CN102078347B - Method for preparing effective part in cumin seed - Google Patents
Method for preparing effective part in cumin seed Download PDFInfo
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- CN102078347B CN102078347B CN2010106145557A CN201010614555A CN102078347B CN 102078347 B CN102078347 B CN 102078347B CN 2010106145557 A CN2010106145557 A CN 2010106145557A CN 201010614555 A CN201010614555 A CN 201010614555A CN 102078347 B CN102078347 B CN 102078347B
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Abstract
The invention relates to a method for preparing an effective part in a cumin seed. The method comprises the following step: obtaining the effective part with bioactivity by virtue of a reflux and extract technology and a rapid preparing chromatographic technique, wherein the effective part can effectively inhibit the activity of fatty acid synthase, and a basis is provided for basic research on the bioactivator of Uyghur edible and pharmaceutical plant cumin and development of a corresponding product. The method is simple in preparation process, the activity of the obtained effective part for inhibiting the fatty acid synthase is high, the heat stability is good, the mass production is easy to realize, and the production cost is lowered, thus the method is ideal for preparing a biological active site. The effective part obtained by the method in the invention can be used for preparing foods and drug additives.
Description
Technical field
The present invention relates to the method for preparing of effective site in a kind of Fructus Cumini Cymini seed, the effective site that obtains through this method has the FAS E.C. 2.3.1.85 of inhibition activity.
Background technology
Since 20th century, the obesity illness that become international, and and hyperlipidemia, fatty liver, major diseases such as type ii diabetes are in close relations.People such as Loftus reported food ration and the body weight that significantly reduces obesity mice with fatty acid and enzyme (FAS) inhibitor in 2000, and its mechanism is for the malonyl CoA being the inhibition hypothalamus feed hormone NPY of intermediary expression.It is closely related to propose FAS and animal feed control, is the new potential target spot of treatment of obesity (T.Loftus et al.Science, 2000).After this this enzyme receives people's concern rapidly, and correlational study gets into fast-developing.And research in recent years shows, and FAS E.C. 2.3.1.85 (fatty acid synthase, FAS) high expressed in tumor tissues, and content is lower in normal structure.The product of FAS effect is the matter and energy source of tumor cell proliferation, and prompting FAS can become the target spot of oncotherapy.Therefore the fatty acid synthase inhibitor of from natural product, seeking a kind of high-efficiency low-toxicity becomes the target of vast researcher.
Fructus Cumini Cymini (Cuminum cyminum L.) belongs to Umbelliferae (umbellifereae); The Fructus Cumini Cymini apium; Having long growth historical in Xinjiang, is the Uygur medicine medicinal herbs most in use, it is documented; Fructus Cumini Cymini acrid in the mouth, warm in nature. have dispersing cold for relieving pain, a effect in transferring of regulating the flow of vital energy, cure mainly coldness and pain in the epigastrium, dyspepsia, colic of cold type and weigh down pain, menoxenia.Though the Fructus Cumini Cymini medicinal history is long, its functional factor is still indeterminate.
The present invention provides the method for preparing of effective site in a kind of Fructus Cumini Cymini seed; This method obtains having the effective site that suppresses FAS E.C. 2.3.1.85 through reflux, extract, and quick preparative hplc technology; Be the bioactive substance basic research of Uigurs medicine food dual purpose plant Fructus Cumini Cymini, and develop corresponding product foundation is provided.It is simple that this method prepares process, and it is active high that gained effective site suppresses FAS E.C. 2.3.1.85, and Heat stability is good is easy to enlargement of scale production, reduces production costs, and is one of preparation biological activity position Perfected process.The effective site that obtains through this method is as the purposes of preparation food, drug additive.
Summary of the invention
The object of the invention is; The method for preparing of effective site in a kind of Fructus Cumini Cymini seed is provided; This method obtains the effective site of biologically active through reflux, extract, and quick preparative hplc technology; This effective site can effectively suppress the FAS E.C. 2.3.1.85 activity, is the bioactive substance basic research of Uigurs medicine food dual purpose plant Fructus Cumini Cymini, and develops corresponding product foundation is provided.It is simple that this method prepares process, and it is active high that the gained position suppresses FAS E.C. 2.3.1.85, and Heat stability is good is easy to enlargement of scale production, reduces production costs, and is one of preparation biological activity position Perfected process.The effective site that obtains through this method is as the purposes of preparation food, drug additive.
