CN105663120A - Application of artemisitene in preparation of antitumor drug - Google Patents
Application of artemisitene in preparation of antitumor drug Download PDFInfo
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- CN105663120A CN105663120A CN201610053766.5A CN201610053766A CN105663120A CN 105663120 A CN105663120 A CN 105663120A CN 201610053766 A CN201610053766 A CN 201610053766A CN 105663120 A CN105663120 A CN 105663120A
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/365—Lactones
- A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
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Abstract
The invention relates to an application of artemisitene in preparation of an antitumor drug. The drug is prepared from artemisitene and pharmaceutically acceptable auxiliary materials, wherein the content of artemisitene is 0.5%-30%. The drug is an injection or a common oral preparation. The artemisitene can inhibit tumor cell growth and induce tumor cell apoptosis, has a higher tumor inhibition effect, and has no toxic and side effects on normal cells.
Description
Technical field
The present invention relates to the new opplication of a kind of compound, prepare the new opplication in antitumor drug in particular to sweet wormwood alkene.
Background technology
Sweet wormwood alkene (Artemisitene) derives from plant Artemisia annua, and its molecular formula is C15H20O5, chemical structural formula isMolecular weight is 280.32, is insoluble to water, dissolves in ethanol and ether and conventional organic solvent. Sweet wormwood alkene is a kind of sesquiterpene lactones compounds with peroxide bridge that extraction and isolation obtains from composite family ends platymiscium Herba Artemisiae annuae leaf, and composite family Chinese mugwort platymiscium is used for the treatment of malaria, early has record in ancient Chinese medical skill. At present, Artemisinin and derivative thereof are own through obtaining generally acknowledging of the whole world as the antimalarial drug of high-efficiency low-toxicity. Sweet wormwood alkene (Artemisitene) is one of precursor component of artemisinin synthesis, and Recent study finds, Artemisinin and derivative thereof have obvious antitumor action. Publication number is the anti-oxidant application that the Chinese patent application of CN104825443A discloses sweet wormwood alkene, and first identified sweet wormwood alkene is activation of Nrf2 in the world, can be used as food auxiliary widespread use. In the prior art, sweet wormwood alkene has specific antineoplastic pharmacologically active not to have experimental data to confirm.
Summary of the invention
Technical problem to be solved by this invention is to provide the purposes of sweet wormwood alkene.
In fact, the purposes of above-mentioned sweet wormwood alkene be sweet wormwood alkene in the application preparing in antitumor drug, the chemical structural formula of wherein said sweet wormwood alkene is shown in lower formula I,
In above-mentioned application, described medicine is made up of sweet wormwood alkene and medically acceptable auxiliary material, and wherein the weight content of sweet wormwood alkene is 0.5～30%. Described medicine can be injection, it is also possible to be common oral preparations, such as tablet and soft capsule.
Above-mentioned injection is the aqueous solution that every 1000ml contains sweet wormwood alkene 500mg and sodium-chlor 9g, and pH value is 3.0～5.5.
The content of above-mentioned soft capsule is made up of the raw material of following weight part: 30 parts, sweet wormwood alkene, edible corn oil 20 parts, thinner 40 parts.
Above-mentioned tablet is made up of sweet wormwood alkene and binder, and wherein the weight content of sweet wormwood alkene is 2.3～2.5%.
Application of the present invention utilizes the growth of sweet wormwood alkene inhibition tumor cell, inducing apoptosis of tumour cell, reach antitumor object, therefore, compound of the present invention, in the application prepared in antitumor drug, specifically refers to the application of described sweet wormwood alkene in the inductor of the inhibitor and apoptosis of tumor cells of preparing growth of tumour cell.
Accompanying drawing explanation
Fig. 1 is Mouse Liver kidney segment HE dyeing Photomicrograph, and wherein the left side is liver, and the right is kidney.
Embodiment
Example 1 (pharmacology, effect experiment)
Test a sweet wormwood alkene to the Inhibition test of human hepatoma cell strain Hep3B2.1-7 subcutaneous transplantation knurl
Experiment purpose: research sweet wormwood alkene is to the restraining effect of rat liver cancer transplanted tumor.
1. experiment material
1.1 laboratory animal
Only, body weight 18～22g, is provided, conformity certification number: 11400700123247 the NODSCID mouse 3O in 6～8 week age of animal by Beijing Wei Tonglihua laboratory animal technology company limited.
1.2 reagent
Peanut oil (Changxing, Guangdong food trade company limited, lot number 090407); Foetal calf serum (Hyclone company); Phosphate buffered saline buffer (PBS, Hyclone company).
1.3 test medicine
Sweet wormwood alkene: be purchased from Tianjin Science and Technology Ltd. of Silan, lot number A777550. Getting sweet wormwood alkene and be placed in beaker, add peanut oil as solvent, heating, is stirred to dissolve, and is 5mg/ml with peanut oil to the mass concentration of sweet wormwood alkene in peanut oil, obtains sweet wormwood alkene oil solution after letting cool.
1.4 instrument
The desk-top self-poise whizzer (DT5-3) of low speed; CO2 incubator (170R); Inverted phase contrast microscope (CKX41 Olympus); Automated cell calculating instrument (ountstarIC-100); Biohazard Safety Equipment (BSC-1000IIAC).
2. method
(1) after being gone down to posterity by human hepatoma cell strain Hep3B2.1-7, with the trypsin digestion and cell of 0.25%, cell count to 10 is adjusted with PBS7Individual/ml, obtains knurl liquid.
(2) getting 30 mouse (18-22g/ is only), the knurl liquid 0.1ml of inoculation step 1 gained is after right upper extremity is subcutaneous respectively, is divided into following 4 groups at random:
1) sweet wormwood alkene low dose group: give the treatment of sweet wormwood alkene, 25mg/kg;
2) sweet wormwood alkene high dose group group: give the treatment of sweet wormwood alkene, 50mg/kg;
3) positive controls: give along platinum (DDP) treatment, 20mg/kg;
4) blank group: peanut oil abdominal injection.
