CN101129497B - Pharmaceutical uses of black-seed grass-seed oil of tuberculate fruit - Google Patents
Pharmaceutical uses of black-seed grass-seed oil of tuberculate fruit Download PDFInfo
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Abstract
The invention discloses a drug application of tumour-fruit black-seed grass-seed oil, which is characterized by the following: squeezing from tumour-fruit black-seed through normal method; extracting in the solvent; decompressing to remove solvent; testing the effect of protein tyrosine phosphatase 1B (PTP1B) inhibitor to resist lipid peroxidization and remove free radical of oxygen. The invention also provides an application in the antidiabetic drug and anti-oxidizing drug and health products.
Description
Technical field
The present invention relates to a kind of Uygur medicine medicinal herbs most in use--black-seed grass-seed oil of tuberculate fruit is as the purposes of medicine and the anti peroxidation of lipid medicine and the health product of protein tyrosine phosphatase inhibitor.The effect of the PTP 1B that it had (PTP1B) inhibitor that confirms by experiment in vitro and the effect of anti peroxidation of lipid.The antidiabetic medicine that it forms and the purposes of anti-oxidation medicine and health product.
Background technology
The Nigella damascena L. platymiscium belongs to ranunculaceae plant Flos Trollii subfamily, three kinds of medicinal plants are wherein arranged, be respectively: nigella glandulifera Freyn (Nigella glandulifera Preyn.), have another name called Semen Nigellae, also has fruit Nigella damascena L. (Nigella sativa), have another name called tame Nigella damascena L. and Nigella damascena L. (Nigella damascena L.), their seed is the medicinal part of using always.External research at present mainly concentrates on the fruit Nigella damascena L..(Nigella glanduliferaFrey, NG), the original producton location is Mediterranean bank and Central Asia to nigella glandulifera Freyn, only cultivation is arranged Xinjiang in China.Its dry mature seed, it is avette to be the rhizoma sparganic shape, long 2.5-3mm, wide about 1.5mm.Surface black, coarse, the top is narrower and sharp, and the lower end is blunt slightly, and irregular projection is arranged.Matter is hard, and section ash soul has oiliness.Feeble QI perfume (or spice), acrid in the mouth.Can stimulate digestion wind dispelling pain relieving, kidney-tonifying and brain tonic, diuresis, diaphoresis, be good for the stomach, anthelmintic, treatment asthma etc., be a kind of custom medication in Uygur's medical science.In recent years, find that after deliberation its congener fruit fennelflower seed (Nigellasativa) has certain effect aspect blood fat reducing, the blood sugar lowering,
Process is to rat oral black race fruit grass seed solid oil (Nigella sativa) 12 weeks, do not find enzyme main in the liver is exerted an influence, simultaneously, cholesterol in the blood, triglyceride, glucose, lymphocyte and number of platelets are compared respectively according to having reduced by 15.5,22,16.5 35 and 32% hematocrit and hemoglobin level have risen 6.4 and 17.4%.
Feed 4 weeks of fennelflower seed ligroin extraction to normal rat with the gastric gavage, ligroin extraction is owing to reduced by 25% food, thereby the body weight short-term alleviates.All do not observe the toxicity of plant and fasting glucose keeps stable in vivo with in the in vitro tests.The rat limosis insulin of treatment group is compared in treatment 4 week backs with matched group and triglyceride levels is lower and the HDL-cholesterol levels is higher.By the Western hybridization analysis show hepatocyte that discovery separates from each treated animal to insulin level reaction risen.The petroleum ether part has played the effect of euglycemic agent by signal transduction path in two kinds of cells that increase hormone receptor in vivo.
