CN101875661A - Anti-tumor compound extracted from house lizard as well as preparation method and application thereof - Google Patents
Anti-tumor compound extracted from house lizard as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an anti-tumor compound extracted from a house lizard, as well as a preparation method of the anti-tumor compound extracted from the house lizard and application of the compound as antitumor drugs. In clinical application, the anti-tumor compound extracted from the house lizard can be dissolved with normal saline and then used in an injection way or can be prepared into preparation for oral use directly; the anti-tumor compound extracted from the house lizard can be used for the chemotherapy of gastric cancer, lung cancer, liver cancer, colon cancer, breast cancer, leucocythemia, genital system tumors, and other common malignant tumors, plays remarkable anti-tumor effect without bone marrow suppression or damage to important organs under conventional dosage; and in addition, since the anti-tumor compound extracted from the house lizard can increase T cellular immunity, the anti-tumor compound has good Aids resistance.
Description
Technical field
The invention belongs to the pharmaceutical chemistry field, be specifically related to from a kind of antineoplastic compound that from gecko, extracts and preparation method thereof and application.
Background technology
At present, still lacking at numerous tumour patients under the prerequisite of effective treatment means, seeking of the treatment of the medicine of high-efficiency low-toxicity, having important significance for theories and using value the tumour cancer.
Gecko is traditional Chinese medicinal materials, and the history of its treatment cancerous swelling of life-time service is arranged in the traditional Chinese medical science.Gecko has another name called house lizard, day dragon, gecko etc., belongs to Reptilia door lizard order Gekkonidae Gekko.Nature and flavor: one-tenth, cold, slightly poisonous; Cure mainly: dispel the wind, arresting convulsion, dissipating bind, detoxifcation.Control paralysis due to windstroke, the severe and migratory arthralgia pain, wind phlegm is shied epilepsy, and scrofula is disliked sore, tetanus, diseases such as carbuncle sore, stomach belch gas.A large amount of in recent ten years clinical reports prove that gecko treatment malignant tumour effect is remarkable.
The Wu Xiongzhi of Tumour Hospital Attached To Tianjin Medical Univ.'s Integrated TCM ﹠ Western Medicine Dept. separates acquisition antitumor activity component---house lizard sulfated polysaccharide from gecko, and finds that it has the characteristic of remarkable inhibition liver cancer cell growth.And research this time with hepatoma cell strain as external trial test, test shows: can induce the liver cancer cell differentiation and maturation, suppress hepatoma cell proliferation, thereby play the effect of treatment liver cancer.At present all fail to obtain the monomeric compound of accurate chemical structure, and the activeconstituents antitumor action that has obtained is not obvious about the correlative study of Chinese medicine gecko.
China Patent No. " ZL200310105343.6 ", patent name: " a kind of preparation method of PTS ", adopting in the method and from the gecko raw material, extract anticancer composition, its anticancer active constituent is 3.0mg/mL, tumour inhibiting rate is 65.9%, and the activeconstituents antitumous effect is remarkable inadequately.
Summary of the invention
The purpose of this invention is to provide the antineoplastic compound that extracts from gecko, another purpose provides the preparation method of above-claimed cpd, and a purpose provides the application of above-claimed cpd again.
In order to realize above purpose, technical scheme of the present invention is:
The invention provides the antineoplastic compound that extracts from gecko, it is characterized in that: compound has the basic skeleton structure shown in (I) formula:
In the formula (I), C
3Be R, C
7Be the S configuration, or C
3Be S, C
7Be the R configuration.
The present invention also provides the derivative of antineoplastic compound (I) formula of extracting from gecko, it is characterized in that: its molecular weight of derivative of this antineoplastic compound (I) formula is 253-341, and this derivative is the compound shown in the general formula (II):
In the formula (II), C
2Be R or S configuration, C
3Be R, C
7Be S, C
8Be R, C
9Be the S configuration, or C
2Be R or S configuration, C
3Be S, C
7Be R, C
8Be S, C
9Be R configuration, substituent R
1Can be-COOCH
3Or-COOC
2H
5, R
2, R
3, R
4, R
5, R
6Identical or inequality, can be respectively H, OH, CH
3, C
2H
5, OCH
3, OC
2H
5
The derivative of relevant general formula (I) formula, promptly each compound such as the following table shown in the general formula (II) is listed:
The all cpds of table 1. formula (II)
| Sequence number | Molecular weight and molecular formula | ??R 1 | ??R 2 | ??R 3 | ??R 4 | ??R 5 | ??R 6 |
| ??Ⅰ | ??253?C 10H 11N 3O 3S | ??COOCH 3 | ?H | ??H | ??H | ??H | ??H |
| ??Ⅱ | ??267?C 11H 13N 3O 3S | ??COOC 2H 5 | ?H | ??H | ??H | ??H | ??H |
| ??Ⅲ | ??295?C 13H 17N 3O 3S | ??COOC 2H 5 | ?CH 3 | ??H | ??H | ??H | ??CH 3 |
| ??Ⅳ | ??309?C 14H 19N 3O 3S | ??COOC 2H 5 | ?H | ??CH 3 | ??C 2H 5 | ??H | ??H |
| ??Ⅴ | ??297?C 12H 15N 3O 4S | ??COOCH 3 | ?CH 3 | ??H | ??H | ??OCH 3 | ??H |
| ??Ⅵ | ??325?C 14H 19N 3O 4S | ??COOC 2H 5 | ?OCH 3 | ??H | ??H | ??CH 3 | ??CH 3 |
| ??Ⅶ | ??327?C 13H 17N 3O 5S | ??COOC 2H 5 | ?OH | ??OCH 3 | ??CH 3 | ??H | ??H |
| ??Ⅷ | ??297?C 12H 15N 3O 4S | ??COOCH 3 | ?H | ??H | ??H | ??CH 3 | ??OCH 3 |
| ??Ⅸ | ??327?C 13H 17N 3O 5S | ??COOC 2H 5 | ?CH 3 | ??H | ??OCH 3 | ??H | ??OH |
| ??Ⅹ | ??323?C 15H 21N 3O 3S | ??COOC 2H 5 | ?C 2H 5 | ??H | ??H | ??H | ??C 2H 5 |
| ??Ⅺ | ??297?C 12H 15N 3O 4S | ??COOCH 3 | ?OCH 3 | ??H | ??H | ??CH 3 | ??H |
| ??Ⅻ | ??313?C 12H 15N 3O 5S | ??COOC 2H 2 | ?H | ??OH | ??H | ??H | ??OCH 3 |
| ??ⅩⅢ | ??325?C 14H 19N 3O 4S | ??COOCH 3 | ?OC 2H 5 | ??C 2H 5 | ??H | ??H | ??H |
| ??ⅩⅣ | ??341?C 14H 19N 3O 5S | ??COOC 2H 5 | ?H | ??OCH 3 | ??H | ??H | ??OC 2H 5 |
| ??ⅩⅤ | ??325?C 14H 19N 3O 4S | ??COOC 2H 5 | ?OCH 3 | ??H | ??H | ??C 2H 5 | ??H |
By last table as can be seen, this antineoplastic compound, promptly the derivative of (I) formula is an imdazole derivatives.
Wherein through Agilent Q-TOF6510 LC-MS instrument mass spectrometric detection, the fragment of formula I molecule is:
Quality is 238,
Quality is 136
The hydrogen spectrum detected result of INOVA-600 nuclear magnetic resonance analyser:
Wherein through Agilent Q-TOF6510 LC-MS instrument mass spectrometric detection, the fragment of formula II molecule is:
Quality is 238,
Quality is 136
The hydrogen spectrum detected result of INOVA-600 nuclear magnetic resonance analyser:
Wherein through Agilent Q-TOF6510 LC-MS instrument mass spectrometric detection, the fragment of formula III molecule is:
Quality is 266,
Quality is 150
The hydrogen spectrum detected result of INOVA-600 nuclear magnetic resonance analyser:
Wherein through Agilent Q-TOF6510 LC-MS instrument mass spectrometric detection, the fragment of formula IV molecule is:
Quality is 280,
Quality is 164
The hydrogen spectrum detected result of INOVA-600 nuclear magnetic resonance analyser:
Wherein through Agilent Q-TOF6510 LC-MS instrument mass spectrometric detection, the fragment of formula V molecule is:
Quality is 282,
Quality is 166
The hydrogen spectrum detected result of INOVA-600 nuclear magnetic resonance analyser:
Wherein through Agilent Q-TOF6510 LC-MS instrument mass spectrometric detection, the fragment of formula VI molecule is:
Quality is 296,
Quality is 165
The hydrogen spectrum detected result of INOVA-600 nuclear magnetic resonance analyser:
Wherein through Agilent Q-TOF6510 LC-MS instrument mass spectrometric detection, the fragment of formula polycationic molecule is:
Quality is 298,
Quality is 150
The hydrogen spectrum detected result of INOVA-600 nuclear magnetic resonance analyser:
Wherein through Agilent Q-TOF6510 LC-MS instrument mass spectrometric detection, the fragment of formula VIII molecule is:
Quality is 282,
Quality is 180
The hydrogen spectrum detected result of INOVA-600 nuclear magnetic resonance analyser:
Wherein through Agilent Q-TOF6510 LC-MS instrument mass spectrometric detection, the fragment of formula IX molecule is:
Quality is 298,
Quality is 182
The hydrogen spectrum detected result of INOVA-600 nuclear magnetic resonance analyser:
Wherein through Agilent Q-TOF6510 LC-MS instrument mass spectrometric detection, the fragment of formula X molecule is:
Quality is 294,
The hydrogen spectrum detected result of INOVA-600 nuclear magnetic resonance analyser:
Wherein through Agilent Q-TOF6510 LC-MS instrument mass spectrometric detection, the fragment of formula XI molecule is:
Quality is 282,
Quality is 150
The hydrogen spectrum detected result of INOVA-600 nuclear magnetic resonance analyser:
Wherein through Agilent Q-TOF6510 LC-MS instrument mass spectrometric detection, the fragment of formula XII molecule is:
Quality is 284,
Quality is 166
The hydrogen spectrum detected result of INOVA-600 nuclear magnetic resonance analyser:
Wherein through Agilent Q-TOF6510 LC-MS instrument mass spectrometric detection, the fragment of formula X III molecule is:
Quality is 136
The hydrogen spectrum detected result of INOVA-600 nuclear magnetic resonance analyser:
Wherein through Agilent Q-TOF6510 LC-MS instrument mass spectrometric detection, the fragment of formula X IV molecule is:
Quality is 312
The hydrogen spectrum detected result of INOVA-600 nuclear magnetic resonance analyser:
Wherein through Agilent Q-TOF6510 LC-MS instrument mass spectrometric detection, the fragment of formula X V molecule is:
Quality is 296,
Quality is 164
The hydrogen spectrum detected result of INOVA-600 nuclear magnetic resonance analyser:
The invention provides a kind of preparation method of the antineoplastic compound that extracts from gecko, the preparation method of the compound shown in the promptly logical formula II of the derivative of logical formula I may further comprise the steps:
(1) cleaning section:
The no web gecko that lives that catches deposited made its digestion in 24-72 hour, drain existing food and waste in the body,, dry with the distilled water cleaning;
(2) pulverizing process:
Gecko with after purifying changes in the steeping tank, adds ethanolic soln, soaks, after the immersion gecko causes death, gecko is changed in the high speed stamp mill, add the soak solution in the steeping tank again, rotating speed is controlled at 10000-20000r/min, and the time was controlled at 2-5 minute, made pulverizing liquid;
(3) soak separation circuit: in the pulverizing liquid that will make, add ethanolic soln, soak and separate, make extracting solution;
(4) extraction process:
A, with the extracting solution that makes, add entry earlier, mix, add non-polar organic solvent again, jolting, standing demix, the water intaking phase;
B, the water that step a is obtained add polar organic solvent, jolting, standing demix, water intaking phase;
(5) concentrated, the freeze-drying operation of underpressure distillation:
With the extraction liquid that makes, carry out underpressure distillation, underpressure distillation is done near, carries out freeze-drying then, makes crude extract, and this crude extract comprises the compound shown in the logical formula II.
Among the above-mentioned preparation method, further improve, the derivative of logical formula I, compound shown in the promptly logical formula II comprises following just purifying and purification step:
(6.1) preparative layer is analysed sample purification procedures just:
With the crude extract that makes, add pure water, after fully dissolving, standing demix, get supernatant liquor, carry out gel permeation chromatography or adsorption chromatography, behind the chromatography, the sample of collecting, carry out underpressure distillation and concentrate, carry out freeze-drying after concentrating, make purifying chromatography sample just after the freeze-drying;
(7) high performance liquid chromatography operation:
A, the first purifying chromatography sample that will make add pure water, and fully after the dissolving, standing demix is got supernatant liquor, with 0.45 μ m filtering with microporous membrane;
B, filtrate is carried out the high performance liquid chromatography (HPLC) chromatography, the elutriant when collecting 6.8～13.0min carries out the underpressure distillation mode and concentrates, and carries out freeze-drying after concentrating, after the freeze-drying promptly.
