CN103127191B - Preparation method and application of apocynum venetum extract - Google Patents
Preparation method and application of apocynum venetum extract Download PDFInfo
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Abstract
The invention discloses a preparation method and application of an apocynum venetum extract. The method comprises the following steps of: crushing apocynum venetum leaves, carrying out reflux extraction at room temperature or heated temperature by using ethanol, filtering, merging extracting solutions, carrying out vacuum concentration until no ethanol smell exists to obtain an ethanol extract; and diluting the ethanol extract by using water, degreasing by using petroleum ether, extracting the ethanol extract by using ethyl acetate or n-butyl alcohol as a solvent, recovering the solvent at reduced pressure, and drying to obtain the apocynum venetum extract. Proved by tests, the apocynum venetum extract obtained by using the method disclosed by the invention is an extract capable of inhibiting the activity of fatty acid synthase so as to be a natural, plant-sourced and nontoxic fatty acid synthase inhibitor.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of preparation method and purposes in suppressing fatty acid synthase activity of Herba Apocyni veneti extract.
Background technology
Fatty acid synthase (FAS) is most important enzyme in the interior synthctic fat process of biological cell.Tian Weixi etc. find the research of poultry FAS, the fat level in poultry abdominal cavity and FAS activity have very high positive correlation, and the theory (Tian Weixi that FAS activity is the effective ways of control body fat level is controlled in proposition, the body lipid level of 1994. animals and the regulation and control of fatty acid synthase activity. the chemistry of life, 14, (1): 184; Tian Weixi etc., the relation of the body lipid level of 1996. different growing stages laying hens and liver fatty acid synthase activity. journal of biological chemistry, 12 (2): 234-236; Mei Li et al., 1999. Factors influencing the levels of fatty acid synthase complex activity in fowl. Biochemistry and Molecular Biology International, 47 (1): 63-69).2000, the Loftus of John Hopkins University etc. proves the result of study of mice FAS, suppresses the accumulation that FAS can cause malonyl CoA, and the latter suppresses the expression with the relevant hypothalamic neuropeptide Y that takes food, in the situation that not affecting mice mobility, make appetite decline, weight loss.T. Loftus etc. points out, FAS can be used as and controls potential treatment target position (Loftus. T.M. et al., 2000. Science, 288 (5475): 2379-2381) of body weight.In addition, much research is found the activity of fatty acid synthase (FAS) and expresses in nearly all non-morbid state adult's tissue to show extremely low level, and permitted eurypalynous cancerous tissue as rectal cancer, breast carcinoma, carcinoma of prostate, carcinoma of endometrium, ovarian cancer, pulmonary carcinoma, hepatocarcinoma etc. in its activity significantly improve, express significantly and raise.Simultaneously, also studies have found that the inhibitor cerulenin (cerulenin) of fatty acid synthase (FAS) can kill the growth of cancerous cell and obstruction xenograft tumor, other FAS inhibitor are as the derivant C75 of cerulenin, orlistat (orlistat, the flavone compound of epigallocatechin gallate (EGCG) (EGCG) and other natural origins is as luteolin (luteolin), Quercetin (quercetin), kaempferol (kaempferol) etc. has also been identified and can have passed through the growth of cell death inducing inhibition tumor cell with antibiotic triclosan (triclosan).Above result shows, fatty acid synthase (FAS) may be potential treatment of obesity and the dual target spot of tumor.Therefore, the inhibitor of development and exploitation fatty acid synthase (FAS) will be of great immediate significance and potentiality to be exploited.
