CN101271089B - Method for identifying allantoin in Cistanche plant variety - Google Patents
Method for identifying allantoin in Cistanche plant variety Download PDFInfo
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- CN101271089B CN101271089B CN2008100091433A CN200810009143A CN101271089B CN 101271089 B CN101271089 B CN 101271089B CN 2008100091433 A CN2008100091433 A CN 2008100091433A CN 200810009143 A CN200810009143 A CN 200810009143A CN 101271089 B CN101271089 B CN 101271089B
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- allantoin
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- tubulosa
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Abstract
An application of allantoin in the variety identification of a desertliving cistanche plant and a new usage for lowering the blood lipid use the absorption and separation technology of macroporous absorption resin and granular activated carbon to obtain the allantoin and components containing the allantoin from the desertliving cistanche by extraction and separation, and the chemical structure thereof is determined by analyzing the physical and chemical data and the spectral data. The allantoin is the component which is firstly discovered in the desertliving cistanche plant, as cistanche tubulosa does not contain the component, the allantoin can be used for identifying the characteristic of the cistanche tubulosa and other desertliving cistanches; adopting the yeast double-hybrid system test of the molecular biology simultaneously, the new medical usage of the allantoin for lowering the blood lipid is determined.
Description
The application is that application number is dividing an application of 200510072549.2 patents of invention.The invention and created name of original application is " preparation allantoin and allantoin extract and their application from saline cistanche ", and the applying date is on May 12nd, 2005.
Technical field
The present invention relates to a kind of extraction separation and structure authenticate technology of Chinese herbal medicine chemical constitution; And relate to natural allantoin or allantoin extract medical usage at aspects such as skin care, reducing blood lipid; The present invention also can be applicable to Cistanche tubulosa powder and the qualitative difference of other saline cistanche powder in the saline cistanche.
Background technology
Over nearly one, 20 year; Both at home and abroad more than the report of the chemical constitution of relevant saline cistanche the pieces of writing existing up to a hundred; The compound quantity of only from Desert Herba Cistanches, getting just reach 69 (Liu Guojun chief editor. saline cistanche and artificial growth thereof. Chinese work social security publishing house; 2003.120), in the saline cistanche of other kind, also separate obtaining more than 40 compound.Wherein most of composition is consistent with Desert Herba Cistanches, the visible KobayashiH of relevant report, et al.ChemPharm.Bull.1984,32 (5); 1984,32 (8); 1985,33 (9); Documents such as 1987,35 (8).Through document Investigation, in the compound that the saline cistanche platymiscium has been reported, do not see the report of allantoin, be the compound of finding first during saline cistanche belongs to so from saline cistanche, separate the allantoin that obtains.
Allantoin (Allantoin) is also claimed urea groups glycolylurea (Ureidehydantoin).It has the effect of skin care, can increase the wettability power of skin keratin cell adhesion matter, owing to can absorb more water, makes keratoprotein dispersion, scales of skin that peel off absent-mindedness, comes off the smooth pliable thereby skin becomes.Have local anesthetic action in addition, abirritation is played in pessimal stimulation, and the effect of the skin wound healing of promotion is arranged.Be used for clinically that dry skin is chapped, skin ulcer, hand dermatitis etc., also orally-ingestible is used for gastric and duodenal ulcer.It also has the effect of alleviating cancer ascites indivedual bibliographical informations.
Do not see that allantois have the report of effect for reducing blood fat.
At present the allantoin of usefulness is synthetic article clinically, and allantoin that obtains with the pure natural extraction or the extract that contains allantoin are not used in cosmetics or treating skin disease agent as yet.The assay value of allantoin is approximately about 0.5～0.8% from tame salt raw meat desert cistanche.
Summary of the invention
Content of the present invention comprises with macroreticular resin and the natural allantoin of Activated Carbon Adsorption Separation or contains the method for allantoin extract; The physicochemical property of gained allantoin and structural confirmation; Contain the preparation of the senior face cream of natural allantoin extract; The effect for reducing blood fat of allantoin; The application of allantoin on saline cistanche platymiscium chemotaxonomy.Below in conjunction with embodiment the present invention is done further detailed description.But do not limit the present invention with this.