The method for preparing of effective site in a kind of Fructus Cumini Cymini seed of the present invention follows these steps to carry out:
A, use pulverizer to pulverize the Fructus Cumini Cymini seed after, with soak degreasing under the petroleum ether room temperature 3 times, 6h at every turn;
B, the Fructus Cumini Cymini seed behind the step a soak degreasing is dried, use concentration to carry as 60-90% ethanol water heat, temperature is 65 ℃, extracts 3 times, and each 4h of extraction time collects extracting solution;
C, the extracting solution that step b is obtained merge, and obtain the Fructus Cumini Cymini seed after distilling under reduced pressure removes and desolvates and extract extractum;
D, step c is extracted extractum disperse, respectively extract 3 times with petroleum ether, dichloromethane, ethyl acetate and n-butyl alcohol successively again, can obtain petroleum ether part, chloroform extract, ethyl acetate extract and n-butanol portion with distilled water;
E, with ethyl acetate extract in the steps d; Use dissolve with methanol, behind the silica gel mixed sample, utilize quick preparative hplc technology; Eluant uses chloroform-methanol system gradient elution, again the eluting that obtains is partly suppressed the FAS E.C. 2.3.1.85 activity experiment to confirm that eluting partly suppresses the FAS E.C. 2.3.1.85 activity.
The petroleum ether that uses among step a, d and the e, dichloromethane, ethyl acetate, n-butyl alcohol, chloroform and methanol are analytical pure.
Concentration of alcohol is 70-80% among the step b.
The methanol volume fraction is 0-100% in the step e eluant chloroform-methanol system.
Method of the present invention through selecting for use in the concentration 70% ethanol extraction extractum, is measured the active not good enough of n-butanol portion 109.24ug/ml, and dichloromethane position＞250ug/ml does not have activity; Petroleum ether part＞250ug/ml does not have activity; Ethyl acetate extract 33.51ug/ml activity is best.According to the active testing result of FAS E.C. 2.3.1.85, it is best to select ethyl acetate extract to suppress the FAS E.C. 2.3.1.85 activity.
The specific embodiment
Embodiment 1
After Fructus Cumini Cymini seed 5.3kg pulverized, with petroleum ether soak degreasing 3 times under room temperature, the time was 6h at every turn;
Fructus Cumini Cymini seed 5kg behind the soak degreasing is dried, and using concentration is that 70% ethanol water heat is carried, and temperature is 65 ℃, extracts 3 times, and the time is each 4h, collects extracting solution;
Obtain the Fructus Cumini Cymini seed after merge extractive liquid,, distilling under reduced pressure remove and desolvate and get extractum 852g;
To extract extractum and disperse, respectively extract 3 times with petroleum ether, dichloromethane and ethyl acetate successively again, obtain petroleum ether part 39g, dichloromethane position 20g and ethyl acetate extract 55.6g, the 55g of n-butyl alcohol portion with distilled water;
With ethyl acetate extract; Use dissolve with methanol, behind the silica gel mixed sample, utilize quick preparative hplc technology; Eluant uses chloroform eluting (the methanol volume fraction is 0), again the eluting that obtains is partly suppressed the FAS E.C. 2.3.1.85 activity experiment and measures the activity that eluting partly suppresses FAS E.C. 2.3.1.85.
Embodiment 2
After Fructus Cumini Cymini seed 5.3kg pulverized, with petroleum ether soak degreasing 3 times under room temperature, the time was 6h at every turn;
Fructus Cumini Cymini seed 5kg behind the soak degreasing is dried, and using concentration is that 70% ethanol water heat is carried, and temperature is 65 ℃, extracts 3 times, and the time is each 4h, collects extracting solution;
Obtain the Fructus Cumini Cymini seed after merge extractive liquid,, distilling under reduced pressure remove and desolvate and get extractum 852g;
To extract extractum and disperse, respectively extract 3 times with petroleum ether, dichloromethane and ethyl acetate successively again, obtain petroleum ether part 39g, dichloromethane position 20g and ethyl acetate extract 55.6g, the 55g of n-butyl alcohol portion with distilled water;
With ethyl acetate extract; Use dissolve with methanol; Behind the silica gel mixed sample, utilize quick preparative hplc technology, eluant is used chloroform successively; Chloroform-methanol system (the methanol volume fraction is 4.9%) gradient elution partly suppresses the FAS E.C. 2.3.1.85 activity experiment to measure the activity that eluting partly suppresses FAS E.C. 2.3.1.85 with the eluting that obtains again.