When transplanted tumor maximum diameter > 5mm time, it is believed that transplanted tumor is inoculated successfully, and strain cell all became knurl when 5 days. Becoming knurl pneumoretroperitoneum drug administration by injection, sweet wormwood alkene group daily once, continuous 10 days, is stopped medicine and is weighed, peel off subcutaneous tumors body after dissection next day, claims knurl weight; The administration next day of positive controls, continuous five times, stops medicine and weighs, peel off subcutaneous tumors body after dissection next day, claims knurl weight; Blank group abdominal injection peanut oil, daily once, continuous 10 days, peels off subcutaneous tumors body after dissection, claim knurl weight. It is calculated as follows the average knurl of tumour inhibiting rate (%)=(control group average knurl weight-administration group average knurl weight)/control group heavy × 100%.
3. result
Table 1 sweet wormwood alkene is on the impact of human hepatoma cell strain Hep3B2.1-7 subcutaneous transplantation knurl size
Result is as shown in table 1, and the restraining effect of human hepatoma cell strain Hep3B2.1-7 subcutaneous transplantation knurl is increased with dosage and increases by sweet wormwood alkene, and effect and the DDP of its Tumor suppression are suitable.
Test two sweet wormwood alkene to the Inhibition test of human hepatoma cell strain QGY-7703 subcutaneous transplantation knurl
Experiment purpose: research sweet wormwood alkene is to the restraining effect of rat liver cancer transplanted tumor.
1. experiment material
1.1 laboratory animal
Only, body weight 18～22g, is provided, conformity certification number: 11400700123247 the NODSCID mouse 3O in 6～8 week age of animal by Beijing Wei Tonglihua laboratory animal technology company limited.
1.2 reagent
Peanut oil (Changxing, Guangdong food trade company limited, lot number 090407); Foetal calf serum (Hyclone company); Phosphate buffered saline buffer (PBS, Hyclone company).
1.3 test medicine
Sweet wormwood alkene: be purchased from Tianjin Science and Technology Ltd. of Silan, lot number A777550.Getting sweet wormwood alkene in beaker, add peanut oil as solvent, heating, is stirred to dissolve, and is that namely 5mg/ml obtains sweet wormwood alkene oil solution with peanut oil to the mass concentration of wormwood artemisia alkene in peanut oil after letting cool.
1.4 instrument
The desk-top self-poise whizzer (DT5-3) of low speed; CO2 incubator (170R); Inverted phase contrast microscope (CKX41 Olympus); Automated cell calculating instrument (ountstarIC-100); Biohazard Safety Equipment (BSC-1000IIAC); Electronic analytical balance (Beijing Sai Duolisi balance company limited); BioTekELx800 microplate reader (BioTek company of the U.S.).
2. method
(1) after being gone down to posterity by human hepatoma cell strain QGY-7703, with the trypsin digestion and cell of 0.25%, cell count to 10 is adjusted with PBS7Individual/ml, obtains knurl liquid.
(2) getting 30 mouse (18-22g/ is only), the knurl liquid 0.1ml of inoculation step 1 gained is after right upper extremity is subcutaneous respectively, is divided into following 4 groups at random
1) sweet wormwood alkene low dose group: give the treatment of sweet wormwood alkene, 25mg/kg;
2) sweet wormwood alkene high dose group group: give the treatment of sweet wormwood alkene, 50mg/kg;
3) positive controls: give along platinum (DDP) treatment, 20mg/kg;
4) blank group: peanut oil abdominal injection.
When transplanted tumor maximum diameter > 5mm time, it is believed that transplanted tumor is inoculated successfully, and strain cell all became knurl when 5 days. Becoming knurl pneumoretroperitoneum drug administration by injection, this compound group daily once, continuous 10 days, is stopped medicine and is weighed, peel off subcutaneous tumors body after dissection next day, claims knurl weight; The administration next day of positive controls, continuous five times, stops medicine and weighs, peel off subcutaneous tumors body after dissection next day, claims knurl weight; Blank group abdominal injection peanut oil, daily once, continuous 10 days, peels off subcutaneous tumors body after dissection, claim knurl weight. It is calculated as follows the average knurl of tumour inhibiting rate (%)=(control group average knurl weight-administration group average knurl weight)/control group heavy × 100%.
3. result
Table 2 sweet wormwood alkene is on the impact of human hepatoma cell strain QGY-7703 subcutaneous transplantation knurl size
Result is as shown in table 2, and the restraining effect of human hepatoma cell strain QGY-7703 subcutaneous transplantation knurl is increased with dosage and increases by sweet wormwood alkene, and effect and the DDP of its Tumor suppression are suitable.
4. discuss
This experiment adopts and builds the external transplanted tumor mouse model of liver cancer, inquires into the antitumor action of sweet wormwood alkene. Result shows, and sweet wormwood alkene can obviously suppress the growth of mouse subcutaneous transplanting knurl, and tumour inhibiting rate increases with the increase of its dosage.
Example 2 (pharmacology, effect experiment)
Test the impact that human hepatoma cell strain Hep3B2.1-7 is bred by a sweet wormwood alkene
Experiment purpose: the impact that human hepatoma cell strain Hep3B2.1-7 is bred by research sweet wormwood alkene.
1. experiment material
1.1 cell strain human hepatoma cell strain Hep3B2.1-7
1.2 medicinal materials, reagent and instrument sweet wormwood alkene: be purchased from Tianjin Science and Technology Ltd. of Silan, lot number A777550. Foetal calf serum (Hyclone company), MEM substratum (Gibco company), DMSO (sigma company), MTT (sigma company); Trypsin Gibco company). Inverted phase contrast microscope (CKX41 Olympus); BioTekELx800 microplate reader (BioTek company of the U.S.); The desk-top self-poise whizzer (DT5-3) of low speed.
2. experimental technique
When 2.1Hep3B2.1-7 cell culture growth is to exponential phase of growth, namely with 0.25% tryptic digestion, the centrifugal 3min of 1000 × g, cell precipitation is adjusted to 5 × 10 with the MEM substratum of 10%FBS4Individual/ml, 200 μ l are inoculated in the 96 every holes of well culture plate, are placed in 37 DEG C, 5%CO2And cultivate 24 hours when saturated humidity.