Herba Apocyni veneti
(Apocynum venetum)for Apocynaceae
(Apocynaceae)apocynum
(Apocynum)a kind of upright fruticuli, it is mainly grown in the sandy ground on salt-soda soil, desert or riverbank, gully, hillside, has growth, the Herba Apocyni veneti performance optimal of deserts in Xinjiang in the most provinces and regions of northern China.Folium Apocyni Veneti is the certified products medical material that < < Pharmacopoeia of People's Republic of China > > (2010 editions) includes, having suppressing the hyperactive liver calms the nerves, the function of clearing away heat and promoting diuresis, for dizziness due to hyperactivity of liver-YANG, palpitation and insomnia, edema oliguria.In the national antiaging agent seminar that the eighties in last century, Chinese Pharmaceutical Association was held, with regard to the research of Folium Apocyni Veneti, carried out academic exchange, according to Beijing planning commission, two Herba Apocyni veneti research cooperative groups of the leader of Jiangsu Province planning commission, Chinese Pharmaceutical Association, Shanghai Pharmaceutical Inst., Chinese Academy of Sciences, Chinese Academy of Sciences's northwest Institute of Zoology, academy of traditional Chinese medicine institute of materia medica, the units such as institute of Materia Medica,Chinese Academy of Medical Sciences and Shaanxi, Beijing, Shanghai, clinical experimental study and the documents and materials of the medical relevant unit such as Nanjing prove, Folium Apocyni Veneti contains a large amount of flavone, triterpene, organic acid, the chemical analysis such as aminoacid, further studying flavone chemical constitution is Quercetin and isoquercitin glycosides, its pharmacological action has blood pressure lowering, blood fat reducing, increase coronary flow, to hypertension, hyperlipidemia, there is good curative effect, especially to dizzy symptom, improving sleep quality has positive effect, has the immunity of enhancing simultaneously, preventing cold, antiasthmatic-antitussive, eliminate depressed, the skin care of invigorating blood circulation, relieving alcoholism and protecting the liver, degraded tobacco poisoning, vessel softening, the effects such as relieving constipation diuresis.Chinese patent CN02135799.4 discloses a kind of tea product that Herba Apocyni veneti and green tea prepared as raw material of take.Application number CN00121863.8 discloses the health tea of a kind of Herba Apocyni veneti.And for example application number CN200310107183.9 discloses the sensitivity of a kind of diabetes patient of increasing to insulin, reduces the dependent kender body fat reducing tea to insulin.Mostly the Herba Apocyni veneti preparation of inventing in above patent, be the traditional medicinal efficacy based on Herba Apocyni veneti, and do not relate to the mechanism of action.
Summary of the invention
The object of the invention is to, a kind of Preparation method and use of Herba Apocyni veneti extract is provided, the method by Folium Apocyni Veneti through after pulverizing, by ethanol room temperature or heating and refluxing extraction, filter merge extractive liquid,, be evaporated to without alcohol taste, obtain ethanol extract, by after ethanol extract dilute with water, with defat with petroleum ether, again with solvent ethyl acetate or n-butanol extraction, decompression and solvent recovery, dry, obtain Herba Apocyni veneti extract.Experimental results show that: the purposes of the Herba Apocyni veneti extract obtaining by the method for the invention in the medicine of preparation inhibition fatty acid synthase.
The preparation method of a kind of Herba Apocyni veneti extract of the present invention, follows these steps to carry out:
A, by Folium Apocyni Veneti through pulverizing, cross 20 mesh sieves, with concentration, be 30-70% ethanol room temperature or heating and refluxing extraction, extraction time is 1-3 hour, extract 1-3 time, filtration, merge extractive liquid,, is evaporated to without alcohol taste, obtains ethanol extract;
B, the ethanol extract that step a is obtained are diluted with water to containing after crude drug 0.2-5g/ml, use defat with petroleum ether 1-5 time;
Water liquid solvent after c, defat that step b is obtained is ethyl acetate or n-butanol extraction 1-5 time, by extract decompression and solvent recovery, dry, obtains Herba Apocyni veneti extract.
In step a, heating and refluxing extraction temperature is 60-90 ℃.
Step b PetroChina Company Limited. ether and ethanol extract aqueous solution volume ratio are 1:1-4.
In step c, ethyl acetate or n-butyl alcohol and defat aqueous solution volume ratio are 1:1-4.
The purposes of the Herba Apocyni veneti extract obtaining by the method for the invention in the medicine of preparation inhibition fatty acid synthase.
