CN102688475A - Anti-cancer extractive of wall gecko - Google Patents
Anti-cancer extractive of wall gecko Download PDFInfo
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- CN102688475A CN102688475A CN2012100108515A CN201210010851A CN102688475A CN 102688475 A CN102688475 A CN 102688475A CN 2012100108515 A CN2012100108515 A CN 2012100108515A CN 201210010851 A CN201210010851 A CN 201210010851A CN 102688475 A CN102688475 A CN 102688475A
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Abstract
The invention discloses an anti-cancer extractive of wall gecko. A preparation method of the extractive comprises the following steps of: firstly, drying and smashing the wall gecko, soaking in a buffer solution with the pH of 7.4 for 12 hours, carrying out ethanol extraction by adding absolute ethyl alcohol, concentrating the extracting solution in an evaporation way, salting out by adding ammonium sulfate in the concentrated solution, obtaining white floccule precipitate in a dialyzing way, and drying the white floccule precipitate to obtain the anti-cancer extractive of the wall gecko. The anti-cancer extractive of the wall gecko disclosed by the invention is obtained by extracting the peptide molecules with the function of tumor resistance from the wall gecko by means of alcohol extraction, salt precipitation and dialysis, so that the extractive is very high in anti-cancer effect, thereby being capable of being taken as the active ingredients of high-efficiency and low-toxicity anti-cancer new drug. The test shows that the anti-cancer extractive of the wall gecko disclosed by the invention is very good in anti-cancer function, which reaches the anti-cancer level of the normal anti-cancer medicine such as fluorouracil and adriamycin, so that the immunity of the human body can be improved, and the extractive of the wall gecko contains the natural wall gecko polypeptide, so that the anti-cancer extractive has the advantages of being low in toxicity, and small in side effects.. Furthermore, the preparation method of the extractive is simple, thereby being easy to popularize and use.
Description
Technical field
The present invention relates to a kind of Gekko Swinhonis anticancer extract.
Background technology
Gekko Swinhonis is the Chinese crude drug of the traditional treatment tumor of China.Lots of clinical report shows that Gekko Swinhonis treatment malignant tumor effect is remarkable, does, bright Gekko Swinhonis, the serum that contains Gekko Swinhonis, Gekko Swinhonis compound preparation, Gekko Swinhonis protease hydrolysis liquid etc. all have curative effect preferably to tumor.Xie Shuan, Wang Xuemei etc. (thank refreshing, Wang Xuemei, Xie Dongze. bright Gekko Swinhonis extract suppresses the research of increment of C6 glioma cell and cell death inducing. and treatment and prevention of tumour is studied; 2003,30:458-461) (get live body and do not have the freezing execution of web Gekko Swinhonis, clear water is cleaned with the bright Gekko Swinhonis extracting solution of variable concentrations; Pulverize homogenate, freeze thawing 10 times, supernatant is got in natural sedimentation; Low-temperature centrifugation 10000r/min, centrifugal 20min gets supernatant) the C6 glioma cell is studied; The bright Gekko Swinhonis extracting solution of result can significantly suppress the growth of C6 glioma cell, and tangible amount-result relation is arranged when 64mg/L, 128mg/L, 256mg/L concentration.(Yang Lihua such as Yang Lihua, Wang Xuemei; Yang Jinxia, Wang Xuemei etc. the Gekko Swinhonis polysaccharide is to the effect research of hepatocarcinoma H22 inside and outside, Mus source. Tianjin Chinese medicine, 2008; 25:494-496) carried out the Gekko Swinhonis polysaccharide to the effect research of hepatocarcinoma H22 inside and outside, Mus source; The result finds that the Gekko Swinhonis polysaccharide is very strong at interaction in vitro, but transplanted tumor in the body is not had effect, and therefore the antitumor action to the Gekko Swinhonis polysaccharide has proposed query.Li Yaohui, Geng Xiufang etc. (Li Yaohui, Sheng Jiwen, Geng Xiufang etc. no web Gekko Swinhonis medicinal ingredient antitumous effect. the Shaanxi traditional Chinese medical science; 2009,30:899-901) insert ethanol to bright Gekko Swinhonis and cause death, smash homogenate to pieces; Alcohol solution dipping, evaporation removes ethanol, removes lipid again; Obtain water extract, last lyophilization obtains dry thing.Then should the drying thing with dissolve with ethanol, through Al