Urinate the inductive diabetes hamster of rhzomorph (STZ) gastric gavage by 400mg/kg to chain and feed Oleum Nigellae (Nigella sativa) 6 weeks, after inducing diabetes, with collagenase digesting and collect the generation that isolating hepatocyte has been determined glycogen.By injected fluorescein emulsion in a period of time, collect peritoneal macrophages after 24 hours to estimate phagocytic activity.The 391+ of treatment group blood glucose before treat/-3.0mg/dl reduces to 325+/-4.7 after first, second, third and fourth week, 246+/-5.9, and 208+/-2.5 and 179+/-3.1mg/dl.The glycogen product that comes from the glyconeogenesis precursor in treatment group hamster obviously reduces the hypoglycemic effect of pointing out it and partly comes from and reduced the synthetic of liver glycogen.In addition, activate the phagocytic capacity and the phagocytic index through Oleum Nigellae treatment posterior peritoneum macrophage obviously rises the increase of peripheral blood medium-sized lymphocyte quantity.This shows that its immanoprotection action is by the vigor of engulfing of direct stimulation macrophage or by stimulating lymphocytic vigor to finish.
Give male spontaneous hyperlipemia rat that is easy to apoplexy and Wistar rat by 800mg/kg oral dose fruit Oleum Nigellae (Nigella sativa) 4 weeks, can obviously reduce the content of T-CHOL in the serum, low density lipoprotein, LDL, triglyceride, simultaneously, high density lipoprotein increasing content.
Induce with STZ and nicotiamide that to have formed rats with diabetes be model.Give the oral Oleum Nigellae of rat (Nigella sativa) 4 weeks, blood glucose obviously reduces, and proves that through enzyme even immunity and glucagon monoclonal antibody insulin level obviously rises.Can observe a large amount of insulin positive reaction districts through immunohistochemical staining in islets of langerhans, the result is presented in the type 2 diabetes mellitus model, and black middle seed oil has the function of similar insulin.
Because the generation of type 2 diabetes mellitus development can cause by the interaction of h and E factor etc., wherein, the important function that insulin signaling transduction obstacle is risen in pathogenesis.PTP 1B (Protein tyrosine phosphatase PTP1B) is crucial negative regulation of insulin signaling transduction.PTP1B is the representative of PTP in the cell, it is first mammalian proteins tyrosine phosphatase of being identified and being purified into, form by 435 amino acid residues, the about 50KD of molecular weight, PTP 1B is ubiquity and expression in each histiocyte of human body, and by making activated Insulin receptor INSR and IRS dephosphorylation inactivations such as (IRs-1) bring into play physiological function.Balance between its tunable tyrosine residue phosphorylation and the dephosphorylation stops the insulin signaling transduction.Protein-tyrosine-phosphatase content and activity special in type 2 diabetes mellitus and the insulin resistant patient body all increase to some extent, and after Protein-tyrosine-phosphatase was disallowable, not only insulin sensitivity increased, and diabetic symptom also makes moderate progress.It is more responsive that the mice that PTP 1B lacks and its wild type control mice compare insulin, and glycemic control is better, and the obesity of diet induced is had better resistant function.Therefore, the effect of Profilin tyrosine phosphatase 1B has become a kind of new treatment insulin resistant (IR), type ii diabetes and fat method.Simultaneously, the high expressed of PTP 1B can cause insulin resistant and leptin opposing, its overexpression can impel apolipoprotein B100 (ApoB100) secretion to increase, blood plasma very low density lipoprotein (VLDL) (VLDL) is generated increase, thereby cause disorders of lipid metabolism, type 2 diabetes mellitus and obesity.Studies show that PTP 1B clpp gene deratization not only insulin sensitivity strengthens, and owing to removed the inhibition of PTP 1B to insulin and leptin signal transduction, makes mice that obesity not take place yet under the high fat diet condition.Be the relation between clear and definite PTP 1B and the disorders of lipid metabolism, Klaman etc. study Protein-tyrosine-phosphatase N1 knock out mice, the result shows that long-term fructose feeds common mice and can cause the blood plasma very low density lipoprotein (VLDL) to raise, and do not see that very low density lipoprotein (VLDL) raises in the fructose nursing PTPN1 knock out mice, the prompting PTP 1B has the excretory effect of the very low density lipoprotein (VLDL) of promotion.Taghi-biglou etc. are to carrier's apolipoprotein B (ApoB100) but lack apolipoprotein B (ApoB)/brown adipose tissue (BAT) deficient mice of brown adipose tissue and find that liver very low density lipoprotein (VLDL)-apolipoprotein B increases and will cause insulin resistant in the hamster Study of model that fructose is raised.There is report to show, the lipoprotein spectrum of the inductive diabetes model animal of the adjustable fructose of PTP 1B, down-regulation protein tyrosine phosphatase 1B expresses can suppress inductive insulin resistant of Hi CHO and associated disorders of lipid metabolism.Therefore PTP 1B one of the target spot of euglycemic agent not only also may be one of treatment target spot of disorders of lipid metabolism simultaneously.