2.2 often group establish 5 multiple holes, every hole adds the sweet wormwood alkene of different concns, continues under above-mentioned condition to cultivate 24 hours.
Negative control group: i.e. not dosing group.
According to the medicine added, experiment is divided into 4 groups:
Experimental group 1: add sweet wormwood alkene, final concentration is 1 μM.
Experimental group 2: add sweet wormwood alkene, final concentration is 5 μMs.
Experimental group 3: add sweet wormwood alkene, final concentration is 10 μMs.
Experimental group 4: add sweet wormwood alkene, final concentration is 20 μMs.
2.3 cell cultures, after 24 hours, siphon away substratum, and every hole adds MTT (the tetramethyl-azo azoles salt) reagent of 100 μ l, 37 DEG C, 5%CO2And continue when saturated humidity to cultivate 4 hours.
2.4 carefully suck liquid in hole, and every hole adds 150ul dimethyl sulfoxide (DMSO), put low-speed oscillation 10min on shaking table, and crystallization is fully dissolved.
It is determined at the absorbance (OD) that wavelength is 450nm place by microplate reader, 570nm is as with reference to wavelength, inhibitory rate of cell growth presses formula: inhibiting rate=(control wells OD value-experimental port OD value)/control wells OD value) × 100% calculating.
3. experimental result
Table 3 sweet wormwood alkene is on the impact of Hep3B2.1-7 cell proliferation
| Grouping | Inhibiting rate (%) |
| Experimental group 4 | 72.64 |
| Experimental group 3 | 50.50 |
| Experimental group 2 | 37.42 |
| Experimental group 1 | 11.07 |
Result is as shown in table 3, and sweet wormwood alkene can effectively suppress Hep3B2.1-7 cell proliferation, below its inhibiting rate strengthens with dosage and increases progressively.
Test the impact that human hepatoma cell strain QGY-7703 is bred by two sweet wormwood alkene
Experiment purpose: the impact that human hepatoma cell strain QGY-7703 is bred by research sweet wormwood alkene.
1. experiment material
1.1 cell strain human hepatoma cell strain QGY-7703
1.2 medicinal materials, reagent and instrument sweet wormwood alkene: be purchased from Tianjin Science and Technology Ltd. of Silan, lot number A777550. Foetal calf serum (Hyclone company), MEM substratum (Gibco company), DMSO (sigma company), MTT (sigma company); Trypsin Gibco company). Inverted phase contrast microscope (CKX41 Olympus); BioTekELx800 microplate reader (BioTek company of the U.S.); The desk-top self-poise whizzer (DT5-3) of low speed.
2. experimental technique
When 2.1QGY-7703 cell culture growth is to exponential phase of growth, namely with 0.25% tryptic digestion, the centrifugal 3min of 1000 × g, cell precipitation is adjusted to 5 × 10 with 1640 substratum of 10%FBS4Individual/ml, 200 μ l are inoculated in the 96 every holes of well culture plate, are placed in 37 DEG C, 5%CO2And cultivate 24 hours when saturated humidity.
2.2 often group establish 5 multiple holes, every hole adds the sweet wormwood alkene of different concns, continues under above-mentioned condition to cultivate 24 hours.
Negative control group: i.e. not dosing group.
According to the medicine added, experiment is divided into 4 groups:
Experimental group 1: add sweet wormwood alkene, final concentration is 1 μM.
Experimental group 2: add sweet wormwood alkene, final concentration is 5 μMs.
Experimental group 3: add sweet wormwood alkene, final concentration is 10 μMs.
Experimental group 4: add sweet wormwood alkene, final concentration is 20 μMs.
2.3 cell cultures, after 24 hours, siphon away substratum, and every hole adds MTT (the tetramethyl-azo azoles salt) reagent of 100 μ l, 37 DEG C, 5%CO2And continue when saturated humidity to cultivate 4 hours.
2.4 carefully suck liquid in hole, and every hole adds 150ul dimethyl sulfoxide (DMSO), put low-speed oscillation 10min on shaking table, and crystallization is fully dissolved.
It is determined at the absorbance (OD) that wavelength is 450nm place by microplate reader, 570nm is as with reference to wavelength, inhibitory rate of cell growth presses formula: inhibiting rate=(control wells OD value-experimental port OD value)/control wells OD value) × 100% calculating.
3. experimental result:
Table 4 sweet wormwood alkene is on the impact of QGY-7703 cell proliferation
Result is as shown in table 4, and sweet wormwood alkene can effectively suppress QGY-7703 cell proliferation, below its inhibiting rate strengthens with dosage and increases progressively.
Test the impact that human hepatoma cell strain Huh7 is bred by three sweet wormwood alkene
Experiment purpose: the impact that human hepatoma cell strain Huh7 is bred by research sweet wormwood alkene.
1. experiment material
1.1 cell strain human hepatoma cell strain Huh7
1.2 medicinal materials, reagent and instrument sweet wormwood alkene: be purchased from Tianjin Science and Technology Ltd. of Silan, lot number A777550. Foetal calf serum (Hyclone company), MEM substratum (Gibco company), DMSO (sigma company), MTT (sigma company); Trypsin Gibco company). Inverted phase contrast microscope (CKX41 Olympus); BioTekELx800 microplate reader (BioTek company of the U.S.); The desk-top self-poise whizzer (DT5-3) of low speed.
2. experimental technique
When 2.1Huh7 cell culture growth is to exponential phase of growth, namely with 0.25% tryptic digestion, the centrifugal 3min of 1000 × g, cell precipitation is adjusted to 5 × 10 with 1640 substratum of 10%FBS4Individual/ml, 200 μ l are inoculated in the 96 every holes of well culture plate, are placed in 37 DEG C, 5%CO2And cultivate 24 hours when saturated humidity.
2.2 often group establish 5 multiple holes, every hole adds the sweet wormwood alkene of different concns, continues under above-mentioned condition to cultivate 24 hours.
