CN1861581A - Preparation of allantoin and allantoin extract from cistanche salsa and their use thereof - Google Patents

Abstract

An allantoin and the component containing said allantoin are extracted from cistanche through separation with macroreticular adsorption resin and granular activated carbon. Said allantoin can be used to decrease blood fat.

Description

Preparation wallantoin and wallantoin extract and their application from Herba Cistanches

Technical field

The present invention relates to a kind of extraction separation and structure authenticate technology of herbal medicine chemical ingredients; And relate to natural wallantoin or wallantoin extract medical usage at aspects such as skin care, reducing blood-fat; The present invention also can be applicable to Cistanche Tubulosa powder and the qualitative difference of other Herba Cistanches powder in the Herba Cistanches.

Background technology

Over nearly one, 20 year, both at home and abroad more than the report of the chemical ingredients of relevant Herba Cistanches 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. Herba Cistanches and artificial growth thereof. Chinese work social security press, 2003.120), in the Herba Cistanches 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).By document Investigation, in the compound that Cistanche Hoffmgg. et Link plants has been reported, do not see the report of wallantoin, be the compound of finding first during Herba Cistanches belongs to so from Herba Cistanches, separate the wallantoin that obtains.

Wallantoin (Allantion) also claims 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, mitigate effects is played in pessimal stimulation, and the effect of the skin wound healing of promotion is arranged.Be used for clinically that xerosis cutis is chapped, skin ulcer, hand dermatitis etc., also can be for oral use in 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 reducing blood lipid.

At present the wallantoin of usefulness is synthetic product clinically, and wallantoin that obtains with the pure natural extraction or the extract that contains wallantoin are not used in makeup or skin diseases treatment agent as yet.The assay value of wallantoin is approximately about 0.5～0.8% from tame Saline Cistanche Herb.

Summary of the invention

Content of the present invention comprises with macroporous resin and the natural wallantoin of Activated Carbon Adsorption Separation or contains the method for wallantoin extract; The physico-chemical property of gained wallantoin and structural confirmation; Contain the preparation of the senior protective skin cream of natural wallantoin extract; The reducing blood lipid of wallantoin; The application of wallantoin on the Cistanche Hoffmgg. et Link plants chemotaxonomy.Below in conjunction with embodiment the present invention is described in further detail.But do not limit the present invention with this.

Embodiment

Embodiment 1: be feedstock production wallantoin extract or wallantoin with the Herba Cistanches

Saline Cistanche Herb Cistanche salsa (C.A.Mey) G.Beck 160g, respectively to extract 2 times under 80 ℃ of conditions of 10 times, 8 times water gaging heating, united extraction liquid, be evaporated to thick paste shape (50 ℃ of density are about 1.1g/ml), add ethanol to containing alcohol amount 60%, standing over night, pure liquid concentrates, and is dissolved in water.Water liquid is by macroporous adsorbent resin SP ₈₂₅Post (the about 500ml of volume); with 3 times of column volume water elutions; collect the stream part that wherein contains wallantoin; by granulated active carbon (about 300g), with the water elution of 3 times of column volumes, use 3 times of column volume 30% ethanol elutions more earlier again; collection contains stream part of wallantoin; be evaporated to thick paste, get the wallantoin extract through lyophilize again, can get colourless crystallization shape wallantoin with ethyl alcohol recrystallization.

Embodiment 2: the structural confirmation of wallantoin

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).With the synthetic wallantoin simultaneously sample introduction in the HPLC instrument, its retention time unanimity.Structural formula is as follows:

Embodiment 3: a kind of preparation of natural wallantoin protective skin cream

Prescription:

Stearic acid Acidi Stearici 100 grams

Hexadecanol Cetylii Alcoholis 45 grams

Lanolin Adeps Lanae 10 grams

150 milliliters of propylene glycol Glycolis Propylenici

Sodium lauryl sulphate Natrii Laurylis Sulfatis 5 grams

Trolamine Triaethanolamini 7 grams

Vitamin-E Vit E 0.5 gram

Natural wallantoin Allantoin 10 grams

Or wallantoin extract or Allantoin extract is equivalent to the extract of 10 gram wallantoins

Soluble perfume Esssenlce is an amount of

Deionized water adds to Aquae and adds to 1000 grams

Method for making:

1. getting stearic acid, hexadecanol, lanolin, vitamin-E places proper container to heat 70～80 ℃;

2. get in another appropriate containers of propylene glycol, sodium lauryl sulphate, trolamine, soluble perfume and water and heat 70～80 ℃.Behind the two liquid isothermals, fluid is added in the entry liquid with thread slowly, with adding with stirring to solidifying promptly;

Purposes: the skin care anticracking, alleviate skin aging.

Embodiment 4: the application of wallantoin on the Cistanche Hoffmgg. et Link plants chemotaxonomy

Cistanche Hoffmgg. et Link plants mainly comprises Cistanche Tubulosa, Saline Cistanche Herb 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 Herba Cistanches below using.

