CN100447129C - Oximated ginger phenol and its synthesis and use - Google Patents
Oximated ginger phenol and its synthesis and use Download PDFInfo
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- CN100447129C CN100447129C CNB2005100329753A CN200510032975A CN100447129C CN 100447129 C CN100447129 C CN 100447129C CN B2005100329753 A CNB2005100329753 A CN B2005100329753A CN 200510032975 A CN200510032975 A CN 200510032975A CN 100447129 C CN100447129 C CN 100447129C
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Abstract
The present invention belongs to the field of the preparation, modification and application of natural products, [and]more specifically relates to a gingerol derivant called a gingerol oxime, a synthetic method thereof and an application thereof. The gingerol oxime is the gingerol derivant produced through oximation reaction of gingerol crude extract or purified gingerol. In the present invention, the semi-product of fresh ginger or ginger (such as dried ginger, oleoresin ginger, etc.) is used as raw materials to produce coarse gingerol, or the gingerol oxime in a crystalline state are obtained by oximation reaction, separation and purification by using pure gingerol, and the bioactivity of antioxidation, antivirus, anti-tumor activity, disinsection, etc. is detected, so that gingerol oximes can be applied to the technical field. Gingerol oximes can be used as a substitute of gingerol, and can be used in the development of novel products such as novel medicine, food additives, green pesticide, etc.
Description
Technical field
The invention belongs to the producing of natural product, modification and Application Areas, specifically utilize the thick gingerol or the pure gingerol that extract in the ginger, produce gingerol oxime, and measure the biological activitys such as anti-oxidant, antiviral, antitumor, desinsection of gingerol oxime by oximation reaction.
Background technology
The eighties in 20th century, external pharmacological testing is verified, the thick gingerol of isolated or chemosynthesis can stimulating mucosal in ginger, promote gastric secretion, in enteron aisle, can suppress abnormal fermentation, promote gaseous emission, pallium and vasomotor center are had excitation, can promote blood circulation.Up-to-date pharmacological evaluation and clinical observation confirm: many-sided biological activitys such as gingerol has cardiac stimulant, reducing blood-fat, prevents and treats cardiovascular disorder, anti-oxidant, anti-ageing, antitumor, preventing or arresting vomiting, relieving fainting, inhibition prostaglandin(PG) are synthetic, anticorrosion desinsection, expelling parasite, cosmetology.
Yet, the natural thick gingerol poor stability that in ginger, obtains, be difficult to separation and purification, be difficult for obtaining, inconvenience is preserved; In addition, the report that does not also have industrialization chemosynthesis gingerol at present.In a word, owing to there is not the gingerol that can use for industry, can't obtain a large amount of gingerols at present is food, protective foods, medicine, makeup, environment friendly agricultural of raw material or the like in order to develop with the gingerol.Therefore, for making full use of, develop the biological activity of gingerol, and the present no gingerol of solution can be for the practical difficulty that utilizes, develop the gingerol derivatives similar such as chemical structure, biological activity, functional property to gingerol, to replace gingerol, can obtain great economic benefit and social benefit as gingerol oxime etc.
Summary of the invention
The object of the present invention is to provide a kind of gingerol derivative---gingerol oxime, can replace the gingerol of present natural extract, develop products such as being similar to the bioactive medicine of gingerol, food, agricultural chemicals, reagent standard specimen.
Gingerol oxime of the present invention, be with gingerol crude extract or pure gingerol by the gingerol derivative that oximation reaction obtains, have following formula:
Wherein: n=4 is the 6-gingerol oxime; N=6 is the 8-gingerol oxime; N=8 is the 10-gingerol oxime; N=10 is the 12-gingerol oxime.
The invention provides the synthetic method of gingerol oxime, with and use.
The present invention is a raw material with fresh ginger or ginger work in-process (as rhizoma zingiberis, oleoresin ginger etc.), obtain thick gingerol, perhaps utilize pure gingerol, obtain the gingerol oxime of crystalline state again by oximation reaction, separation and purification, and measure biological activitys such as it is anti-oxidant, antiviral, antitumor, desinsection, thereby make gingerol oxime be applied to above-mentioned field.