Negative control group: i.e. not dosing group.
According to the medicine added, experiment is divided into 4 groups:
Experimental group 1: add sweet wormwood alkene, final concentration is 1 μM.
Experimental group 2: add sweet wormwood alkene, final concentration is 5 μMs.
Experimental group 3: add sweet wormwood alkene, final concentration is 10 μMs.
Experimental group 4: add sweet wormwood alkene, final concentration is 20 μMs.
2.3 cell cultures, after 24 hours, siphon away substratum, and every hole adds MTT (the tetramethyl-azo azoles salt) reagent of 100 μ l, 37 DEG C, 5%CO2And continue when saturated humidity to cultivate 4 hours.
2.4 carefully suck liquid in hole, and every hole adds 150ul dimethyl sulfoxide (DMSO), put low-speed oscillation 10min on shaking table, and crystallization is fully dissolved.
It is determined at the absorbance (OD) that wavelength is 450nm place by microplate reader, 570nm is as with reference to wavelength, inhibitory rate of cell growth presses formula: inhibiting rate=(control wells OD value-experimental port OD value)/control wells OD value) × 100% calculating.
3. experimental result
Table 5 sweet wormwood alkene is on the impact of Huh7 cell proliferation
| Grouping | Inhibiting rate (%) |
| Experimental group 4 | 63.85 |
| Experimental group 3 | 49.27 |
| Experimental group 2 | 33.71 |
| Experimental group 1 | 25.46 |
Result is as shown in table 5, and sweet wormwood alkene can effectively suppress Huh7 cell proliferation, below its inhibiting rate strengthens with dosage and increases progressively.
Test the impact that human hepatoma cell strain sk-hep1 is bred by four sweet wormwood alkene
Experiment purpose: the impact that human hepatoma cell strain sk-hep1 is bred by research sweet wormwood alkene.
1. experiment material
1.1 cell strain human hepatoma cell strain sk-hep1
1.2 medicinal materials, reagent and instrument sweet wormwood alkene: be purchased from Tianjin Science and Technology Ltd. of Silan, lot number A777550. Foetal calf serum (Hyclone company), MEM substratum (Gibco company), DMSO (sigma company), MTT (sigma company); Trypsin Gibco company). Inverted phase contrast microscope (CKX41 Olympus); BioTekELx800 microplate reader (BioTek company of the U.S.); The desk-top self-poise whizzer (DT5-3) of low speed.
2. experimental technique
When 2.1sk-hep1 cell culture growth is to exponential phase of growth, namely with 0.25% tryptic digestion, the centrifugal 3min of 1000 × g, cell precipitation is adjusted to 5 × 10 with 1640 substratum of 10%FBS4Individual/ml, 200 μ l are inoculated in the 96 every holes of well culture plate, are placed in 37 DEG C, 5%CO2And cultivate 24 hours when saturated humidity.
2.2 often group establish 5 multiple holes, every hole adds the sweet wormwood alkene of different concns, continues under above-mentioned condition to cultivate 24 hours.
Negative control group: i.e. not dosing group.
According to the medicine added, experiment is divided into 4 groups:
Experimental group 1: add sweet wormwood alkene, final concentration is 1 μM.
Experimental group 2: add sweet wormwood alkene, final concentration is 5 μMs.
Experimental group 3: add sweet wormwood alkene, final concentration is 10 μMs.
Experimental group 4: add sweet wormwood alkene, final concentration is 20 μMs.
2.3 cell cultures, after 24 hours, siphon away substratum, and every hole adds MTT (the tetramethyl-azo azoles salt) reagent of 100 μ l, 37 DEG C, 5%CO2And continue when saturated humidity to cultivate 4 hours.
2.4 carefully suck liquid in hole, and every hole adds 150ul dimethyl sulfoxide (DMSO), put low-speed oscillation 10min on shaking table, and crystallization is fully dissolved.
It is determined at the absorbance (OD) that wavelength is 450nm place by microplate reader, 570nm is as with reference to wavelength, inhibitory rate of cell growth presses formula: inhibiting rate=(control wells OD value-experimental port OD value)/control wells OD value) × 100% calculating.
3. experimental result:
Table 6 sweet wormwood alkene is on the impact of sk-hep1 cell proliferation
| Grouping | Inhibiting rate (%) |
| Experimental group 4 | 87.26 |
| Experimental group 3 | 54.95 |
| Experimental group 2 | 30.29 |
| Experimental group 1 | 18.88 |
Result is as shown in table 6, and sweet wormwood alkene can effectively suppress sk-hep1 cell proliferation, below its inhibiting rate strengthens with dosage and increases progressively.
Test the impact that human hepatoma cell strain QGY-7703 is withered and dies by five sweet wormwood alkene
Experiment purpose: the impact that human hepatoma cell strain QGY-7703 is withered and dies by research sweet wormwood alkene.
1. experiment material
1.1 cell strain human hepatoma cell strain QGY-7703
1.2 medicinal materials, reagent and instrument sweet wormwood alkene: be purchased from Tianjin Science and Technology Ltd. of Silan, lot number A777550. Foetal calf serum (Hyclone company), MEM substratum (Gibco company), DMSO (sigma company); Apoptosis kit (BD company); Trypsin Gibco company). Inverted phase contrast microscope (CKX41 Olympus); BioTekELx800 microplate reader (BioTek company of the U.S.); The desk-top self-poise whizzer (DT5-3) of low speed; Flow cytometer (BekmanFC500).
2. experimental technique
When 2.1QGY-7703 cell culture growth is to exponential phase of growth, namely with 0.25% tryptic digestion, the centrifugal 3min of 1000 × g, cell precipitation is adjusted to 5 × 10 with 1640 substratum of 10%FBS5Individual/ml, 6 well culture plate every hole inoculation 2ml, is placed in 37 DEG C, 5%CO2And cultivate 24 hours when saturated humidity.
2.2 often group establish 3 multiple holes, every hole adds the sweet wormwood alkene of different concns, continues under above-mentioned condition to cultivate 24 hours.
