CN100556428C - A kind of fatty acid synthase inhibitor and preparation method thereof and application - Google Patents
A kind of fatty acid synthase inhibitor and preparation method thereof and application Download PDFInfo
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- CN100556428C CN100556428C CNB2005100564537A CN200510056453A CN100556428C CN 100556428 C CN100556428 C CN 100556428C CN B2005100564537 A CNB2005100564537 A CN B2005100564537A CN 200510056453 A CN200510056453 A CN 200510056453A CN 100556428 C CN100556428 C CN 100556428C
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Abstract
The invention discloses a kind of fatty acid synthase inhibitor and manufacture method thereof and application, its objective is provides a kind of fatty acid synthase inhibitor and manufacture method and its thereof with the fatty acid synthase being the prevention of relevant disease of target spot and the application in the auxiliary treatment.This fatty acid synthase inhibitor obtains Folium Camelliae sinensis warm macerating extract or catechin compounds through acid treatment.The preparation method of fatty acid synthase inhibitor provided by the present invention is that Folium Camelliae sinensis warm macerating extract or catechin compounds and acid reaction under 25-120 ℃ of condition are obtained fatty acid synthase inhibitor.The present invention in preparation is aspect the prevention and auxiliary treatment of disease of target spot with the fatty acid synthase, particularly has great potential using value in the medicine of controlling body weight, prevention and auxiliary treatment obesity-related disease and prevention and auxiliary for treating cancer and health product.
Description
Technical field
The present invention relates to inhibitor and the manufacture method and the application of enzyme, particularly relate to a kind of fatty acid synthase (former title fatty acid synthetase) inhibitor and manufacture method thereof and its and with the fatty acid synthase be the prevention of relevant disease of target spot and the application in the auxiliary treatment.
Background technology
At present, no matter in developed country or developing country, the sickness rate of obesity is all in rising trend.Obesity is considered to a kind of and is easy to find, obviously, by the Metabolic disorder disease of overdetermined complexity, can influence the physiological process (George L.Wolff.The Journal of Nutrition.127,9:1871-1873 (1997)) of whole machine body normal function.The fat probability (Hubert, H.B.Annu.Rev.Public.Health.7:493-502 (1986)) that can increase trouble type ii diabetes and cardio-cerebrovascular diseases.The consequence that spreads the aspects of being brought such as health, economy and social mentality of this pertinacious disease is serious.
2000, people such as Loftus go up report fatty acid synthase (former title fatty acid synthetase at " Science ", Fattyacid synthase, EC.2.3.1.85, be abbreviated as FAS) inhibitor be intermediary with the malonyl coenzyme A, suppress the expression of the feed hormone nerve signal peptide Y (NPY) in the hypothalamus, thereby but appetite-suppressing, the food ration and the body weight of obesity mice significantly reduced (Loftus T.M.et al.Science, 288:2379-2381 (2000)).This article proposes FAS may have substantial connection with the control of animal feed, is the potential target spot of treatment of obesity.The activity that suppresses FAS can either block living fat path, and the body that reduces fat is interior synthetic, can cause the rising of malonyl coenzyme A concentration again, thereby reach the purpose that reduces appetite, has double effects.The suitable fatty acids synthase inhibitor can be regulated the metabolism and the storage of fat, thereby can reduce the intake that appetite reduces food again, and its effect and do not rely on the special genes defective.
In recent years, scientist finds the excessive existence of fatty acid synthase in multiple cancerous cell, and in most of normal cells lower (Alo P.L., Visca P., Marci A., et al.Cancer, 77:474 (1996) of the content of this enzyme; Pizer E., Lax S., Kuhajda F., et al.Cancer, 83:528 (1998) etc.); Experiment in vitro shows that fatty acid synthase inhibitor can stop growth (Pizer E.S., Wood F.D., HeineH.S., et al.Cancer Res, the 56:1189 (1996) of multiple cancerous cell; Furuya Y., Akimoto S., YasadaK., et al.Anticancer Res, 17:4589 (1997) etc.), mouse model with this enzyme inhibitor processing inoculated tumour can make tumor growth be obstructed, and reduces mortality of mice (Pizer E.S., Wood F.D., Heine H.S., et al.Cancer Res, 56:1189 (1996)).2000, U.S. scientist F.Kuhajda has delivered summary at the achievement in research in this field, and proposition inhibition fatty acid synthase might become new chemotherapy approach (Kuhajda F.P. in the oncotherapy, Nutrition, 16:202-208 (2000)), make fatty acid synthase inhibitor that application prospect widely arranged.
The current research that medical journals " The New England Journal of Medicine " (New England Journal of Medicine) is reported, 900,000 U.S. healthy adult men and women are carried out the follow up survey in 16 years, show that cancer mortality and obese degree (body-mass index) have closely related relation, also high (the Call e E.E. of the cancer mortality of obese people, Rodriguez C., Kimberly W-T., et al.The New England Journal of Medicine, 348 (17): 1625-1638 (2003)).The research has disclosed cancer with science data for the first time and obesity is dependency relation.
