) change.
Absorbance value (OD value) behind Figure 12 variable concentrations safflower loranthus parasiticus extract-treated NCI-446 cell strain changes.
DNA ladder collection of illustrative plates behind Figure 13 variable concentrations safflower loranthus parasiticus extract-treated HL-60 cell strain 24h
Among Figure 13: the 1-negative control; 2-1 μ g/mL group; 3-0.5 μ g/mL group; 4-0.25 μ g/mL group
The specific embodiment:
The following examples and biological activity test are used for further setting forth the present invention.
Embodiment 1: adopt the heating and refluxing extraction method to prepare safflower loranthus parasiticus (Scurrula parasitica L.) the branch and leaf extract that parasitizes on the Folium seu Cortex Nerii (Nerium indicum)
(1) plucks leaf and the twig that parasitizes the safflower loranthus parasiticus on the Folium seu Cortex Nerii, clean cool (or baking) dried back and pulverize, cross 60 mesh sieves.Get 1000 gram coarse powder and add 15 times 80% ethanol and divide reflux, extract, three times, each 4 hours, extract 65 ℃ of temperature.Add water to that to contain alcohol about 70%, place in the refrigerator (4 ℃) and spend the night, cross the elimination precipitation, filtrate reuse petroleum ether extraction is further removed pigment and oils and fats one time.Get ethanol liquid decompression recycling ethanol, be concentrated into extractum.
(2) extractum is mixed thoroughly in about 1: 1 ratio with Silon, 60 ℃ of oven dry, be milled into powder, cross polyamide column (100～200 order), with distilled water, 10% ethanol, 30% ethanol, 50% ethanol, 70% ethanol and 90% ethanol substep gradient elution, the eluant of every kind of gradient is eluted to and changes a gradient when not having color substantially.The difference concentrating under reduced pressure.Distilled water and 10% ethanol elution thing are tough colloid substance, mainly contain polysaccharide (about 56%) through composition detection, also contain glycoprotein, polypeptide, aminoacid and other material; And mainly contain Flavonoid substances (about 54%) in 30%, 50%, the 70% and 90% ethanol elution thing, also contain cardiotonic glycoside, alkaloid and other material.Above-mentioned eluate is mixed or application separately in varing proportions, be the Herba Taxilli extract of indication of the present invention.
Embodiment two: adopt the heating and refluxing extraction method to prepare safflower loranthus parasiticus (Scurrula parasitica L.) the branch and leaf extract that parasitizes on the Folium seu Cortex Nerii (Nerium indicum)
(1) plucks leaf and the twig that parasitizes the Ramulus Scurmlae Parasificae on the Folium seu Cortex Nerii, clean cool (or baking) dried back and pulverize, cross 60 mesh sieves.Get 1000 gram coarse powder and add 15 times 80% ethanol and divide three backflow, each 4 hours, 65 ℃ of extraction temperature.Add water to that to contain alcohol about 70%, place in the refrigerator (4 ℃) and spend the night, cross the elimination precipitation, filtrate reuse petroleum ether extraction further discolor for one time element and oils and fats.Get ethanol liquid decompression recycling ethanol, along with the reduction gradually of concentration of alcohol in the concentrated solution, constantly have precipitation to separate out in this process, precipitate and separate is taken out; Continue to be concentrated into extractum.
(2) extractum is mixed thoroughly in about 1: 1 ratio with Silon, 60 ℃ of oven dry, be milled into powder, cross polyamide column (100～200 order), with distilled water, 10% ethanol, 30% ethanol, 50% ethanol, 70% ethanol and 90% ethanol substep gradient elution, every kind of eluant is eluted to changes a gradient when not having color substantially.The difference concentrating under reduced pressure.Distilled water and 10% ethanol elution thing are tough colloid substance, mainly contain polysaccharide (about 56%) after testing, also contain glycoprotein, polypeptide, aminoacid and other material; And mainly contain Flavonoid substances (about 54%) in 30%, 50%, the 70% and 90% ethanol elution thing, also contain cardiotonic glycoside, alkaloid and other material.Above-mentioned eluate is mixed or application separately in varing proportions, be the Herba Taxilli extract of indication of the present invention.