According to the medicine added, experiment is divided into 6 groups:
Positive controls: adding along platinum, final concentration is 10 μ g/ml.
Negative control group: i.e. not dosing group.
According to the medicine added, experiment is divided into 4 groups:
Experimental group 1: add sweet wormwood alkene, final concentration is 1 μM.
Experimental group 2: add sweet wormwood alkene, final concentration is 5 μMs.
Experimental group 3: add sweet wormwood alkene, final concentration is 10 μMs.
Experimental group 4: add sweet wormwood alkene, final concentration is 20 μMs.
After 2.3 cell cultures 24 h before harvest cells, PBS washes 2 cells, 1000rpm, and 4 DEG C centrifugal, 10min.
2.4 by 1 × 106Individual/ml cell is resuspended in damping fluid.
2.5 get 500ul cell suspension adds in test tube.
2.6 add 5ulannexinv-FITC, and 5ul propidium iodide enters in test tube.
2.7 lucifuges, room temperature 10min.
Machine testing on 2.8.
3. experimental result
Table 7 sweet wormwood alkene is on the impact of QGY-7703 apoptosis
| Group | Necrosis rate (%) | Apoptosis rate (%) |
| Negative control group | 0.1 | 0 |
| Positive controls | 4.8 | 8.9 |
| Experimental group 1 | 0.4 | 1.3 |
| Experimental group 2 | 0.9 | 2.1 |
| Experimental group 3 | 0.9 | 18.7 |
| Experimental group 4 | 34.6 | 21.5 |
Result is as shown in table 7, and when the final final concentration of sweet wormwood alkene is 20 μMs, QGY-7703 cell obtains apoptosis rate up to more than 50%, it is seen that sweet wormwood alkene energy inducing apoptosis of tumour cell.
Example 3 (pharmacology, effect experiment)
Test a sweet wormwood alkene to be tested by Mouse Liver injury of the kidney
Experiment purpose: research sweet wormwood alkene is to the damage of Mouse Liver kidney.
1. experiment material
1.1 laboratory animal
Only, body weight 18～22g, is provided, conformity certification number: 11400700123247 the NODSCID mouse 3O in 6～8 week age of animal by Beijing Wei Tonglihua laboratory animal technology company limited.
1.2 reagent
Peanut oil (Changxing, Guangdong food trade company limited, lot number 090407); Foetal calf serum (Hyclone company); Phosphate buffered saline buffer (PBS, Hyclone development company); HE dye liquor (Google is biological); Dehydrated alcohol (luxuriant greatly); Dimethylbenzene (luxuriant greatly).
1.3 test medicine
Sweet wormwood alkene: be purchased from Tianjin Science and Technology Ltd. of Silan, lot number A777550. Getting sweet wormwood alkene in beaker, add peanut oil as solvent, heating, is stirred to dissolve, and is 5mg/ml with peanut oil to the mass concentration of wormwood artemisia alkene in peanut oil, obtains sweet wormwood alkene oil solution after letting cool.
1.4 instrument
The desk-top self-poise whizzer (DT5-3) of low speed; CO2 incubator (170R); Inverted phase contrast microscope (CKX41 Olympus); Automated cell calculating instrument (ountstarIC-100); Biohazard Safety Equipment (BSC-1000IIAC); Full-automatic dyeing envelope sheet industrial workstation (LEICAST5020), tissue embedding machine (HisTOSTAR), Full automatic closed tissue dewatering machine (SHANDONPATACENTRE), paraffin section machine (LEICARM2245).
2. method
Hematoxylin-eosin (Hematoxylin-eosin, HE) dye: the section carrying out paraffin sample with paraffin section machine, thickness 3.5 μm, serial section, dyeing procedure is roasting sheet drying 20 minutes, dimethylbenzene 2 times × 10 minutes, dehydrated alcohol 2 times × 2 minutes, 95% ethanol 1 minute, 80% ethanol 1 minute, 70% ethanol 1 minute, wash 1 minute, Hematorylin 8 minutes, Hematorylin 10 minutes, wash 2 times × 1 minute, 0.5% hydrochloride alcohol 10 seconds, wash 10 minutes, 2 minutes, Yihong, wash 1 minute, 80% ethanol 5 seconds, 85% ethanol 5 seconds, 90% ethanol 5 seconds, 95% ethanol 1 minute, dehydrated alcohol 2 times × 2 minutes, dehydrated alcohol 3 minutes, dimethylbenzene 2 times × 2 minutes, direct neutral tree rubber seal sheet after terminating dyeing.
2.1 experimental result
As shown in Figure 1, animal liver and kidney is had no significant effect result by sweet wormwood alkene. Wherein, the visible sinus hepaticus of liver section, liver rope morphological structure are complete, and liver has no nucleus oedema, and tenuigenin is without pathology; The visible renal glomerulus structural integrity of Kidney sections, and quantity is consistent with control group, tenuigenin, nucleus are showed no pathological change form.
Example 4 (pharmacology, effect experiment)
Test the impact that people's tire liver inoblast (Liverfibroblast) is bred by a sweet wormwood alkene
Experiment purpose: the impact that people's tire liver inoblast (Liverfibroblast) is bred by research sweet wormwood alkene.
1. experiment material
1.1 cell strains people's tire liver inoblast (Liverfibroblast)
1.2 medicinal materials, reagent and instrument sweet wormwood alkene: be purchased from Tianjin Science and Technology Ltd. of Silan, lot number A777550.Foetal calf serum (Hyclone company), MEM substratum (Gibco company), DMSO (sigma company), MTT (sigma company); Trypsin Gibco company). Inverted phase contrast microscope (CKX41 Olympus); BioTekELx800 microplate reader (BioTek company of the U.S.); The desk-top self-poise whizzer (DT5-3) of low speed.
2. experimental technique
When 2.1 people's tire liver inoblast (Liverfibroblast) incubation growth are to exponential phase of growth, namely with 0.25% tryptic digestion, the centrifugal 3min of 1000 × g, cell precipitation is adjusted to 5 × 10 with the DMEM substratum of 10%FBS4Individual/ml, 200 μ l are inoculated in the 96 every holes of well culture plate, are placed in 37 DEG C, 5%CO2And cultivate 24 hours when saturated humidity.