So far, the fatty acid synthase inhibitor of having reported is less, has only synthetic C75 (Kuhajda F.P., Pizer E.S., Li J.N., et al.Proc Natl Acad Sci USA, 97,3450-3454 (2000)), natural product cerulenin (cerulenin) (Vance D., the Goldberg I. that early finds, Mitsuhashi O.et al.Biochem Biophys Res Commun.48,649-656 (1972)) and the ester catechin EGCG in the green tea (Wang X., Tian W.X..Biochem Biophys Res Commun, 288 (5): 1200-1206 (2001)) and ECG (Wang X., Song K.S., Guo Q.X., et al.Biochem Pharmacol, 66:2039-2047 (2003)).The common drawback that these inhibitor had is to suppress active not high enough, and using value is limited to, and the inhibition activity that therefore improves fatty acid synthase inhibitor has important application value.
Tea is the popular drink of extensively being drunk in the world today, also is to be subjected to a lot of sanitarians to recommend one of health beverage of drinking.Drink tea in the existing history more than 2,000 years of China, develop into daily drinking beverage by a kind of medical herbs to Han dynasty tea.Green tea has the health care of fat-reducing, controlling body weight and prophylaxis of cancer.Studies show that the extract of green tea and black tea has stronger inhibitory action to fatty acid synthase, its inhibit feature even catechin EGCG and the ECG more contained than green tea are also strong; On the other hand, tea extract is to loss of weight and but food function and its inhibition ability positive correlation (Zhang Rui, Xiao Wenping, Tian Weixi to fatty acid synthase of model mice.Yunnan University's journal (natural science edition), 26 (6A): 42-47 (2004)).The present inventor has applied for relevant patent " the new purposes of Folium Camelliae sinensis warm macerating extract " February 23 calendar year 2001, and the patent No. is: 01104308.3, and this patent is illustrated, and Folium Camelliae sinensis warm macerating extract has the active effect of the fatty acid synthase of inhibition.
Summary of the invention
The purpose of this invention is to provide a kind of active higher fatty acid synthase inhibitor that suppresses.
Fatty acid synthase inhibitor provided by the present invention obtains Folium Camelliae sinensis warm macerating extract or catechin compounds through acid treatment.
Described catechin compounds is ester catechin and several theine of monomer.
In actual applications, the acid that is used for handling Folium Camelliae sinensis warm macerating extract or catechin compounds is preferably strong acid, strong acid or organic acid.Wherein, strong acid can be preferably sulphuric acid or hydrochloric acid for nontoxic non-oxidizable strong acid; Middle strong acid can be the nontoxic non-oxidizable middle strong acid of 1-2 for the PKa value, is preferably phosphoric acid; Organic acid can be the nontoxic non-oxidizable organic acid of 1-5 for the PKa value, is preferably oxalic acid, malic acid, citric acid, tartaric acid or acetic acid.
In order to make the better effects if of processing, acid-treated temperature is generally 25-120 ℃.
The described processing time can be by the power and the temperature decision of used acid, acidity is strong more, the high more required processing time of temperature is short more, when using strong acid, during as sulfuric acid treatment, after at room temperature (25 ℃) handled the long period (19 hours), products therefrom still had certain raising (for 2.6 times of original activity) to the inhibition activity of fatty acid synthase.
Second purpose of the present invention provides a kind of preparation method of above-mentioned fatty acid synthase inhibitor.
The preparation method of fatty acid synthase inhibitor provided by the present invention is that Folium Camelliae sinensis warm macerating extract or catechin compounds and acid reaction under 25-120 ℃ of condition are obtained fatty acid synthase inhibitor.
The described acid that is used for Folium Camelliae sinensis warm macerating extract or catechin compounds processing is strong acid, strong acid or organic acid, and described strong acid can be preferably hydrochloric acid for sulphuric acid, hydrochloric acid or other nontoxic non-oxidizable strong acid, and concentration is 0.2-3.0mol/L; Middle strong acid can be the nontoxic non-oxidizable middle strong acid of 1-2 for the PKa value, is preferably phosphoric acid; Organic acid can be the nontoxic non-oxidizable organic acid of 1-5 for the PKa value, is preferably oxalic acid, malic acid, citric acid, tartaric acid or acetic acid, and concentration is 0.25-2.0mol/L, is preferably 1mol/L.Temperature high processing rate more is fast more, and effect is obvious more, and temperature is low more, and then required time is long more, and described heating-up temperature is 25-120 ℃, is preferably 70 ℃-100 ℃, is preferably 30-170 minute heat time heating time.
Described Folium Camelliae sinensis warm macerating extract obtains the Folium Camelliae sinensis warm macerating under 20 ℃ of-40 ℃ of conditions with organic solvent; Described organic solvent is the ethanol water of 10%-95%, is preferably the ethanol water of 50%-70%; The described warm macerating time is 2-5 hour, is preferably 3 hours.
Described catechin compounds comprises ester catechin and catechin, needs before mixing with acid pure or blended catechin compounds are dissolved in pure water.
For ease of packing and carrying, reaction can be carried out dried to the fatty acid synthase inhibitor that obtains through said method after finishing.
The composition catechin compounds (comprising ester catechin and catechin) that the present invention has significantly improved Folium Camelliae sinensis extract and Folium Camelliae sinensis suppresses the ability of fatty acid synthase, thereby provides a class to have the active fatty acid synthase inhibitor of higher inhibition.The present invention in preparation is aspect the prevention and auxiliary treatment of disease of target spot with the fatty acid synthase, particularly is significant in the medicine of aspects such as preparation controlling body weight, control disease relevant with obesity and anti-curing cancers.Simultaneously, fatty acid synthase inhibitor of the present invention also has bigger using value in the production of health product and cosmetics.