The synthetic method of gingerol oxime of the present invention is characterized in that, may further comprise the steps:
A) oximation reaction: the gingerol with gingerol crude extract or purification is a raw material, add 1～3 times of mole number to the oxammonium hydrochloride of gingerol, be under 3.5～5.5 the condition at 0～100 ℃, pH, carry out oximation reaction, in 1～3 hour reaction times, the solvent that reaction is used is selected from a kind of in 10～100% methyl alcohol, ethanol, ether, the ethyl acetate;
B) produce thick gingerol oxime: oximation reaction liquid is concentrated into 10～50% of original volume, uses organic solvent extraction 2～3 times; Institute's organic solution that obtains is successively washed, is washed through overpickling, salt mutually, repeats this process 1～3 time; Reclaim solvent again, get thick gingerol oxime; Described organic solvent is selected from ether, ethyl acetate, normal hexane;
C) rough segmentation of gingerol oxime: carry out chromatographic separation with sorbent material as stationary phase, elutriant through concentrate reclaim thick gingerol oxime;
Among the step C, described sorbent material is a silica gel, when silica gel chromatography separates, collects normal hexane and ether volume ratio and be 1: 0.5～2 wash-out part.
Among the step C, described sorbent material is a polymeric amide, during the polymeric amide chromatographic separation, collects 20%～60% methyl alcohol, ethanol or acetone wash-out part.
Among the step C, described sorbent material is a macroporous resin, during the macroporous resin chromatographic separation, collects 20%～50% methyl alcohol, ethanol or acetone wash-out part.
Behind step C, the gingerol oxime concentrated solution is left standstill crystallization under 0～40 ℃, can obtain the coarse crystallization gingerol oxime.
D) segmentation of gingerol oxime: use dextrane gel as stationary phase, with Virahol: chloroform=1～8: 1, for moving phase is carried out continuous wash-out, collect Virahol respectively: chloroform is 1.5: 1 to 5: 1 a wash-out part, after removing moving phase, get 6-gingerol oxime, 8-gingerol oxime, 10-gingerol oxime, 12-gingerol oxime respectively.
The raw material of oximation reaction---gingerol crude extract prepares approach from following two kinds:
(1) described gingerol crude extract is the thick gingerol of natural extract, prepares by following steps:
1) raw material is handled: fresh ginger is cleaned earth, be cut into shredded ginger or thin ginger splices, dry, wear into 100 order ginger powder then in air seasoning place, and stand-by, or directly the dried ginger slice that seasoning obtained is ground into 100 order ginger powder, stand-by;
2) obtain the gingerol extraction liquid: with 40%～100% organic solvent extraction ginger powder, adopt measures such as solvent cycle or diacolation between soak period, to improve the percentage extraction of gingerol, soak time is no less than 12 hours; Every batch of ginger powder extracts 3～5 times; Used extraction liquid is collected in supplies next step usefulness together; Described organic solvent is selected from a kind of of ethanol, methyl alcohol, acetone, ethyl acetate;
In the described step 2, can adopt percolation to obtain the gingerol extraction liquid, be about to ginger powder dress post, make 40%～100% organic solvent, obtain the gingerol extraction liquid slowly by Jiang Fenzhu; Described organic solvent is selected from a kind of of ethanol, methyl alcohol, acetone, ethyl acetate, normal hexane.
3) reclaim solvent: under 40～70 ℃, the gingerol extraction liquid of distillation or fractionating step 2 gained reclaims solvent, gets the gingerol crude extract;
4) extracting and separating gingerol crude extract: in the gingerol crude extract, add the normal hexane of 0.5～1 part of volume, extraction gingerol, continuous extraction 2～5 times; After merging normal hexane extraction liquid, reclaim 75～90% normal hexanes, resistates is used isopyknic extracted with diethyl ether gingerol again, and continuous extraction 2～3 times gets thick gingerol behind the recovery ether.
(2) described gingerol crude extract is various types of oleoresin gingers, prepares by following steps: the water-containing organic solvent with 20～80% extracts oleoresin ginger, fractionates out the ginger essential oil, gets the gingerol crude extract; Described organic solvent is selected from ethanol, methyl alcohol, acetone, ether.
Beneficial effect of the present invention is:
(1) develops a kind of gingerol derivative---gingerol oxime, be the gingerol derivatives similar such as a kind of chemical structure, biological activity, functional property to gingerol, can make full use of, develop the biological activity of gingerol, the gingerol that replaces present natural extract, develop products such as being similar to the bioactive medicine of gingerol, food, agricultural chemicals, reagent standard specimen, solving at present, no gingerol can be expected to obtain great economic benefit and social benefit for the practical difficulty that utilizes.
(2) provide gingerol oxime synthetic method feasible, that yield is high, easy to utilize, make the industrial application prospect of gingerol oxime wide.
(3) the present invention's gingerol that will be difficult to obtain at present makes that modification is easy to get, the similar gingerol oxime of biological activity, for other natural products that are difficult to obtain provide a kind of feasible solution thinking.
The present invention also provides gingerol oxime to be used to prepare the purposes that preventing respiratory closes kitchen virus drugs, antitumor drug, and the purposes that is used to prepare antioxidant, sterilant.