The Herba Apocyni veneti extract obtaining by the method for the invention is suppressing that fatty acid synthase or preparation suppress fatty acid synthase product or for losing weight product or for the purposes of antineoplastic product.
The experiment proved that: the present invention compared with prior art has the following advantages:
(1), the suppressible fatty acid synthase of the Herba Apocyni veneti extract that obtains by the method for the invention is active, this extract is a kind of natural, plant source, nontoxic fatty acid synthase inhibitor;
(2), the Herba Apocyni veneti extract that obtains by the method for the invention can suppress the activity of fatty acid synthase significantly, its active size is better than internationally recognized several fatty acid synthase inhibitors at present, as cerulenin, EGCG etc.;
(3), the power due to fatty acid synthase activity directly has influence on the number of synthctic fat in biological cell and the ability of tumor cell synthetic fatty acid, therefore, the activity that suppresses fatty acid synthase, can make on the one hand the process of synthctic fat in biological cell weaken, fat content can controlledly even reduce; Can make on the other hand the ability of tumor cell synthetic fatty acid decline, thus growth that can inhibition tumor cell; Therefore, the Herba Apocyni veneti extract obtaining by the method for the invention as active component make the slimming medicine of various dosage forms or cancer therapy drug and have that effect is active significantly, the mechanism of action is clear, the target spot feature such as clearly, meet the requirement of pharmaceutical preparation exploitation;
(4), due to Herba Apocyni veneti, be a kind of plant resources of traditional medicine-food two-purpose, the extract of producing from Herba Apocyni veneti with the present invention is made fat-reducing or antineoplastic health food, function cosmetics, bread and cheese etc. as raw material, meet country for restriction and the laws and regulations requirement of the raw material of health food and bread and cheese, therefore, the extract with inhibition fatty acid synthase activity of the present invention has more wide application and development prospect.
In a word, the present invention has not only enriched the source of pure natural fatty acid synthase inhibitor, has also opened up new effect and the using value of Herba Apocyni veneti plant with region feature and resources advantage.
The specific embodiment
In embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
Embodiment is to further description of the present invention, but does not mean that any limitation of the invention.In the situation that not departing from the above-mentioned thought of the present invention, various substitute modes or the change according to ordinary skill knowledge and conventional means, made, within being all included in the present invention.
Embodiment 1,
A, by 5 kilograms of Folium Apocyni Venetves through pulverizing, cross 20 mesh sieves, by concentration, be that 30% ethanol room temperature is extracted, extraction time is 3 hours, extracts 1 time, filtration, is evaporated to without alcohol taste, obtains ethanol extract;
B, the ethanol extract that step a is obtained are diluted with water to containing after crude drug 0.2g/ml, use defat with petroleum ether 1 time, and wherein petroleum ether and ethanol extract aqueous solution volume ratio are 1:1;
Water liquid solvent after c, defat that step b is obtained is ethyl acetate, extracts 1 time, and ethyl acetate and defat aqueous solution volume ratio are 1:1, by extract decompression and solvent recovery, dry, obtains Herba Apocyni veneti extract 60g.
Embodiment 2
A, by 5 kilograms of Folium Apocyni Venetves through pulverizing, cross 20 mesh sieves, by concentration, be that 50% alcohol heating reflux extracts, extracting temperature is 60 ℃, extraction time is 2 hours, extract 3 times, filtration, merge extractive liquid,, is evaporated to without alcohol taste, obtains ethanol extract;
B, the ethanol extract that step a is obtained are diluted with water to containing after crude drug 1g/ml, use defat with petroleum ether 2 times, and wherein petroleum ether and ethanol extract aqueous solution volume ratio are 1:2;
Water liquid solvent after c, defat that step b is obtained is n-butanol extraction 2 times, and n-butyl alcohol and defat aqueous solution volume ratio are 1:2, by extract decompression and solvent recovery, dry, obtains Herba Apocyni veneti extract 250g.