The specific embodiment
Among the following embodiment, used fatty acid synthase is with conventional method (W.X.Tian et al., 1985.J.Biol.Chem., 260 (20): 11375-11387; Tian Weixi etc., body lipid level of 1996. different growing stages laying hens and the active relation of liver fatty acid synthetase. journal of biological chemistry, 12 (2): 234-236) make a fresh start to separate in the freshly-slaughtered poultry liver and purify, and detect through polyacrylamide gel electrophoresis and to be single band.
Active S-acetyl-coenzyme-A, malonyl coenzyme A, the NADPH of adopting of fatty acid synthase is standard measuring method for activity mensuration (W.X.Tian et al., 1985.J.Biol.Chem., 260 (20): 11375-11387 of substrate; Tian Weixi etc., body lipid level of 1996. different growing stages laying hens and the active relation of liver fatty acid synthetase. journal of biological chemistry, 12 (2): 234-236).
Embodiment 1, green tea warm macerating extract use 1M sulphuric acid at 100 ℃ of following Processing of Preparation fatty acid synthase inhibitors
The green tea of pulverizing of learning from else's experience joins in the ethanol of 10 times of weight 60%, and warm macerating is 2 hours under 30 ℃, stirring condition, and the centrifuging and taking supernatant obtains green tea warm macerating extract.In green tea warm macerating extract, add isopyknic 2M sulphuric acid, mixing back sulphuric acid final concentration is 1M, handle at 100 ℃ of following constant temperature, get at regular intervals in 50% ethanol that 50 μ l are put into 950 μ l and (add up to diluted 40 times of green tea warm macerating extract), get 3 μ l and add in the fatty acid synthase activity determining system, measure the active A i of this enzyme.Contrast is the green tea warm macerating extract through diluting 40 times, gets 3 μ l and adds in the fatty acid synthase activity determining system, measures the active A O of this enzyme.With the activity of blank determination as 1.0, A
i/ AO is the relative surplus activity of enzyme under the treatment fluid effect, and the low more treatment fluid inhibitory action that shows of this activity is strong more.The inhibition activity of measurement result display process liquid (the relative surplus activity with repressed enzyme is represented) increased and strengthens along with the processing time, but the variation of inhibition degree is not obvious when 24min arrives 27min, illustrates that its activity has reached the highest substantially.To the treatment fluid behind the processing 24min, add the inhibition degree of quantitative determination with different disposal liquid to fatty acid synthase, the curve that can be inhibited, and thus in the curve residual activity 50% place (just suppressing degree 50% place) of enzyme can obtain the 503nhibiting concentration (IC of treatment fluid
50) be 1.8 μ g tea dry weight/ml, and the 503nhibiting concentration of green tea warm macerating extract is 37 μ g tea dry weight/ml after measured before handling, thereby handle back IC
50Value has reduced about 20 times, has promptly suppressed increased activity about 20 times.
Embodiment 2, green tea warm macerating extract use 1M sulphuric acid at 70 ℃ of following Processing of Preparation fatty acid synthase inhibitors
Obtain green tea warm macerating extract with the method identical with embodiment 1, in green tea warm macerating extract, add isopyknic 2M sulphuric acid, handle at 70 ℃ of following constant temperature, get at regular intervals in 50% ethanol that 50 μ l are put into 450 μ l and (add up to diluted 20 times of green tea warm macerating extract), get 5 μ l and add in the fatty acid synthase activity determining system, measure the active A i of this enzyme.Contrast is the green tea warm macerating extract of 20 times of dilutions, gets 5 μ l and adds in the fatty acid synthase activity determining system, measures the active A O of this enzyme.As 1.0, Ai/AO is the relative surplus activity of enzyme under the treatment fluid effect with the activity of blank determination.Measurement result has shown that the inhibition activity of treatment fluid increased and strengthens along with the processing time, and the inhibition degree reaches the highest when 170min.To handling the treatment fluid behind the 170min, add the inhibition degree of quantitative determination with different disposal liquid to fatty acid synthase, the curve that can be inhibited, and can obtain the IC of this treatment fluid thus in the curve
50Be 1.3 μ g tea dry weight/ml, thereby handle back IC
50Value has reduced about 28 times, has promptly suppressed increased activity about 28 times.This result shows that 70 ℃ of following sulfuric acid treatment are still very effective, but required time is handled long down than 100 ℃.
Embodiment 3, green tea warm macerating extract use 1M sulphuric acid at 40 ℃ of following Processing of Preparation fatty acid synthase inhibitors
Obtain green tea warm macerating extract with the method identical with embodiment 1, in green tea warm macerating extract, add isopyknic 2M sulphuric acid, handle at 40 ℃ of following constant temperature, get at regular intervals in 50% ethanol that 50 μ l are put into 450 μ l and (add up to diluted 20 times of green tea warm macerating extract), get 10 μ l and add in the fatty acid synthase activity determining system, measure the active A i of this enzyme.Contrast is the green tea warm macerating extract of 20 times of dilutions, gets 10 μ l and adds in the fatty acid synthase activity determining system, measures the active A O of this enzyme.As 1.0, Ai/AO is the relative surplus activity of enzyme under the treatment fluid effect with the activity of blank determination.Measurement result has shown that the inhibition activity of treatment fluid increased and strengthens along with the processing time, but enhanced speed is obviously slow, still has very big distance from maximum inhibition degree during to 390 minutes.Measure the 503nhibiting concentration that obtains this moment is 13.6 μ g tea dry weight/ml, suppresses activity and only brings up to 2.7 times.Therefore 40 ℃ of following sulfuric acid treatment are still effective, handle down but the active speed that improves of inhibition has been significantly less than 70 ℃.