Embodiment
Embodiment 1: be feedstock production allantoin extract or allantoin with the saline cistanche
Salt raw meat desert cistanche Cistanche salsa (C.A.Mey) G.Beck 160g; To extract 2 times under 80 ℃ of conditions of 10 times, 8 times water gaging heating, merge extract respectively, be evaporated to thick paste shape (50 ℃ of density are about 1.1g/ml); Add ethanol to containing alcohol amount 60%; Hold over night, pure liquid concentrates, and is dissolved in water.Water liquid is through macroporous absorbent resin SP
825Post (the about 500ml of volume) with 3 times of bed volume water elutions, is collected the stream part that wherein contains allantoin; Through granular activated carbon (about 300g), with the water elution of 3 times of bed volumes, use 3 times of bed volume 30% ethanol elutions more earlier again; Collection contains stream part of allantoin; Be evaporated to thick paste, get the allantoin extract through freeze drying again, can get colourless crystallization shape allantoin with ethyl alcohol recrystallization.
Embodiment 2: the structural confirmation of allantoin
By the crystallization of embodiment 1 gained, mp243～245 ℃, soluble in water and rare alcohol, the alkaloid reaction is positive.IR (KBr) cm
-1: 3437,3344 (NH
2); 3207,3062 (NH); 1779,1568 (C=O); 1602,1528,1430,1183 (C-N); MS:158 (M
+) point out molecular structure that even number N is arranged;
1H-NMR (DMSO-d
6) δ ppm:5.25 (1H, d, J=8.1Hz, H-4), 5.76 (2H, s, H-8), 6.86 (1H, d, J=8.1Hz, H-6), 8.03 (1H, s, H-3), 10.51 (1H, s, H-1);
13C-NMR (DMSO-d
6) δ ppm:62.6 (C-4), 156.9 (C-2), 157.5 (C-7), 173.7 (C-5).Sample introduction is in the HPLC appearance simultaneously with synthetic allantoin, and its retention time is consistent.Structural formula is following:
Embodiment 3: a kind of preparation of natural allantoin face cream
Prescription:
Stearic acid Acidi Stearici 100 grams
Cetanol Cetylii Alcoholis 45 grams
Sheep oil Adeps Lanae 10 grams
150 milliliters of propylene glycol Glycolis Propylenici
Lauryl sodium sulfate Natrii Laurylis Sulfatis 5 grams
Triethanolamine Triaethanolamini 7 grams
Vitamin E Vit E 0.5 gram
Natural allantoin Allantoin 10 grams
Or allantoin extract or Allantoin extract is equivalent to the extract of 10 gram allantoins
Soluble perfume Esssenlce is an amount of
Deionized water adds to Aquae and adds to 1000 grams
Method for making:
1. getting stearic acid, cetanol, sheep oil, vitamin E places proper container to heat 70～80 ℃;
2. get in another appropriate containers of propylene glycol, lauryl sodium sulfate, triethanolamine, soluble perfume and water and heat 70～80 ℃.Behind the two liquid isothermals, fluid is added in the entry liquid with thread slowly, get with adding with stirring to solidifying promptly;
Purposes: the skin care anticracking, alleviate skin aging.
Embodiment 4: the application of allantoin on saline cistanche platymiscium chemotaxonomy
The saline cistanche platymiscium mainly comprises Cistanche tubulosa, salt raw meat desert cistanche and Desert Herba Cistanches.Wherein Cistanche tubulosa accounts for the over half of medicinal material market, Japanese health ministry expressly provided Cistanche tubulosa list in Japan plant health food scope within.Method can be distinguished Cistanche tubulosa and other saline cistanche below using.