Application of the present invention is based on following gingerol oxime biological activity determination:
(1) estimates anti-oxidant activity with hemolytic action: suppress the size that hemolytic action that the oxidation of cytolemma material causes is estimated its resistance of oxidation by gingerol oxime.
(2) evaluation of lipoid peroxidization resistant: the size that promptly reduces its antioxygenation of merit rating of fatty superoxide formation by gingerol oxime.
(3) mensuration of killing ability: take in its insecticidal action of effect assessment that gingerol oxime causes death by measuring cotton lamp worm (Spilosoma boiqua).
(4) mensuration of anti respiratory syncytial virus (RSV) ability: the effect that suppresses its anti-RSV of vigor effect assessment of infection RSV cell by gingerol oxime.
(5) mensuration of anti-tumor activity: estimate its anti-tumor activity by measuring the viability that suppresses tumour cell, measurement result shows that it is suitable with gingerol.
The measurement result of above index sees Table 1.
Table 1: the biological activity of gingerol oxime
-1
Annotate:
1) IC of sample
50All in 6-gingerol oxime (the 6-gingerol oxime in the sample, 8-gingerol oxime, 10-gingerol oxime, 12-gingerol oxime content are respectively 58%, 15%, 22%, 3%);
2) IC
50The expression inhibiting rate is 50% o'clock a dosage;
3) reference substance is the 6-gingerol.
Said determination is the result show: compare with the 6-gingerol, the biological activity of gingerol oxime with it quite or slightly high: its antioxygenation, antitumor and insecticidal action are higher than gingerol, but anti-RSV effect is slightly less than gingerol.
Description of drawings
Fig. 1 is preparation technology's schema of crystallization gingerol oxime.
Fig. 2 is the UV, visible light scan light spectrogram of crystallization gingerol oxime, condition determination: methyl alcohol; Instrument: UnicamUV504, Britain Thermo Spectronic company produces.
Fig. 3 is the infrared spectrogram of crystallization gingerol oxime, the German Bruker Equinox of company 55 type Fourier transformation infrared spectrometers, KBr compressing tablet.
Fig. 4 is the mass spectrum of 6-gingerol oxime, Finnigan MAT TSQ-70 mass spectrograph.
Fig. 5 is the mass spectrum of 8-gingerol oxime, Finnigan MAT TSQ-70 mass spectrograph.
Fig. 6 is the mass spectrum of 10-gingerol oxime, Finnigan MAT TSQ-70 mass spectrograph.
Fig. 7 is the mass spectrum of 12-gingerol oxime, Finnigan MAT TSQ-70 mass spectrograph.
Embodiment
Embodiment one:
The first step, produce the gingerol crude extract: according to shown in Figure 1, take by weighing 1 kilogram of fresh ginger, soak half an hour, take out impurity such as earth, drench the solid carbon dioxide branch, dry, be cut into 2～4 millimeters thin slice, dry in the sun with tap water.Put in the pulverizer and pulverize, cross 100 mesh sieves.According to the ginger powder: the ratio of 40% alcohol=1: 3～6 (volume/volume), the ginger powder is joined in the alcohol, soaked 24 hours, between soak period, stirred once every 2 hours; Repeat to soak three times, merge alcohol-pickled liquid.Concentrate recovery ethanol down at 65 ℃.Resistates is with isopyknic n-hexane extraction three times, reclaim the normal hexane of about 3/4 volume after, resistates is used 1: 1 extracted with diethyl ether gingerol again, continuous extraction three times, reclaim behind the ether thick gingerol extract 60 grams.
Second step, oximation reaction: with thick gingerol extract 10 grams (containing gingerol about 14%), be dissolved in an amount of 10% ethanolic soln, add 1.5 gram oxammonium hydrochlorides, regulate pH value 5.5,100 ℃ of following stirring reactions 1 hour.
The processing of the 3rd step, oximation reaction liquid: after reaction finishes, boil off 90% ethanol, use ethyl acetate extraction resistates 3 times, wash with 1M hydrochloric acid, 1M salt solution, deionized water again, repeat this process three times, reclaim ethyl acetate; Resistates separates as next step silicagel column.
The 4th the step, the silicagel column rough segmentation: silicagel column on the resistates, collecting normal hexane and ether volume ratio is 1: 0.5～2 wash-out parts, get final product the gingerol oxime coarse crystallization.Gingerol oxime coarse crystallization efficiency of pcr product is 10% (in thick gingerol extract).