In view of fruit fennelflower seed and nigella glandulifera Freyn seed are congeners, and in traditional medicine, have blood fat reducing, blood sugar reducing function that similar effect and fruit fennelflower seed have, the present invention has studied the function of polysaccharide of black-seed grass-seed oil of tuberculate fruit, utilize conventional method to obtain black-seed grass-seed oil of tuberculate fruit, and at external effect and the lipoid peroxidization resistant that confirms its Protein-tyrosine-phosphatase protein-tyrosine phosphatase 1B inhibitor.
Summary of the invention
The object of the invention is, provides medicinal herbs most in use-black-seed grass-seed oil of tuberculate fruit in a kind of Uygur medical science as the purposes of the medicine of preparation treatment anti-diabetic protein tyrosine phosphatase inhibitor or health product and as the purposes of preparation anti-oxidation medicine or health product.Removal of solvent under reduced pressure obtains this black-seed grass-seed oil of tuberculate fruit adopts conventional squeezing or solvent extraction from dimension medicine nigella glandulifera Freyn seed after, and in the external effect that confirms its Protein-tyrosine-phosphatase PTP1B inhibitor, the ability of anti peroxidation of lipid, and the anti-diabetic of its formation and the purposes of antioxidative medicine and health product.
Black-seed grass-seed oil of tuberculate fruit of the present invention is as the medicine of preparation treatment anti-diabetic protein tyrosine phosphatase inhibitor or the purposes of health product.
Black-seed grass-seed oil of tuberculate fruit of the present invention is as the purposes of preparation anti-oxidation medicine or health product.
The medicinal usage of black-seed grass-seed oil of tuberculate fruit of the present invention, wherein black-seed grass-seed oil of tuberculate fruit is from the Uygur medicine medicinal herbs most in use--the nigella glandulifera Freyn seed, take the black-seed grass-seed oil of tuberculate fruit that conventional squeezing or conventional solvent extraction method, microwave-assisted solvent extract, the extraction of ultrasound wave secondary solvent obtains, the concrete operations step is undertaken by following:
Comprise petroleum ether with little polar solvent, hexane, heptane, dichloromethane, chloroform, ether and their various mixed solvents directly soak the nigella glandulifera Freyn seed, and distilling under reduced pressure removes to desolvate and promptly gets black-seed grass-seed oil of tuberculate fruit then;
Or the solvent such as the methanol of use high polarity, the methanol of ethanol or 0%-30% concentration, alcoholic acid aqueous solution, extract the nigella glandulifera Freyn seed down in 20-98 ℃ of temperature conditions, this extract concentrates the back to be disperseed with concentration 0%-30% methanol or dissolve with ethanol, again with organic solvent such as petroleum ether, hexane, heptane, dichloromethane, chloroform extraction concentrates the dried black-seed grass-seed oil of tuberculate fruit that promptly gets with extract.