Embodiment 4, green tea warm macerating extract 1M sulphuric acid Processing of Preparation fatty acid synthase inhibitor under room temperature (25 ℃)
Obtain green tea warm macerating extract with the method identical with embodiment 1, in green tea warm macerating extract, add isopyknic 2M sulphuric acid, handle at 25 ℃ of following constant temperature, get at regular intervals in 50% ethanol that 50 μ l are put into 450 μ l and (add up to diluted 20 times of green tea warm macerating extract), get 5 μ l and add in the fatty acid synthase activity determining system, measure the active A i of this enzyme.Contrast is the green tea warm macerating extract of 20 times of dilutions, gets 5 μ l and adds in the fatty acid synthase activity determining system, measures the active A O of this enzyme.As 1.0, Ai/AO is the relative surplus activity of enzyme under the treatment fluid effect with the activity of blank determination.Measurement result has shown that the inhibition activity of treatment fluid increased and strengthens along with the processing time, but the speed that increases is very slow.Handle the IC of 19 hours treatment fluids
50Value is 14.3 μ g tea dry weight/ml, with handle before compare and be strengthened to 2.5 times, sulfuric acid treatment improves still effectively under the visible room temperature to the activity of green tea warm macerating extract, but speed is very slow.
Embodiment 5, green tea warm macerating extract use 1M hydrochloric acid at 100 ℃ of following Processing of Preparation fatty acid synthase inhibitors
Obtain green tea warm macerating extract with the method identical with embodiment 1, in green tea warm macerating extract, add isopyknic 2M hydrochloric acid, handle at 100 ℃ of following constant temperature, get at regular intervals in 50% ethanol that 50 μ g are put into 950 μ l and (add up to diluted 40 times of green tea warm macerating extract), get 5 μ l and add in the fatty acid synthase activity determining system, measure the active A i of this enzyme.Contrast is the green tea warm macerating extract of 40 times of dilutions, gets 5 μ g and adds in the fatty acid synthase activity determining system, measures the active A O of this enzyme.As 1.0, Ai/AO is the relative surplus activity of enzyme under the treatment fluid effect with the activity of blank determination.Measurement result has shown that the inhibition activity of treatment fluid increased and strengthens fast along with the processing time, and the inhibition degree reaches very high when 35min.To handling the treatment fluid behind the 35min, add the inhibition degree of quantitative determination with different disposal liquid to fatty acid synthase, the curve that can be inhibited, and can obtain the IC of this treatment fluid thus in the curve
50Be 1.7 μ g tea dry weight/ml, thereby handle back IC
50Value has reduced about 21 times, has promptly suppressed increased activity about 21 times.This result shows that the acid treatment of 100 ℃ of following 1M salt is similar with the result of 1M sulfuric acid treatment.
Embodiment 6, green tea warm macerating extract use 1M acetic acid (pKa=4.76) at 100 ℃ of following Processing of Preparation fatty acid synthase inhibitors
Obtain green tea warm macerating extract with the method identical with embodiment 1, in green tea warm macerating extract, add isopyknic 2M acetic acid, handle at 100 ℃ of following constant temperature, get at regular intervals in 50% ethanol that 50 μ l are put into 950 μ l and (add up to diluted 40 times of green tea warm macerating extract), get 10 μ l and add in the fatty acid synthase activity determining system, measure the active A i of this enzyme.Contrast is the green tea warm macerating extract of 40 times of dilutions, gets 10 μ l and adds in the fatty acid synthase activity determining system, measures the active A O of this enzyme.As 1.0, Ai/AO is the relative surplus activity of enzyme under the treatment fluid effect with the activity of blank determination.Measurement result has shown that the inhibition activity of treatment fluid is along with the processing time increases inhibition and slowly increase.To handling the treatment fluid behind the 200min, add the inhibition degree of quantitative determination with different disposal liquid to fatty acid synthase, the curve that can be inhibited, and can obtain the IC of this treatment fluid thus in the curve
50Be 14.2 μ g tea dry weight/ml, thereby handle back IC
50Value has reduced about 2.6 times, has promptly suppressed increased activity about 2.6 times.This result shows 100 ℃ of following 1M acetic acid treatment, and to suppress the ability of fatty acid synthase still effective to improving green tea green tea warm macerating extract, and to suppress active ability obviously weak much but the such weak acid of acetic acid improves than hydrochloric acid, the such strong acid of sulphuric acid.
Embodiment 7, green tea warm macerating extract use 1M phosphoric acid at 100 ℃ of following Processing of Preparation fatty acid synthase inhibitors
Obtain green tea warm macerating extract with the method identical with embodiment 1, in green tea warm macerating extract, add isopyknic 2M phosphoric acid, at 100 ℃ of constant temperature, 20,40,60 and get during 100min in 50% ethanol that 50 μ l are put into 450 μ l (adding up to diluted 20 times of green tea warm macerating extract), get 10 μ l and add in the fatty acid synthase activity determining system, measure the active A i of this enzyme.Contrast is the green tea warm macerating extract of 20 times of dilutions, gets 10 μ l and adds in the fatty acid synthase activity determining system, measures the active A O of this enzyme.As 1.0, Ai/AO is the relative surplus activity of enzyme under the treatment fluid effect with the activity of blank determination.Measurement result shows that treatment fluid increases along with processing time increase inhibition is active and the result of same temperature sulphuric acid and acetic acid treatment compares, and the effect the when effect of processing 20 and 40min and sulfuric acid treatment 4min and 8min is similar, but effect is much higher than acetic acid treatment.Effect reaches maximum, the IC that the treatment fluid of this moment is measured after handling 100min
50Be 2.4 μ g tea dry weight/ml, thereby handle back IC
50Value has reduced about 15 times, has promptly suppressed increased activity about 15 times.This result shows that 100 ℃ of following 1M phosphoric acid handle that to suppress the ability of fatty acid synthase significantly effective to improving green tea green tea warm macerating extract, than acetic acid improve fast, than hydrochloric acid, sulphuric acid improve slow.