Embodiment three: adopt the percolation extraction method to prepare safflower loranthus parasiticus (Scurrulaparasitica L.) the branch and leaf extract that parasitizes on the Folium seu Cortex Nerii (Nerium indicum)
(1) plucks leaf and the twig that parasitizes the Ramulus Scurmlae Parasificae on the Folium seu Cortex Nerii, clean cool (or baking) dried back and pulverize, cross 60 mesh sieves.Get 3000 gram coarse powder, be loaded in the percolator, add 80% soak with ethanol after 10 days, emit percolate, add 80% ethanol at any time and all be soaked in the ethanol liquid, think that to percolate extraction finishes when colourless substantially to keep medical material with the speed of the about 1mL of per minute.Regulate behind the extracting solution reclaim under reduced pressure part ethanol and contain the alcohol amount to about 70%.Other step is with embodiment 2.
Embodiment four: the pharmacodynamic study of Ramulus Scurmlae Parasificae extract and the anti-people's acute leukemia of amycin coupling transplanted tumor
1. experiment material:
The tumor strain: high tumorigenesis human promyelocytic leukemia cell strain HL-60 is from Fujian Province Concord Hospital hematopathy institute, nude mice: BALB/c, nu/nu, Mus 6～8w in age, body weight 18～22g available from Chinese Academy of Sciences's Shanghai Experimental Animal Center, raises in constant temperature (25-270C), in the lamina air flow cabinet of constant humidity (45%-50%) SPF level, male and female are separately raised.Free pickuping food and water, drinking water, feedstuff, bedding and padding and other nude mices contact article are handled by autoclaving.Amycin (ADR), Hualian Pharmaceutical Co., Ltd., Shanghai's product, lot number 04-04-30.
2. experimental technique:
(1) get the HL-60 cell that is in exponential phase and make cell suspension, the adjustment cell concentration is 1 * 107ml-1, and every nude inoculation 0.2ml is subcutaneous in the right hind dorsal part.Inoculate the 5th day, tumor long to about Semen phaseoli radiati drug treatment when big or small.Nude mice is weighed, and measures the tumor footpath, random packet, and experiment is established 5 groups, 5 of every treated animals altogether.Observe and write down diet and activity situation and the growth of xenografted situation of nude mice every day, per 2 days with precise electronic balance measurement nude mouse quality, carry out the drug toxicity preliminary assessment, be that opisthosoma quality (FBW)/first body constitution amount (IBW) 〉=0.8 is non-toxic reaction,＜0.8 is toxic reaction. measure gross tumor volume weekly, and with vernier caliper measurement tumor major diameter (a), minor axis (b), calculate gross tumor volume according to formula V=ab2/2, draw the gross tumor volume growth curve.Treatment finishes back second day disconnected neck and puts to death nude mice, gets tumor and weighs, and calculates tumour inhibiting rate.Tumour inhibiting rate (IR)=(the average tumor of the average tumor weight/matched group of 1-experimental group is heavy) * 100%.The computing formula of drug interaction index (CDI) is: (A * B), in this experiment, AB is two coupling groups and the heavy ratio of matched group tumor to CDI=AB/, and A or B are private medicine group of each prescription and the heavy ratio of matched group tumor.When CDI＜1, two medicines have synergism, when CDI＜0.75, and the synergism highly significant.
Grouping: 1. normal saline group: lumbar injection contains the normal saline of 1%DMSO; 2. ADR organizes: the 1.5mg/ml amycin; 3. extract abdominal cavity group: lumbar injection 1mg/ml safflower loranthus parasiticus extract; 4. extract tumor week is organized: tumor week injection 1mg/ml safflower loranthus parasiticus extract is extract+ADR group 5.: lumbar injection 1mg/ml safflower loranthus parasiticus extract, tail vein injection 1.5mg/ml amycin.Injected dose is the 0.1ml/10g body weight.The disposable drug administration by injection of amycin tail vein, safflower loranthus parasiticus extract and normal saline intraperitoneal injection every day, once a day, totally 14 days.
(2) pathologic finding: dissect animal, perusal internal organs pathological changes situation when experiment finishes; Take out the heart, liver,kidney,spleen, normal saline flushing is placed in 10% formalin and fixes 24 hours, does pathological examination.