The 5th step, sephadex column segmentation: get 1 gram gingerol oxime coarse crystallization, be dissolved in methyl alcohol, be splined on sephadex column, use Virahol: chloroform mixed solvent wash-out, collect Virahol: chloroform=1.5～5: 1 wash-out part, room temperature leaves standstill, obtain the 6-gingerol oxime respectively (as shown in Figure 4, the mass-to-charge ratio of 6-gingerol oxime is 309, the molecular weight that shows the 6-gingerol is 309), the 8-gingerol oxime (as shown in Figure 5, the mass-to-charge ratio of 8-gingerol oxime is 337, the molecular weight that shows the 8-gingerol is 337), 10-gingerol oxime (as shown in Figure 6, the mass-to-charge ratio of 10-gingerol oxime is 365, and the molecular weight that shows the 10-gingerol is 365), the 12-gingerol oxime (as shown in Figure 7, the mass-to-charge ratio of 12-gingerol oxime is 393, and the molecular weight that shows the 12-gingerol is 393); Their ultra-violet absorption spectrum as shown in Figure 2,227,282 absorption peaks show that gingerol oxime contains phenyl ring.Their infrared absorption spectrum as shown in Figure 3,3241cm-1 hydroxyl (O-H) stretching vibration peak, 2934cm-1 methylene radical (C-H) stretching vibration peak, 1672cm-1 hydroxyl (O-H) rotational vibration peak, the scissoring vibration peak of 1458cm-1 methylene radical (C-H), 1606 and 1515 phenyl ring charateristic avsorption bands.Its yield is respectively: 6-gingerol oxime 53%, 8-gingerol oxime 10%, 10-gingerol oxime 15%, 12-gingerol oxime 3% (in thick gingerol oxime).
Embodiment two:
Take by weighing 300g ginger powder, cross 100 mesh sieves.According to the ginger powder: the ratio of acetone=1: 1～3 (volume/volume), the ginger powder is joined in the acetone, soaked 12～24 hours, between soak period, stirred once every 2 hours; Repeat to soak five times, merge the acetone soak solution.Reclaim acetone at 70 ℃ of following vacuum concentration.Resistates is with the n-hexane extraction of 1/2 volume five times, reclaim the normal hexane of about 80% volume after, resistates is used 1: 1 extracted with diethyl ether gingerol again, continuous extraction twice, reclaim behind the ether thick gingerol extract 80 grams.
With thick gingerol extract 10 grams, be dissolved in an amount of 10% methanol solution, add 20 gram oxammonium hydrochlorides, regulate pH value 3.5,50 ℃ of following stirring reactions 2 hours.After reaction finishes, boil off an amount of methyl alcohol, oximation reaction liquid is concentrated into 50% of original volume, with extracted with diethyl ether resistates 2 times, wash with 1M hydrochloric acid, 1M salt solution, deionized water again, repeat this process three times, carry out the vacuum concentration drying again, behind the recovery ether, with 10 gram Silons absorption resistatess.
The rough segmentation of gingerol oxime: the Silon that will adsorb gingerol oxime is splined on the polymeric amide chromatographic column, reclaims through concentrating with 0～100% methyl alcohol (or ethanol, acetone) elutriant, collects 20%～60% methyl alcohol, ethanol or acetone wash-out part, thick gingerol oxime.The gingerol oxime concentrated solution is placed under 40 ℃, leaves standstill crystallization, can obtain the coarse crystallization gingerol oxime.Gingerol oxime coarse crystallization efficiency of pcr product is 15% (in thick gingerol extract).
The segmentation of gingerol oxime: get 1 gram gingerol oxime coarse crystallization, be dissolved in methyl alcohol, be splined on sephadex column, use Virahol: the mixed solvent of chloroform=4: 1 volume ratio is that moving phase is carried out chromatographic separation, collects Virahol: chloroform=1.5～5: 1 wash-out part, and room temperature leaves standstill, obtain 6-gingerol oxime (as Fig. 4) respectively, 8-gingerol oxime (as Fig. 5), 10-gingerol oxime (as Fig. 6), 12-gingerol oxime (as Fig. 7); Their ultraviolet and infrared absorption spectrum are respectively as shown in Figures 2 and 3.Its yield is respectively: 6-gingerol oxime 54%, 8-gingerol oxime 9%, 10-gingerol oxime 14%, 12-gingerol oxime 5% (in thick gingerol oxime).
Embodiment three:
Take by weighing rhizoma zingiberis 200 grams, put in the pulverizer and pulverize, cross 100 mesh sieves.According to the ginger powder: the ratio of methyl alcohol=1: 1～3 (volume/volume), the ginger powder is joined in the methyl alcohol, soaked 24 hours, after waiting to soak into, the pillar of packing into methyl alcohol diacolation slowly, is collected about 400 milliliters of percolate again.Reclaim methyl alcohol at 40 ℃ of following vacuum concentration, resistates is with isopyknic n-hexane extraction three times, reclaim the normal hexane of about 90% volume after, resistates is used 1: 1 extracted with diethyl ether gingerol again, continuous extraction three times, 40 ℃ get thick gingerol extract after reclaiming ether down.