Among the above-mentioned preparation method, the another kind of improvement, the compound shown in the logical formula II comprises following just purifying and purification step:
(6.2) the first purification procedures of high performance liquid chromatography:
A, with the crude extract that makes, fully after the dissolving, standing demix is got supernatant liquor, with sample introduction behind the 0.45 μ m filtering with microporous membrane;
B, filtrate is carried out high performance liquid chromatography partly prepare the C18 column chromatography;
Chromatographic condition is: column temperature: 35 ℃, moving phase: pure water and acetonitrile, stationary phase is selected the C18 filler for use, detect wavelength: 210nm, UV spectrum wavelength region: 200～380nm, gradient elution: 0～5min, the ratio of acetonitrile is 0～0%, 5～10min, the ratio of acetonitrile is 0%～5%, 10～15min, 5%～10%, 15～20min, 10%～20%, 20～21min, 20%～30%, 21～27min, 30%～30%, 27～28min, 30%～0.00%, 28～38min, 0.00%, the C18 reversed-phase column of chromatography column: 10mm * 250mm, flow velocity: 4.00mL/min, the application of sample amount is 100 μ L;
C, carry out chromatography by above-mentioned chromatographic condition, the elutriant when being collected in 6～10min carries out freeze-drying after concentrating, and makes the first step high performance liquid chromatography purifying chromatography sample just after the freeze-drying;
(7) high performance liquid chromatography operation:
A, the first purifying chromatography sample that will make add pure water, and fully after the dissolving, standing demix is got supernatant liquor, with 0.45 μ m filtering with microporous membrane;
B, filtrate is carried out the high performance liquid chromatography (HPLC) chromatography, the elutriant when collecting 6.8～13.0min carries out the underpressure distillation mode and concentrates, and carries out freeze-drying after concentrating, after the freeze-drying promptly.
Further scheme, in the described step (3), immersion and isolating number of times are four times, each concrete operations are as follows:
Under the room temperature, add ethanolic soln, soak and suction filtration, isolate filtrate and filter residue,,, carry out concentrating under reduced pressure, be evaporated to and remove ethanol, make extracting solution,, mix No. four extracting solutions through Rotary Evaporators with isolated filtrate.
But above-mentioned immersion and isolating number of times are not limited to four times, essence this further the purpose of scheme four times be as far as possible effective constituent to be extracted, can adopt in the practice 1 time or repeatedly.
In the described step (2), described ethanolic soln, concentration of volume percent are 60%-95%, and its add-on is 1: 1～3 for the ratio of no web gecko raw materials quality and the volume of ethanolic soln.
In the step (2), the soak solution in the steeping tank of adding, its add-on is the ratio 1: 0.5～1.5 of no web gecko raw materials quality with the volume of soak solution.
In the step (3), the ethanolic soln that described immersion adds, concentration of volume percent is 60%-95%, the ratio that its add-on is respectively no web gecko raw materials quality and the volume of ethanolic soln is 1: 0.5～3.
In the step (4), describedly add entry, add-on be extracting liquid volume 1-4 doubly;
In the step (4), the non-polar organic solvent of adding, each consumption are respectively 1-2 that extracting solution adds cumulative volume after the entry doubly.
In the step (4), the polar organic solvent of adding, each consumption is respectively 1-2 times of water volume.
Described gel permeation chromatography, the kind of gel are dextrane gel and hydroxypropyl dextrane gel;
Described adsorption chromatography, the kind of sorbent material is: aluminum oxide and silica gel.
The actual conditions that in described step (7) the high performance liquid chromatography operation filtrate is carried out the high performance liquid chromatography (HPLC) chromatography is as follows:
High performance liquid chromatography in the described high performance liquid chromatography operation, chromatographic condition is: column temperature: 32-36 ℃, moving phase: pure water and acetonitrile, stationary phase are selected C18, C8, C18-EPS, C8-EPS, C18-AQ, C8-AQ, C18-NH for use
2Or C8-NH
2In a kind of, detect wavelength: 200-280nm, UV spectrum wavelength region: 200～380nm, the ratio of gradient elution: 0～2.0min, acetonitrile is 0～8%, 0.5～12min, is 2%～10%, 8～15min, 8%～30%, 15～25min, 20%～35%, 18～25min, 30%～0.00%, 20～35min, 0.00%, chromatography column: internal diameter is 10mm, and length is C18, C8, C18-EPS, C8-EPS, C18-AQ, C8-AQ, the C18-NH of 150-300mm
2Or C8-NH
2In a kind of reversed-phase column, flow velocity: 4.0-5.0mL/min, the application of sample amount is 50-200 μ L.
Described compound is as the application of antitumor drug.
Compound of the present invention can be prepared into it conventional solid preparation such as tablet, pulvis, granula, capsule etc., also can make the solution of injection.
The clinical application of the antineoplastic compound that from gecko, extracts of the present invention's preparation: can use physiological saline solution, use or directly make oral preparations with injection system again.Concrete using dosage uses as one feels fit according to factors such as the disease type and the state of an illness, and its per daily dose is: 2-10mg, natural gift are taken for 1-3 time.
1. be mainly used in the chemotherapy of multiple common cancers such as treatment cancer of the stomach, lung cancer, liver cancer, colorectal carcinoma, mammary cancer, leukemia, genital system tumor, antitumor action is obvious, and conventional amount used does not have bone marrow depression and important organ infringement; In addition, because this compound can increase T cellular immunization, anti-AIDS effect is preferably arranged.
2. have good water-solubility, under the normal temperature, its solubleness can reach 200 mg/ml, and dissolves in 95% alcoholic acid white crystal.
3. its antitumor action have efficiently, the characteristics of wide spectrum, low toxicity.
4. compare with other antitumour drug, antitumor action is stronger.
Description of drawings
Accompanying drawing 1, negative control apoptosis of tumor cells rate figure;
Accompanying drawing 2, gecko activeconstituents apoptosis of tumor cells rate figure;
Accompanying drawing 3: positive control (5-FU) apoptosis of tumor cells rate figure.
Embodiment
Below in conjunction with embodiment the present invention is elaborated.
Embodiment 1: a kind of preparation method of the antineoplastic compound that from gecko, extracts, and the compound shown in the both logical formula II of the derivative of logical formula I may further comprise the steps:
(1) cleaning section: the no web gecko that lives that will catch is deposited made its digestion in 24-72 hour, drain existing food and waste in the body, cleaned with distilled water, dried;
(2) pulverizing process: the gecko after will purifying, change in the steeping tank, add ethanolic soln, described ethanolic soln, concentration of volume percent is 95%, soaks, and after the immersion gecko causes death, gecko and soak solution is changed in the high speed stamp mill, makes pulverizing liquid;
The volumes of aqueous ethanol percentage concentration is 95% in the above-mentioned steps, and under the constant prerequisite of other processing condition and step, the volumes of aqueous ethanol percentage concentration all can reach the purpose of preparation in the 60%-95% interval through evidence.
(3) soak separation circuit: in the pulverizing liquid that will make, add ethanolic soln, soak and separate, make extracting solution;
The present embodiment employing is soaked and isolating number of times is four times in concrete operating in this operation, and each concrete operations are as follows: under the room temperature, add ethanolic soln, its concentration of volume percent is 80%, soaks and suction filtration, isolates filtrate and filter residue, with isolated filtrate, through Rotary Evaporators, pressure-0.095～-0.099Mpa between, temperature is controlled at 30～50 ℃, carry out concentrating under reduced pressure, be evaporated to and remove ethanol, make extracting solution, with No. four extracting solutions, mix.
But above-mentioned immersion and isolating number of times are not limited to four times, and four times purpose is as far as possible effective constituent to be extracted in fact, can adopt in the practice 1 time or repeatedly also can.
(4) extraction process:
A, with the extracting solution that makes, add entry earlier, mix, add the non-polar organic solvent normal hexane again, the non-polar organic solvent that adds, each consumption is respectively extracting solution and adds 2 times of cumulative volume after the entry, jolting, standing demix, water intaking phase, add for the second time non-polar organic solvent again, jolting, standing demix water intaking phase add non-polar organic solvent more for the third time, jolting, standing demix water intaking phase;
Present embodiment adopts the water intaking phase three times, its purpose for extraction fully, whether abundant 1-2 time or other time do not influence the final purpose of preparation, and just extract the whether simple of or leaching process.
Non-polar organic solvent normal hexane in the present embodiment also can adopt hexanaphthene, heptane all can reach the purpose of preparation.
D, the water that step a is obtained, add the polar organic solvent methylene dichloride, the polar organic solvent of adding, each consumption is respectively 1-2 times of water volume, jolting, standing demix water intaking phase, for the second time add methylene dichloride, standing demix, water intaking phase, add methylene dichloride more for the third time, standing demix, the water intaking phase makes extraction liquid;
Present embodiment adopts the water intaking phase three times, its purpose for extraction fully, whether abundant 1-2 time or other time do not influence the final purpose of preparation, and just extract the whether simple of or leaching process.
Non-polar organic solvent methylene dichloride in the present embodiment also can adopt trichloromethane, ether all can reach the purpose of preparation.
(5) concentrated, the freeze-drying operation of underpressure distillation:
With the extraction liquid that makes, carry out underpressure distillation, pressure-controlling-0.095～-0.099Mpa between, temperature is controlled at 40～55 ℃, underpressure distillation is done near, again with pressure-controlling at-0.099Mpa, temperature is controlled at-45 ℃～-55 ℃ and carries out freeze-drying, carries out freeze-drying then, makes crude extract, comprise the compound shown in the logical formula II in the crude extract, as follows:
This compound molecular weight is 253-341;
In the formula II, C
2Be R or S configuration, C
3Be R, C
7Be S, C
8Be R, C
9Be the S configuration, or C
2Be R or S configuration, C
3Be S, C
7Be R, C
8Be S, C
9Be R configuration, substituent R
1Can be H ,-COOCH
3Or-COOC
2H
5, R
2, R
3, R
4, R
5, R
6Identical or inequality, can be respectively H, OH, CH
3, C
2H
5, OCH
3Or OC
2H
5
This compound has the basic skeleton structure shown in (I) formula:
In the formula I, C
3Be R, C
7Be the S configuration, or C
3Be S, C
7Be the R configuration.
Present embodiment adopts above-mentioned technology to carry out, should be within protection scope of the present invention based on known variation of technical solution of the present invention or simple improvement.
Compound shown in embodiment 2, the logical formula II comprises following just purifying and purification step:
(6.1) preparative layer is analysed sample purification procedures just:
Crude extract with embodiment 1 makes adds pure water, fully after the dissolving, standing demix makes the aqueous solution that concentration is 1.0g/mL, gets supernatant liquor, after carrying out chromatographic separation,, carry out underpressure distillation and concentrate the sample of collecting, pressure-controlling-below the 0.096Mpa, temperature is controlled at 40 ℃～50 ℃, carries out freeze-drying after concentrating, freeze dried pressure-controlling-0.099～-0.100Mpa, temperature is controlled at-45 ℃～-55 ℃, makes purifying chromatography sample just after the freeze-drying;
(7) high performance liquid chromatography operation:
A, the first purifying chromatography sample that will make add pure water, and fully after the dissolving, standing demix makes the aqueous solution that concentration is 1.0g/mL, discards insoluble solids, gets supernatant liquor, with 0.45 μ m filtering with microporous membrane;
B, filtrate is carried out the high performance liquid chromatography (HPLC) chromatography, the actual conditions of chromatography is as follows:
High performance liquid chromatography in the described high performance liquid chromatography operation, chromatographic condition is: column temperature: 32-36 ℃, moving phase: pure water and acetonitrile, stationary phase are selected C18, C8, C18-EPS, C8-EPS, C18-AQ, C8-AQ, C18-NH for use
2Or C8-NH
2In a kind of, detect wavelength: 200-280nm, UV spectrum wavelength region: 200～380nm, the ratio of gradient elution: 0～2.0min, acetonitrile is 0～8%, 0.5～12min, is 2%～10%, 8～15min, 8%～30%, 15～25min, 20%～35%, 18～25min, 30%～0.00%, 20～35min, 0.00%, chromatography column: internal diameter is 10mm, and length is C18, C8, C18-EPS, C8-EPS, C18-AQ, C8-AQ, the C18-NH of 150～300mm
2Or C8-NH
2In a kind of reversed-phase column, flow velocity: 4.0-5.0mL/min, the application of sample amount is 50-200 μ L.
Elutriant when collecting 6.8～13.0min carries out the underpressure distillation mode and concentrates, pressure-controlling-below the 0.096Mpa, temperature is controlled at about 40 ℃～50 ℃, carries out freeze-drying after concentrating, freeze dried pressure-controlling-0.099～-0.100Mpa, temperature is controlled at-45 ℃～-55 ℃, after the freeze-drying promptly.