In traditional Uygur medical science, the nigella glandulifera Freyn seed is in uighur medicine, Nigella damascena L. has diuresis, supplementing the brain, the kidney invigorating, invigorates blood circulation, detoxifcation, stimulating milk secretion, stimulate the menstrual flow, cough-relieving, black hair, the anti-ageing effect of waiting for a long time, be a kind of custom medication in Uygur's medical science, black-seed grass-seed oil of tuberculate fruit is made into composite siyadan oil for promoting hair growth at present, this inferior dawn hair growth promoting tincture of compound recipe is mainly used in black hair and hair growth promoting.Up to the present, only limiting to priorities such as Hao Haifeng, Liu Yuming, Ni Junjun about the research of nigella glandulifera Freyn separates from the nigella glandulifera Freyn seed and has obtained 16 chemical compounds, and investigation such as Tian Ze is from wherein separating the anti-tumor activity of two kinds of Hederagenins that obtain, but its hypoglycemic activity and Study on oxidation resistance are not appeared in the newspapers as yet.
Referring to accompanying drawing
Fig. 1 is the electrophoretogram of phosphorylation AKT in the Chinese hamster ovary celI of western blotting method analysis overexpression PTP1B of the present invention, and wherein 1 blank contrasts behind 2 insulin stimulatings, 3 positive controls, 4 specimen.
The specific embodiment
Get dry nigella glandulifera Freyn seed (Nigella glandulifera Preyn.) 10Kg, utilize the method for conventional squeezer squeezing, obtain nigella glandulifera Freyn oil.
Get exsiccant nigella glandulifera Freyn seed (Nigella glandulifera Preyn.) 10Kg, after the pulverizing, utilized the petroleum ether soak at room temperature 7 days, remove by filter grass seed, filtrate is utilized Rotary Evaporators, removes petroleum ether and obtains black-seed grass-seed oil of tuberculate fruit.
Embodiment 3:
Get the dry seed 200g of nigella glandulifera Freyn (Nigella glandulifera Preyn.), pulverize, place 2L tool plug flask, flask is put the ultrasonic extraction instrument, with petroleum ether room temperature supersound extraction 3 times, each 1.5L, each extraction time is 0.5 hour, and merge extractive liquid, filters, in Rotary Evaporators, be evaporated to driedly, obtain grease 75g.
Get the dry seed 10Kg of nigella glandulifera Freyn (Nigella glandulifera Preyn.), pulverize, place the 50L extraction pot, extract 4 times with 70% ethanol room temperature merceration, each ethanol consumption is 30L, and each extraction time is 7 days, merge extractive liquid,, in Rotary Evaporators, be evaporated to driedly, add 10% methanol aqueous solution 2L dissolving, and with petroleum ether (60-90 ℃) extraction 5 times, each 2L, combining extraction liquid is evaporated to driedly in Rotary Evaporators, obtains oily sample 3560g.
Embodiment 5
Get the dry seed 100g of nigella glandulifera Freyn (Nigella glandulifera Preyn.), pulverize, place 1L tool plug flask, flask is put in the microwave oven, with 95% ethanol microwave auxiliary extraction 3 times, each 600mL, each extraction time is 5min; Merge extractive liquid, filters, and is evaporated to driedly in Rotary Evaporators, obtains grease 18g.
Embodiment 6
Black-seed grass-seed oil of tuberculate fruit is as the bioactivity screening of the purposes of the medicine of preparation treatment anti-diabetic protein tyrosine phosphatase inhibitor or health product:
Screening technique: with right-nitrobenzophenone disodium hydrogen phosphate (pNPP) as substrate, being contrast at the positive drug vanadic acid sodium, utilize microplate reader to carry out the high flux screening of PTP1B enzyme inhibitor, produce the activity that color reaction is measured PTP1B according to the phosphate group of PTP1B hydrolysis pNPP.The enzyme reaction system is composed as follows: buffer (50mM HEPES, pH7.3,100mM sodium chloride, 0.1% bovine serum albumin, and 1mM dithiothreitol dithio), PTP1B fusion rotein, pNPP, the special inhibitor vanadate of PTP1B (100 μ g/mL).Place 30rnin at 37 ℃ behind the reaction system mixing, add 1M sodium hydroxide cessation reaction, put the absorption value of measuring on the colour comparatour under the 405 wavelength conditions (A), measurement result is calculated enzymatic activity after deducting background values.