Embodiment 3
A, by 5 kilograms of Folium Apocyni Venetves through pulverizing, cross 20 mesh sieves, by concentration, be that 60% alcohol heating reflux extracts, extracting temperature is 75 ℃, extraction time is 3 hours, extract 2 times, filtration, merge extractive liquid,, is evaporated to without alcohol taste, obtains ethanol extract;
B, the ethanol extract that step a is obtained are diluted with water to containing after crude drug 3g/ml, use defat with petroleum ether 4 times, and wherein petroleum ether and ethanol extract aqueous solution volume ratio are 1:3;
Water liquid solvent after c, defat that step b is obtained is ethyl acetate extraction 4 times, and ethyl acetate and defat aqueous solution volume ratio are 1:3, by extract decompression and solvent recovery, is dried, and obtains Herba Apocyni veneti extract 70g.
Embodiment 4
A, by 5 kilograms of Folium Apocyni Venetves through pulverizing, cross 20 mesh sieves, by concentration, be that 70% alcohol heating reflux extracts, extracting temperature is 90 ℃, extraction time is 1 hour, extract 3 times, filtration, merge extractive liquid,, is evaporated to without alcohol taste, obtains ethanol extract;
B, the ethanol extract that step a is obtained are diluted with water to containing after crude drug 5g/ml, use defat with petroleum ether 5 times, and petroleum ether and ethanol extract aqueous solution volume ratio are 1:4;
Water liquid solvent after c, defat that step b is obtained is n-butanol extraction 1-5 time, and n-butyl alcohol and defat aqueous solution volume ratio are 1:4, by extract decompression and solvent recovery, dry, obtains Herba Apocyni veneti extract 260g.
Embodiment 5
The purposes of the Herba Apocyni veneti extract obtaining by the method for the invention in the medicine of preparation inhibition fatty acid synthase:
The detection of Herba Apocyni veneti extract:
Positive control cerulenin (cerulenin), epigallocatechin gallate (EGCG) (EGCG) are all purchased from Sigma company;
The preparation of fatty acid synthase (Fatty acid synthase, FAS):
Fatty acid synthase (FAS) slightly carries: fresh chicken liver weigh after by 1:1.8(g/mL) add the extraction buffer 0.1 mol/L KH of 4 ℃ of temperature
2pO
4-K
2hPO
4, 1 mmol/L ethylenediaminetetraacetic acid (EDTA), 1 mmol/L dithiothreitol, DTT (DTT), 0.07 mol/L KHCO
3pH 8.0), homogenate 50 seconds, measures after homogenate volume with 8000 r ∕ min speed, centrifugal 15min at 4 ℃ of temperature, supernatant is through filtered through gauze, after measurement volumes, then with 35000 r ∕ min speed, centrifugal 30min at 4 ℃ of temperature, supernatant is sub-packed in low-temperature resistant plastic bottle after filtered through gauze, spends the night in temperature-20 ℃, and residue is placed in temperature-80 ℃ and saves backup;
Ammonium sulfate fractional precipitation: freezing high speed centrifugation supernatant is put to water-bath and thaw, thaw and be placed in ice chest, under magnetic agitation state, by the saturated ammonium sulfate of the pH 7.0 of 4 ℃ of three/a volume of dropping temperatures, contain 1 mmol/L dithiothreitol, DTT (DTT) to 25% saturation, stir after 10min by 10000 r/min speed, centrifugal 15min at 4 ℃ of temperature, discard precipitation, the fatty acid synthase of sampling and measuring supernatant (FAS) activity;
The saturated ammonium sulfate that drips again 4 ℃ of pH 7.0 of temperature of supernatant 1/4th volumes under stirring contains 1 mmol/L dithiothreitol, DTT (DTT) to 40% saturation, stir after 10min with the centrifugal 15min of 10000 r/min, if do not contain in mensuration supernatant or only contain, seldom fatty acid synthase FAS is active, supernatant discarded, retains precipitation;
Ion-exchange chromatography: diethylaminoethyl cellulose cellulose DE52 uses 4 ℃ of buffer A (5mmol/L KH of temperature after treatment
2pO
4-K