This derivative is the compound shown in the logical formula II:
In the formula II, C
2Be R or S configuration, C
3Be R, C
7Be S, C
8Be R, C
9Be the S configuration, or C
2Be R or S configuration, C
3Be S, C
7Be R, C
8Be S, C
9Be R configuration, substituent R
1Can be-COOCH
3Or-COOC
2H
5, R
2, R
3, R
4, R
5, R
6Identical or inequality, can be respectively H, OH, CH
3, C
2H
5, OCH
3, OC
2H
5
Embodiment 3, present embodiment provide the another kind of preparation method who is different from embodiment 2, and the compound shown in the logical formula II comprises following just purifying and purification step:
(6.2) the first purification procedures of high performance liquid chromatography:
A, the crude extract that embodiment 1 is made, fully after the dissolving, standing demix makes the aqueous solution that concentration is 1.0g/mL, discards insoluble solids, gets supernatant liquor, with sample introduction behind the 0.45 μ m filtering with microporous membrane;
B, filtrate is carried out high performance liquid chromatography partly prepare the C18 column chromatography, chromatographic condition is: column temperature: 35 ℃, moving phase: pure water and acetonitrile, stationary phase is selected the C18 filler for use, detect wavelength: 210nm, UV spectrum wavelength region: 200～380nm, gradient elution: 0～5min, the ratio of acetonitrile is 0～0%, 5～10min, the ratio of acetonitrile is 0%～5%, 10～15min, 5%～10%, 15～20min, 10%～20%, 20～21min, 20%～30%, 21～27min, 30%～30%, 27～28min, 30%～0.00%, 28～38min, 0.00%, the C18 reversed-phase column of chromatography column: 10mm * 250mm, flow velocity: 4.00mL/min, the application of sample amount is 100 μ L;
C, carry out chromatography by above-mentioned chromatographic condition, elutriant when being collected in 6～10min, pressure-controlling is at-0.099Mpa, temperature is controlled at 40 ℃, carry out freeze-drying after concentrating, freeze dried pressure-controlling is at-0.100Mpa, and temperature is controlled at-50 ℃, makes the first step high performance liquid chromatography purifying chromatography sample just after the freeze-drying;
(7) high performance liquid chromatography operation:
A, the first purifying chromatography sample that will make add pure water, and fully after the dissolving, standing demix makes the aqueous solution that concentration is 1.0g/mL, discards insoluble solids, gets supernatant liquor, with 0.45 μ m filtering with microporous membrane;
B, filtrate is carried out the high performance liquid chromatography (HPLC) chromatography, the actual conditions that in described step (7) the high performance liquid chromatography operation filtrate is carried out the high performance liquid chromatography (HPLC) chromatography is as follows:
High performance liquid chromatography in the described high performance liquid chromatography operation, chromatographic condition is: column temperature: 32-36 ℃, moving phase: pure water and acetonitrile, stationary phase are selected C18, C8, C18-EPS, C8-EPS, C18-AQ, C8-AQ, C18-NH for use
2Or C8-NH
2In a kind of, detect wavelength: 200-280nm, UV spectrum wavelength region: 200～380nm, the ratio of gradient elution: 0～2.0min, acetonitrile is 0～8%, 0.5～12min, is 2%～10%, 8～15min, 8%～30%, 15～25min, 20%～35%, 18～25min, 30%～0.00%, 20～35min, 0.00%, chromatography column: internal diameter is 10mm, and length is C18, C8, C18-EPS, C8-EPS, C18-AQ, C8-AQ, the C18-NH of 150～300mm
2Or C8-NH
2In a kind of reversed-phase column, flow velocity: 4.0-5.0mL/min, the application of sample amount is 50-200 μ L.
Elutriant when collecting 6.8～13.0min, carrying out the underpressure distillation mode concentrates, pressure-controlling-below the 0.096Mpa, temperature is controlled at about 40 ℃～50 ℃, carry out freeze-drying after concentrating, freeze dried pressure-controlling-0.099～-0.100Mpa, temperature is controlled at-45 ℃～-55 ℃, promptly, this derivative is the compound shown in the logical formula II after the freeze-drying:
In the formula II, C
2Be R or S configuration, C
3Be R, C
7Be S, C
8Be R, C
9Be the S configuration, or C
2Be R or S configuration, C
3Be S, C
7Be R, C
8Be S, C
9Be R configuration, substituent R
1Can be-COOCH
3Or-COOC
2H
5, R
2, R
3, R
4, R
5, R
6Identical or inequality, can be respectively H, OH, CH
3, C
2H
5, OCH
3, OC
2H
5
Embodiment 4, present embodiment provide a kind of preparation method of the antitumor derivative that extracts from gecko, this derivative is the antineoplastic compound derivative II, and molecular weight is 267, and molecular formula is C
11H
13N
3O
3S.
May further comprise the steps:
(1) cleaning section:
The no web gecko that lives that will catch autumn was deposited 24-72 hour, made its digestion, drained existing food and waste in the body, and distilled water cleans, and dries, and weighs;
(2) pulverizing process:
The gecko of back 1kg will be purified, change in the steeping tank, add concentration of volume percent and be 1.5 liters of 90% ethanolic solns, soak, after the immersion gecko causes death, gecko is changed in the high speed stamp mill, add 1 liter of soak solution in the steeping tank again, rotating speed is controlled at 10000r/min, and the time was controlled at 2 minutes, made pulverizing liquid;
(3) soak separation circuit:
In a, the pulverizing liquid that will make, adding concentration of volume percent is 1.5 liters of 90% ethanolic solns, under the room temperature, carry out the first time and soak, soak after 24 hours, carry out suction filtration, isolate filtrate and filter residue, with isolated filtrate, through Rotary Evaporators, pressure-controlling is at-0.099Mpa, temperature is controlled at 45 ℃, carry out concentrating under reduced pressure, be evaporated to and remove ethanol, make extracting solution No. one time;
B, with the isolated filter residue of step (a), adding concentration of volume percent is 2.5 liters of 90% ethanolic solns, under the room temperature, carry out the second time and soak, soaked 24 hours, carry out suction filtration, isolate filtrate and filter residue, with isolated filtrate, through Rotary Evaporators, pressure is at-0.099Mpa, temperature is controlled at 40 ℃, carry out concentrating under reduced pressure, be evaporated to and remove ethanol, make secondary raffinate;
C, with the isolated filter residue of step (b), adding concentration of volume percent is 2.5 liters of 90% ethanolic solns, under the room temperature, soak for the third time, soak after 25 hours, carry out suction filtration, isolate filtrate and filter residue, with isolated filtrate, through Rotary Evaporators, pressure is at-0.099Mpa, temperature is controlled at 40 ℃, carry out concentrating under reduced pressure, be evaporated to and remove ethanol, make extracting solution No. three times;
D, with the isolated filter residue of step (c), adding concentration of volume percent is 2.5 liters of 90% ethanolic solns, under the room temperature, carry out the 4th time and soak, soak after 26 hours, carry out suction filtration, isolate filtrate and filter residue, with isolated filtrate, through Rotary Evaporators, pressure is between-0.099Mpa, temperature is controlled at 40 ℃, carry out concentrating under reduced pressure, be evaporated to and remove ethanol, make extracting solution No. four times;
E, an extracting solution that will make, secondary raffinate, No. three extracting solutions and No. four extracting solutions mix, and make extracting solution.
(4) extraction process:
A, get 0.15 liter of extracting solution, 0.15 liter in water mixes, add 0.3 liter of normal hexane again, jolting 3 minutes, standing demix, the water intaking phase, for the second time add 0.3 liter of jolting of normal hexane 2 minutes again, standing demix, water intaking phase, add 0.3 liter of normal hexane more for the third time, jolting 2 minutes, standing demix, water intaking phase;
D, with the water that step a obtains, add 0.3 liter of methylene dichloride again, jolting 3 minutes, standing demix, the water intaking phase adds 0.3 liter of methylene dichloride more for the second time, jolting 2 minutes, standing demix, the water intaking phase adds 0.3 liter of methylene dichloride more for the third time, jolting 2 minutes, standing demix, the water intaking phase makes extraction liquid;
(5) concentrated, the freeze-drying operation of underpressure distillation:
With the extraction liquid that makes, carry out underpressure distillation, pressure-controlling is at-0.095Mpa, temperature is controlled at 50 ℃, is concentrated near doing, again with pressure-controlling at-0.099Mpa, temperature is controlled at-50 ℃, carries out freeze-drying, after the freeze-drying, makes crude extract;
(6) dextran G-25 gel chromatography:
A, with the crude extract that makes, add pure water, stirred 8 minutes, after fully dissolving, standing demix, make the aqueous solution that concentration is 1.0g/mL, get supernatant liquor, application of sample is in previously prepd dextran G-25 gel chromatography column, and the chromatography column specification is 26 * 600mm, the consumption of dextran G-25 gel is 90g, and application of sample concentration is the aqueous solution 12mL of 1.0g/mL;
B, carry out wash-out with distilled water, the elution speed of elutriant is 2～3 droplets/second, discards the 70mL elutriant that begins to collect, and after this every collection 30mL is numbered by collecting sequencing successively as a sample, collects 20 samples altogether;
C, the 12～14# sample that will collect carry out underpressure distillation respectively and concentrate, and pressure-controlling is at-0.096Mpa, temperature is controlled at 45 ℃, carries out freeze-drying after concentrating, and freeze dried pressure-controlling is at-0.099Mpa, temperature is controlled at-50 ℃, makes the sample behind the chromatography after the freeze-drying;
(7) high performance liquid chromatography operation:
A, with the sample that makes after the freeze-drying behind the chromatography, add pure water, stirred 10 minutes, after fully dissolving, standing demix is made the aqueous solution that concentration is 0.8g/mL, discards insoluble solids, gets supernatant liquor, with 0.45 μ m filtering with microporous membrane;
B, filtrate is carried out high performance liquid chromatography partly prepare the C18 column chromatography, chromatographic condition is: column temperature: 35 ℃, moving phase: pure water and acetonitrile, stationary phase is selected C18 for use, detect wavelength: 210nm, UV spectrum wavelength region: 200～380nm, the ratio of gradient elution: 0～0.5min, acetonitrile is 0～5%, 0.5 the ratio of～10min, acetonitrile is 5%～10%, 10～13min, 10%～30%, 13～19min, 30%～30%, 19～20min, 30%～0.00%, 20～28min, 0.00%, chromatography column: internal diameter is the C of 10mm * 250mm
18Reversed-phase column, flow velocity: 4.76mL/min, the application of sample amount is 200 μ l;
C, carry out chromatography by above-mentioned chromatographic condition, the elutriant when being collected in 8.8～9.9min, the concentrate drying pressure-controlling is at-0.092Mpa, temperature is controlled at 40 ℃, carries out freeze-drying after concentrating, and freeze dried pressure-controlling is at-0.099Mpa, temperature is controlled at-45 ℃, after the freeze-drying promptly.
The antineoplastic compound II that present embodiment obtains, molecular weight are 267, and molecular formula is C
11H
13N
3O
3S.
Embodiment 5, present embodiment provide a kind of preparation method of the antineoplastic compound derivative that extracts from gecko:
(1) cleaning section:
The no web gecko that lives that will catch autumn was deposited 36 hours, made its digestion, drained existing food and waste in the body, and distilled water cleans, and dries;
(2) pulverizing process:
The gecko of back 1kg weight will be purified, change in the steeping tank, add concentration of volume percent and be 1 liter of 95% ethanolic soln, soak, after the immersion gecko causes death, gecko is changed in the high speed stamp mill, add 0.5 liter of soak solution in the steeping tank again, rotating speed is controlled at 20000r/min, and the time was controlled at 5 minutes, made pulverizing liquid;
(3) soak separation circuit:
In a, the pulverizing liquid that will make, adding concentration of volume percent is 1.0 liters of 95% ethanolic solns, under the room temperature, carry out the first time and soak, soak after 25 hours, carry out suction filtration, isolate filtrate and filter residue, with isolated filtrate, through Rotary Evaporators, pressure-controlling is at-0.097Mpa, temperature is controlled at 35 ℃, carry out concentrating under reduced pressure, be evaporated to and remove ethanol, make extracting solution No. one time;
B, with the isolated filter residue of step (a), adding concentration of volume percent is 2.0 liters of 95% ethanolic solns, under the room temperature, carry out the second time and soak, soaked 26 hours, carry out suction filtration, isolate filtrate and filter residue, with isolated filtrate, through Rotary Evaporators, pressure is at-0.098Mpa, temperature is controlled at 30 ℃, carry out concentrating under reduced pressure, be evaporated to and remove ethanol, make secondary raffinate;
C, with the isolated filter residue of step (b), adding concentration of volume percent is 2.0 liters of 95% ethanolic solns, under the room temperature, soak for the third time, soak after 26 hours, carry out suction filtration, isolate filtrate and filter residue, with isolated filtrate, through Rotary Evaporators, pressure is at-0.099Mpa, temperature is controlled at 30 ℃, carry out concentrating under reduced pressure, be evaporated to and remove ethanol, make extracting solution No. three times;
D, with the isolated filter residue of step (c), adding concentration of volume percent is 2.0 liters of 90% ethanolic solns, under the room temperature, carry out the 4th time and soak, soak after 26 hours, carry out suction filtration, isolate filtrate and filter residue, with isolated filtrate, through Rotary Evaporators, pressure is at-0.098Mpa, temperature is controlled at 30 ℃, carry out concentrating under reduced pressure, be evaporated to and remove ethanol, make extracting solution No. four times;
E, an extracting solution that will make, secondary raffinate, No. three extracting solutions and No. four extracting solutions mix, and make extracting solution.
(4) extraction process:
A, get 0.05 liter of extracting solution, add 0.2 liter of entry earlier, mix, add 0.5 liter of normal hexane again, jolting 2 minutes, standing demix, the water intaking phase adds 0.5 liter of normal hexane, jolting 2 minutes more for the second time, standing demix, the water intaking phase adds 0.5 liter of normal hexane, jolting 1 minute more for the third time, standing demix, the water intaking phase;
D, with the water that step a obtains, add 0.5 liter of methylene dichloride again, jolting 2 minutes, standing demix, the water intaking phase adds 0.5 liter of methylene dichloride more for the second time, jolting 2 minutes, standing demix, the water intaking phase adds 0.5 liter of methylene dichloride more for the third time, jolting 2 minutes, standing demix, the water intaking phase makes extraction liquid;
(5) concentrated, the freeze-drying operation of underpressure distillation:
With the extraction liquid that makes, carry out underpressure distillation, pressure-controlling is at-0.099Mpa, and temperature is controlled at 55 ℃, is concentrated near doing, and at-0.099Mpa, temperature is controlled at-55 ℃, carries out freeze-drying, after the freeze-drying, makes crude extract with pressure-controlling;
(6) preparative layer is analysed the sample operation:
A, with the crude extract that makes, add pure water, stirred 2 minutes, after fully dissolving, standing demix, make the aqueous solution that concentration is 1.5g/mL, get supernatant liquor, application of sample is in previously prepd dextran LH-20 gel chromatography column, and the chromatography column specification is 26 * 600mm, the consumption of dextran LH-20 gel is 100g, and application of sample concentration is the aqueous solution 12mL of 1.5g/mL;
B, carry out wash-out with distilled water, elution speed is 3～4 droplets/second, in the time of wash-out and collect elutriant, discard the 70mL elutriant that begins to collect, regather the 30mL elutriant as sample 1#, after collecting sample 1#, regather the 30mL elutriant as sample 2#, collect sample 2# after, be that 50% ethanolic soln carries out wash-out with concentration of volume percent again, elution speed is 3～4 droplets/second, in the time of wash-out and collect elutriant, every collection 30mL is numbered the sample of collecting by the sequencing of collecting successively as a sample, collect 20 samples altogether
C, the 11～13# sample that will collect, carrying out underpressure distillation respectively concentrates, pressure-controlling-below the 0.096Mpa, temperature is controlled at 40 ℃-50 ℃, carry out freeze-drying after concentrating, freeze dried pressure-controlling is at-0.099--0.100Mpa, and temperature is controlled at-45 ℃--55 ℃, make the chromatography sample after the freeze-drying.