The selection result:
Conclusion:
Show that from experimental result black-seed grass-seed oil of tuberculate fruit has the protein-tyrosine phosphatase 1B inhibitor effect.
Immunoblot experiment:
1) liposome transfection: hamster ovarian cancer cell (CHO) is 30h is cultivated in the back of going down to posterity in F12+10% hyclone (FBS) culture medium after, the pJ3H plasmid DNA that to carry total length-PTP1B gene according to the method on the Lipofectamine 2000TM operation instructions is mixed the back transfection and is advanced in the Chinese hamster ovary celI with liposome, after 6 hours, remove the culture medium that contains liposome and change F12+10%FBS culture medium continuation overnight incubation;
2) change the F12+10%FBS culture medium that contains specimen for the cell of handling well in the first step, add an amount of insulin stimulating 5min, harvesting after 3 hours.In ice bath, scrape cell with cell, the collecting cell lysate.
3) after electrophoresis finishes, take out gel, the rinsing several seconds in the Tris/ glycine buffer.Get gel method: with two glass plates separately with blade, unnecessary gel is scratched, top is as the criterion with concentrated glue and all discards, a bit all scratch with molecular weight standard smallest molecule leukorrhagia the bottom, gets a 10ml syringe and fill with the transfer printing buffer, inserts water filling between glass plate and the gel, the pressure of water is separated both naturally, advance while pushing away, the repeated multiple times water filling is got off until gel landing from the glass plate.Open electric transfer printing folder, the transfer printing liquid of using of every side pad lastblock special use soaks saturating sea pad, respectively put the filter paper that a transfer printing liquid soaks into again, filter paper and sea pad size identical or and pvdf membrane, gel size identical all can, gel is lain on the cathode side filter paper, at last film is lain on the gel, remove bubble, clip electric transfer printing folder.Electrophoresis tank is filled it up with electric transfer printing liquid, inserts electric transfer printing folder, and electrophoresis tank is put into refrigerator (will put into the refrigerator pre-cooling before the electric transfer printing liquid), connects electrode, turn-on current, the positive pole of the film reply electrophoresis tank of transfer printing folder.Utilize electroporation that the protein transduction on the SDS-PAGE is moved on on the film.Condition: 400mA 1h or 280mA, 1.5-2h;
4) sealing: before carrying out antibody hybridization, need earlier transfer film to be sealed, to prevent the non-specific adsorption of immunoreagent.1. wash transfer film: 3 times * 10min of room temperature rinsing with the SDS on the flush away transfer film of trying one's best, prevents to influence the antibodies of back.Get the transfer film of rinsing, put into the confining liquid that contains 5% defatted milk powder, the shaking table vibrations, room temperature sealing 2h also can spend the night at 4 ℃.3. use buffer, 3 times * 10min. of PH7.6 room temperature rinsing
5) antibody hybridization: the main indirect method that adopts in antibody surface.1. the hybond membrane after the sealing is put into hybridization bag, adds antibody diluent, seal, 4 ℃ of overnight incubation or room temperature (22-25 ℃) shake hatch 2h. 2. liquid wash 3 times * 10min of film and 3. add two anti-diluents, room temperature 1h washes film 3 * 10min)
6) detect: adopting enhanced chemiluminescence (ECL) to detect is to utilize the horseradish peroxidase enzyme catalytic chemiluminescent substance, generate a kind of unsettled intermediate material, in the darkroom, form tangible macroscopic chemiluminescence band during its decay, utilize X line light reaching the film principle, outcome record is got off: ECM developer preparation: press 1mlH
2O adds each 1 of developer A, B, mixing.To hybridize the back blotting membrane in the darkroom and put into the colour developing box, add the colour developing liquid that mixes, about 1-5 minute with, napkin is inhaled and is removed blotting membrane edge or the unnecessary colour developing liquid of corner part, one transparent cellophane covered smooth, and determine that the surface of doing contacts with film. blotting membrane was made the film exposure 1-5 minute in the darkroom, develop photographic film to determine the antigenic correct exposure time of being surveyed, exposure time can be as short as several seconds and also can grow to a few hours. step develops photographic film: in developer solution, wash earlier occur to the film band or film transparent till, putting into the fixative solution rinsing, getting final product in ten seconds.The result shows: the content of cells phosphorylation AKT in can increasing, this makes black-seed grass-seed oil of tuberculate fruit to transport by PI3K/AKT pathway stimulation GLUT4, thereby reaches the purpose of blood sugar lowering.