2.2 often group establish 5 multiple holes, every hole adds the sweet wormwood alkene of different concns, continues under above-mentioned condition to cultivate 24 hours.
Negative control group: i.e. not dosing group.
According to the medicine added, experiment is divided into 4 groups:
Experimental group 1: add sweet wormwood alkene, final concentration is 1 μM.
Experimental group 2: add sweet wormwood alkene, final concentration is 5 μMs.
Experimental group 3: add sweet wormwood alkene, final concentration is 10 μMs.
Experimental group 4: add sweet wormwood alkene, final concentration is 20 μMs.
2.3 cell cultures, after 24 hours, siphon away substratum, and every hole adds MTT (the tetramethyl-azo azoles salt) reagent of 100 μ l, 37 DEG C, 5%CO2And continue when saturated humidity to cultivate 4 hours.
2.4 carefully suck liquid in hole, and every hole adds 150ul dimethyl sulfoxide (DMSO), put low-speed oscillation 10min on shaking table, and crystallization is fully dissolved.
It is determined at the absorbance (OD) that wavelength is 450nm place by microplate reader, 570nm is as with reference to wavelength, inhibitory rate of cell growth presses formula: inhibiting rate=(control wells OD value-experimental port OD value)/control wells OD value) × 100% calculating.
3. experimental result
Table 8 sweet wormwood alkene is on the impact of people's tire liver fibroblast proliferation
| Grouping | Survival rate (%) |
| Experimental group 4 | 126.34 |
| Experimental group 3 | 115.32 |
| Experimental group 2 | 107.60 |
| Experimental group 1 | 102.62 |
Result is as shown in table 8, and sweet wormwood alkene is to people's tire liver inoblast (Liverfibroblast) nontoxicity.
Test the impact that the neural stem cell (NSC) that ipsc is differentiation-inducing is bred by two sweet wormwood alkene
Experiment purpose: the impact that the neural stem cell (NSC) that ipsc is differentiation-inducing is bred by research sweet wormwood alkene
1. experiment material
The neural stem cell (NSC) that 1.1 cell strain ipsc are differentiation-inducing
1.2 medicinal materials, reagent and instrument sweet wormwood alkene: be purchased from Tianjin Science and Technology Ltd. of Silan, lot number A777550. Foetal calf serum (Hyclone company), MEM substratum (Gibco company), DMSO (sigma company), MTT (sigma company); Trypsin Gibco company). Inverted phase contrast microscope (CKX41 Olympus); BioTekELx800 microplate reader (BioTek company of the U.S.); The desk-top self-poise whizzer (DT5-3) of low speed.
2. experimental technique
When 2.1NSC cell culture growth is to exponential phase of growth, namely with 0.25% tryptic digestion, the centrifugal 3min of 1000 × g, cell precipitation is adjusted to 5 × 10 with nerve stem cell culture medium4Individual/ml, 200 μ l are inoculated in the 96 every holes of well culture plate, are placed in 37 DEG C, 5%CO2And cultivate 24 hours when saturated humidity.
2.2 often group establish 5 multiple holes, every hole adds the sweet wormwood alkene of different concns, continues under above-mentioned condition to cultivate 24 hours.
Negative control group: i.e. not dosing group.
According to the medicine added, experiment is divided into 4 groups:
Experimental group 1: add sweet wormwood alkene, final concentration is 1 μM.
Experimental group 2: add sweet wormwood alkene, final concentration is 5 μMs.
Experimental group 3: add sweet wormwood alkene, final concentration is 10 μMs.
Experimental group 4: add sweet wormwood alkene, final concentration is 20 μMs.
2.3 cell cultures, after 24 hours, siphon away substratum, and every hole adds MTT (the tetramethyl-azo azoles salt) reagent of 100 μ l, 37 DEG C, 5%CO2And continue when saturated humidity to cultivate 4 hours.
2.4 carefully suck liquid in hole, and every hole adds 150ul dimethyl sulfoxide (DMSO), put low-speed oscillation 10min on shaking table, and crystallization is fully dissolved.
It is determined at the absorbance (OD) that wavelength is 450nm place by microplate reader, 570nm is as with reference to wavelength, inhibitory rate of cell growth presses formula: inhibiting rate=(control wells OD value-experimental port OD value)/control wells OD value) × 100% calculating.
3. experimental result:
Table 9 sweet wormwood alkene is on the impact of NSC cell proliferation
| Grouping | Survival rate (%) |
| Experimental group 4 | 67.68 |
| Experimental group 3 | 93.05 |
| Experimental group 2 | 109.39 |
| Experimental group 1 | 113.45 |
Result is as shown in table 9, and sweet wormwood alkene lower concentration can promote NSC cell proliferation, and high density can suppress the propagation of NCS cell, but to the inhibiting rate of NSC cell lower than the inhibiting rate to tumour cell when sweet wormwood alkene concentration is 20 μMs. Therefore sweet wormwood alkene is to NSC cytotoxic.
Test the impact that people's renal cells (HK2) is bred by three sweet wormwood alkene
Experiment purpose: the impact that people's renal cells (HK2) is bred by research sweet wormwood alkene
1. experiment material
1.1 cell strains people's renal cells (HK2)
1.2 medicinal materials, reagent and instrument sweet wormwood alkene: be purchased from Tianjin Science and Technology Ltd. of Silan, lot number A777550. Foetal calf serum (Hyclone company), MEM substratum (Gibco company), DMSO (sigma company), MTT (sigma company); Trypsin Gibco company). Inverted phase contrast microscope (CKX41 Olympus); BioTekELx800 microplate reader (BioTek company of the U.S.); The desk-top self-poise whizzer (DT5-3) of low speed.
2. experimental technique
When 2.1HK2 cell culture growth is to exponential phase of growth, namely with 0.25% tryptic digestion, the centrifugal 3min of 1000 × g, cell precipitation is adjusted to 5 × 10 with the DMEM substratum of 10%FBS4Individual/ml, 200 μ l are inoculated in the 96 every holes of well culture plate, are placed in 37 DEG C, 5%CO2And cultivate 24 hours when saturated humidity.