(7) high performance liquid chromatography operation:
A, will make the chromatography sample after the freeze-drying, add pure water, stirred 10 minutes, after fully dissolving, standing demix is made the aqueous solution that concentration is about 0.5g/mL, discards insoluble solids, gets supernatant liquor, with 0.45 μ m filtering with microporous membrane;
B, filtrate is carried out high performance liquid chromatography partly prepare the C8 column chromatography, chromatographic condition is: column temperature: 32 ℃, moving phase: pure water and acetonitrile, stationary phase is selected C8 for use, detect wavelength 250nm, UV spectrum wavelength region: 200～380nm, the ratio of gradient elution: 0～2min, acetonitrile is 0～2%, the ratio of 2～12min, acetonitrile is 2%～8%, 12～15min, 8%～15%, 13～19min, 15%～30%, 19～20min, 30%～0.00%, 20～30min, 0.00%, chromatography column: internal diameter is the C of 10mm * 300mm
8Reversed-phase column, flow velocity: 4.0mL/min, the application of sample amount is 150 μ l;
C, carry out chromatography by above-mentioned chromatographic condition, the elutriant when being collected in 6.8～7.4min, concentrate drying, promptly.
The derivative I that present embodiment obtains, molecular weight are that 253 molecular formula are C
10H
11N
3O
3S.
Embodiment 6, present embodiment provide a kind of preparation method of the antineoplastic compound derivative that extracts from gecko:
(1) cleaning section:
The no web gecko that lives that catches deposited made its digestion in 24 hours, drain existing food and waste in the body,, dry with the distilled water cleaning;
(2) pulverizing process:
Back one kilogram weight gecko will be purified, change in the steeping tank, add concentration of volume percent and be 1 liter of 60% ethanolic soln, soak, after the immersion gecko causes death, gecko is changed in the high speed stamp mill, be added into 0.5 liter of the interior soak solution of steeping tank, rotating speed is controlled at 15000r/min, and the time was controlled at 2 minutes, made pulverizing liquid;
(3) soak separation circuit:
In a, the pulverizing liquid that will make, add remaining soak solution and replenish that to add concentration of volume percent be 1 liter of 60% ethanolic soln, under the room temperature, carry out the first time and soak, soak after 20 hours, carry out suction filtration, isolate filtrate and filter residue, with isolated filtrate, through Rotary Evaporators, pressure-controlling is at-0.095Mpa, temperature is controlled at 55 ℃, carry out concentrating under reduced pressure, be evaporated to and remove ethanol, make extracting solution No. one time;
B, with the isolated filter residue of step (a), adding concentration of volume percent is 2 liters of 60% ethanolic solns, under the room temperature, carry out the second time and soak, soaked 21 hours, carry out suction filtration, isolate filtrate and filter residue, with isolated filtrate, through Rotary Evaporators, pressure is at-0.095Mpa, temperature is controlled at 50 ℃, carry out concentrating under reduced pressure, be evaporated to and remove ethanol, make secondary raffinate;
C, with the isolated filter residue of step (b), adding concentration of volume percent is 2 liters of 60% ethanolic solns, under the room temperature, soak for the third time, soak after 22 hours, carry out suction filtration, isolate filtrate and filter residue, with isolated filtrate, through Rotary Evaporators, pressure is at-0.097Mpa, temperature is controlled at 50 ℃, carry out concentrating under reduced pressure, be evaporated to and remove ethanol, make extracting solution No. three times;
D, with the isolated filter residue of step (c), adding concentration of volume percent is 2 liters of 60% ethanolic solns, under the room temperature, carry out the 4th time and soak, soak after 23 hours, carry out suction filtration, isolate filtrate and filter residue, with isolated filtrate, through Rotary Evaporators, pressure is between-0.098Mpa, temperature is controlled at 50 ℃, carry out concentrating under reduced pressure, be evaporated to and remove ethanol, make extracting solution No. four times;
E, an extracting solution that will make, secondary raffinate, No. three extracting solutions and No. four extracting solutions mix, and make extracting solution.
(4) extraction process:
A, get 0.1 liter of extracting solution, add 0.2 liter of entry earlier, mix, add 0.6 liter of normal hexane again, jolting 1 minute, standing demix, the water intaking phase adds 0.6 liter of normal hexane, jolting 1 minute more for the second time, standing demix, the water intaking phase adds 0.6 liter of normal hexane, jolting 2 minutes more for the third time, standing demix, the water intaking phase;
D, with the water that step a obtains, add 0.6 liter of methylene dichloride again, jolting 2 minutes, standing demix, the water intaking phase adds 0.6 liter of methylene dichloride more for the second time, jolting 1 minute, standing demix, the water intaking phase adds 0.6 liter of methylene dichloride more for the third time, jolting 1 minute, standing demix, the water intaking phase makes extraction liquid;
(5) concentrated, the freeze-drying operation of underpressure distillation:
With the extraction liquid that makes, carry out underpressure distillation, pressure-controlling is at-0.096Mpa, and temperature is controlled at 40 ℃, is concentrated near doing, and at-0.099Mpa, temperature is controlled at-45 ℃, carries out freeze-drying, after the freeze-drying, makes crude extract with pressure-controlling;
(6) preparative layer is analysed the sample operation:
A, with the crude extract that makes, add 95% ethanol, stirred 15 minutes, fully after the dissolving, standing demix makes the ethanolic soln that concentration is 0.5g/mL, gets supernatant liquor, application of sample is in previously prepd Al
2O
3Chromatography column, chromatography column specification are 26 * 600mm, the Al of 100～200 order specifications
2O
3Solid 300g, application of sample concentration is the aqueous solution 16mL of 0.5g/mL;
B, Al
2O
3The filling of chromatography column: Al
2O
3Use soaked in absolute ethyl alcohol, fully stirred 10 minutes, the amount of pursuing in the stirring is with Al
2O
3Add in the chromatography pipe, with the alcoholic acid suspension until the chromatography pipe is filled;
C, beginning are carried out wash-out with 95% ethanol earlier, the yellow of sample moves down gradually in the elution process, when yellow moves to chromatography pipe bottom, approximately received 95% ethanol eluate of 200mL this moment, discard this part elutriant, begin to receive the elutriant as sample, every reception 200mL is as a sample, number consecutively; After the 1# sample reception finishes, use 90% ethanol instead and carry out wash-out, after the 2# sample reception finishes, use 85% ethanol instead and carry out wash-out, after the 3# sample reception finishes, use 80% ethanol instead and carry out wash-out, after the 5# sample reception finishes, use 70% ethanol instead and carry out wash-out, after the 7# sample reception finishes, use 60% ethanol instead and carry out wash-out, after the 9# sample reception finishes, use 50% ethanol instead and carry out wash-out, after the 11# sample reception finishes, use distilled water instead and carry out wash-out, after the 13# sample reception finishes, continue to carry out wash-out with 1000mL distilled water, after this 1000mL elutriant of Jie Shouing is collected 14 samples altogether as the 14# sample;
D, 10～12# sample of collecting carry out underpressure distillation respectively and concentrate, and pressure-controlling is at-0.096Mpa, and temperature is controlled at-50 ℃, carries out freeze-drying after concentrating, and freeze dried pressure-controlling is-0.099, and temperature is controlled at-55 ℃, makes the chromatography sample after the freeze-drying;
(7) high performance liquid chromatography operation:
A, with the Al that makes
2 O
310～12# sample behind the adsorption chromatography adds pure water, stirs 10 minutes, and fully after the dissolving, standing demix makes the aqueous solution that concentration is about 1.5g/mL, discards insoluble solids, gets supernatant liquor, with 0.45 μ m filtering with microporous membrane;
B, filtrate is carried out high performance liquid chromatography partly prepare the C18-EPS column chromatography, chromatographic condition is: column temperature: 38 ℃, moving phase: pure water and acetonitrile, stationary phase is selected C18-EPS for use, detect wavelength: 254nm, UV spectrum wavelength region: 200～380nm, gradient elution: 0～2min, the ratio of acetonitrile is 0～8%, 2～10min, the ratio of acetonitrile is 8%～10%, 10～13min, 10%～30%, 13～19min, 30%～30%, 19～20min, 30%～0.00%, 20～28min, 0.00%, chromatography column: internal diameter is the C18-EPS reversed-phase column of 10mm * 150mm, flow velocity: 4.2mL/min, the application of sample amount is 100 μ L;
C, carry out chromatography by above-mentioned chromatographic condition, the elutriant when being collected in 9.1～9.9min, pressure-controlling is at-0.099Mpa, temperature is controlled at 40 ℃, carries out freeze-drying after concentrating, freeze dried pressure-controlling is at-0.100Mpa, temperature is controlled at-50 ℃, after the freeze-drying promptly.
The compound IV that present embodiment obtains, molecular weight 309, molecular formula is C
14H
19N
3O
3S,
Embodiment 7, present embodiment provide a kind of preparation method of the antitumor derivative that extracts from gecko:
(1) cleaning section:
The no web gecko that lives that catches deposited made its digestion in 60 hours, drain existing food and waste in the body,, dry with the distilled water cleaning;
(2) pulverizing process:
The gecko of back 1kg weight will be purified, change in the steeping tank, add concentration of volume percent and be 1.5 liters of 80% ethanolic solns, soak, after the immersion gecko causes death, gecko is changed in the high speed stamp mill, add 1 liter of the interior soak solution of steeping tank again, rotating speed is controlled at 15000r/min, and the time was controlled at 2 minutes, made pulverizing liquid;
(3) soak separation circuit:
In a, the pulverizing liquid that will make, adding concentration of volume percent is 1.5 liters of 80% ethanolic solns, under the room temperature, carry out the first time and soak, soak after 22 hours, carry out suction filtration, isolate filtrate and filter residue, with isolated filtrate, through Rotary Evaporators, pressure-controlling is at-0.095Mpa, temperature is controlled at 35 ℃, carry out concentrating under reduced pressure, be evaporated to and remove ethanol, make extracting solution No. one time;
B, with the isolated filter residue of step (a), adding concentration of volume percent is 3.0 liters of 80% ethanolic solns, under the room temperature, carry out the second time and soak, soaked 24 hours, carry out suction filtration, isolate filtrate and filter residue, with isolated filtrate, through Rotary Evaporators, pressure is at-0.095Mpa, temperature is controlled at 36 ℃, carry out concentrating under reduced pressure, be evaporated to and remove ethanol, make secondary raffinate;
C, with the isolated filter residue of step (b), adding concentration of volume percent is 3.0 liters of 80% ethanolic solns, under the room temperature, soak for the third time, soak after 25 hours, carry out suction filtration, isolate filtrate and filter residue, with isolated filtrate, through Rotary Evaporators, pressure is at-0.097Mpa, temperature is controlled at 40 ℃, carry out concentrating under reduced pressure, be evaporated to and remove ethanol, make extracting solution No. three times;
D, with the isolated filter residue of step (c), adding concentration of volume percent is 3.0 liters of 80% ethanolic solns, under the room temperature, carry out the 4th time and soak, soak after 26 hours, carry out suction filtration, isolate filtrate and filter residue, with isolated filtrate, through Rotary Evaporators, pressure is at-0.098Mpa, temperature is controlled at 50 ℃, carry out concentrating under reduced pressure, be evaporated to and remove ethanol, make extracting solution No. four times;
E, an extracting solution that will make, secondary raffinate, No. three extracting solutions and No. four extracting solutions mix, and make extracting solution.