2hPO
4containing 1 mmol/L ethylenediaminetetraacetic acid (EDTA), 1 mmol/L dithiothreitol, DTT (DTT), pH 7.0) balance, after above-mentioned precipitation control is dry, be dissolved in 4 ℃ of buffer A of temperature, mensuration electric conductivity value is not more than 5.5 ms/cm and gets final product loading, after loading with 4 ℃ of buffer A of temperature wash to foreign protein be absorbed as 0, then use successively 4 ℃ of 50 mmol/L of temperature, 160 mmol/L, 250 mmol/L, 1 mol/L KH
2pO
4-K
2hPO
4buffer is (all containing 1 mmol/L EDTA, 1 mmol/L dithiothreitol, DTT (DTT), pH 7.0) eluting, collect respectively eluting peak, measurement volumes, protein content and fatty acid synthase (FAS) activity, chromatography process is used LKB albumen monitor and monitor monitoring;
Affinity chromatograph: 4 ℃ of buffer B of temperature (50 mmol/L KH for blue-sepharose gel Sepharose 6B resin
2pO
4-K
2hPO
4containing 1 mmol/L ethylenediaminetetraacetic acid (EDTA), 1 mmol/L dithiothreitol, DTT (DTT), pH 7.0) balance, by 250 mmol/L KH of DEAE chromatography
2pO
4-K
2hPO
4buffer solution elution is collected liquid loading, after loading, by 4 ℃ of buffer B of temperature, wash, by 4 ℃ of temperature, contain 0.35 mol/L, 2 mol/L NaCl buffer B eluting successively again, collect respectively eluting peak, measurement volumes, protein content and fatty acid synthase (FAS) activity;
Concentrated preservation: slowly add 4 ℃ of saturated ammonium sulfate solution to 40% saturations of temperature with precipitated fatty acid synthase FAS by containing 0.35mol/LNaCl buffer B eluting collection liquid 2/3rds volumes under stirring, stir after 10min with 10000r/min speed, centrifugal 15min at 4 ℃ of temperature, abandoning supernatant, is dissolved in a small amount of store buffer liquid (0.1 mol/L KH after the dry precipitation of control
2pO
4-K
2hPO
41 mmol/L ethylenediaminetetraacetic acid (EDTA), 10 mmol/L dithiothreitol, DTTs (DTT), 20% glycerol, pH 7.0) in, make fatty acid synthase (FAS) concentration in 10 mg/mL left and right, sample determination protein concentration and the fatty acid synthase of taking a morsel (FAS) activity, after all the other subpackages, after spending the night in temperature-20 ℃, transposition temperature-80 ℃ freezing saving backup, obtains fatty acid synthase (FAS);
Fatty acid synthase is active adopts S-acetyl-coenzyme-A, malonyl CoA, NADPH (NADPH) to measure (W.X.Tian et al. for the standard measuring method for activity of substrate, 1985. J.Biol.Chem., 260 (20): 11375-11387; Tian Weixi etc., the relation of the body lipid level of 1996. different growing stages laying hens and liver fatty acid synthase activity. journal of biological chemistry, 12 (2): 234-236);
The institute of above separating-purifying process all carries out in steps under 4-8 ℃ of low temperature, and whole sample fatty acid synthase detects as single band through polyacrylamide gel electrophoresis, and molecular weight is 270kD;
The inhibitory action of Herba Apocyni veneti extract to fatty acid synthase:
Get 10.9 mg Herba Apocyni veneti extracts, be dissolved in 218 ml dimethyl sulfoxide as given the test agent solution;
Fatty acid synthase is active adopts the standard measuring method for activity that S-acetyl-coenzyme-A, malonyl CoA, NADPH are substrate to measure (W.X.Tian et al., 1985. J.Biol.Chem., 260 (20): 11375-11387; Tian Weixi etc., the relation of the body lipid level of 1996. different growing stages laying hens and liver fatty acid synthase activity. journal of biological chemistry, 12 (2): 234-236);
Testing sample solution is added and surveyed in live body system, and 37 ℃ of constant temperature of temperature, then add fatty acid synthase FAS to start reaction, and recording enzymatic activity is A
i.Adding the enzymatic activity that sample solvent control records is A
0, A
i/ A