2O
3Column chromatography obtains 15 chromatography drug samples.Find that through experimentation the 3rd to the 9th part in these samples has the effect that suppresses HepG-2, the remainder effect is not obvious.And (Li Yaohui such as Li Yaohui; Liu Dongmei, Sheng Jiwen etc. the extraction of no web Gekko Swinhonis antineoplastic component and to the inhibitory action of CT-26 mice adenocarcinoma of colon. The Fourth Military Medical University's journal, 2009; 30:1103-1106) in the extraction and research thereof of no web Gekko Swinhonis antineoplastic component to CT-26 mice adenocarcinoma of colon; Report has 19 chromatography compositions, and wherein the 11st to the 19th sample do not have effect, and the 1st to the 10th sample all has certain antitumor action, and wherein the 1st to the 6th sample effect is the strongest.(Kang Jiangong such as Kang Jiangong, Li Yaohui; Zhang Shizhuan; Li Yaohui etc. bright no web Gekko Swinhonis anti-tumor active ingredient suppresses CT-26 growth of tumour cell experimentation. the Chinese Hospitals pharmaceutical journal; 2007,27:441-444) also compared the effect of bright Gekko Swinhonis active component and liposome thereof to CT-26, find that the effect in vivo of active component liposome is stronger.
Above-mentioned great deal of research results shows; The Gekko Swinhonis extract has sure antitumor action; And aspect treatment of solid tumors, good prospects for application is arranged; But the method for distilling of present Gekko Swinhonis extract is comparatively complicated, and the Gekko Swinhonis extract to the tumor killing effect of tumor be difficult to reach fluorouracil and amycin but the tumor level.
Summary of the invention
The purpose of this invention is to provide the good Gekko Swinhonis anticancer extract of a kind of tumor killing effect.
In order to realize above purpose, the technical scheme that the present invention adopted is: a kind of Gekko Swinhonis anticancer extract, and it adopts following method to extract and forms:
1) Gekko Swinhonis is cleaned, dries, be broken into powder, placing pH is the buffer solution of Tris-hydrochloric acid of 7.4, soaks 12 hours;
2) it is broken to soak the back, and it is 55% to extract that the centrifuging and taking supernatant adds dehydrated alcohol to ethanol final concentration, and the centrifuging and taking supernatant promptly gets the Gekko Swinhonis alcohol extract then;
3) with Gekko Swinhonis alcohol extract evaporation and concentration, adding ammonium sulfate to its concentration is 25%, adjustment pH to 7, and 4 ℃ left standstill 3～4 hours, and every separated 30min stirs once, and the centrifuging and taking supernatant gets supernatant then; It is 50% that the supernatant that obtains is added ammonium sulfate to its concentration, adjustment PH to 4.6, and 4 ℃ left standstill 3～4 hours, and every separated 30min stirs once, the centrifugal then deposition that obtains;
4) be in the 3.5KDa bag filter with the deposition molecular cut off of packing into, place 0 ℃ of distilled water to dialyse bag filter, white floccule in bag filter, occurs;
5) take out bag filter white floccule is carried out vacuum lyophilization, obtain the Gekko Swinhonis anticancer extract.
Step 2) and centrifugal described in the step 3) be centrifugal 20 minutes of 7000g.
Step 2) saidly is broken for ultrasonic cell disintegration, colloid mill homogenate.
Step 2) behind the said adding dehydrated alcohol, left standstill 3～4 hours in 4 ℃, every at a distance from stirring in 30 minutes once.
What the said adjustment of step 3) pH adopted is hydrochloric acid.