3. experimental result:
(1) after the Drug therapy, each organizes the FBW/IBW value all＞0.8, visible non-toxic reaction. and (table 1)
Table 1 drug toxicity evaluation table
| Drug toxicity is estimated | The normal saline group | The ADR group | Extract abdominal cavity group | Extract tumor week is organized | Extract+ADR group |
| First body constitution amount (IBW) | 22.3±1.7 | 22.6±1.2 | 23.1±1.5 | 19.7±1.1 | 20.2±0.6 |
| Opisthosoma quality (FBW) | 30.1±4.8 | 25.7±2.1 | 28.9±2.4 | 24.8±3.2 | 20.8±0.7 |
| FBW/IBW | 1.35 | 1.13 | 1.24 | 1.25 | 1.02 |
(2) HL-60 nude mice subcutaneous transplantation tumor change in volume situation (table 2):
Table 2 HL-60 nude mice subcutaneous transplantation tumor change in volume situation (unit: cm
3)
| | 0 day | 7 days | 14 days |
| Matched group | 0.025±0.026 | 1.267±0.776 | 8.939±2.942 |
| The ADR group | 0.028±0.025 | 0.197±0.111 | 3.527±1.822 |
| Extract abdominal cavity group | 0.024±0.033 | 0.889±0.517 | 7.041±3.964 |
| Extract tumor week is organized | 0.035±0.017 | 0.237±0.144 | 5.085±3.094 |
| Extract+ADM group | 0.037±0.018 | 0.026±0.016 | 0.767±0.539 |
Annotate: 0 day expression beginning time of administration
As can be seen, each processed group of extract all significantly suppressed the tumor bulk-growth in the time of the 7th day, but the lumbar injection group is not good to tumor bulk-growth later stage inhibitory action; And the injection of tumor week also has certain inhibitory action to tumor body late growing stage.Extract and amycin coupling can suppress the tumor bulk-growth by highly significant.
(3) HL-60 nude mice subcutaneous transplantation tumor weight situation of change (table 3):
Table 3 HL-60 nude mice subcutaneous transplantation tumor weight situation of change
| Group | Number of animals (only) | Remove the tumor body weight change | Tumor is heavy | Tumour inhibiting rate | P | CDI |
| Beginning/end | (g) | (x±s，g) | (％) | |||
| | 5/5 | +1.394 | 7.52±1.36 | |||
| The | 5/5 | +0.532 | 2.57±1.38 | 65.8 | 0.001＜0.01 * | |
| Extract | 5/5 | +0.360 | 5.48±2.46 | 27.1 | 0.184＞0.05 * | |
| Extract tumor week is organized | 5/5 | +1.510 | 3.55±2.29 | 52.8 | 0.019＜0.05 * | |
| Extract+ | 5/4 | +0.145 | 0.46±0.23 | 93.9 | 0.002＜0.01 * 0.026＜0.05 ** 0.009＜0.01 *** | 0.24 |
Annotate:
*Experimental group and matched group (normal saline group) compare,
*Experimental group and amycin list compare with group,
* *Experimental group and lumbar injection group are relatively.
Safflower loranthus parasiticus extract 10mg/kg lumbar injection tumour inhibiting rate was 27.1% (comparing P＞0.05 with matched group), and is all not remarkable with matched group difference.Yet this dosage tumor week injection tumor-inhibiting action is remarkable, and tumour inhibiting rate was 52.8% (comparing P＜0.05 with matched group).This dosage and amycin coupling can significantly strengthen the tumor-inhibiting action of amycin, in the experiment, the tumor control rate of the independent medication group of ADR15mg/kg was 65.8% (comparing P＜0.05 with matched group), the suppression ratio of two medicine coupling groups is 93.8%, compare P＜0.01 with normal saline group and lumbar injection group, difference all reaches extremely significant level; More also there were significant differences with the amycin group, the P＜remarkable CDI=0.24 of 0.05, two medicine synergism＜0.75.
(4) each processed group internal organs of macroscopy all do not have obvious change; Pathological section shows that there is pathological changes in amycin individual processing group heart tissue, the myocardial cell enlargement, and the cell arrangement disorder, and normal with Herba Taxilli coupling group myocardial cell.
Embodiment five: Ramulus Scurmlae Parasificae extracts the research of anti-murine sarcoma S180 in the object
1. experiment material:
Murine sarcoma S180 ascitic type comes free Fujian Province pharmaceutical college of medical university, and Kunming kind white mice is available from the Fujian Experimental Animal Center, body weight 18～22g, and, in 6～8 ages in week, the male and female dual-purpose is available from Fujian medical university Experimental Animal Center.Amycin (ADR), Hualian Pharmaceutical Co., Ltd., Shanghai's product, lot number 04-04-30.