2.2 often group establish 5 multiple holes, every hole adds the sweet wormwood alkene of different concns, continues under above-mentioned condition to cultivate 24 hours.
Negative control group: i.e. not dosing group.
According to the medicine added, experiment is divided into 4 groups:
Experimental group 1: add sweet wormwood alkene, final concentration is 1 μM.
Experimental group 2: add sweet wormwood alkene, final concentration is 5 μMs.
Experimental group 3: add sweet wormwood alkene, final concentration is 10 μMs.
Experimental group 4: add sweet wormwood alkene, final concentration is 20 μMs.
2.3 cell cultures, after 24 hours, siphon away substratum, and every hole adds MTT (the tetramethyl-azo azoles salt) reagent of 100 μ l, 37 DEG C, 5%CO2And continue when saturated humidity to cultivate 4 hours.
2.4 carefully suck liquid in hole, and every hole adds 150ul dimethyl sulfoxide (DMSO), put low-speed oscillation 10min on shaking table, and crystallization is fully dissolved.
It is determined at the absorbance (OD) that wavelength is 450nm place by microplate reader, 570nm is as with reference to wavelength, inhibitory rate of cell growth presses formula: inhibiting rate=(control wells OD value-experimental port OD value)/control wells OD value) × 100% calculating.
3. experimental result:
Table 10 sweet wormwood alkene is on the impact of HK2 cell proliferation
| Grouping | Survival rate (%) |
| Experimental group 4 | 88.88 |
| Experimental group 3 | 105.97 |
| Experimental group 2 | 105.80 |
| Experimental group 1 | 104.98 |
Result is as shown in table 10, and sweet wormwood alkene is to people's renal cells (HK2) nontoxicity.
Test the impact that human bronchial epithelial cell (beas-2b) is bred by four sweet wormwood alkene
Experiment purpose: the impact that human bronchial epithelial cell (beas-2b) is bred by research sweet wormwood alkene
1. experiment material
1.1 cell strain human bronchial epithelial cells (beas-2b)
1.2 medicinal materials, reagent and instrument sweet wormwood alkene: be purchased from Tianjin Science and Technology Ltd. of Silan, lot number A777550. Foetal calf serum (Hyclone company), MEM substratum (Gibco company), DMSO (sigma company), MTT (sigma company); Trypsin Gibco company). Inverted phase contrast microscope (CKX41 Olympus); BioTekELx800 microplate reader (BioTek company of the U.S.); The desk-top self-poise whizzer (DT5-3) of low speed.
2. experimental technique
When 2.1beas-2b cell culture growth is to exponential phase of growth, namely with 0.25% tryptic digestion, the centrifugal 3min of 1000 × g, cell precipitation is adjusted to 5 × 10 with 1640 substratum of 10%FBS4Individual/ml, 200 μ l are inoculated in the 96 every holes of well culture plate, are placed in 37 DEG C, 5%CO2And cultivate 24 hours when saturated humidity.
2.2 often group establish 5 multiple holes, every hole adds the sweet wormwood alkene of different concns, continues under above-mentioned condition to cultivate 24 hours.
Negative control group: i.e. not dosing group.
According to the medicine added, experiment is divided into 4 groups:
Experimental group 1: add sweet wormwood alkene, final concentration is 1 μM.
Experimental group 2: add sweet wormwood alkene, final concentration is 5 μMs.
Experimental group 3: add sweet wormwood alkene, final concentration is 10 μMs.
Experimental group 4: add sweet wormwood alkene, final concentration is 20 μMs.
2.3 cell cultures, after 24 hours, siphon away substratum, and every hole adds MTT (the tetramethyl-azo azoles salt) reagent of 100 μ l, 37 DEG C, 5%CO2And continue when saturated humidity to cultivate 4 hours.
2.4 carefully suck liquid in hole, and every hole adds 150ul dimethyl sulfoxide (DMSO), put low-speed oscillation 10min on shaking table, and crystallization is fully dissolved.
It is determined at the absorbance (OD) that wavelength is 450nm place by microplate reader, 570nm is as with reference to wavelength, inhibitory rate of cell growth presses formula: inhibiting rate=(control wells OD value-experimental port OD value)/control wells OD value) × 100% calculating.
3. experimental result:
Table 11 sweet wormwood alkene is on the impact of beas-2b cell proliferation
| Grouping | Survival rate (%) |
| Experimental group 4 | 67.09 |
| Experimental group 3 | 76.13 |
| Experimental group 2 | 93.96 |
| Experimental group 1 | 92.11 |
Result is as shown in table 11, and sweet wormwood alkene is to human bronchial epithelial cell (beas-2b) nontoxicity.
Test the impact that Human normal hepatocyte (LO-2) is bred by five sweet wormwood alkene
Experiment purpose: the impact that Human normal hepatocyte (LO-2) is bred by research sweet wormwood alkene
1. experiment material
1.1 cell strain Human normal hepatocytes (LO-2)
1.2 medicinal materials, reagent and instrument sweet wormwood alkene: be purchased from Tianjin Science and Technology Ltd. of Silan, lot number A777550. Foetal calf serum (Hyclone company), MEM substratum (Gibco company), DMSO (sigma company), MTT (sigma company); Trypsin Gibco company). Inverted phase contrast microscope (CKX41 Olympus); BioTekELx800 microplate reader (BioTek company of the U.S.); The desk-top self-poise whizzer (DT5-3) of low speed.
2. experimental technique
When 2.1LO-2 cell culture growth is to exponential phase of growth, namely with 0.25% tryptic digestion, the centrifugal 3min of 1000 × g, cell precipitation is adjusted to 5 × 10 with the DMEM substratum of 10%FBS4Individual/ml, 200 μ l are inoculated in the 96 every holes of well culture plate, are placed in 37 DEG C, 5%CO2And cultivate 24 hours when saturated humidity.