Gekko Swinhonis anticancer extract of the present invention; The method of adopt alcohol extraction, saltouing, dialysing; From Gekko Swinhonis, extract peptide molecule, make extract have very strong antitumous effect, can be used as the active component of the antineoplastic new medicine of efficient, low toxicity with antitumor action.Has good antitumaous effect through evidence Gekko Swinhonis anticancer extract of the present invention; Reached the anticancer level of conventional cancer therapy drug fluorouracil and amycin; Can improve the immunity of body; And contain natural Gekko Swinhonis polypeptide in the Gekko Swinhonis extract, have the advantage that toxicity is low, side effect is little.Method for preparing of the present invention in addition is simple, is easy to apply.
The specific embodiment
Embodiment
1, Gekko Swinhonis dries and pulverizes: after drying the dried no web Gekko Swinhonis 1000g that cleaned, use pulverizer to pulverize and be coarse powder.
2, immersion, fragmentation and homogenate: place 4000mL Tris-HCl buffer (pH=7.4), soaked overnight, ultrasonic cell disintegration, colloid mill homogenate is also collected homogenate.
3, centrifugal and alcohol extraction: 7000g (11000rpm) is centrifugal, and 20min gets supernatant 3800ml; The limit slowly adds dehydrated alcohol (99.7%) 4676ml to make the ethanol final concentration is 55% in supernatant solution; 4 ℃ left standstill 3～4 hours, and every separated 30min stirs once, and 7000g (11000rpm) is centrifugal then; 20min gets supernatant and promptly gets Gekko Swinhonis ethanol extract aqueous solution.
4, rotary evaporation concentrates: 55 ℃, concentrated ethanol extract aqueous solution is to 200ml under the 0.085-0.1MPa.
5, salt dissolves and saltouts: in Gekko Swinhonis ethanol extract aqueous solution, slowly add 66.7ml saturated ammonium sulfate solution; Making the ammonium sulfate saturated concentration is 25%, transfers PH to 7 with HCl solution, and 4 ℃ left standstill 3～4 hours; Every separated 30min stirs once; 7000g (11000rpm) is centrifugal then, and 20min gets supernatant and gets supernatant 260ml; In gained supernatant solution, add 133ml saturated ammonium sulfate solution, making the ammonium sulfate saturated concentration is 50%, and reuse HCl transfers PH to 4.6, and 4 ℃ left standstill 3～4 hours, and every at a distance from the 30min stirring once 7000g (11000rpm) is centrifugal then, and 20min must precipitate 222.6g.
6, dialysis: uses molecular cut off to be the 3.5KDa bag filter, the thing of saltouing in right amount of packing into places 0 ℃ of distilled water, magnetic agitation, and every 30min changes first water, a large amount of white floccules of appearance in Dissolve things inside.
7, lyophilizing: getting that solution is put in the lyophilizing bottle in the bag filter, is 20Pa in vacuum, temperature be under-50 ℃ of conditions on vacuum freeze drier at dry 48H, obtain dry thing 62.1g.
Experimental example
1, the Gekko Swinhonis anticancer extract of variable concentrations is to the inhibited proliferation of human liver cancer cell HepG2
Mtt assay detects the propagation inhibition of Gekko Swinhonis anticancer extract to human liver cancer cell HepG2: with the every hole 1 * 10 of the HCC HepG2 of RPMI1640 culture medium culturing
4/ mL is inoculated in 96 orifice plates, with the Gekko Swinhonis anticancer extract of variable concentrations (0.625,0.75,1.5,3mg/ml), handles 24,48 respectively, 72h.The every hole of 4h adds fresh 5mg/mLMTT liquid 20 μ L before finishing, and continues to cultivate 4h, discards culture medium; Every hole adds dimethyl sulfoxide (DMSO) 150 μ L; After treating to dissolve fully, ELIASA is measured the absorbance A that the 490nm wavelength is measured each hole, and is calculated as follows suppression ratio.Suppression ratio (%)=[1-(A experimental group/A negative control)] * 100%.Experiment repetition 3 times.The result sees table 1
The Gekko Swinhonis anticancer extract of table 1 variable concentrations is to the inhibited proliferation (
n=5) of human liver cancer cell HepG2
Annotate:
*Compare p＜0.05 with matched group;
*Compare p＜0.01 with matched group;
#Compare p＜0.05 with the fluorouracil group
As shown in table 1; Compare with matched group; 0.375,0.75,1,1.5, the thick polypeptide of 3mg/mL Gekko Swinhonis can significantly suppress the growth (p＜0.01 or p＜0.05) of HepG2 cell, its inhibitory action is constantly strengthened with the increase of concentration, time, is doses and time dependence.Be respectively 1,1.5 at 24h dosage, the growth inhibited effect of the thick polypeptide pair cell of 3mg/mL Gekko Swinhonis is superior to positive group (p＜0.05); At 48h dosage is that the growth inhibited effect of the thick polypeptide pair cell of 3mg/mL Gekko Swinhonis is superior to positive group (p＜0.05); 72h dosage is that the growth inhibited effect of the thick polypeptide pair cell of 3mg/mL Gekko Swinhonis does not have significance (p＞0.05) with positive group comparing difference.