Present embodiment adopts percolation to obtain the gingerol extraction liquid, and available ethanol, acetone, ethyl acetate, normal hexane instead of methanol are carried out diacolation, and its concentration range can be 40%～100%.
With thick gingerol extract 10 grams, be dissolved in an amount of diethyl ether solution, add 3 gram oxammonium hydrochlorides, regulate pH value 4.5,70 ℃ of following stirring reactions 2 hours.After reaction finishes, boil off 70% ether,, after the water washing desalination, reclaim normal hexane with n-hexane extraction resistates 3 times, get final product thick gingerol oxime crystallization.Efficiency of pcr product is 94% (in gingerol).
The rough segmentation of gingerol oxime: the Silon that will adsorb gingerol oxime is splined on macroporous resin chromatography, reclaims through concentrating with 0～100% methyl alcohol (or ethanol, acetone) elutriant, collects 20%～60% methyl alcohol, ethanol or acetone wash-out part, thick gingerol oxime.The gingerol oxime concentrated solution is placed under 0 ℃, leaves standstill crystallization, can obtain the coarse crystallization gingerol oxime.Gingerol oxime coarse crystallization efficiency of pcr product is 18% (in thick gingerol extract).
The segmentation of gingerol oxime: get 1 gram gingerol oxime coarse crystallization, be dissolved in 10～50% methyl alcohol, be splined on polyamide column, with the continuous wash-out of 0～95% ethanol, collect 20～60% ethanol elution part, room temperature leaves standstill, obtain 6-gingerol oxime (as Fig. 4) respectively, 8-gingerol oxime (as Fig. 5), 10-gingerol oxime (as Fig. 6), 12-gingerol oxime (as Fig. 7); Their ultraviolet and infrared absorption spectrum are respectively as shown in Figures 2 and 3.Its yield is respectively: 6-gingerol oxime 50%, 8-gingerol oxime 8%, 10-gingerol oxime 15%, 12-gingerol oxime 3% (in thick gingerol oxime).
Embodiment four:
Take by weighing 20 gram oleoresin gingers,, get thick gingerol extract behind the recovery methyl alcohol with 80% methanol extraction gingerol, continuous extraction three times, combining extraction liquid.
With thick gingerol extract 50 grams, be dissolved in an amount of 90% ethanolic soln, add 5 gram oxammonium hydrochlorides, regulate pH value 5,0 ℃ of following stirring reaction 3 hours.After reaction finishes, boil off ethanol, use ethyl acetate extraction resistates 3 times, after reclaiming ethyl acetate, with sephadex chromatography post on the resistates, with Virahol, chloroform (1～8: 1, V/V) be moving phase, collect Virahol, chloroform (3～5: 1) wash-out part, remove moving phase after, under room temperature, leave standstill elutriant, obtain crystalline 6-gingerol oxime respectively, the 8-gingerol oxime, 10-gingerol oxime, 12-gingerol oxime.The yield of 6-gingerol oxime, 8-gingerol oxime, 10-gingerol oxime, 12-gingerol oxime is respectively 11%, 5%, 9% and 1% (in oleoresin ginger).
Embodiment five:
Take by weighing 10 gram oleoresin gingers, the ether continuous extraction with 20% five times merges ether extraction liquid, gets thick gingerol extract behind the recovery ether.
With thick gingerol extract 50 grams, be dissolved in an amount of 50% ethyl acetate solution, add 5 gram oxammonium hydrochlorides, regulate pH value 5, at room temperature stirring reaction is 2 hours.After reaction finishes, boil off ethyl acetate, use ethyl acetate extraction resistates 3 times, after the recovery ethyl acetate, with macroporous adsorbent resin chromatography post on the resistates, with the continuous wash-out of 0～95% ethanol, collect 20～50% ethanol elution part, put in 0 ℃ of refrigerator and leave standstill, obtain 6-gingerol oxime (as Fig. 4) respectively, 8-gingerol oxime (as Fig. 5), 10-gingerol oxime (as Fig. 6), 12-gingerol oxime (as Fig. 7); Their ultraviolet and infrared absorption spectrum are respectively as shown in Figures 2 and 3.Its yield is respectively: 6-gingerol oxime 56%, 8-gingerol oxime 10%, 10-gingerol oxime 15%, 12-gingerol oxime 3% (in thick gingerol oxime).