2. experimental technique:
2.1 medicine preparation: 30% ethanol eluate powder, 50% ethanol eluate powder, 70% each 100mg of ethanol eluate powder presses 1: 1: 1 mixed, is diluted to the storing solution of 10.0mg/ml with normal saline.Taking by weighing safflower loranthus parasiticus distilled water eluate powder 5g is dissolved in the 200ml normal saline and is configured to 25.0mg/ml distilled water eluate group.
Negative control group (A): normal saline;
Positive controls (B): 1.5mg/ml amycin, disposable celiac injection;
Ethanol elution thing group (C): storing solution is diluted to 1.0mg/ml with normal saline;
Ethanol elution thing+distilled water eluate group (D): contain (ethanol elution thing 1.0mg+ distilled water eluate 25.0mg) to every milliliter with distilled water eluate dilution storing solution;
Distilled water eluate group (E): 25.0mg/ml distilled water eluate.
2.2 experimental technique: get the ascitogenous sarcoma SI80 ascites that went down to posterity 7 days, become the tumor cell suspension by 1: 3 dilution proportion, be inoculated in mouse peritoneal, every Mus 0.2ml with physiological saline solution.Inoculate next day, be divided into A, B, C, D, E, 5 groups at random, every group 10, press the administration volume intraperitoneal injection of 10g body weight 0.1ml, every day 1 time, successive administration 14 days, statistics mice survival natural law (exceed 28 days and calculated) by 28 days, calculate the mean survival time of each group, obtain increase in life span.
2.3 experimental result: see Table 4.
As can be seen from the results, positive controls, ethanol elution thing group, distilled water eluate group, ethanol elution thing+distilled water eluate group, to the increase in life span of lotus S180 mice be respectively 25.5%, 24.0%, 23.0%h and 43%.Distilled water eluate group has the effect that improves the prolongation of mice life, and the mixing of distilled water eluate and ethanol eluate is used mice life prolongation effect is reached extremely remarkable, and is more single with having anti-mice S180 sarcoma effect in the stronger body than the two.
Table 4 safflower loranthus parasiticus extract is to the influence of tumor-bearing mice increase in life span
| Grouping | The animal number of elements | Time-to-live (d) | Increase in life span (%) |
| | 10 10 10 10 10 | 16.5±1.2 20.7±4.7 * 20.5±3.6 * 23.6±4.0 ** 20.3±4.6 * | - 25.5 24.0 43.0 23.0 |
Annotate: compare with matched group,
*P＜0.05,
*P＜0.01
Embodiment six: the safflower loranthus parasiticus extract is to the influence of NB4, Jurkat cell colony formation rate
Tumor system by different ratios, propagation and the different oncocyte group of differentiation capability constitute.Wherein only fraction has the self renewal ability, and promptly so-called stem cell is about 1% of whole cell mass.Stem cell is the target cell of radiotherapy and cancer drug therapy, and is close with healing, recurrence or the transfer relationship of tumor.It is generally acknowledged and have only stem cell just to have the ability of differentiation, formation colony.So the colony method can be used as one of vitro detection cancer therapy drug sensitivity method.
1. experiment material: methylcellulose is available from sigma, and other is with embodiment 6.
2. experimental technique:
(1) preparation cell suspension, culture fluid is the RPMI1640 culture fluid that contains 20% hyclone.Counting dilutes for 0.45ml and contains about 300 cells.
(2) inoculating cell: add 0.45ml1.6% methylcellulose (with 1640 preparations) in the every hole of 24 orifice plates, the bubble of rushing; The cell suspension of NB4, the Jurkat that step 1 is prepared adds every hole 0.45ml, about 300 cells then.
(3) dosing: the every hole of suspension cell adds medicine 0.1ml, and 6 drug level are established two repeating holes and established no medicine parallel control group in addition.
(4) place 37 ℃, 5%CO2 cultivated 7 days in the incubator of saturated humidity, meter CFU cell number (〉=30 is a colony).Calculate the colony suppression ratio according to formula, that is: colony suppression ratio=(1-T/C) * 100%.