2, the Gekko Swinhonis extract is to the tumor-inhibiting action of H22 tumor-bearing mice
Rat liver cancer H
22The foundation of model: with frozen H22 cell recovery, go down to posterity, will be in exponential phase H22 cell with physiological saline solution, the adjustment cell concentration is 1.0 * 10
7/ ml gets 0.2m l and injects the kunming mice abdominal cavity, 6～8 days one-tenth ascites; Aseptic condition removes 1～2ml milk shape, more heavy-gravity ascites with the 5ml syringe pump down; Earlier expect blue dyeing with 0.4%, living cell counting rate＞90%, reuse are sterilized, and to adjust concentration be 1 * 10 to the dilution of 0.9% normal saline
7/ ml, every mice is processed solid type mice with tumor model in right fore axillary fossa subcutaneous vaccination 0.2ml.Be controlled within 2 hours to having transplanted last mice time from extracting ascites.
Divide into groups, medicine preparation and medication: with 50 tumor-bearing mices (male and female half and half, body weight 18～22g) are divided into 5 groups at random: high, normal, basic group of model group, ADM group, Gekko Swinhonis extract, weigh and every mices of label record for every group each 10.Blank control group is irritated stomach normal saline 0.2ml/10g; The ADM group, with 2mg/kg dosage, lumbar injection 0.2ml/10g, two days are once, totally 5 times; The high, normal, basic dose groups of Gekko Swinhonis extract is irritated stomach with 80mg/kg, 40mg/kg, 20mg/kg respectively, every day 1 time, 10d continuously.Each is organized in drug withdrawal in the 11st day and after 24 hours, weighs, and the dislocation of mice cervical vertebra is put to death, and dissects, and peels off the tumor piece, calculates tumour inhibiting rate.Tumor control rate=[1-(the average tumor of the average tumor weight/matched group of experimental group is heavy)] * 100%.
The result sees shown in the table 2:
Table 2 Gekko Swinhonis extract is to the tumor-inhibiting action of H22 tumor-bearing mice
In the experiment in vitro, claim that tumor re-computation tumour inhibiting rate confirms that its tumor-inhibiting action is more close with positive drug.
3, the Gekko Swinhonis extract is to the influence of H22 tumor-bearing mice immunization
Rat liver cancer H
22The foundation of model and grouping, medicine preparation and medication are with embodiment 2.Before putting to death white mice, get blood from eye socket, to carry out cytometry.Get thymus and spleen simultaneously and weigh, to calculate index and spleen index and thymus index.Thymus index=thymic weight (mg)/10g body weight; Index and spleen index=spleen weight (mg)/10g body weight.
The result sees shown in the table 3:
Table 3 Gekko Swinhonis extract is to the influence of H22 tumor-bearing mice immunization
Annotate: with the blank control group contrast,
*P＜0.05; Amycin: positive control.
Under the close prerequisite of tumour inhibiting rate, the thick polypeptide of Gekko Swinhonis can significantly improve H22 tumor-bearing mice immune organ weight, improves its immunity, and body body constitution is increased, and resistance strengthens.