Embodiment six:
Take by weighing 15 gram oleoresin gingers, with 50% acetone or alcohol extraction gingerol, continuous extraction three times, combining extraction liquid gets thick gingerol extract behind the recovery acetone or alcohol.
With thick gingerol extract 50 grams, be dissolved in an amount of 50% diethyl ether solution, add 5 gram oxammonium hydrochlorides, regulate pH value 5, at room temperature stirring reaction is 2 hours.After reaction finishes, boil off ethanol, use n-hexane extraction resistates 3 times, after reclaiming normal hexane, with sephadex chromatography post on the resistates, with Virahol, chloroform (1～8: 1, V/V) be moving phase, collect Virahol, chloroform (3～5: 1) wash-out part, remove moving phase after, under room temperature, leave standstill elutriant, obtain crystalline 6-gingerol oxime respectively, the 8-gingerol oxime, 10-gingerol oxime, 12-gingerol oxime.The yield of 6-gingerol oxime, 8-gingerol oxime, 10-gingerol oxime, 12-gingerol oxime is respectively 11%, 5%, 9% and 1% (in oleoresin ginger).
Embodiment seven:
The purity of getting purchase is 95% 6-gingerol 30mg, be dissolved in the 2ml ether, add 0.2ml normal hexane azanol (hydroxylamine content is 50%) solution, mixing, reaction is 2 hours under room temperature, add 2ml1M salt acid elution three times respectively, 2ml1M salt solution washing three times, 2ml deionized water washing three times is behind the anhydrous sodium sulfate dehydration, leave standstill, treat to obtain after ether volatilizees the 6-gingerol oxime 25mg of crystalline state.
The biological activity determination of the gingerol oxime of the foregoing description gained:
The measuring method of leading indicator is as follows:
(1) estimate anti-oxidant activity with hemolytic action:
Get 200 gram BALB/c male mouses, put to death, get posterior vein pipe blood rapidly and put into the Glass tubing that heparin sodium was handled, the centrifugal red corpuscle that gets cleans three times with phosphoric acid buffer (PBS), inhales dehematize slurry and pale brown look erythrocyte sedimentation rate with suction pipe at every turn, centrifugal 10 minutes then in 1000g, discard supernatant liquid, suspend again, and be mixed with 20% red blood cell suspension with PBS.
Haemolysis is estimated: get the 0.1ml20% red blood cell suspension respectively, add 0.1mlPBS respectively or contain the PBS and the 0.2ml AAPH[2 of different concns gingerol oxime sample, 2 '-Azobis (2-amidino-propane) dihydrochloride] mixing is in 37 ℃ of water bath heat preservations after 3 hours, add 8ml PBS (A) again, another arm adds 8ml distilled water (B) so that erythrocyte fragmentation haemolysis, two kinds of reactants all under 1000g centrifugal 10 minutes are measured its absorbancy at 540nm respectively.Absorbancy is high more, illustrates that hemolytic action is serious more, and the antioxygenation of used gingerol oxime is weak more.With vitamins C as positive control.Calculate inhibiting rate according to following formula: the % inhibiting rate=[(1-A)/B] * 100%
Gingerol oxime concentration during with 50% inhibiting rate is as median effective dose (IC
50).
(2) evaluation of lipid peroxidation:
The making of brain homogenate: get the male BALB/c mouse of 150g brain, rapidly with 0 ℃ of Tris-hydrochloride buffer of 20mM (pH7.4) thorough washing, put homogeneous on Polytron PT 3000 refiners, under 3000g centrifugal 10 minutes again, get supernatant liquor and carry out the lipid peroxidation evaluation.
The mensuration of lipid peroxidation:, use FeSO4-Vit.C system induction lipid peroxidation with the index that mda (MDA) is estimated as lipid peroxidation.Get 0.1ml mouse brain homogenate supernatant liquor, 0.1ml 10uMFeSO4,0.1ml 0.1mM Vit.C, 0.2ml different concns gingerol oxime sample solution, mixing, 37 ℃ of water bath heat preservations are after 1 hour, add 0.5ml 28% trichoroacetic acid(TCA) (TCA) stopped reaction, the MDA that adds the generation of 0.38ml 2% thiobarbituricacid (TBA) and mouse brain homogenate lipid peroxidation lipid reactant is in 80 ℃ of reactions 20 minutes, after the cooling, centrifugal 10 minutes, the absorbancy of the MDA-TBA reactant under 532nm in the mensuration supernatant liquor.With BHA as positive control.Be calculated as follows inhibiting rate:
The % inhibiting rate=[(1-A)/B] * 100%
In the formula: A is the absorbancy of sample; B is the absorbancy of contrast.Gingerol oxime concentration during with 50% inhibiting rate is as median effective dose (IC
50).