3. experimental result:
Table 5 safflower loranthus parasiticus extract forms suppression ratio to the tumor cell colony
| Group | Dosing group colony number (individual) | Matched group colony number (individual) | Colony forms suppression |
| NB4 cell | |||
| 5 μ g/ml 2.5 μ g/ | 0 0 1 1 9 26 | 48 47 45 75 29 87 | 100％ 100％ 97.78％ 98.67％ 68.97％ 70.12 |
| Jurkqat cell | |||
| 5 μ g/ml 2.5 μ g/ | 0 1 1 2 4 23 | 32 38 45 56 42 38 | 100％ 97.37％ 97.78％ 96.43％ 91.00％ 39.47％ |
Compare with the MTT experiment, it is higher that the medicine of same concentration forms suppression ratio to colony, illustrates that the safflower loranthus parasiticus extract is more responsive to the proliferative cell effect in the tumor cell.
Embodiment seven: the research of the various people of safflower loranthus parasiticus extract vitro inhibition source tumor cell
1. experiment material: people source tumor cell line: NB4 (people's acute promyelocytic leukemia cell strain M3), Jurket (human T lymphocyte's leukemia), U266 (human myeloma cell strain), HL-60 (people's acute promyelocytic leukemia cell strain M2), K562 (human chronic myelogenous leukemia's cell strain), NCI-H446 (human lung carcinoma cell line) Hela (human cervical carcinoma cell strain), CNE (human nasopharyngeal carcinoma cell line), from Fujian Province's hematopathy institute; The former foster chronic myeloid leukemia cell of being commissioned to train is taken from slow granulosis people's bone marrow; RPMI1640 culture fluid (GIBCO), hyclone (Hangzhou Ilex purpurea Hassk.[I.chinensis Sims), MTT (sigma), safflower loranthus parasiticus extract.
2. experimental technique: mtt assay, the cell adjustment density of getting the exponential phase of cultivation contains 1 * 104～2 * 104 viable cell densities to per 180 μ l, be inoculated in 96 well culture plates (3 every group multiple holes, repeat 3 times, average), suspension cell has been inoculated and has been got final product dosing, attached cell was then cultivated one day, treated dosing again behind the cell attachment, and every Kong Jun adds 20 μ l medicines, and no medicine matched group and blank group be set, place 37 ℃ then, 5%CO2, saturated humidity the CO2 incubator in hatch 24h respectively, 48h, 72h, behind 96h and the 120h, add 20 μ l MTT, cultivate 4h and remove supernatant, add 150 μ lDMSO, go up vibration 3-4 minute in microplate reader (StatFax-2100), survey the OD value with dual wavelength 492nm and 630nm, calculate suppression ratio.Suppression ratio=(1-experimental group OD value/untreated fish group OD value) * 100%.
3. experimental result: shown in Fig. 1～9, the safflower loranthus parasiticus extract still is effect and the lethal effect that the former foster chronic myeloid leukemia cell of being commissioned to train all has intensive inhibition propagation to human tumor cell line, various tumor cell lines are all had good effect, are a kind of broad-spectrum anti-cancer drugs.Microexamination finds that most of cell volume increases, visible a large amount of cavitys in the endochylema.Some cell volumes dwindle cell and are the shape change of sprouting, and nuclear concentrates, and cellular morphology is complete.Adopt the dna ladder band to detect and find that the safflower loranthus parasiticus extract is cell death inducing (Figure 13) to the lethal effect of tumor cell.
Embodiment eight: the safflower loranthus parasiticus extract is external to the Normocellular effect research in people source
1. experiment material: normal person's embryonic kidney fibroblast strain 293T derives from life sciences institute of Fujian Normal University; Other together
Embodiment 6.
2. experimental technique: with embodiment 6.
3. experimental result: the safflower loranthus parasiticus extract is in external propagation also inhibited (Figure 10) to normal person's embryonic kidney fibroblast strain 293T, but there is no lethal effect, and cytoactive is normal, just can't be adherent.Promptly use up to the absorbance value constant substantially (Figure 11) after the drug level processing of 50 μ g/ml, the essentially no death of visible cell does not have propagation yet, and tumor cell for example lung cancer cell line NCI-446 promptly use the drug level of 2.5 μ g/ml to handle, its absorbance value also is (Figure 12) that descends, and lethal effect has been described.Above presentation of results safflower loranthus parasiticus extract is less to Normocellular influence, and cytotoxicity is low.
Claims (14)
1. a Herba Taxilli extract is characterized in that wherein containing the cardiotonic glycoside constituents, and the host of Herba Taxilli plant is an apocynaceae plant.