2.2 often group establish 5 multiple holes, every hole adds the sweet wormwood alkene of different concns, continues under above-mentioned condition to cultivate 24 hours.
Negative control group: i.e. not dosing group.
According to the medicine added, experiment is divided into 4 groups:
Experimental group 1: add sweet wormwood alkene, final concentration is 1 μM.
Experimental group 2: add sweet wormwood alkene, final concentration is 5 μMs.
Experimental group 3: add sweet wormwood alkene, final concentration is 10 μMs.
Experimental group 4: add sweet wormwood alkene, final concentration is 20 μMs.
2.3 cell cultures, after 24 hours, siphon away substratum, and every hole adds MTT (the tetramethyl-azo azoles salt) reagent of 100 μ l, 37 DEG C, 5%CO2And continue when saturated humidity to cultivate 4 hours.
2.4 carefully suck liquid in hole, and every hole adds 150ul dimethyl sulfoxide (DMSO), put low-speed oscillation 10min on shaking table, and crystallization is fully dissolved.
It is determined at the absorbance (OD) that wavelength is 450nm place by microplate reader, 570nm is as with reference to wavelength, inhibitory rate of cell growth presses formula: inhibiting rate=(control wells OD value-experimental port OD value)/control wells OD value) × 100% calculating.
3. experimental result:
Table 12 sweet wormwood alkene is on the impact of LO-2 cell proliferation
| Grouping | Inhibiting rate (%) |
| Experimental group 4 | 110.51 |
| Experimental group 3 | 116.11 |
| Experimental group 2 | 115.94 |
| Experimental group 1 | 104.64 |
Result is as shown in table 12, and sweet wormwood alkene is to Human normal hepatocyte (LO-2) nontoxicity.
3. conclusion
Sweet wormwood alkene both can the propagation of inhibition tumor cell, again to not normal cells side effect, thus show good antineoplastic action. There is the dose-effect relationship in name Xi'an between its restraining effect and its concentration, along with sweet wormwood alkene concentration increases, the ability of its inhibition tumor cell propagation strengthens. This experiment adopts the method for inside and outside combination, the antitumor action of mouse experiment checking sweet wormwood alkene in cell in vitro and body.
Embodiment
Example 1 (injection)
Get sweet wormwood alkene 500mg, add sodium-chlor 9g, add water to 1000ml, with 1mol/ml sodium hydroxide solution adjust pH to 3.0-5.5, filter, filtrate be potted in 2,5 or 10ml ampoule in, 100 DEG C of sterilizing 30min, obtain injection liquid. Injection is used for tumour particularly liver cancer patient. This product can intravenous injection or intramuscular injection, each 1～50ml, 1～3 time on the one, 10～20 days is a course for the treatment of, can use for 2～3 courses for the treatment of continuously.
Example 2 (soft capsule)
Get sweet wormwood alkene 30g to mix with edible corn oil 20g, fully stir even, add thinner 40g and make content, using appropriate Liquid Paraffin as lubricant, adopt spinning block platen press, being loaded by content in automatic rotary transformation of ownership capsule machine, suppressing specification is every soft capsule 500 containing sweet wormwood alkene 60mg, every net weight 0.18g; Wherein said thinner is gelatin by mass ratio: glycerine: water=1: the gelatin of 0.5: 1, glycerine and water are made. Oral it is applicable to tumour particularly liver cancer patient. Each 1～3,3 times on the one, within 10～20 days, it is a course for the treatment of, can use for 2～3 courses for the treatment of continuously.
Example 3 (tablet)
Get sweet wormwood alkene 40g, add lactose 480g, starch 1100g mix, the starch slurry 300g with 7% as tackiness agent wet granulation, is dried, is added Magnesium Stearate 16g and mix, is pressed into 10000, the tablet that every sheet contains sweet wormwood alkene 4mg, every sheet net weight 0.17g. Oral it is applicable to tumour particularly liver cancer patient. Each 3～9,3 times on the one, within 10～20 days, it is a course for the treatment of, can use for 2～3 courses for the treatment of continuously.
Claims (6)
1. sweet wormwood alkene is in the application prepared in antitumor drug, and the chemical structural formula of wherein said sweet wormwood alkene is shown in lower formula I,
2. application according to claim 1, it is characterised in that, described medicine is made up of sweet wormwood alkene and medically acceptable auxiliary material, and wherein the weight content of sweet wormwood alkene is 0.5～30%.
3. application according to claim 1 and 2, it is characterised in that, described medicine is injection or common oral preparations.
4. application according to claim 3, it is characterised in that, described injection is the aqueous solution that every 1000ml contains sweet wormwood alkene 500mg and sodium-chlor 9g, and pH value is 3.0～5.5.
5. application according to claim 3, it is characterised in that, described oral preparations is soft capsule, and the content of this soft capsule is made up of the raw material of following weight part: 30 parts, sweet wormwood alkene, edible corn oil 20 parts, thinner 40 parts.
6. application according to claim 3, it is characterised in that, described oral preparations is tablet, and this tablet is made up of sweet wormwood alkene and binder, and wherein the weight content of sweet wormwood alkene is 2.3～2.5%.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107157984A (en) * | 2017-06-20 | 2017-09-15 | 山东省中医药研究院 | Application of the Artemisitene in treatment liver disease drug is prepared |
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Non-Patent Citations (3)
| Title |
|---|
| BEEKMAN, AC等: "Cytotoxicity of Artemisinin, a Dimer of Dihydroartemisinin, Artemisitene and Eupatoriopicrin as Evaluated by the MTT and Clonogenic Assay", 《PHYTOTHERAPY RESEARCH》 * |
| THOMAS EFFERTH等: "Cytotoxic activity of secondary metabolites derived from Artemisia annua L. towards cancer cells in comparison to its designated active constituent artemisinin", 《PHYTOMEDICINE》 * |
| 张强等: "《药剂学》", 31 January 2005, 北京大学医学出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107157984A (en) * | 2017-06-20 | 2017-09-15 | 山东省中医药研究院 | Application of the Artemisitene in treatment liver disease drug is prepared |
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