(4) extraction process:
A, get 0.1 liter of extracting solution, add 0.3 liter of entry earlier, mix, add 0.4 liter of normal hexane again, jolting 2 minutes, standing demix, the water intaking phase adds 0.4 liter of normal hexane, jolting 2 minutes more for the second time, standing demix, the water intaking phase adds 0.4 liter of normal hexane, jolting 1 minute more for the third time, standing demix, the water intaking phase;
D, with the water that step a obtains, add 0.4 liter of methylene dichloride again, jolting 3 minutes, standing demix, the water intaking phase adds 0.4 liter of methylene dichloride more for the second time, jolting 2 minutes, standing demix, the water intaking phase adds 0.4 liter of methylene dichloride more for the third time, jolting 1 minute, standing demix, the water intaking phase makes extraction liquid;
(5) concentrated, the freeze-drying operation of underpressure distillation:
With the extraction liquid that makes, carry out underpressure distillation, pressure-controlling is at-0.098Mpa, and temperature is controlled at 45 ℃, is concentrated near doing, and at-0.099Mpa, temperature is controlled at-55 ℃, carries out freeze-drying, after the freeze-drying, makes crude extract with pressure-controlling;
(6) preparative layer is analysed the sample operation:
A, with the crude extract that makes, add 95% ethanol, stirred 14 minutes, after fully dissolving, standing demix, make the ethanolic soln that concentration is 0.5g/mL, get supernatant liquor, application of sample is in the previously prepd silica gel column chromatography, and the chromatography column specification is 26 * 600mm, the silica gel 300g of 160～200 order specifications, application of sample concentration is that concentration is the ethanolic soln 10mL of 0.5g/mL;
The filling of b, silica gel column chromatography: silica gel 95% alcohol immersion, fully stirred 10 minutes, the amount of pursuing in the stirring is with Al
2O
3Add in the chromatography pipe, with the suspension of ethanolic soln until the chromatography pipe is filled;
C, beginning are carried out wash-out with 95% ethanol earlier, the yellow of sample moves down gradually in the elution process, when yellow moves to chromatography pipe bottom, approximately received 95% ethanol eluate of 200mL this moment, discard this part elutriant, begin to receive the elutriant as sample, every reception 200mL is as a sample, number consecutively; After the 1# sample reception finishes, use 90% ethanol instead and carry out wash-out, after the 2# sample reception finishes, use 85% ethanol instead and carry out wash-out, after the 3# sample reception finishes, use 80% ethanol instead and carry out wash-out, after the 5# sample reception finishes, use 70% ethanol instead and carry out wash-out, after the 7# sample reception finishes, use 60% ethanol instead and carry out wash-out, after the 9# sample reception finishes, use 50% ethanol instead and carry out wash-out, after the 11# sample reception finishes, use distilled water instead and carry out wash-out, after the 13# sample reception finishes, continue to carry out wash-out with 800mL distilled water, after this 800mL elutriant of Jie Shouing is collected 14 samples altogether as the 14# sample;
D, 10～11# sample of collecting carry out underpressure distillation respectively and concentrate, and pressure-controlling is at-0.096Mpa, and temperature is controlled at-50 ℃, carries out freeze-drying after concentrating, and freeze dried pressure-controlling is-0.099, and temperature is controlled at-55 ℃, makes the chromatography sample after the freeze-drying;
(7) high performance liquid chromatography operation:
A, with the 10～11# sample behind the silica gel adsorption chromatography that makes, add pure water, stirred 10 minutes, fully after the dissolving, standing demix makes the aqueous solution that concentration is about 1.5g/mL, discard insoluble solids, get supernatant liquor, with sample introduction behind the 0.45 μ m filtering with microporous membrane;
B, filtrate is carried out high performance liquid chromatography partly prepare the C8-EPS column chromatography, chromatographic condition is: column temperature: 35 ℃, moving phase: pure water and acetonitrile, stationary phase is selected C8-EPS for use, detect wavelength: 250nm, UV spectrum wavelength region: 200～380nm, gradient elution: 0～2min, the ratio of acetonitrile is 0～3%, 2～10mi n, the ratio of acetonitrile is 3%～7%, 10～13min, 7%～20%, 13～19min, 20%～30%, 19～20min, 30%～0.00%, 20～30min, 0.00%, the C8-EPS reversed-phase column of chromatography column: 10mm * 250mm, flow velocity: 4.00mL/min, the application of sample amount is 80 μ l;
C, carry out chromatography by above-mentioned chromatographic condition, the elutriant when being collected in 7.5～8-3min, pressure-controlling is at-0.099Mpa, temperature is controlled at 40 ℃, carries out freeze-drying after concentrating, and freeze dried pressure-controlling is at-0.100Mpa, temperature is controlled at-50 ℃, makes the chromatography sample after the freeze-drying.
The compound VII that present embodiment obtains, molecular weight are 327, and molecular formula is C
13H
17N
3O
5S,
Embodiment 8, present embodiment provide a kind of preparation method of the antitumor derivative that extracts from gecko:
(1) cleaning section:
The no web gecko that lives that catches deposited made its digestion in 72 hours, drain existing food and waste in the body,, dry with the distilled water cleaning;
(2) pulverizing process:
Back one kilogram weight gecko will be purified, change in the steeping tank, add concentration of volume percent and be 1.5 liters of 80% ethanolic solns, soak, after the immersion gecko causes death, gecko is changed in the high speed stamp mill, add 1 liter of the interior soak solution of steeping tank again, rotating speed is controlled at 15000r/min, and the time was controlled at 2 minutes, made pulverizing liquid;
(3) soak separation circuit:
In a, the pulverizing liquid that will make, adding concentration of volume percent is 1.5 liters of 80% ethanolic solns, under the room temperature, carry out the first time and soak, soak after 22 hours, carry out suction filtration, isolate filtrate and filter residue, with isolated filtrate, through Rotary Evaporators, pressure-controlling is at-0.095Mpa, temperature is controlled at 35 ℃, carry out concentrating under reduced pressure, be evaporated to and remove ethanol, make extracting solution No. one time;
B, with the isolated filter residue of step (a), adding concentration of volume percent is 3.0 liters of 80% ethanolic solns, under the room temperature, carry out the second time and soak, soaked 24 hours, carry out suction filtration, isolate filtrate and filter residue, with isolated filtrate, through Rotary Evaporators, pressure is at-0.095Mpa, temperature is controlled at 36 ℃, carry out concentrating under reduced pressure, be evaporated to and remove ethanol, make secondary raffinate;
C, with the isolated filter residue of step (b), adding concentration of volume percent is 3.0 liters of 80% ethanolic solns, under the room temperature, soak for the third time, soak after 25 hours, carry out suction filtration, isolate filtrate and filter residue, with isolated filtrate, through Rotary Evaporators, pressure is at-0.097Mpa, temperature is controlled at 40 ℃, carry out concentrating under reduced pressure, be evaporated to and remove ethanol, make extracting solution No. three times;
D, with the isolated filter residue of step (c), adding concentration of volume percent is 3.0 liters of 80% ethanolic solns, under the room temperature, carry out the 4th time and soak, soak after 26 hours, carry out suction filtration, isolate filtrate and filter residue, with isolated filtrate, through Rotary Evaporators, pressure is at-0.098Mpa, temperature is controlled at 50 ℃, carry out concentrating under reduced pressure, be evaporated to and remove ethanol, make extracting solution No. four times;
E, an extracting solution that will make, secondary raffinate, No. three extracting solutions and No. four extracting solutions mix, and make extracting solution.
(4) extraction process:
A, get 0.1 liter of extracting solution, add 0.3 liter of entry earlier, mix, add 0.4 liter of normal hexane again, jolting 2 minutes, standing demix, the water intaking phase adds 0.4 liter of normal hexane, jolting 2 minutes more for the second time, standing demix, the water intaking phase adds 0.4 liter of normal hexane, jolting 1 minute more for the third time, standing demix, the water intaking phase;
D, with the water that step a obtains, add 0.4 liter of methylene dichloride again, jolting 3 minutes, standing demix, the water intaking phase adds 0.4 liter of methylene dichloride more for the second time, jolting 2 minutes, standing demix, the water intaking phase adds 0.4 liter of methylene dichloride more for the third time, jolting 1 minute, standing demix, the water intaking phase makes extraction liquid;
(5) concentrated, the freeze-drying operation of underpressure distillation:
With the extraction liquid that makes, carry out underpressure distillation, pressure-controlling is at-0.098Mpa, and temperature is controlled at 45 ℃, is concentrated near doing, and at-0.099Mpa, temperature is controlled at-55 ℃, carries out freeze-drying, after the freeze-drying, makes crude extract with pressure-controlling;
(6) high performance liquid chromatography prepurification operation:
A, with the crude extract that makes, add pure water and stirred 5～10 minutes, fully after the dissolving, standing demix makes the aqueous sample that concentration is about 1.0g/mL, discards insoluble solids, gets supernatant liquor, with sample introduction behind the 0.45 μ m filtering with microporous membrane;
B, filtrate is carried out high performance liquid chromatography partly prepare the C18 column chromatography, chromatographic condition is: column temperature: 35 ℃, moving phase: pure water and acetonitrile, stationary phase is selected the C18 filler for use, detect wavelength: 210nm, UV spectrum wavelength region: 200～380nm, gradient elution: 0～5min, the ratio of acetonitrile is 0～0%, 5～10min, the ratio of acetonitrile is 0%～5%, 10～15min, 5%～10%, 15～20min, 10%～20%, 20～21min, 20%～30%, 21～27min, 30%～30%, 27～28min, 30%～0.00%, 28～38min, 0.00%, the C18 reversed-phase column of chromatography column: 10mm * 250mm, flow velocity: 4.00mL/min, the application of sample amount is 100 μ l;
C, carry out chromatography by above-mentioned chromatographic condition, elutriant when being collected in 6～10min is the 3# sample, pressure-controlling is at-0.099Mpa, temperature is controlled at 40 ℃, carry out freeze-drying after concentrating, freeze dried pressure-controlling is at-0.100Mpa, and temperature is controlled at-50 ℃, makes the first step high performance liquid chromatography prepurification sample after the freeze-drying.
(7) high performance liquid chromatography operation:
A, the high performance liquid chromatography prepurification sample that obtains is added pure water, stirred 5 minutes, after fully dissolving, standing demix makes the aqueous solution that concentration is about 1.0g/mL, discards insoluble solids, gets supernatant liquor, with sample introduction behind the 0.45 μ m filtering with microporous membrane;
B, filtrate is carried out high performance liquid chromatography partly prepare the C18-EPS column chromatography, chromatographic condition is: column temperature: 35 ℃, moving phase: pure water and acetonitrile, stationary phase is selected the C18-EPS filler for use, detect wavelength: 210nm, UV spectrum wavelength region: 200～380nm, gradient elution: 0～0.5min, the ratio of acetonitrile is 0～5%, 0.5～10min, the ratio of acetonitrile is 5%～8%, 10～13min, 8%～10%, 13～14min, 10%～30%, 14～20min, 30%～30%, 20～21min, 30%～0.00%, 21～31min, 0.00%, the C18-EPS reversed-phase column of chromatography column: 10mm * 250mm, flow velocity: 4.00mL/min, the application of sample amount is 100 μ l;
C, carry out chromatography by above-mentioned chromatographic condition, the elutriant when being collected in 8.6～9.5min, pressure-controlling is at-0.099Mpa, and temperature is controlled at 40 ℃, carries out freeze-drying after concentrating, and freeze dried pressure-controlling is at-0.100Mpa, and temperature is controlled at-50 ℃, makes after the freeze-drying.
The compound VIII that present embodiment makes, molecular weight are 297, and molecular formula is C
12H
15N
3O
4S.
Embodiment 9, present embodiment provide a kind of preparation method of the antitumor derivative that extracts from gecko:
(1) cleaning section:
The no web gecko that lives that will catch autumn was deposited 72 hours, made its digestion, drained existing food and waste in the body, and distilled water cleans, and dries, and weighs.
(2) pulverizing process:
Back one kilogram weight gecko will be purified, change in the steeping tank, add concentration of volume percent and be 1.5 liters of 90% ethanolic solns, soak, after the immersion gecko causes death, gecko is changed in the high speed stamp mill, add 1 liter of soak solution in the steeping tank again, rotating speed is controlled at 10000r/min, and the time was controlled at 2 minutes, made pulverizing liquid;
(3) soak separation circuit:
In a, the pulverizing liquid that will make, adding concentration of volume percent is 1.5 liters of 90% ethanolic solns, under the room temperature, carry out the first time and soak, soak after 24 hours, carry out suction filtration, isolate filtrate and filter residue, with isolated filtrate, through Rotary Evaporators, pressure-controlling is at-0.099Mpa, temperature is controlled at 45 ℃, carry out concentrating under reduced pressure, be evaporated to and remove ethanol, make extracting solution No. one time;
B, with the isolated filter residue of step (a), adding concentration of volume percent is 2.5 liters of 90% ethanolic solns, under the room temperature, carry out the second time and soak, soaked 24 hours, carry out suction filtration, isolate filtrate and filter residue, with isolated filtrate, through Rotary Evaporators, pressure is at-0.099Mpa, temperature is controlled at 40 ℃, carry out concentrating under reduced pressure, be evaporated to and remove ethanol, make secondary raffinate;
C, with the isolated filter residue of step (b), adding concentration of volume percent is 2.5 liters of 90% ethanolic solns, under the room temperature, soak for the third time, soak after 25 hours, carry out suction filtration, isolate filtrate and filter residue, with isolated filtrate, through Rotary Evaporators, pressure is at-0.099Mpa, temperature is controlled at 40 ℃, carry out concentrating under reduced pressure, be evaporated to and remove ethanol, make extracting solution No. three times;
D, with the isolated filter residue of step (c), adding concentration of volume percent is 2.5 liters of 90% ethanolic solns, under the room temperature, carry out the 4th time and soak, soak after 26 hours, carry out suction filtration, isolate filtrate and filter residue, with isolated filtrate, through Rotary Evaporators, pressure is between-0.099Mpa, temperature is controlled at 40 ℃, carry out concentrating under reduced pressure, be evaporated to and remove ethanol, make extracting solution No. four times;
E, an extracting solution that will make, secondary raffinate, No. three extracting solutions and No. four extracting solutions mix, and make extracting solution.