Embodiment 3
After Fructus Cumini Cymini seed 5.3kg pulverized, with petroleum ether soak degreasing 3 times under room temperature, the time was 6h at every turn;
Fructus Cumini Cymini seed 5kg behind the soak degreasing is dried, and using concentration is that 70% ethanol water heat is carried, and temperature is 65 ℃, extracts 3 times, and the time is each 4h, collects extracting solution;
Obtain the Fructus Cumini Cymini seed after merge extractive liquid,, distilling under reduced pressure remove and desolvate and get extractum 852g;
To extract extractum and disperse, respectively extract 3 times with petroleum ether, dichloromethane and ethyl acetate successively again, obtain petroleum ether part 39g, dichloromethane position 20g and ethyl acetate extract 55.6g, the 55g of n-butyl alcohol portion with distilled water;
With ethyl acetate extract; Use dissolve with methanol; Behind the silica gel mixed sample, utilize quick preparative hplc technology, it is 4.9% that eluant uses chloroform, chloroform-methanol system methanol volume fraction; Reuse chloroform-methanol system (the methanol volume fraction is 14.9%) gradient elution partly suppresses the FAS E.C. 2.3.1.85 activity experiment to measure the activity that eluting partly suppresses FAS E.C. 2.3.1.85 with the eluting that obtains again.
Embodiment 4
After Fructus Cumini Cymini seed 5.3kg pulverized, with petroleum ether soak degreasing 3 times under room temperature, the time was 6h at every turn;
Fructus Cumini Cymini seed 5kg after soaking is dried, and using concentration is that 70% ethanol water heat is carried, and temperature is 65 ℃, extracts 3 times, and the time is each 4h, collects extracting solution;
Obtain the Fructus Cumini Cymini seed after merge extractive liquid,, distilling under reduced pressure remove and desolvate and get extractum 852g;
To extract extractum and disperse, respectively extract 3 times with petroleum ether, dichloromethane and ethyl acetate successively again, obtain petroleum ether part 39g, dichloromethane position 20g and ethyl acetate extract 55.6g, the 55g of n-butyl alcohol portion with distilled water;
With ethyl acetate extract; Use dissolve with methanol, behind the silica gel mixed sample, utilize quick preparative hplc technology; Eluant uses chloroform; Chloroform-methanol system methanol volume fraction is 4.9%, 14.9%, and reuse chloroform-methanol system (the methanol volume fraction is 35.1%) gradient elution partly suppresses the FAS E.C. 2.3.1.85 activity experiment to measure the activity that eluting partly suppresses FAS E.C. 2.3.1.85 with the eluting that obtains again.
Embodiment 5
After Fructus Cumini Cymini seed 5.3kg pulverizing, with petroleum ether soak degreasing under room temperature, to soak 3 times, each time is 6h;
Fructus Cumini Cymini seed 5kg after soaking is dried, and using concentration is that 70% ethanol water heat is carried, and temperature is 65 ℃, extracts 3 times, and the time is each 4h, collects extracting solution;
Obtain the Fructus Cumini Cymini seed after merge extractive liquid,, distilling under reduced pressure remove and desolvate and get extractum 852g;
To extract extractum and disperse, respectively extract 3 times with petroleum ether, dichloromethane and ethyl acetate successively again, obtain petroleum ether part 39g, dichloromethane position 20g and ethyl acetate extract 55.6g, the 55g of n-butyl alcohol portion with distilled water;
With ethyl acetate extract; Use dissolve with methanol; Behind the silica gel mixed sample, utilize quick preparative hplc technology, it is 4.9%, 14.9%, 35.1% that eluant uses chloroform, chloroform-methanol system methanol volume fraction successively; Reuse chloroform-methanol system methanol volume fraction is 64.9% eluant gradient elution, and the eluting that will finally obtain again partly suppresses the FAS E.C. 2.3.1.85 activity experiment to measure the activity that eluting partly suppresses FAS E.C. 2.3.1.85.