Method: get Cistanche tubulosa, each 0.2 gram of salt raw meat desert cistanche (or Desert Herba Cistanches) powder respectively, put in the 25ml measuring bottle, add an amount of ultrasonic Extraction of 20% methyl alcohol 30 minutes; Add 20% methyl alcohol to scale; Shake up, miillpore filter (0.45 μ m) filters, as need testing solution.Other gets allantoin reference substance 1mg, puts in the 10ml measuring bottle, adds 20% dissolve with methanol and is added into scale, as reference substance solution.(" using octadecylsilane chemically bonded silica is filling agent (this experiment use Symmetry ShieldR for one one of Chinese pharmacopoeia version in 2005, appendix (VID) test according to high performance liquid chromatography
P8 chromatographic column 4.6mm * 150mm, 5 μ m), acetonitrile-water (1: 99) is a moving phase, detects wavelength 224nm, flow velocity 0.5ml/min, and 25 ℃ of column temperatures, accurate respectively the absorption supplies examination solution and each 5 μ l of contrast solution, injects liquid chromatograph, the record chromatogram.In the HPLC chromatogram of salt raw meat desert cistanche (or Desert Herba Cistanches); With the corresponding position of reference substance allantoin chromatogram on; Show the identical chromatographic peak of retention time; And in the HPLC chromatogram of Cistanche tubulosa test sample, no allantoin reference substance peak, the saline cistanche that prompting does not contain allantoin possibly be Cistanche tubulosa.
The effect for reducing blood fat of embodiment 5 allantoins
Invention realizes through following method: yeast two-hybrid method (yeast two-hybrid) is the method for in cell, studying protein interaction that developed recently gets up; We select for use Clontech company to be able to GAL4 is experimental system for basic two-hybrid system III; Usefulness accounts for the apoAI of rat HDL65% and the Full-length cDNA Construction of HDL acceptor SR-BI becomes corresponding Yeast expression carrier; To have interactional power between the expressed beta galactosidase enzyme of reporter gene apoAI that lived detection by quantitative and SR-BI; If a certain compound promoted this interaction to albumen; Just must improve the activity of reporter gene, promptly detect the rising of reporter gene vigor and just explain that this compound possibly promote this interaction to albumen, has certain effect for reducing blood fat.It is research object that the present invention's report separates the natural allantoin that obtains with extraction from the saline cistanche medicinal material, from the molecular biology research angle, disclose it and can strengthen the interaction between apoAI and the SR-BI, thus the blood fat reducing function of prompting allantoin.
Experimental technique:
The inoculation: cotransformation apo-AI and the AH109 bacterial strain of SR-BI or the dliploid bacterial strain of mating;
2. add medicine to be measured in the nutrient culture media respectively, cultivate under the same conditions;
3. measure OD
600Judging the toxicity of medicine, is blank when little (have only could with this system detect the yeast strain growth effect) with the cotransformation bacterial strain that does not add the medicine component.
4. the vigor detection method of the sweet enzyme of beta galactose:
With nitrobenzene galactopyranose (ONPG) is that the fluid analysis method of substrate detects vigor that the vigor of beta galactosidase can record the yeast expressed beta galactosidase that contains pGBADT7-SR-BI, pGBKBT7-apoAI plasmid about 20-80U.Concrete grammar is following:
1) adding contains pGBADT7-SR-BI, pGBKBT7-apoAI plasmid bacterial plaque in the SD/-Leu/-Trp/-His/-Ade fluid nutrient medium, shakes (250rpm) 24-30h activated spawn at 30 ℃.
2) take out bacterium liquid 20-50 μ l and mix back 30 ℃ in the 5mlYPDA nutrient culture media and shake (250rpm) 16-18h, at this moment, OD
600=0.4-1.2.
3) get 0.5-1.5ml bacterium liquid in the 1.5ml centrifuge tube, centrifugal after, clean once with Z-buffer, centrifugal again, abandon supernatant.
4) use the 0.1mlZ-buffer re-suspended cell again.
5) cracking more than 5 times in liquid nitrogen.
6) 30 ℃ of insulations were clocked to the solution flavescence after adding 0.7ml Z-buffer dredged basic ethanol, 0.16ml ONPG (4mg/ml) mixing.
7) adding 0.4ml 1M Na
2CO
3The centrifugal 10min of 14000rpm behind the mixing.
8) with supernatant colorimetric estimation OD
420Value, the vigor of calculating beta galactosidase.