(4) extraction process:
A, get 0.15 liter of extracting solution, 0.15 liter in water mixes, add 0.3 liter of normal hexane again, jolting 3 minutes, standing demix, the water intaking phase, for the second time add 0.3 liter of jolting of normal hexane 2 minutes again, standing demix, water intaking phase, add 0.3 liter of normal hexane more for the third time, jolting 2 minutes, standing demix, water intaking phase;
D, with the water that step a obtains, add 0.3 liter of methylene dichloride again, jolting 3 minutes, standing demix, the water intaking phase adds 0.3 liter of methylene dichloride more for the second time, jolting 2 minutes, standing demix, the water intaking phase adds 0.3 liter of methylene dichloride more for the third time, jolting 2 minutes, standing demix, the water intaking phase makes extraction liquid;
(5) concentrated, the freeze-drying operation of underpressure distillation:
With the extraction liquid that makes, carry out underpressure distillation, pressure-controlling is-0.095, temperature is controlled at 50 ℃, is concentrated near doing, again with pressure-controlling at-0.099Mpa, temperature is controlled at-50 ℃, carries out freeze-drying, after the freeze-drying, makes crude extract;
(6) dextran LH-20 gel chromatography:
A, with the crude extract that makes, add pure water, stirred 2 minutes, after fully dissolving, standing demix, make the aqueous solution that concentration is 0.8g/mL, get supernatant liquor, application of sample is in previously prepd dextran LH-20 gel chromatography column, and the chromatography column specification is 26 * 600mm, the consumption of dextran LH-20 gel is 90g, and the application of sample amount is that concentration is the aqueous solution 15mL of 0.8g/mL;
B, carry out wash-out with distilled water, elution speed is 3～4 droplets/second, in the time of wash-out and collect elutriant, discard the 70mL elutriant that begins to collect, regather the 20mL elutriant as sample 1#, after collecting sample 1#, regather the 20mL elutriant as sample 2#, collect sample 2# after, be that 50% ethanolic soln carries out wash-out with concentration of volume percent again, elution speed is 3～4 droplets/second, in the time of wash-out and collect elutriant, every collection 20mL is numbered the sample of collecting by the sequencing of collecting successively as a sample, collect 30 samples altogether
C, the 13～15# sample that will collect, carrying out underpressure distillation respectively concentrates, pressure-controlling-below the 0.096Mpa, temperature is controlled at 40 ℃-50 ℃, carry out freeze-drying after concentrating, freeze dried pressure-controlling is at-0.099--0.100Mpa, and temperature is controlled at-45 ℃--55 ℃, make the chromatography sample after the freeze-drying.
(7) high performance liquid chromatography operation:
A, with the sample that makes after the freeze-drying behind the chromatography, add pure water, stirred 10 minutes, after fully dissolving, standing demix is made the aqueous solution that concentration is about 0.8g/mL, discards insoluble solids, gets supernatant liquor, with 0.45 μ m filtering with microporous membrane;
B, filtrate is carried out high performance liquid chromatography partly prepare the C18 column chromatography, chromatographic condition is: column temperature: 35 ℃, moving phase: pure water and acetonitrile, stationary phase is selected C18 for use, detect wavelength: 210nm, UV spectrum wavelength region: 200～380nm, the ratio of gradient elution: 0～0.5min, acetonitrile is 0～5%, 0.5 the ratio of～10min, acetonitrile is 5%～10%, 10～13min, 10%～30%, 13～19min, 30%～30%, 19～20min, 30%～0.00%, 20～28min, 0.00%, chromatography column: internal diameter is the C of 10mm * 250mm
18Reversed-phase column, flow velocity: 4.76mL/min, the application of sample amount is 200 μ L;
C, carry out chromatography by above-mentioned chromatographic condition, the elutriant when being collected in 11.6～12.6min, the concentrate drying pressure-controlling is at-0.092Mpa, temperature is controlled at 40 ℃, carries out freeze-drying after concentrating, and freeze dried pressure-controlling is at-0.099Mpa, temperature is controlled at-45 ℃, after the freeze-drying promptly.
The compound X that present embodiment makes, molecular weight are 323, and molecular formula is C
15H
21N
3O
3S,
(1) cleaning section:
The no web gecko that lives that will catch autumn was deposited 72 hours, made its digestion, drained existing food and waste in the body, and distilled water cleans, and dries, and weighs.
(2) pulverizing process:
Back one kilogram weight gecko will be purified, change in the steeping tank, add concentration of volume percent and be 1.5 liters of 90% ethanolic solns, soak, after the immersion gecko causes death, gecko is changed in the high speed stamp mill, add 1 liter of soak solution in the steeping tank again, rotating speed is controlled at 10000r/min, and the time was controlled at 2 minutes, made pulverizing liquid;
(3) soak separation circuit:
In a, the pulverizing liquid that will make, adding concentration of volume percent is 1.5 liters of 90% ethanolic solns, under the room temperature, carry out the first time and soak, soak after 24 hours, carry out suction filtration, isolate filtrate and filter residue, with isolated filtrate, through Rotary Evaporators, pressure-controlling is at-0.099Mpa, temperature is controlled at 45 ℃, carry out concentrating under reduced pressure, be evaporated to and remove ethanol, make extracting solution No. one time;
B, with the isolated filter residue of step (a), adding concentration of volume percent is 2.5 liters of 90% ethanolic solns, under the room temperature, carry out the second time and soak, soaked 24 hours, carry out suction filtration, isolate filtrate and filter residue, with isolated filtrate, through Rotary Evaporators, pressure is at-0.099Mpa, temperature is controlled at 40 ℃, carry out concentrating under reduced pressure, be evaporated to and remove ethanol, make secondary raffinate;
C, with the isolated filter residue of step (b), adding concentration of volume percent is 2.5 liters of 90% ethanolic solns, under the room temperature, soak for the third time, soak after 25 hours, carry out suction filtration, isolate filtrate and filter residue, with isolated filtrate, through Rotary Evaporators, pressure is at-0.099Mpa, temperature is controlled at 40 ℃, carry out concentrating under reduced pressure, be evaporated to and remove ethanol, make extracting solution No. three times;
D, with the isolated filter residue of step (c), adding concentration of volume percent is 2.5 liters of 90% ethanolic solns, under the room temperature, carry out the 4th time and soak, soak after 26 hours, carry out suction filtration, isolate filtrate and filter residue, with isolated filtrate, through Rotary Evaporators, pressure is between-0.099Mpa, temperature is controlled at 40 ℃, carry out concentrating under reduced pressure, be evaporated to and remove ethanol, make extracting solution No. four times;
E, an extracting solution that will make, secondary raffinate, No. three extracting solutions and No. four extracting solutions mix, and make extracting solution.
(4) extraction process:
A, get 0.15 liter of extracting solution, 0.15 liter in water mixes, add 0.3 liter of normal hexane again, jolting 3 minutes, standing demix, the water intaking phase, for the second time add 0.3 liter of jolting of normal hexane 2 minutes again, standing demix, water intaking phase, add 0.3 liter of normal hexane more for the third time, jolting 2 minutes, standing demix, water intaking phase;
D, with the water that step a obtains, add 0.3 liter of methylene dichloride again, jolting 3 minutes, standing demix, the water intaking phase adds 0.3 liter of methylene dichloride more for the second time, jolting 2 minutes, standing demix, the water intaking phase adds 0.3 liter of methylene dichloride more for the third time, jolting 2 minutes, standing demix, the water intaking phase makes extraction liquid;
(5) concentrated, the freeze-drying operation of underpressure distillation:
With the extraction liquid that makes, carry out underpressure distillation, pressure-controlling is-0.095, temperature is controlled at 50 ℃, is concentrated near doing, again with pressure-controlling at-0.099Mpa, temperature is controlled at-50 ℃, carries out freeze-drying, after the freeze-drying, makes crude extract;
(6) dextran LH-20 gel chromatography:
A, with the crude extract that makes, add pure water, stirred 2 minutes, after fully dissolving, standing demix, make the aqueous solution that concentration is 0.8g/mL, get supernatant liquor, application of sample is in previously prepd dextran LH-20 gel chromatography column, and the chromatography column specification is 26 * 600mm, the consumption of dextran LH-20 gel is 90g, and the application of sample amount is that concentration is the aqueous solution 15mL of 0.8g/mL;
B, carry out wash-out with distilled water, elution speed is 3～4 droplets/second, in the time of wash-out and collect elutriant, discard the 70mL elutriant that begins to collect, regather the 20mL elutriant as sample 1#, after collecting sample 1#, regather the 20mL elutriant as sample 2#, collect sample 2# after, be that 50% ethanolic soln carries out wash-out with concentration of volume percent again, elution speed is 3～4 droplets/second, in the time of wash-out and collect elutriant, every collection 20mL is numbered the sample of collecting by the sequencing of collecting successively as a sample, collect 30 samples altogether
C, the 13～15# sample that will collect, carrying out underpressure distillation respectively concentrates, pressure-controlling-below the 0.096Mpa, temperature is controlled at 40 ℃-50 ℃, carry out freeze-drying after concentrating, freeze dried pressure-controlling is at-0.099--0.100Mpa, and temperature is controlled at-45 ℃--55 ℃, make the chromatography sample after the freeze-drying.
(7) high performance liquid chromatography operation:
A, with the sample that makes after the freeze-drying behind the chromatography, add pure water, stirred 10 minutes, after fully dissolving, standing demix is made the aqueous solution that concentration is about 0.8g/mL, discards insoluble solids, gets supernatant liquor, with 0.45 μ m filtering with microporous membrane;
B, filtrate is carried out high performance liquid chromatography partly prepare C18-NH
2Column chromatography, chromatographic condition is: column temperature: 35 ℃, moving phase: pure water and acetonitrile, stationary phase is selected C18-NH for use
2Detect wavelength: 210nm, UV spectrum wavelength region: 200～380nm, the ratio of gradient elution: 0～0.5min, acetonitrile is 0～5%, and the ratio of 0.5～10min, acetonitrile is 5%～10%, 10～13min, 10%～30%, 13～19min, 30%～30%, 19～20min, 30%～0.00%, 20～28min, 0.00%, chromatography column: internal diameter is the C of 10mm * 250mm
18-NH
2Reversed-phase column, flow velocity: 4.76mL/min, the application of sample amount is 200 μ L;
C, carry out chromatography by above-mentioned chromatographic condition, the elutriant when being collected in 6.8～7.5min, the concentrate drying pressure-controlling is at-0.092Mpa, temperature is controlled at 40 ℃, carries out freeze-drying after concentrating, and freeze dried pressure-controlling is at-0.099Mpa, temperature is controlled at-45 ℃, after the freeze-drying promptly.
The compound XII that present embodiment makes, molecular weight are 313, and molecular formula is C
12H
15N
3O
5S,
Embodiment 11, present embodiment provide a kind of preparation method of the antitumor derivative that extracts from gecko:
(1) cleaning section:
The no web gecko that lives that catches deposited made its digestion in 48 hours, drain existing food and waste in the body,, dry with the distilled water cleaning;
(2) pulverizing process:
Back one kilogram weight gecko will be purified, change in the steeping tank, add concentration of volume percent and be 1 liter of 60% ethanolic soln, soak, after the immersion gecko causes death, gecko is changed in the high speed stamp mill, be added into 0.5 liter of the interior soak solution of steeping tank, rotating speed is controlled at 15000r/min, and the time was controlled at 2 minutes, made pulverizing liquid;
(3) soak separation circuit:
In a, the pulverizing liquid that will make, add remaining soak solution and replenish that to add concentration of volume percent be 1 liter of 60% ethanolic soln, under the room temperature, carry out the first time and soak, soak after 20 hours, carry out suction filtration, isolate filtrate and filter residue, with isolated filtrate, through Rotary Evaporators, pressure-controlling is at-0.095Mpa, temperature is controlled at 55 ℃, carry out concentrating under reduced pressure, be evaporated to and remove ethanol, make extracting solution No. one time;
B, with the isolated filter residue of step (a), adding concentration of volume percent is 2 liters of 60% ethanolic solns, under the room temperature, carry out the second time and soak, soaked 21 hours, carry out suction filtration, isolate filtrate and filter residue, with isolated filtrate, through Rotary Evaporators, pressure is at-0.095Mpa, temperature is controlled at 50 ℃, carry out concentrating under reduced pressure, be evaporated to and remove ethanol, make secondary raffinate;
C, with the isolated filter residue of step (b), adding concentration of volume percent is 2 liters of 60% ethanolic solns, under the room temperature, soak for the third time, soak after 22 hours, carry out suction filtration, isolate filtrate and filter residue, with isolated filtrate, through Rotary Evaporators, pressure is at-0.097Mpa, temperature is controlled at 50 ℃, carry out concentrating under reduced pressure, be evaporated to and remove ethanol, make extracting solution No. three times;
D, with the isolated filter residue of step (c), adding concentration of volume percent is 2 liters of 60% ethanolic solns, under the room temperature, carry out the 4th time and soak, soak after 23 hours, carry out suction filtration, isolate filtrate and filter residue, with isolated filtrate, through Rotary Evaporators, pressure is between-0.098Mpa, temperature is controlled at 50 ℃, carry out concentrating under reduced pressure, be evaporated to and remove ethanol, make extracting solution No. four times;
E, an extracting solution that will make, secondary raffinate, No. three extracting solutions and No. four extracting solutions mix, and make extracting solution.