Embodiment 7:
Purposes function as preparation anti-oxidation medicine or health product:
The Lipid Peroxidation model reaction that with the lipovitellinin is substrate comprises: yolk suspension (the isopyknic PH7.45 of yolk of dilution in 1: 25,0.1M phosphate buffer (PBS) is made into, play stirring 10min with preceding magnetic agitation) 0.15mL, 25mm copperas solution 0.15mL, at last, supply 1.2mL with PBS, do not add sample in the control tube, sample volume is with the PBS polishing and add 20%TCA solution 0.675mL in advance, places 37 ℃ water-bath to place 15min simultaneously in above-mentioned two kinds of test tubes after on-test.After the taking-up, medicine is transferred in the 1.5mL centrifuge tube after closing and adding the static 10min of 20%TCA solution 0.675mL, the centrifugal 7-8min of 7000rmp/min gets supernatant and adds thiobarbituricacid solution (0.8%) 0.5mL respectively, behind the mixing, be divided into two parts, in boiling water bath, place 15min, take out cooling.With the zeroing of PBS solution, 721 type spectrophotometric determination A
532Value, the antioxidation degree of sample is used the suppression ratio of lipovitellinin lipid peroxidation is represented:
AOA%=(control tube A
532Value-sample cell A
532Value)/control tube A
532Value * 100%
The result shows: black-seed grass-seed oil of tuberculate fruit has the ability of anti peroxidation of lipid.
Claims (1)
1. a black-seed grass-seed oil of tuberculate fruit is in the medicine of preparation diabetes protein-tyrosine phosphatase 1B inhibitor or the purposes of health product.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104688843A (en) * | 2015-02-11 | 2015-06-10 | 中国科学院新疆理化技术研究所 | Application of ethyl acetate extract of nigella glandulifera |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101926863B (en) * | 2010-09-29 | 2012-06-27 | 新疆维吾尔自治区药物研究所 | Method for identifying Nigella glandulifera Freyn medicinal material |
| CN102657693B (en) * | 2012-05-24 | 2014-08-13 | 王乐观 | SH2-containing tyrosine phosphatase 1(SHP-1) inhibitor and preparation thereof |
| CN103086967B (en) * | 2013-02-05 | 2015-01-07 | 中国科学院新疆理化技术研究所 | Alkaloid having type 1 skeleton in Nigellaglandulifera Freyn grass seeds, and its preparation method |
| CN103288826B (en) * | 2013-06-03 | 2016-01-06 | 中国科学院新疆理化技术研究所 | Alkaloidal constituent in nigella glandulifera Freyn seed and its production and use |
| CN103285000B (en) * | 2013-06-03 | 2015-01-07 | 中国科学院新疆理化技术研究所 | Use of alkaloid with framework types I and II in Nigella glandulifera Freyn seeds |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104688843A (en) * | 2015-02-11 | 2015-06-10 | 中国科学院新疆理化技术研究所 | Application of ethyl acetate extract of nigella glandulifera |
| CN104688843B (en) * | 2015-02-11 | 2017-12-12 | 中国科学院新疆理化技术研究所 | A kind of purposes of nigella glandulifera Freyn seed ethyl acetate extract |
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