Embodiment 8, green tea warm macerating extract use 1M malic acid (pKa=3.40) at 100 ℃ of following Processing of Preparation fatty acid synthase inhibitors
Obtain green tea warm macerating extract with the method identical with embodiment 1, in green tea warm macerating extract, add isopyknic 2M malic acid, handle at 100 ℃ of following constant temperature, 20,40,60 and get during 100min in 50% ethanol that 50 μ l are put into 450 μ l (adding up to diluted 20 times of green tea warm macerating extract), get 10 μ l and add in the fatty acid synthase activity determining system, measure the active A i of this enzyme.Contrast is the green tea warm macerating extract of 20 times of dilutions, gets 10 μ l and adds in the fatty acid synthase activity determining system, measures the active A O of this enzyme.As 1.0, Ai/AO is the relative surplus activity of enzyme under the treatment fluid effect with the activity of blank determination.Measurement result shows that treatment fluid increases along with the processing time increase suppresses active, and effect is similar with the phosphoric acid processing when handling 20min, and effect no longer increases but after this suppress activity also a little more than phosphoric acid during 40min, and final effect is handled not as phosphoric acid.IC to the treatment fluid mensuration behind the processing 40min
50Be 5 μ g tea dry weight/ml, thereby handle back IC
50Value has reduced by 7.4 times, has promptly suppressed increased activity 7.4 times.This result shows that 100 ℃ of following 1M malic acids handle that to suppress the ability of fatty acid synthase still effective to improving green tea green tea warm macerating extract, obviously than acetic acid improve fast, more approaching than the effect slow and phosphoric acid that hydrochloric acid, sulphuric acid improve.
Embodiment 9, green tea warm macerating extract use 1M citric acid (pKa=3.13) at 100 ℃ of following Processing of Preparation fatty acid synthase inhibitors
Obtain green tea warm macerating extract with the method identical with embodiment 1, in green tea warm macerating extract, add isopyknic 2M citric acid, handle at 100 ℃ of following constant temperature, 20,40,60 and get during 100min in 50% ethanol that 50 μ l are put into 450 μ l (adding up to diluted 20 times of green tea warm macerating extract), get 10 μ l and add in the fatty acid synthase activity determining system, measure the active A i of this enzyme.Contrast is the green tea warm macerating extract of 20 times of dilutions, gets 10 μ l and adds in the fatty acid synthase activity determining system, measures the active A O of this enzyme.As 1.0, Ai/AO is the relative surplus activity of enzyme under the treatment fluid effect with the activity of blank determination.Measurement result shows that treatment fluid increase to suppress active and increases along with the processing time, but the active speed that improves is lower slightly when handling than malic acid and phosphoric acid, and than active improve fast of acetic acid treatment.Activity reaches maximum, the IC that the treatment fluid of this moment is measured after handling 60min
50Be 8.5 μ g tea dry weight/ml, thereby handle back IC
50Value has reduced about 4.3 times, has promptly suppressed increased activity about 4.3 times.This result shows 100 ℃ of following 1M citric acid treatment, and to suppress the ability of fatty acid synthase still effective to improving green tea warm macerating extract, than acetic acid improve fast, than hydrochloric acid, sulphuric acid and phosphoric acid improve slow.
Embodiment 10, green tea warm macerating extract use the 0.5M citric acid at 100 ℃ of following Processing of Preparation fatty acid synthase inhibitors
Obtain green tea warm macerating extract with the method identical with embodiment 1, in green tea warm macerating extract, add isopyknic 1M citric acid, handle at 100 ℃ of following constant temperature, 20,40,100,150 and get during 210min in 50% ethanol that 50 μ l are put into 950 μ l (adding up to diluted 40 times of green tea warm macerating extract), get 10 μ l and add in the fatty acid synthase activity determining system, measure the active A i of this enzyme.Contrast is the green tea warm macerating extract of 40 times of dilutions, gets 10 μ l and adds in the fatty acid synthase activity determining system, measures the active A O of this enzyme.As 1.0, Ai/AO is the relative surplus activity of enzyme under the treatment fluid effect with the activity of blank determination.Measurement result shows that inhibition is active to be increased treatment fluid along with the processing time increases, and is close when active speed that improves and 1M citric acid treatment.The treatment fluid IC that measures when handling 210min
50Be 6.25 μ g tea dry weight/ml, thereby handle back IC
50Value has reduced about 5.9 times, has promptly suppressed increased activity about 5.9 times.This result shows that down to suppress the ability of fatty acid synthase still effective to improving green tea warm macerating extract with the 0.5M citric acid treatment at 100 ℃, and the whole activity than with the 1M citric acid treatment time is also slightly high.