(3) mensuration of killing ability:
Preparation 0.2% (W/V) crystallization gingerol oxime ethanol mother liquor: the crystallization of 0.2g gingerol oxime is dissolved in the 10ml20% ethanol.Time spent respectively with 0.01% molecule distillating monoglyceride solution dilution become 10,7,5,3,2,1g/L solution.The solution that the prepares dry Folium Ricini with homalographic is soaked into, with this feed as cotton lamp worm.Contrast the dried Folium Ricini of the feed of cotton lamp worm with 0.01% molecule distillating monoglyceride solution soaking homalographic.
Third-instar larvae (6～8 days) and one of Folium Ricini soaking into gingerol oxime solution are put into bottle, feed after 24 hours the normal Folium Ricini of feeding again.In the process of feeding, changed feed, and removed dregs such as ight soil in per 24 hours.Add up the mortality ratio of larva, unusual larva rate, pupa mortality ratio etc., and calculate the correction inhibiting rate according to following formula:
% correction growth inhibition ratio=[(T-C)/100-C] * 100
In the formula: T---handle the total growth-inhibiting amount of cotton moths attracted by lamplight;
C---contrast the total growth-inhibiting amount of cotton moths attracted by lamplight.
Gingerol oxime concentration during with 50% inhibiting rate is as median effective dose (IC
50).
(4) mensuration of anti respiratory syncytial virus (RSV) ability:
Virus and cell: RSV, monkey kidney dermoid cancer cell (Hep-2) and Madin-Darby canine kidney(cell line) (MDCK) (MDCK) cell are all available from ATCC (American Type Culture Collection).
Cell growth medium and keep liquid: growth medium: DMEM (Dulbecco ' s modifiedEagle ' s Medium).During the preparation growth medium, every liter is added 10% bovine serum albumin, 10000U penicillin, 2mM gentamicin, 2mM glutamine.When liquid is kept in the growth of preparation cell, only add 1% bovine serum albumin, other is prepared with growth medium.Other reagent is Sigma company product.
The active mensuration of anti-RSV
The gingerol oxime sample is all used an amount of dimethyl sulfoxide (DMSO) (control its content in the substratum mother liquor and be lower than 1%) dissolving, and to be diluted to the mother liquor that contains sample 4-8mg/ml standby with the substratum of keeping for preparing again.
The growth medium that will contain well-grown Hep-2 cell is sub-packed on the 96 hole plastic culture plates, at 37 ℃, contain in the incubator of 5% carbonic acid gas and cultivated 3 days, behind cell growth formation monolayer, remove growth medium, add 0.1 milliliter of RSV suspension and 0.1 milliliter of liquid of keeping that contains the proper concn sample that contains 100TCID50 (half infective virus amount).Three concentration of each sample determination, with sample to the maximal non-toxic dosage of culturing cell as full test concentration, other two concentration are doubling dilution concentration.Each sample concentration triplicate.Do not add sample in the contrast.With the positive contrast of virazole.96 orifice plates are put 37 ℃, 5%CO
2Incubator was cultivated after 2 days, and microscopy cytopathy situation is with the activity of cytopathy variability (CPE) reflection sample.Utilize CPE value under the different sample concentrations to obtain the median effective dose (IC of sample
50), with the anti-RSV effect of more different samples.
Cell toxicant is measured: poison the positive error that culturing cell causes for avoiding enriched sample, the maximal non-toxic dosage of first working sample before the working sample activity.Concrete measurement operation is with the determination of activity of aforementioned sample.Just utilize pure growth medium to replace viral suspension.Sample concentration when measurement result unusual (forming bubble etc. as cell rounding, contraction, protoplastis) occur with 50% culturing cell is expressed as the maximal non-toxic dosage of this sample.
(5) mensuration of anti-tumor activity:
Cell: blood cell (K562); Monkey-kidney cells (Vero), leukemia tumour cell (HL-60).
The mensuration of antiviral vitality: get cultured HL-60 and K-562 tumour cell, be seeded in respectively on 96 orifice plates, 1 * 10-4 cell of every hole inoculation, add gingerol oxime solution more respectively, make that its final concentration is 2.5,5,10,20ug/L, under 37 ℃, 5%CO2,95% relative humidity, cultivated 72 hours again.After cultivating end, add and an amount of 0.4%Tryphan blue solution, the quantity of microscopy dead cell and viable cell is calculated inhibition rate of tumor cell according to following formula.
% inhibiting rate=(1-sample preparation viable count/contrast viable count) * 100
The mensuration of gingerol oxime pair cell virulence: with the active mensuration of anti-RSV.