Method: get Cistanche Tubulosa, each 0.2 gram of Saline Cistanche Herb (or Desert Herba Cistanches) powder respectively, put in the 25ml measuring bottle, add an amount of supersound extraction of 20% methyl alcohol 30 minutes, add 20% methyl alcohol to scale, shake up, millipore filtration (0.45 μ m) filters, as need testing solution.Other gets wallantoin reference substance 1mg, puts in the 10ml measuring bottle, adds 20% dissolve with methanol and is added into scale, in contrast product solution.According to high performance liquid chromatography (" one one of Chinese pharmacopoeia version in 2005, appendix (VID) test, with octadecylsilane chemically bonded silica is weighting agent (Symmetry ShieldRP8 chromatographic column 4.6mm * 150mm, 5 μ m are used in this experiment), and acetonitrile-water (1: 99) is a moving phase, detect wavelength 224nm, flow velocity 0.5ml/min, 25 ℃ of column temperatures, accurate respectively test solution and each 5 μ l of contrast solution of drawing, inject liquid chromatograph, the record color atlas.In the HPLC color atlas of Saline Cistanche Herb (or Desert Herba Cistanches), with the corresponding position of reference substance wallantoin chromatogram on, show the identical chromatographic peak of retention time, and in the HPLC color atlas of Cistanche Tubulosa trial-product, no wallantoin reference substance peak, the Herba Cistanches that prompting does not contain wallantoin may be Cistanche Tubulosa.

The reducing blood lipid of embodiment 5 wallantoins

Invention realizes by the following method: yeast two-hybrid method (yeast two-hybrid) is the method for studying protein interaction in cell 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, Full-length cDNA Construction with apoAI that accounts for rat HDL65% and HDL acceptor SR-BI becomes corresponding Yeast expression carrier, to have interactional power between the expressed beta-galactosidase enzymes enzyme of reporter gene apoAI that lived detection by quantitative and SR-BI, if a certain compound promoted this to proteic interaction, just must improve the activity of reporter gene, promptly detect the rising of reporter gene vigor and just illustrate that this compound may promote this to proteic interaction, has certain reducing blood lipid.The natural wallantoin that the present invention report obtains with extraction separation from the Herba Cistanches medicinal material is a research object, from the molecular biology research angle, discloses it and can strengthen interaction between apoAI and the SR-BI, thus the blood fat reducing function of prompting wallantoin.

Experimental technique:

The inoculation: cotransformation apo-AI and the AH109 bacterial strain of SR-BI or the diploid bacterial strain of mating;

2. add medicine to be measured in the substratum 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 oil of mirbane galactopyranose (ONPG) is that the vigor that the fluid analysis method of substrate detects beta-galactosidase enzymes can record the vigor of the yeast expressed beta-galactosidase enzymes that contains pGBADT7-SR-BI, pGBKBT7-apoAI plasmid about 20-80U.Concrete grammar is as follows:

1) adding contains pGBADT7-SR-BI, pGBKBT7-apoAI plasmid bacterial plaque in the SD/-Leu/-Trp/-His/-Ade liquid 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 substratum 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 enzymes.

Calculation formula is as follows:

The beta-galactosidase enzymes energy value ^[1]=1000 * OD ₄₂₀/ (t * V * OD ₆₀₀)

The t=reaction begins the time V=0.1ml * enrichment factor OD 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 enzymes, 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 enzymes energy value (x)

Sample	Concentration (mg/ml)	Beta-galactosidase enzymes energy value (n=3)
			Wallantoin ginkgo blade 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 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 wallantoin group is 0.052, under two concentration of 0.104mg/ml, the puerarin group is 0.396, under two concentration of 0.792mg/ml, compare with the blank group respectively, the t value is all greater than 4.587, show that wallantoin group, puerarin group have statistical significance, p＜0.001 (t with the beta-galactosidase enzymes energy value difference of blank group _0.001,10=4.587).

Adopting the yeast strain AH109-030613 carry rat lipophorin 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 lipophorin apo-AI and the scavenger receptor SR-BI.

Beta-galactosidase enzymes energy value from table 1 can find out when wallantoin 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 (4)

1. the present invention relates to a natural compounds or contain the extract of this compound.Its preparation process comprises: be raw material with the Herba Cistanches; alcohol-water mixed solvent through water or any ratio extracts; extracting solution is through concentrating; add behind the ethanol sedimentation impurity or and directly separate by macroporous adsorbent resin without ethanol sedimentation impurity; collect the water elution part; the component that wherein contains wallantoin is again by the granulated active carbon fractionation by adsorption; earlier with water elution; again with 10～50% ethanol elution; collection contains stream part of wallantoin; lyophilize must contain the extract of wallantoin, gets wallantoin with ethanol or recrystallizing methanol.

2. as the right 1 described natural wallantoin that contains the wallantoin extract or derive from Herba Cistanches, make an addition in protective skin cream, skin care ointment or the health-care nutrient liquor with any ratio.

3. as right 1 described wallantoin extract or the wallantoin of containing, application or other lipopenicillinase composition combined utilization are in the medical treatment or health-care preparation of reducing blood-fat separately.

4. with the wallantoin identification reagent, Cistanche Tubulosa and other kind during the difference Herba Cistanches belongs to.