Embodiment 11, green tea warm macerating extract use 0.25M oxalic acid (pKa=1.27) at 100 ℃ of following Processing of Preparation fatty acid synthase inhibitors
Obtain green tea warm macerating extract with the method identical with embodiment 1, in green tea warm macerating extract, add isopyknic 0.5M oxalic acid, handle at 100 ℃ of following constant temperature, 20,40,80 and get during 120min in 50% ethanol that 50 μ l are put into 950 μ l (adding up to diluted 40 times of green tea warm macerating extract), get 10 μ l and add in the fatty acid synthase activity determining system, measure the active A i of this enzyme.Contrast is the green tea warm macerating extract of 40 times of dilutions, gets 10 μ l and adds in the fatty acid synthase activity determining system, measures the active A O of this enzyme.As 1.0, Ai/AO is the relative surplus activity of enzyme under the treatment fluid effect with the activity of blank determination.Measurement result shows that treatment fluid increase to suppress active and increases along with the processing time, and the active speed that improves is slightly high during than malic acid and citric acid treatment.The IC that treatment fluid behind the processing 120min is measured
50Be 5.4 μ g tea dry weight/ml, thereby handle back IC
50Value has reduced about 6.8 times, has promptly suppressed increased activity about 6.8 times.This result shows that 100 ℃ of following 0.25M oxalic acid treatment are still effective to the ability that improves green tea green tea warm macerating extract inhibition fatty acid synthase, handle improve many than malic acid and citric acid, than lacking that hydrochloric acid, sulphuric acid and phosphoric acid improve.
Embodiment 12, green tea warm macerating extract use 0.2M hydrochloric acid at 100 ℃ of following Processing of Preparation fatty acid synthase inhibitors
Obtain green tea warm macerating extract with the method identical with embodiment 1, in green tea warm macerating extract, add isopyknic 0.4M hydrochloric acid, handle at 100 ℃ of following constant temperature, 20,40,60 and get during 90min in 50% ethanol that 50 μ l are put into 950 μ l (adding up to diluted 40 times of green tea warm macerating extract), get 10 μ l and add in the fatty acid synthase activity determining system, measure the active A i of this enzyme.Contrast is the green tea warm macerating extract of 40 times of dilutions, gets 10 μ l and adds in the fatty acid synthase activity determining system, measures the active A O of this enzyme.As 1.0, Ai/AO is the relative surplus activity of enzyme under the treatment fluid effect with the activity of blank determination.Measurement result shows that treatment fluid increase to suppress active and increases along with the processing time, and the active speed that improves is slow during than the acid treatment of 1M salt.The IC that treatment fluid behind the processing 90min is measured
50Be 6.13 μ g tea dry weight/ml, thereby handle back IC
50Value has reduced about 6 times, has promptly suppressed increased activity about 6 times.This result shows the acid treatment of 100 ℃ of following 0.2M salt, and to suppress the ability of fatty acid synthase still effective to improving green tea green tea warm macerating extract, but improve during than the acid treatment of 1M salt slow.
Embodiment 13, green tea warm macerating extract use 3M hydrochloric acid at 70 ℃ of following Processing of Preparation fatty acid synthase inhibitors
Obtain green tea warm macerating extract with the method identical with embodiment 1, in green tea warm macerating extract, add isopyknic 6M hydrochloric acid, handle at 100 ℃ of following constant temperature, 3,10, get during 20min in 50% ethanol that 50 μ l are put into 950 μ l and (add up to diluted 40 times of green tea warm macerating extract), get 10 μ l and add in the fatty acid synthase activity determining system, measure the active A i of this enzyme.Contrast is the green tea warm macerating extract of 40 times of dilutions, gets 10 μ l and adds in the fatty acid synthase activity determining system, measures the active A O of this enzyme.As 1.0, Ai/AO is the relative surplus activity of enzyme under the treatment fluid effect with the activity of blank determination.Measurement result shows treatment fluid along with the processing time increase suppresses active increase, height during the speed 1M salt acid treatment of active raising.The IC that treatment fluid behind the processing 3min is measured
50Be 5.44 μ g tea dry weight/ml, thereby handle back IC
50Value has reduced about 6.8 times, has promptly suppressed increased activity about 6.8 times.The IC that treatment fluid behind the processing 20min is measured
50Be 0.99 μ g tea dry weight/ml, thereby handle back IC
50Value has reduced by 37 times, has promptly suppressed increased activity 37 times.This result shows the acid treatment of 100 ℃ of following 3M salt, and to suppress the ability of fatty acid synthase very effective to improving green tea green tea warm macerating extract, than the acid treatment of 1M salt improve fast.
Embodiment 14, green tea warm macerating extract use 1M hydrochloric acid at 120 ℃ of following Processing of Preparation fatty acid synthase inhibitors
Obtain green tea warm macerating extract with the method identical, in green tea warm macerating extract, add isopyknic 2M hydrochloric acid, handle 30min, the IC that treatment fluid is measured at 120 ℃ of following constant temperature with embodiment 1
50Be 0.97 μ g tea dry weight/ml, thereby handle back IC
50Value has reduced about 38.1 times, has promptly suppressed increased activity about 38.1 times.This result shows that the acid treatment of 120 ℃ of following 1M salt is very effective to the ability that improves green tea green tea warm macerating extract inhibition fatty acid synthase, suppresses the faster, higher of active raising when handling down than 100 ℃ of 1M hydrochloric acid.