0be residual activity (the Remaining Activity of fatty acid synthase FAS, R.A.), this value is less, show that sample is stronger to the inhibition ability of fatty acid synthase (FAS), the inhibitory action that this method is measured is generally reversible, and the combination that represents inhibitor and enzyme is non-covalent bond;
The degree IC50(503nhibiting concentration of fatty acid synthase (FAS) activity inhibited) represent, its computational methods are: take only add in substrate corresponding extraction solvent as contrast, measure its fatty acid synthase activity value, this value A
0represent, and to set it be 100%, what in substrate, add fatty acid synthase inhibitor extracting solution is experimental group, and the numerical value that records its fatty acid synthase activity is A
050% time, the concentration of inhibitor is IC
50value, this value is less, shows that sample is stronger to the inhibition ability of fatty acid synthase FAS;
Use said determination method, record result as shown in the table:
The inhibitory action of table Herba Apocyni veneti extract to fatty acid synthase
| Test event | Embodiment 1 | Embodiment 2 | Embodiment 3 | Embodiment 4 | Cerulenin | EGCG |
| 503nhibiting concentration (IC 50）(μg/mL) | 1.3 | 2.7 | 1.5 | 3.2 | 20 | 24 |
From table, result can show, Herba Apocyni veneti extract has strong inhibitory action to the activity of fatty acid synthase, and apparently higher than current internationally recognized several fatty acid synthase inhibitor cerulenin and EGCG, and required effective dose is very low.
Claims (1)
1. the purposes of Herba Apocyni veneti extract in the medicine of preparation inhibition fatty acid synthase, is characterized in that Herba Apocyni veneti extract is obtained by following preparation method, and preparation method follows these steps to carry out:
A, by Folium Apocyni Veneti through pulverizing, cross 20 mesh sieves, the ethanol room temperature or the heating and refluxing extraction that by concentration, are 30-70%, extraction time is 1-3 hour, extract 1-3 time, filter merge extractive liquid,, be evaporated to without alcohol taste, obtain ethanol extract, wherein heating and refluxing extraction temperature is 60-90 ℃;
B, the ethanol extract that step a is obtained are diluted with water to containing after crude drug 0.2-5g/ml, use defat with petroleum ether 1-5 time, and wherein petroleum ether and ethanol extract volume ratio are 1:1-4;
Aqueous solution solvent after c, defat that step b is obtained is ethyl acetate or n-butanol extraction 1-5 time, and ethyl acetate or n-butyl alcohol and defat aqueous solution volume ratio are 1:1-4, by extract decompression and solvent recovery, dry, obtain Herba Apocyni veneti extract.
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| CN106038636A (en) * | 2016-06-03 | 2016-10-26 | 通化师范学院 | Application of apocynum venetum to preparation of anti-tumor medicine |
| CN108341716A (en) * | 2018-04-02 | 2018-07-31 | 青岛元汇丰企业管理咨询服务有限公司 | A kind of antibacterial growth-promoting elongated biological pesticide of plant source |
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| CN116036141B (en) * | 2023-02-10 | 2024-04-19 | 中国农业科学院麻类研究所 | Apocynum venetum extract with three-high reducing function as well as preparation method and application thereof |
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Effective date of registration: 20161115 Address after: 836509, Xinjiang, Aletai, Saline Lake, Aletai, Ma Bao Ma base Patentee after: Al tiger Bao tea Limited by Share Ltd Address before: 833307, the Xinjiang Uygur Autonomous Region, Bortala, Mongolia Autonomous Prefecture, Jinghe County toto Ma Bao Ma Industrial Park Patentee before: Xinjiang Ebinur Lake Gaubau Hemp Co., Ltd. Patentee before: Al tiger Bao tea Limited by Share Ltd Patentee before: Gaubau Green Industry (Shenzhen) Co., Ltd. |