(4) extraction process:
A, get 0.1 liter of extracting solution, add 0.2 liter of entry earlier, mix, add 0.6 liter of normal hexane again, jolting 1 minute, standing demix, the water intaking phase adds 0.6 liter of normal hexane, jolting 1 minute more for the second time, standing demix, the water intaking phase adds 0.6 liter of normal hexane, jolting 2 minutes more for the third time, standing demix, the water intaking phase;
D, with the water that step a obtains, add 0.6 liter of methylene dichloride again, jolting 2 minutes, standing demix, the water intaking phase adds 0.6 liter of methylene dichloride more for the second time, jolting 1 minute, standing demix, the water intaking phase adds 0.6 liter of methylene dichloride more for the third time, jolting 1 minute, standing demix, the water intaking phase makes extraction liquid;
(5) concentrated, the freeze-drying operation of underpressure distillation:
With the extraction liquid that makes, carry out underpressure distillation, pressure-controlling is at-0.096Mpa, and temperature is controlled at 40 ℃, is concentrated near doing, and at-0.099Mpa, temperature is controlled at-45 ℃, carries out freeze-drying, after the freeze-drying, makes crude extract with pressure-controlling;
(6) preparative layer is analysed the sample operation:
A, with the crude extract that makes, add 95% ethanol, stirred 15 minutes, fully after the dissolving, standing demix makes the ethanolic soln that concentration is 0.5g/mL, gets supernatant liquor, application of sample is in previously prepd Al
2O
3Chromatography column, chromatography column specification are 26 * 600mm, the Al of 100～200 order specifications
2O
3Solid 300g, application of sample concentration is the aqueous solution 10mL of 0.5g/mL;
B, Al
2O
3The filling of chromatography column: Al
2O
3Use soaked in absolute ethyl alcohol, fully stirred 10 minutes, the amount of pursuing in the stirring is with Al
2O
3Add in the chromatography pipe, with the alcoholic acid suspension until the chromatography pipe is filled;
C, beginning are carried out wash-out with 95% ethanol earlier, the yellow of sample moves down gradually in the elution process, when yellow moves to chromatography pipe bottom, approximately received 95% ethanol eluate of 200mL this moment, discard this part elutriant, begin to receive the elutriant as sample, every reception 200mL is as a sample, number consecutively; After the 1# sample reception finishes, use 90% ethanol instead and carry out wash-out, after the 2# sample reception finishes, use 85% ethanol instead and carry out wash-out, after the 3# sample reception finishes, use 80% ethanol instead and carry out wash-out, after the 5# sample reception finishes, use 70% ethanol instead and carry out wash-out, after the 7# sample reception finishes, use 60% ethanol instead and carry out wash-out, after the 9# sample reception finishes, use 50% ethanol instead and carry out wash-out, after the 11# sample reception finishes, use distilled water instead and carry out wash-out, after the 13# sample reception finishes, continue to carry out wash-out with 1000mL distilled water, after this 1000mL elutriant of Jie Shouing is collected 14 samples altogether as the 14# sample;
D, 10～12# sample of collecting carry out underpressure distillation respectively and concentrate, and pressure-controlling is at-0.096Mpa, and temperature is controlled at-50 ℃, carries out freeze-drying after concentrating, and freeze dried pressure-controlling is-0.099, and temperature is controlled at-55 ℃, makes the chromatography sample after the freeze-drying;
(7) high performance liquid chromatography operation:
A, with the Al that makes
2 O
310～12# sample behind the adsorption chromatography adds pure water, stirs 10 minutes, and fully after the dissolving, standing demix makes the aqueous solution that concentration is about 1.5g/mL, discards insoluble solids, gets supernatant liquor, with 0.45 μ m filtering with microporous membrane;
B, filtrate is carried out high performance liquid chromatography partly prepare the C18-AQ column chromatography, chromatographic condition is: column temperature: 38 ℃, moving phase: pure water and acetonitrile, stationary phase is selected C18-AQ for use, detect wavelength: 254nm, UV spectrum wavelength region: 200～380nm, gradient elution: 0～2min, the ratio of acetonitrile is 0～8%, 2～10min, the ratio of acetonitrile is 8%～10%, 10～13min, 10%～30%, 13～19min, 30%～30%, 19～20min, 30%～0.00%, 20～28min, 0.00%, chromatography column: internal diameter is the C18-AQ reversed-phase column of 10mm * 150mm, flow velocity: 4.2mL/min, the application of sample amount is 100 μ L;
C, carry out chromatography by above-mentioned chromatographic condition, the elutriant when being collected in 10.1～10.9min, pressure-controlling is at-0.099Mpa, temperature is controlled at 40 ℃, carries out freeze-drying after concentrating, and freeze dried pressure-controlling is at-0.100Mpa, temperature is controlled at-50 ℃, after the freeze-drying promptly.
The compound X III that present embodiment obtains, molecular weight 325, molecular formula is C
14H
19N
3O
4S,
The clinical efficacy of medicine observing of compound among embodiment 12, the embodiment 1
From between year March in October, 1997 to 2000, in the advanced lung cancer case of treatment, 86 routine lung cancer in non-cellule type are reported as follows: hereinafter the gecko activeconstituents is meant the compound of preparation among the embodiment 1.
The male sex's 71 examples, women 15 examples; Age is at 28-81 between year; Clinical stages: III a49 example, III b29 example, IV 8 examples;
Histological type: squama cancer 46 examples, gland cancer 40 examples; Gecko activeconstituents+doctor trained in Western medicine is 53 examples that classical western medical treatment poor effect is then used the treatment of gecko activeconstituents instead, and is single with gecko activeconstituents 33 examples.
Diagnosis: all gecko activeconstituents treatment case must have that reliable image is qualitative, level diagnosis and pathological diagnosis, and obtaining of Pathologic specimen mainly is bronchoscope and lung puncture, and perspective guiding and CT guide.Diagnosis for the first time must have the CT sheet and carry out by stages with reference to the lung cancer clinical stages standard that 1988 american cancer joint committees deliver.
Treatment: oral gecko activeconstituents liposome particles agent 10mg/ time, 1 time/day.
Check: 10 days checks plain film or CT after the drug withdrawal; Liver function, kidney merit, routine blood test etc.
Therapeutic evaluation: remission rate: according to national antitumor drug efficacy assessment standard in 1978, with reference to external pharmacological agent cancer efficacy evaluation method and standard authorization curative effect.Quality of life is marked with the Karnofsky method.
The result: one, lump changes:
Table 10 liang treatment group lump size change (CR, PR, MR, P, S)
Annotate: be the percentage ratio that accounts for total routine number in (1), the bracket.
(2), size be according to take medicine 10 days CT of drug withdrawal after two courses of treatment or X-ray film with former contrast.
The lump Changing Pattern is to find through the repeatedly CT examination of some cases: lump does not at first dwindle, and only shows as the CT value and descends, and then lump diminishes gradually.And gecko activeconstituents II+doctor trained in Western medicine group more singly uses gecko activeconstituents group more obvious.
Two, quality of life:
The Karnofsky scoring:
Gecko activeconstituents+doctor trained in Western medicine group: being 41.7 ± 3.4 minutes before the medication, was 69.6 ± 5.0 fens both contrast there were significant differences property (P＜0.005) after taking medicine 30 days.
Gecko activeconstituents group: being 59.5 ± 5.3 minutes before the medication, was 73.6 ± 4.2 fens both contrast there were significant differences property (P＜0.005) after taking medicine 30 days.
Three, survival time:
Gecko activeconstituents+doctor trained in Western medicine group survival time is 15.6 ± 2.2 months; Single is 13.6 ± 1.8 with gecko activeconstituents group.
Conclusion: advanced lung cancer is clinical common, and the treatment group survival time is higher than non-treatment group, and the mean survival time of treatment group is not wait in 2.5 months-8.6 months.Two groups of (gecko activeconstituents+doctor trained in Western medicine group and single with a sky gecko activeconstituents) mean survival times are respectively 15.6 and 13.6 months, all are higher than the chemicotherapy group.Classical western medical treatment is many when prolonging patient's survival time to be cost to reduce patient's quality of life, especially in the recent period life quality.Patient is when tumour is controlled for the treatment of gecko activeconstituents, and patient's physical condition is many to take a turn for the better synchronously.Patient's quality of life can be used as the reference index of tumour control situation.The complete remission rate of gecko activeconstituents treatment tumour knurl body size is more than 7.0%, and efficient is more than 96.6%.Above clinical observation proves: the antineoplastic compound crude extract has obvious antitumor action.
Embodiment 13,8 kind of antineoplastic compound are tested the external knurl that presses down of three different tumour cells
Method: with tumour cell with containing RPMI 1640 culture medium culturing of 10% new-born calf serum, the cell in the vegetative period of taking the logarithm, behind 0.25% tryptic digestion with 3 * 10
3Individual/hole is inoculated in 96 orifice plates, replenishes nutrient solution to every hole 200 μ L, places 37 ℃, 5%CO
2Cultivated 24 hours in the incubator, experimental group adds 10 μ g monomeric compounds, with serum free medium dilution, the aseptic filtering with microporous membrane of 0.22 μ m.If adding equivalent PBS is the blank group.After the Measuring Time of drafting is promptly cultivated 24 hours, 48 hours, 72 hours, inverted microscope observation of cell form and taking pictures at first, every then hole adds the MTT solution 20 μ L of 5mg/mL, places 37 ℃, 5%CO
2Continue to cultivate in the incubator 4 hours, and stopped cultivating, the careful suction abandoned the culture supernatant hole in, and every hole adds 150 μ L DMSO, shakes 10 minutes, crystallisate is fully dissolved after, with automatic microplate reader measurement 570mn place absorbancy.
Growth of tumour cell inhibiting rate calculation formula is as follows:
The OD value of the actual OD value/negative control hole in tumour cell survival rate (%)=dosing hole
Growth of tumour cell inhibiting rate (%)=100%-cell survival rate
Result: as following table
All cpds is to the inhibiting rate of 7402 liver cancer cells
| ??Ⅰ | ??Ⅱ | ??Ⅳ | ??Ⅶ | ??Ⅷ | ??Ⅹ | ??Ⅻ | ??ⅩⅢ | |
| ??24h | ??63.7 | ??71.2 | ??52.6 | ??65.6 | ??43.6 | ??41.9 | ??51.7 | ??36.8 |
| ??48h | ??84.9 | ??91.4 | ??71.9 | ??83.3 | ??65.2 | ??61.8 | ??68.3 | ??58.3 |
| ??72h | ??92.3 | ??94.5 | ??85.2 | ??91.4 | ??83.4 | ??80.3 | ??84.7 | ??79.5 |
All cpds is to the inhibiting rate of PG lung carcinoma cell
| ??Ⅰ | ??Ⅱ | ??Ⅳ | ??Ⅶ | ??Ⅷ | ??Ⅹ | ??Ⅻ | ??ⅩⅢ | |
| ??24h | ??64.5 | ??73.2 | ??58.1 | ??65.7 | ??52.4 | ??47.1 | ??56.3 | ??39.4 |
| ??48h | ??85.3 | ??92.1 | ??75.9 | ??82.6 | ??68.2 | ??63.8 | ??73.0 | ??56.8 |
| ??72h | ??93.9 | ??97.5 | ??86.2 | ??91.9 | ??82.3 | ??81.2 | ??83.9 | ??78.6 |
All cpds is to the inhibiting rate of HT-29 colon cancer cell
| ??Ⅰ | ??Ⅱ | ??Ⅳ | ??Ⅶ | ??Ⅷ | ??Ⅹ | ??Ⅻ | ??ⅩⅢ | |
| ??24h | ??65.5 | ??69.7 | ??54.1 | ??62.6 | ??41.6 | ??41.9 | ??53.7 | ??36.5 |
| ??48h | ??84.7 | ??90.8 | ??70.9 | ??81.4 | ??65.7 | ??61.8 | ??69.4 | ??55.3 |
| ??72h | ??92.1 | ??93.5 | ??84.8 | ??90.7 | ??83.3 | ??80.3 | ??82.9 | ??77.2 |
Conclusion: as seen from the table, the external knurl that presses down experimental results show that 8 kinds of different compounds all have the obvious suppression effect to three kinds of different tumour cells.
Embodiment 14, large bowel cancer chick chorioallantoic membrane transplantation model proof antineoplastic compound derivative II blood vessel formation against function
(1) observes the dynamic change of chick chorioallantoic membrane vascular development, clearly set up the optimum condition of large bowel cancer chick chorioallantoic membrane transplantation model.The minimum quantity of inoculation HT-29 cell is as the desirable inoculating cell quantity of modelling when selecting the highest tumor formation rate; The HT-29 cell inoculation of identical ideal quantity in the relative avascular area of different days chick chorioallantoic membrane, is determined best inoculation embryo age; With the cell inoculation of ideal quantity relative avascular area, understand the knurl bulk-growth and the vasculogenesis Changing Pattern of large bowel cancer chick chorioallantoic membrane transplantation model to the suitableeest age in days chick chorioallantoic membrane.
(2) antitumor mechanism is inquired into:
The antineoplastic compound II is established high, medium and low 3 concentration administration groups, and PBS does the blank group, the positive control group of grace degree, the negative control group of VEGF, totally 6 groups.Select to have inoculated the one-tenth knurl chicken embryo of HT-29 cell after 3 days, pretreated gelfoam is placed at the place in the one-tenth knurl, experimental group medicine and control group PBS are respectively got on gelfoam that 10 μ L are added on each group respectively, hatched 5 days, fixedly get film, observe different experiments group vascularization feature, calculate institute's favored area blood vessel area ratio, and calculate the vasculogenesis inhibiting rate.Get knurl body tissue and fix, paraffin embedding, routine paraffin wax section, the HE staining is observed the histologic characteristics of knurl body, adopts immunohistochemistry technology to detect the expression of organizing proliferating cell nuclear antigen PCNA.
The antineoplastic compound II can suppress the vasculogenesis of large bowel cancer chick chorioallantoic membrane transplantation model, and the antineoplastic mechanism of antineoplastic compound II may not be the propagation by direct inhibition tumour cell.Therefore, the mechanism of antineoplastic compound II inhibition tumour is the propagation by the inhibition vascular endothelial cell, and then suppresses the tumor tissues vascularization, also further suppresses tumor growth.