Embodiment 15, green tea warm macerating extract use 0.5M tartaric acid at 100 ℃ of following Processing of Preparation fatty acid synthase inhibitors
Obtain green tea warm macerating extract with the method identical with embodiment 1, in green tea warm macerating extract, add isopyknic 1M tartaric acid, handle at 100 ℃ of following constant temperature, 20,40,80 and get during 150min in 50% ethanol that 50 μ l are put into 950 μ l (adding up to diluted 40 times of green tea warm macerating extract), get 10 μ g and add in the fatty acid synthase activity determining system, measure the active A i of this enzyme.Contrast is the green tea warm macerating extract of 40 times of dilutions, gets 10 μ g and adds in the fatty acid synthase activity determining system, measures the active A O of this enzyme.As 1.0, Ai/AO is the relative surplus activity of enzyme under the treatment fluid effect with the activity of blank determination.Measurement result shows that inhibition is active to be increased treatment fluid along with the processing time increases, and is similar when the active speed that improves and 0.5M and 1M citric acid treatment.The IC that treatment fluid behind the processing 150min is measured
50Be 7.27 μ g tea dry weight/ml, thereby handle back IC
50Value has reduced about 5 times, has promptly suppressed increased activity about 5 times.This result shows that 100 ℃ of following 0.5M tartaric acid processing are still effective to the ability that improves green tea green tea warm macerating extract inhibition fatty acid synthase, and the slow effect with citric acid treatment that improves during than the acid treatment of 1M salt is close.
Embodiment 16, green tea warm macerating extract use 0.25M tartaric acid at 100 ℃ of following Processing of Preparation fatty acid synthase inhibitors
Obtain green tea warm macerating extract with the method identical with embodiment 1, in green tea warm macerating extract, add isopyknic 0.5M tartaric acid, handle at 100 ℃ of following constant temperature, 20,40,80 and get during 150min in 50% ethanol that 50 μ l are put into 950 μ l (adding up to diluted 40 times of green tea warm macerating extract), get 10 μ l and add in the fatty acid synthase activity determining system, measure the active A i of this enzyme.Contrast is the green tea warm macerating extract of 40 times of dilutions, gets 10 μ l and adds in the fatty acid synthase activity determining system, measures the active A O of this enzyme.As 1.0, Ai/AO is the relative surplus activity of enzyme under the treatment fluid effect with the activity of blank determination.Measurement result shows that inhibition is active to be increased treatment fluid along with the processing time increases, and is slow when the active speed that improves is handled than 0.5M tartaric acid.The IC that treatment fluid behind the processing 1500min is measured
50Be 9.8 μ g tea dry weight/ml, thereby handle back IC
50Value has reduced about 3.7 times, has promptly suppressed increased activity about 3.7 times.This result shows that 100 ℃ of following 0.5M tartaric acid handle that to suppress the ability of fatty acid synthase still effective to improving green tea green tea warm macerating extract, but improve when handling than 1M tartaric acid slowly.
(epigallocatechin gallate EGCG) uses 1M sulphuric acid at 60 ℃ of following Processing of Preparation fatty acid synthase inhibitors for embodiment 17, ester catechin
In the EGCG of 6.8mg/ml aqueous solution, add isopyknic 2M sulphuric acid, 60 ℃ of constant temperature 18 hours, get in 50% ethanol that 50 μ l are put into 450 μ l (being equivalent to diluted 20 times), get 10 μ l and add in the fatty acid synthase activity determining system, measure the active A i of this enzyme.Contrast is the EGCG aqueous solution of 20 times of dilutions, gets 10 μ l and adds in the fatty acid synthase activity determining system, measures the active A O of this enzyme.As 1.0, Ai/AO is the relative surplus activity of enzyme under the treatment fluid effect with the activity of blank determination.Handle the IC that the treatment fluid after 18 hours is measured
50Being 0.48 μ g/ml, is 24 μ g/ml before the processing.Thereby IC after handling
50Value has reduced by 50 times, has promptly suppressed increased activity 50 times.This result shows that 60 ℃ of following 1M sulfuric acid treatment can increase substantially the ability that EGCG suppresses fatty acid synthase.
Embodiment 18, catechin D-(+) catechin use 1M sulphuric acid at 100 ℃ of following Processing of Preparation fatty acid synthase inhibitors
In the D-of 10mg/ml (+) catechin aqueous solution, add isopyknic 2M sulphuric acid, handle 30min, get in 50% ethanol that 50 μ l are put into 450 μ l (adding up to diluted 20 times) at 100 ℃ of following constant temperature, get 10 μ l and add in the fatty acid synthase activity determining system, measure the active A i of this enzyme.Contrast is D-(+) the catechin aqueous solution of 20 times of dilutions, gets 10 μ l and adds in the fatty acid synthase activity determining system, measures the active A O of this enzyme.As 1.0, Ai/AO is the relative surplus activity of enzyme under the treatment fluid effect with the activity of blank determination.The IC that treatment fluid behind the processing 30min is measured
50Being 10 μ g/ml, is 1600 μ g/ml before the processing.Thereby IC after handling
50Value has reduced about 160 times, has promptly suppressed increased activity about 160 times.This result shows that 100 ℃ of following 1M sulfuric acid treatment can increase substantially the ability that D-(+) catechin suppresses fatty acid synthase.