2. Herba Taxilli extract as claimed in claim 1 is characterized in that wherein said Herba Taxilli is meant the Loranthaceae plant.
3. Herba Taxilli extract as claimed in claim 1 is characterized in that wherein said apocynaceae plant is meant Folium seu Cortex Nerii (Nerium indicum) and Semen Thevetiae (Thevetia peruviana).
4. Herba Taxilli extract as claimed in claim 1 is characterized in that containing flavone compound in the chemical constituent of this extract.
5. Herba Taxilli extract as claimed in claim 1 is characterized in that containing polysaccharide compound in the chemical constituent of this extract.
6. Herba Taxilli extract as claimed in claim 2 is characterized in that wherein said Loranthaceae plant is meant safflower loranthus parasiticus (Scurrula parasitica L.), Herba Taxilli (Taxillus chinensis) and refers to Sichuan parasitism (Taxillussutchuenensis Danser).
7. the method for preparing the described Herba Taxilli extract of claim 1 is characterized in that this method includes following steps:
(1) pulverizes after the medical material clean dry,, cross leaching filtrate with solvent extraction 1～10 time; Medical material can be used earlier organic solvent extraction, filter, discard filtrate and keep filtering residue with liposoluble substances such as removal pigments, filtering residue reuse said method extracts;
(2) it is about 50～70% that filtrate being adjusted to contains alcohol, puts refrigerator cold-storage more than one hour, so that pigment and oils and fats are separated out, removes by filter pigment and oils and fats; But the reuse petroleum ether extraction is further removed pigment and oils and fats; Get filtrate and reclaim solvent, concentrate;
(3) concentrated solution is crossed the polyamide column purification, and water, 10%～90% gradient alcohol liquid eluting are collected eluent, reclaims solvent and concentrates, and this concentrated solution is through the further dry moisture of removing, Herba Taxilli extract.
8. the method for preparing Herba Taxilli extract as claimed in claim 7 is characterized in that wherein said solvent is meant 1%～99% alcohols solvent.
9. the method for preparing Herba Taxilli extract as claimed in claim 8 is characterized in that wherein said alcohols solvent is meant 1～99% ethanol.
10. the method for preparing Herba Taxilli extract as claimed in claim 7 is characterized in that wherein said solvent is meant water.
11. the method for preparing Herba Taxilli extract as claimed in claim 7 is characterized in that wherein said drying means is meant lyophilization, spray drying, evaporation drying.
12. the purposes of Herba Taxilli extract as claimed in claim 1 is characterized in that its cell proliferation disorders to humans and animals has therapeutical effect.
13. as the purposes of the Herba Taxilli extract of claim 12, it is characterized in that when wherein said therapeutical effect is meant this medicine of independent use therapeutical effect, the therapeutical effect when being used as the antitumous effect of sensitizer enhanced sensitivity other drug, as the therapeutical effect of the compositions of effective ingredient and other medicines, pharmaceutical carrier or excipient.
14. the purposes as the Herba Taxilli extract of claim 12 is characterized in that wherein said therapeutical effect can reduce the toxic action of other antitumor drug when being meant with other antitumor drug couplings.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101250233B (en) * | 2008-03-27 | 2010-06-02 | 福建师范大学 | Use of safflower loranthus parasiticus polysaccharides in pharmaceuticals |
| CN112174848A (en) * | 2020-11-04 | 2021-01-05 | 吉林大学 | Oleoylethanolamide compound with antibacterial activity in parasitic loranthus, preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1163253C (en) * | 2000-06-27 | 2004-08-25 | 中国医学科学院医药生物技术研究所 | Mulberry mistletoe extract for preventing or treating anaphylactic reaction, medicinal composition containing it and its preparing process |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101250233B (en) * | 2008-03-27 | 2010-06-02 | 福建师范大学 | Use of safflower loranthus parasiticus polysaccharides in pharmaceuticals |
| CN112174848A (en) * | 2020-11-04 | 2021-01-05 | 吉林大学 | Oleoylethanolamide compound with antibacterial activity in parasitic loranthus, preparation method and application thereof |
| CN112174848B (en) * | 2020-11-04 | 2021-11-12 | 吉林大学 | Oleoylethanolamide compound with antibacterial activity in parasitic loranthus, preparation method and application thereof |
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