Embodiment 6
After Fructus Cumini Cymini seed 5.3kg pulverized, with petroleum ether soak degreasing 3 times under room temperature, the time was 6h at every turn;
Fructus Cumini Cymini seed 5kg after soaking is dried, and using concentration is that 70% ethanol water heat is carried, and temperature is 65 ℃, extracts 3 times, and the time is each 4h, collects extracting solution;
Obtain the Fructus Cumini Cymini seed after merge extractive liquid,, distilling under reduced pressure remove and desolvate and get extractum 852g;
To extract extractum and disperse, respectively extract 3 times with petroleum ether, dichloromethane and ethyl acetate successively again, obtain petroleum ether part 39g, dichloromethane position 20g and ethyl acetate extract 55.6g, the 55g of n-butyl alcohol portion with distilled water;
With ethyl acetate extract; Use dissolve with methanol, behind the silica gel mixed sample, utilize quick preparative hplc technology; Eluant uses chloroform successively; Chloroform-methanol system methanol volume fraction is 4.9%, 14.9%, 35.1%, 64.9%, and reuse methanol (the methanol volume fraction is 100%) gradient elution, the eluting that will finally obtain again partly suppress the FAS E.C. 2.3.1.85 activity experiment to measure the fatty acid synthase restraining activity of eluting part.
Embodiment 7
Determination of activity
Activity measurement principle:
Surveying live body system introduces:
Protoenzyme 50 μ l (be stored in-20 ℃, room temperature was thawed more than 1 hour before using) with after surveying the work buffer and diluting (being generally 15 or 20 times) by required multiple, at room temperature stablize more than 1 hour.
1m M PH=7.0K-Pi buffer 40ml
0.1M disodium EDTA 4ml
Distilled water 320ml
The determination of activity system cumulative volume of FAS E.C. 2.3.1.85 is 2ml; With buffer 1.8ml, S-acetyl-coenzyme-A 25 μ l, malonyl coenzyme A 50 μ l; Two nucleoside of nicotinamide adenine phosphoric acid 50 μ l add in the cuvette successively, and METHOD FOR CONTINUOUS DETERMINATION two nucleoside of nicotinamide adenine concentration of phosphoric acid changes under 340nm.
Measure earlier the full response vigor, and as the standard value of enzyme activity, the last amount that adds sample that changes successively; Obtain a series of energy value; Residue vigor after obtaining enzyme according to this and inhibitor combining is drawn inhibition concentration-residue energy value curve, calculates 503nhibiting concentration IC according to this curve
50Value is seen table;
Table:
From table, can find out: chloroform and methanol volume fraction are 4.9% eluting position inhibition FAS E.C. 2.3.1.85 best results.
The active active testing result of inhibition FAS E.C. 2.3.1.85 of 60-90% different concentration ethanol aqueous solution extractum is as shown in the table:
Table:
Table can be found out thus: it is best that 70% concentration ethanol extractum suppresses the active result of FAS E.C. 2.3.1.85.
Claims (3)
1. the method for preparing of effective site in the Fructus Cumini Cymini seed is characterized in that following these steps to carrying out:
A, use pulverizer to pulverize the Fructus Cumini Cymini seed after, with soak degreasing under the petroleum ether room temperature 3 times, 6h at every turn;
B, the Fructus Cumini Cymini seed behind the step a soak degreasing is dried, using concentration is that 70% ethanol water heat is carried, and temperature is 65 ℃, extracts each 4h of extraction time, collection extracting solution 3 times;
C, the extracting solution that step b is obtained merge, and obtain the Fructus Cumini Cymini seed after distilling under reduced pressure removes and desolvates and extract extractum;
D, step c is extracted extractum disperse, respectively extract 3 times with petroleum ether, dichloromethane, ethyl acetate and n-butyl alcohol successively again, can obtain petroleum ether part, chloroform extract, ethyl acetate extract and n-butanol portion with distilled water;
E, with ethyl acetate extract in the steps d; Use dissolve with methanol; Behind the silica gel mixed sample; Utilize quick preparative hplc technology, eluant uses chloroform-methanol system gradient elution, and the eluting that will finally obtain again partly suppresses the FAS E.C. 2.3.1.85 activity experiment to confirm that eluting partly suppresses the FAS E.C. 2.3.1.85 activity.
2. method according to claim 1 is characterized in that the petroleum ether, dichloromethane, ethyl acetate, n-butyl alcohol, chloroform and the methanol that use among step a, d and the e are analytical pure.
3. method according to claim 1 is characterized in that the methanol volume fraction is 0-100% in the step e eluant chloroform-methanol system.
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