Table 2: the antineoplastic compound II is intervened the large bowel cancer chick chorioallantoic membrane and is suppressed the experiment grouping that model vessel generates.
Table 3: different concns antineoplastic compound II is at the cell inhibitory rate of different time mtt assay measuring and calculating.
Annotate: to HT-29MTT test-results OD value (570nm).
Table 4: different concns antineoplastic compound II is to the influence of chick chorioallantoic membrane transplantation model inductive vasculogenesis
Annotate: through the variance analysis check, * P＜0.05 of comparing with the PBS control group.
Embodiment 15, flow cytometer detect antineoplastic compound II, IV, the effect of VII inducing apoptosis of tumour cell
The HepG-2 cell in vegetative period of taking the logarithm is made into 1 * 10 with the PRMI-1640 nutrient solution that contains 10% calf serum with cell
5Individual L
-1, every culturing bottle adds the good cell 4mL of dilution, and positive control adds 1mL 5-FU250mg/mL, experimental group respectively adds 1mL II, IV, VII compound, making its final concentration is 0.2mg/mL, establishes negative control simultaneously, adds 5 μ LAnnexin-V-FITC with 195 μ L cell suspensions, the mixing mark, the room temperature temperature is bathed 20min, uses PBS damping fluid washed cell 1 time, uses 190 μ LBindingBuffer damping fluids resuspended again, and then adding 5 μ L propidium iodides, the upflowing cell instrument carries out apoptosis and detects.Detected result sees the following form.Flow cytometer is the FACSCalibur type that U.S. Becton Dickinson company produces.Annexin-V-FITC and PI jack to jack adapter are viable cell; The Annexin-V-FITC positive and the PI feminine gender is a viable apoptotic cell; The two positives of Annexin-V-FITC and PI are non-viable apoptotic cell; Annexin-V-FITC feminine gender and the PI positive is a non-viable non-apoptotic cell.
The apoptosis rate of each active compound and control group during flow cytometer detects
| Group | Negative control | ??5-FU | The II compound | The IV compound | The VII compound |
| Apoptosis rate | ??3.73 | ??15.74 | ??38.97 | ??30.41 | ??34.65 |
The flow cytometer detected result proves: the gecko activeconstituents has the ability of very strong cell death inducing; Its antitumor mechanism is relevant with cell death inducing.
Press down the knurl experiment in the body of embodiment 16, antineoplastic compound I, II, VII compound
With the conventional CT-26 cell of cultivating of the RPMI-1640 substratum that contains 10% calf serum, contain 5%CO in 37 ℃
2Be cultured to mouse junction cancer CT-26 cell strain logarithmic phase in the constant incubator and carry out cell inoculation, with 1.5 * 10
6It is subcutaneous that the density 0.2mL of/mL is inoculated in BALB/c mouse right side armpit.After the lotus knurl 5 days, 30 mouse see that all grain of rice size tumour grows, 30 tumor-bearing mices are divided into 5 groups at random, in lotus knurl the 5th day respectively in left side armpit subcutaneous injection physiological saline (blank group), Endostatin (positive controls) (10 μ g/ days/only), (I, II, VII compound experimental group) low dosage (0.1 μ g/ days/only), middle dosage (0.2 μ g/ days/only) and high dosage (0.5 μ g/ days/only) each 0.1mL.Behind the continuous use 14 days, MR somatoscopy tumor growth situation is estimated result of treatment.After testing end in 16 days, put to death mouse, take by weighing knurl and heavily reach measurement tumour size and carry out pathology and the immunohistochemical methods detection.
Tumor control rate: each organize mouse treatment after 16 days each experimental group gross tumor volume and the heavy inhibiting rate of knurl see the following form.
The tumour inhibiting rate of I, II, VII compound various dose medicine group and Endostatin group
Learn by statistics and handle, the tumor control rate of gecko activeconstituents senior middle school low dose group and antineoplastic vascular generative capacity all are higher than Endostatin group (remarkable statistical significance is arranged between group).
Claims (21)
1. the antineoplastic compound that extracts from gecko is characterized in that: this compound has the basic skeleton structure shown in (I) formula:
In the formula (I), C
3Be R, C
7Be the S configuration, or C
3Be S, C
7Be the R configuration.
2. the antineoplastic compound that extracts from gecko according to claim 1 is characterized in that: this compound is the compound shown in the general formula (II):
This compound molecular weight is 253-341;
In the formula (II), C
2Be R or S configuration, C
3Be R, C
7Be S, C
8Be R, C
9Be the S configuration, or C
2Be R or S configuration, C
3Be S, C
7Be R, C
8Be S, C
9Be R configuration, substituent R
1Can be H ,-COOCH
3Or-COOC
2H
5, R
2, R
3, R
4, R
5, R
6Identical or inequality, can be respectively H, OH, CH
3, C
2H
5, OCH
3Or OC
2H
5
3. the antineoplastic compound that extracts from gecko according to claim 1 and 2 is characterized in that: this compound is the compound shown in the formula I:
4. the antineoplastic compound that extracts from gecko according to claim 1 and 2 is characterized in that: this compound is the compound shown in the formula II:
5. the antineoplastic compound that extracts from gecko according to claim 1 and 2 is characterized in that: this compound is the compound shown in the formula IV:
6. the antineoplastic compound that extracts from gecko according to claim 1 and 2 is characterized in that: this compound is the compound shown in the formula VII:
7. the antineoplastic compound that extracts from gecko according to claim 1 and 2 is characterized in that: this compound is the compound shown in the formula VIII:
8. the antineoplastic compound that extracts from gecko according to claim 1 and 2 is characterized in that: this compound is the compound shown in the formula X:
9. the antineoplastic compound that extracts from gecko according to claim 1 and 2 is characterized in that: this compound is the compound shown in the formula XII:
10. the antineoplastic compound that extracts from gecko according to claim 1 and 2 is characterized in that: this compound is the compound shown in the formula XIII:
11. the preparation method according to claim 1 or 2 described a kind of antineoplastic compound that extract from gecko is characterized in that: the compound shown in the preparation general formula (II) may further comprise the steps:
(1) cleaning section:
The no web gecko that lives that catches deposited made its digestion in 24-72 hour, drain existing food and waste in the body,, dry with the distilled water cleaning;
(2) pulverizing process:
Gecko with after purifying changes in the steeping tank, adds ethanolic soln, soaks, and after the immersion gecko causes death, gecko and soak solution is changed in the high speed stamp mill, and rotating speed is controlled at 10000-20000r/min, and the time was controlled at 2-5 minute, made pulverizing liquid;
(3) soak separation circuit: in the pulverizing liquid that will make, add ethanolic soln, soak and separate, make extracting solution;
(4) extraction process:
A, with the extracting solution that makes, add entry earlier, mix, add non-polar organic solvent again, jolting, standing demix, the water intaking phase;
B, the water that step a is obtained add polar organic solvent, jolting, standing demix, water intaking phase;
(5) concentrated, the freeze-drying operation of underpressure distillation:
With the extraction liquid that makes, carry out underpressure distillation, underpressure distillation is done near, carries out freeze-drying then, makes crude extract, promptly comprises the mixture of compound shown in the general formula (II).
12. the preparation method according to the described a kind of antineoplastic compound that extracts from gecko of claim 11 is characterized in that: the compound shown in the preparation general formula (II) comprises following just purifying and purification step:
(6.1) preparative layer is analysed sample purification procedures just:
With the crude extract that makes, add pure water, fully after the dissolving, standing demix is got supernatant liquor, carries out chromatography, behind the chromatography, with the sample of collecting, carries out underpressure distillation and concentrates, and carries out freeze-drying after concentrating, and makes purifying chromatography sample just after the freeze-drying;
(7) high performance liquid chromatography operation:
A, the first purifying chromatography sample that will make add pure water, and fully after the dissolving, standing demix is got supernatant liquor, uses filtering with microporous membrane;
B, filtrate is carried out the high performance liquid chromatography (HPLC) chromatography, the elutriant when collecting 6.8～13.0min carries out the underpressure distillation mode and concentrates, and carries out freeze-drying after concentrating, after the freeze-drying promptly.
13. the preparation method according to the described a kind of antineoplastic compound that extracts from gecko of claim 11 is characterized in that: the compound shown in the preparation general formula (II) comprises following just purifying and purification step:
(6.2) the first purification procedures of high performance liquid chromatography:
A, with the crude extract that makes, fully after the dissolving, standing demix is got supernatant liquor, with sample introduction behind the filtering with microporous membrane;
B, filtrate is carried out high performance liquid chromatography partly prepare the C18 column chromatography; Its chromatographic condition is: column temperature: 35 ℃, moving phase: pure water and acetonitrile, stationary phase is selected the C18 filler for use, detect wavelength: 210nm, UV spectrum wavelength region: 200～380nm, gradient elution: 0～5min, the ratio of acetonitrile is 0～0%, 5～10min, the ratio of acetonitrile is 0%～5%, 10～15min, 5%～10%, 15～20min, 10%～20%, 20～21min, 20%～30%, 21～27min, 30%～30%, 27～28min, 30%～0.00%, 28～38min, 0.00%, the C18 reversed-phase column of chromatography column: 10mm * 250mm, flow velocity: 4.00mL/min, the application of sample amount is 100 μ L;
C, carry out chromatography by above-mentioned chromatographic condition, the elutriant when being collected in 6～10min carries out freeze-drying after concentrating, and makes the first step high performance liquid chromatography purifying chromatography sample just after the freeze-drying;
(7) high performance liquid chromatography operation:
A, the first purifying chromatography sample that will make add pure water, and fully after the dissolving, standing demix is got supernatant liquor, uses filtering with microporous membrane;
B, filtrate is carried out the high performance liquid chromatography (HPLC) chromatography, the elutriant when collecting 6.8～13.0min carries out the underpressure distillation mode and concentrates, and carries out freeze-drying after concentrating, after the freeze-drying promptly.
14. the preparation method of a kind of antineoplastic compound that extracts from gecko according to claim 11 is characterized in that: in the described step (3), immersion and isolating number of times are four times, and each concrete operations are as follows:
Under the room temperature, add ethanolic soln, soak and suction filtration, isolate filtrate and filter residue,,, carry out concentrating under reduced pressure, be evaporated to and remove ethanol, make extracting solution,, mix No. four extracting solutions through Rotary Evaporators with isolated filtrate.
15. the preparation method of a kind of antineoplastic compound that extracts from gecko according to claim 11 is characterized in that: in the described step (2), described ethanolic soln, concentration of volume percent are 60%-95%.
16. the preparation method of a kind of antineoplastic compound that extracts from gecko according to claim 11 is characterized in that: in the step (2), the soak solution in the steeping tank of adding.
17. the preparation method of a kind of antineoplastic compound that extracts from gecko according to claim 11 is characterized in that: in the step (3), the ethanolic soln that described immersion adds, concentration of volume percent is 60%-95%.
18. the preparation method of a kind of antineoplastic compound that extracts from gecko according to claim 11 is characterized in that:
In the step (4), describedly add entry, add-on be extracting liquid volume 1-4 doubly;
In the step (4), the non-polar organic solvent of adding, each consumption are respectively 1-2 that extracting solution adds cumulative volume after the entry doubly.
In the step (4), the polar organic solvent of adding, each consumption is respectively 1-2 times of water volume.
19. the preparation method of a kind of antineoplastic compound that extracts from gecko according to claim 12 is characterized in that: described gel permeation chromatography, the kind of gel are dextrane gel and hydroxypropyl dextrane gel;
Described adsorption chromatography, the kind of sorbent material is: aluminum oxide and silica gel.
20. the preparation method according to claim 12 or 13 described a kind of antineoplastic compound that extract from gecko is characterized in that: the actual conditions that in described step (7) the high performance liquid chromatography operation filtrate is carried out the high performance liquid chromatography (HPLC) chromatography is as follows:
High performance liquid chromatography in the described high performance liquid chromatography operation, chromatographic condition is: column temperature: 32-36 ℃, moving phase: pure water and acetonitrile, stationary phase are selected C18, C8, C18-EPS, C8-EPS, C18-AQ, C8-AQ, C18-NH for use
2Or C8-NH
2In a kind of, detect wavelength: 200-280nm, UV spectrum wavelength region: 200～380nm, the ratio of gradient elution: 0～2.0min, acetonitrile is 0～8%, 0.5～12min, is 2%～10%, 8～15min, 8%～30%, 15～25min, 20%～35%, 18～25min, 30%～0.00%, 20～35min, 0.00%, chromatography column: internal diameter is 10mm, and length is C18, C8, C18-EPS, C8-EPS, C18-AQ, C8-AQ, the C18-NH of 150～300mm
2Or C8-NH
2In a kind of reversed-phase column, flow velocity: 4.0-5.0mL/min, the application of sample amount is 50-200 μ L.
21. according to one of them described antineoplastic compound that extracts from gecko of claim 1-10, it is characterized in that: described compound is as the application of antitumor drug.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102688475A (en) * | 2012-01-14 | 2012-09-26 | 河南科技大学 | Anti-cancer extractive of wall gecko |
| CN110269869A (en) * | 2019-07-30 | 2019-09-24 | 宁夏医科大学 | A kind of anti-tumor drug and verification method based on lizard |
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2010
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102688475A (en) * | 2012-01-14 | 2012-09-26 | 河南科技大学 | Anti-cancer extractive of wall gecko |
| CN102688475B (en) * | 2012-01-14 | 2014-10-29 | 河南科技大学 | Anti-cancer extractive of wall gecko |
| CN110269869A (en) * | 2019-07-30 | 2019-09-24 | 宁夏医科大学 | A kind of anti-tumor drug and verification method based on lizard |
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