The zoopery of embodiment 19, controlling body weight and feed
Select female CD-1 mice for use, 8 ages in week, body weight 25-27 gram, 3 groups of (6 every group) sub-cage rearings, free choice feeding contain the high energy feed of high sugar, raise continuously 7 days.Give every injected in mice 0.2ml test sample solution respectively at first day, the 4th day and the 6th day.Test group 1 injected sample is the fatty acid synthase inhibitor of embodiment 1 preparation, and through ethyl acetate extraction, drying, be dissolved in pure water again, concentration is 12.2mg/ml.Test group 2 injected sample are the fatty acid synthase inhibitor of embodiment 18 preparation, and through ethyl acetate extraction, drying, be dissolved in pure water again, concentration is 10mg/ml.The sample of matched group injection is a pure water.The result as shown in Table 1 and Table 2, table 1 is the changing value of test group mice average weight every day, the average weight of duration of test control group mice does not have significant change.Table 2 is 24 hours an average food ration changing value behind the test group injected in mice fatty acid synthase inhibitor of the present invention, and the average food ration of duration of test control group mice every day changes little.Second day body weight and food ration all obviously descend after the test group 1 mice per injection as a result, stop then to recover after the injection, wherein inject for the first time back 24 hours food rations descend and surpassed 50% (inject preceding 24 hourly average food rations be 3.15 restrain).Body weight and food ration all obviously descend after test group 2 injected in mice, are in all the time under the original value, wherein inject for the first time back 24 hours food rations and descend near 50%, recover slower and stop to inject the back.Above-mentioned experimental result shows that fatty acid synthase inhibitor of the present invention has the effect of tangible reduction feed and controlling body weight.
Mice average body weight change (unit: gram) every day behind the table 1 injection fatty acid synthase inhibitor sample of the present invention
Variation (the unit: gram) of mice 24 hourly average food rations behind the table 2 injection fatty acid synthase inhibitor of the present invention
The fatty acid synthase inhibitor that embodiment 20, strong acid heat treated green tea warm macerating extract obtain is to the effect of growth of cancer cells
Test group 1 sample is the fatty acid synthase inhibitor of embodiment 1 preparation, through ethyl acetate extraction, drying, be dissolved in dimethyl sulfoxide again.Test group 2 samples are the fatty acid synthase inhibitor of embodiment 17 preparation, through ethyl acetate extraction, drying, be dissolved in dimethyl sulfoxide again.Control sample is an absolute dimethyl sulfoxide.Hepatoma cell strain Bel-7402 is carried out In vitro culture, add quantitative sample in the test group, add the equivalent dimethyl sulfoxide in the matched group.Detect the hyperplasia level with tetrazolium bromide reducing process (mtt assay), obtain cell inhibitory rate.Be 5,10,20,40 with final concentration respectively, test group 1 sample and test group 2 samples of 60 μ g/mL are handled hepatoma cell strain.Table 3 is under each sample concentration, compares the cancerous cell suppression ratio of test group with matched group.Along with the increase of sample concentration, the suppression ratio of growth of cancer cells improves, and shows that fatty acid synthase inhibitor of the present invention has the effect of significant anticancer growth.
Table 3 fatty acid synthase inhibitor of the present invention is to the suppression ratio (%) of lung cancer cell line growth
Claims (7)
1, a kind of fatty acid synthase inhibitor obtains Folium Camelliae sinensis warm macerating extract, ester catechin or catechin through acid treatment; Wherein, described acid treatment temperature is 70-100 ℃, and the time is 30-170 minute, and the final concentration of acid is 0.2-3.0mol/L; Described acid is that nontoxic non-oxidizable strong acid, PKa value is the nontoxic non-oxidizable organic acid of 1-5 for nontoxic non-oxidizable middle strong acid or the PKa value of 1-2; Described Folium Camelliae sinensis warm macerating extract is that the ethanol water with 10%-95% obtained the Folium Camelliae sinensis warm macerating under 20 ℃ of-40 ℃ of conditions in 2-5 hour.
2, fatty acid synthase inhibitor according to claim 1 is characterized in that: described strong acid is sulphuric acid or hydrochloric acid; Strong acid is phosphoric acid in described; Described organic acid is oxalic acid, malic acid, citric acid, tartaric acid or acetic acid.
3, the preparation method of the described fatty acid synthetase inhibitor of a kind of claim 1 is that Folium Camelliae sinensis warm macerating extract, ester catechin or catechin are carried out acid treatment, obtains fatty acid synthetase inhibitor; Wherein,
Described acid treatment temperature is 70-100 ℃, and the time is 30-170 minute, and the final concentration of acid is 0.2-3.0mol/L; Described acid is that nontoxic non-oxidizable strong acid, PKa value is the nontoxic non-oxidizable organic acid of 1-5 for nontoxic non-oxidizable middle strong acid or the PKa value of 1-2;
Described Folium Camelliae sinensis warm macerating extract is that the ethanol water with 10%-95% obtained the Folium Camelliae sinensis warm macerating under 20 ℃ of-40 ℃ of conditions in 2-5 hour.
4, preparation method according to claim 3 is characterized in that: described strong acid is sulphuric acid or hydrochloric acid; Strong acid is phosphoric acid in described; Described organic acid is oxalic acid, malic acid, citric acid, tartaric acid or acetic acid.
5, the application of the fatty acid synthetase inhibitor described in the claim 1 or 2 in the medicine of preparation controlling body weight.
6, the fatty acid synthetase inhibitor described in the claim 1 or 2 is application in the medicine of target spot with the fatty acid synthetase in preparation.
7, the fatty acid synthetase inhibitor described in the claim 1 or 2 is in preparation prevention and the medicine of auxiliary for treating cancer or the application in the health product.
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