Claims (5)
1. Gekko Swinhonis anticancer extract is characterized in that: it adopts following method to extract and forms:
1) Gekko Swinhonis is cleaned, dries, be broken into powder, placing pH is the buffer solution of Tris-hydrochloric acid of 7.4, soaks 12 hours;
2) it is broken to soak the back, and it is 55% to extract that the centrifuging and taking supernatant adds dehydrated alcohol to ethanol final concentration, and the centrifuging and taking supernatant promptly gets the Gekko Swinhonis alcohol extract then;
3) with Gekko Swinhonis alcohol extract evaporation and concentration, adding ammonium sulfate to its concentration is 25%, adjustment pH to 7, and 4 ℃ left standstill 3～4 hours, and every separated 30min stirs once, and the centrifuging and taking supernatant gets supernatant then; It is 50% that the supernatant that obtains is added ammonium sulfate to its concentration, adjustment PH to 4.6, and 4 ℃ left standstill 3～4 hours, and every separated 30min stirs once, the centrifugal then deposition that obtains;
4) be in the 3.5KDa bag filter with the deposition molecular cut off of packing into, place 0 ℃ of distilled water to dialyse bag filter, white floccule in bag filter, occurs;
5) take out bag filter white floccule is carried out vacuum lyophilization, obtain the Gekko Swinhonis anticancer extract.
2. Gekko Swinhonis anticancer extract according to claim 1 is characterized in that: centrifugally said step 2) and in the step 3) be centrifugal 20 minutes of 7000g.
3. Gekko Swinhonis anticancer extract according to claim 1 is characterized in that: step 2) saidly be broken for ultrasonic cell disintegration, colloid mill homogenate.
4. Gekko Swinhonis anticancer extract according to claim 1 is characterized in that: step 2) behind the said adding dehydrated alcohol, left standstill 3～4 hours in 4 ℃, every at a distance from stirring in 30 minutes once.
5. Gekko Swinhonis anticancer extract according to claim 1 is characterized in that: what the said adjustment of step 3) pH adopted is hydrochloric acid.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112755053A (en) * | 2019-11-05 | 2021-05-07 | 湖南许丰现代农业股份有限公司 | Processing technology of traditional Chinese medicine tianlong |
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| CN1256101C (en) * | 2003-10-21 | 2006-05-17 | 张仕状 | Method for preparing novel anti-cancer medicine |
| CN1977863A (en) * | 2005-12-09 | 2007-06-13 | 中国科学院化学研究所 | Use of gekko powder for preparing medicine for treating colon adeno carcinoma |
| CN101810826A (en) * | 2010-04-13 | 2010-08-25 | 邵梦扬 | Chinese medicament for treating liver cancer |
| CN101875661A (en) * | 2010-05-25 | 2010-11-03 | 张仕状 | Anti-tumor compound extracted from house lizard as well as preparation method and application thereof |
| CN102206647A (en) * | 2011-05-04 | 2011-10-05 | 南通大学 | Method of in-vitro expression of gekko japonicus Hoxc10 (homebox gene c10) protein and preparation of polyclonal antibody |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1256101C (en) * | 2003-10-21 | 2006-05-17 | 张仕状 | Method for preparing novel anti-cancer medicine |
| CN1977863A (en) * | 2005-12-09 | 2007-06-13 | 中国科学院化学研究所 | Use of gekko powder for preparing medicine for treating colon adeno carcinoma |
| CN101810826A (en) * | 2010-04-13 | 2010-08-25 | 邵梦扬 | Chinese medicament for treating liver cancer |
| CN101875661A (en) * | 2010-05-25 | 2010-11-03 | 张仕状 | Anti-tumor compound extracted from house lizard as well as preparation method and application thereof |
| CN102206647A (en) * | 2011-05-04 | 2011-10-05 | 南通大学 | Method of in-vitro expression of gekko japonicus Hoxc10 (homebox gene c10) protein and preparation of polyclonal antibody |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112755053A (en) * | 2019-11-05 | 2021-05-07 | 湖南许丰现代农业股份有限公司 | Processing technology of traditional Chinese medicine tianlong |
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