Computing formula is following:
The beta galactosidase energy value
[1]=1000 * OD
420/ (t * V * OD
600) the time V=0.1ml * enrichment factor OD of t=reaction beginning the to the solution flavescence
600=600nm light absorption value OD
420=420nm light absorption value
[1]Miller，J.H.In?a?short?course?in?bacterial?genetics.Cold?spring?harbor?laboratory?press，cold?spring?harbor；p74
Test sample is to the influence of reporter gene beta galactosidase, thereby judges that it is to existing interactional influence between apolipoprotein AI (apoAI) and scavenger receptor (SR-BI).
5. experimental result:
Table 1 beta galactosidase energy value
| Sample | Concentration (mg/ml) | Beta galactosidase energy value (n=3) |
| Allantoin ginkgo blade is blank | 0.052 0.104 0.396 0.792 | 46.39±1.41 54.71±0.78 46.74±1.86 55.16±1.97 40.67±1.20 |
Blank control group sample n=3, sample sets sample n=9, degree of freedom v=10
Annotate: blank for not adding sample, add and sample same amount solvent.
Check through t; The allantoin group is 0.052, under two concentration of 0.104mg/ml; The Folium Ginkgo group is compared with blank control group respectively 0.396, under two concentration of 0.792mg/ml, and the t value is all greater than 4.587; The beta galactosidase energy value difference that shows allantoin group, Folium Ginkgo group and blank control group has statistical significance, p＜0.001 (t
0.001,10=4.587).
Adopting the yeast strain AH109-030613 carry rat apolipoprotein apo-AI and scavenger receptor SR-BI gene plasmid in the test process is test strain, in blank, added and test specimens solution equivalent solvent with the deduction solvent to interactional influence between rat apolipoprotein apo-AI and the scavenger receptor SR-BI.
Beta galactosidase energy value from table 1 can find out when allantoin is 0.052mg/ml, 0.104mg/ml in concentration, interactional effect between the apo-AI of enhancing and the SR-BI is arranged, and prompting has certain effect for reducing fat.
Claims (1)
1. a method of differentiating saline cistanche platymiscium kind is an object with the allantoin, Cistanche tubulosa and other kind during the difference saline cistanche belongs to.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008100091433A CN101271089B (en) | 2005-05-12 | 2005-05-12 | Method for identifying allantoin in Cistanche plant variety |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008100091433A CN101271089B (en) | 2005-05-12 | 2005-05-12 | Method for identifying allantoin in Cistanche plant variety |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2005100725492A Division CN100402505C (en) | 2005-05-12 | 2005-05-12 | Preparation of allantoin and allantoin extract from cistanche salsa and their use thereof |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011103235560A Division CN102406638A (en) | 2005-05-12 | 2005-05-12 | Single application of allantoin as active ingredient or application thereof |
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| CN101271089A CN101271089A (en) | 2008-09-24 |
| CN101271089B true CN101271089B (en) | 2012-07-25 |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105121457B (en) * | 2013-02-22 | 2019-04-30 | 新加坡科技研究局 | For removing endotoxic material and method from protein formulation |
| CN108607000A (en) * | 2018-05-30 | 2018-10-02 | 新疆医科大学第附属医院 | The application of allantois extract and allantoin monomer in preparing blood lipid-lowering medicine |
| CN113234020A (en) * | 2020-03-24 | 2021-08-10 | 南京益唯森生物科技有限公司 | Process for extracting allantoin from cistanche |
| CN114470868B (en) * | 2022-02-22 | 2023-04-25 | 瑞昌市渝瑞实业有限公司 | System for separating allantoin from yam mucus |
Citations (4)
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| CN1379036A (en) * | 2002-05-10 | 2002-11-13 | 北京大学药学院 | Phenylethanol boschnaloside compounds |
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| CN1443476A (en) * | 2003-03-05 | 2003-09-24 | 中国农业大学 | Chinese yam product and its preparation method |
| CN1548138A (en) * | 2003-05-19 | 2004-11-24 | 广东明珠药业有限公司 | Chinese medicine composition for treating consumptive disease and its quality control method |
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| 吴凤薇 |
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