The measurement result of above index sees Table 1.
Table 1: the biological activity of gingerol oxime
-1
Annotate:
1) IC of sample
50All in 6-gingerol oxime (the 6-gingerol oxime in the sample, 8-gingerol oxime, 10-gingerol oxime, 12-gingerol oxime content are respectively 58%, 15%, 22%, 3%);
2) IC
50The expression inhibiting rate is 50% o'clock a dosage;
3) reference substance is the 6-gingerol.
Said determination is the result show: compare with the 6-gingerol, the biological activity of gingerol oxime with it quite or slightly high: its antioxygenation, antitumor and insecticidal action are higher than gingerol, but anti-RSV effect is slightly less than gingerol.
Claims (12)
1, gingerol oxime is characterized in that, be with gingerol crude extract or pure gingerol by the gingerol derivative that oximation reaction obtains, have following formula:
Wherein: n=6 is the 8-gingerol oxime; N=8 is the 10-gingerol oxime; N=10 is the 12-gingerol oxime.
2, the synthetic method of gingerol oxime as claimed in claim 1 is characterized in that, may further comprise the steps:
A) oximation reaction: the gingerol with gingerol crude extract or purification is a raw material, add 1～3 times of mole number to the oxammonium hydrochloride of gingerol, be under 3.5～5.5 the condition at 0～100 ℃, pH, carry out oximation reaction, in 1～3 hour reaction times, the solvent that reaction is used is selected from a kind of in 10～100% methyl alcohol, ethanol, ether, the ethyl acetate;
B) produce thick gingerol oxime: oximation reaction liquid is concentrated into 10～50% of original volume, uses organic solvent extraction 2～3 times; Institute's organic solution that obtains is successively washed, is washed through overpickling, salt mutually, repeats this process 1～3 time; Reclaim solvent again, get thick gingerol oxime; Described organic solvent is selected from ether, ethyl acetate, normal hexane;
C) rough segmentation of gingerol oxime: carry out chromatographic separation with sorbent material as stationary phase, elutriant through concentrate reclaim thick gingerol oxime;
D) segmentation of gingerol oxime: use dextrane gel as stationary phase, with Virahol: chloroform=1～8: 1, for moving phase is carried out continuous wash-out, collect Virahol respectively: chloroform is 1.5: 1 to 5: 1 a wash-out part, after removing moving phase, get 6-gingerol oxime, 8-gingerol oxime, 10-gingerol oxime, 12-gingerol oxime respectively.
3, synthetic method according to claim 2 is characterized in that: among the described step C, described sorbent material is a silica gel, when silica gel chromatography separates, collects normal hexane and ether volume ratio and be 1: 0.5～2 wash-out part.
4, synthetic method according to claim 2 is characterized in that: among the described step C, described sorbent material is a polymeric amide, during the polymeric amide chromatographic separation, collects 20%～60% methyl alcohol, ethanol or acetone wash-out part.
5, synthetic method according to claim 2 is characterized in that: among the described step C, described sorbent material is a macroporous resin, during the macroporous resin chromatographic separation, collects 20%～50% methyl alcohol, ethanol or acetone wash-out part.
6, synthetic method according to claim 2 is characterized in that: behind step C, the gingerol oxime concentrated solution is left standstill crystallization under 0～40 ℃, can obtain the coarse crystallization gingerol oxime.
7, synthetic method according to claim 2 is characterized in that, described gingerol crude extract is the thick gingerol of natural extract, prepares by following steps:
1) raw material is handled: fresh ginger is cleaned earth, be cut into shredded ginger or thin ginger splices, dry, wear into 100 order ginger powder then in air seasoning place, and stand-by, or directly the dried ginger slice that seasoning obtained is ground into 100 order ginger powder, stand-by;
2) obtain the gingerol extraction liquid: with 40%～100% organic solvent extraction ginger powder, adopt solvent cycle or diacolation measure between soak period, to improve the percentage extraction of gingerol, soak time is no less than 12 hours; Every batch of ginger powder extracts 3～5 times; Used extraction liquid is collected in supplies next step usefulness together; Described organic solvent is selected from a kind of of ethanol, methyl alcohol, acetone, ethyl acetate;
3) reclaim solvent: under 40～70 ℃, the gingerol extraction liquid of distilation steps 2 gained reclaims solvent, gets the gingerol crude extract;
4) extracting and separating gingerol crude extract: in the gingerol crude extract, add the normal hexane of 0.5～1 part of volume, extraction gingerol, continuous extraction 2～5 times; After merging normal hexane extraction liquid, reclaim 75～90% normal hexanes, resistates is used isopyknic extracted with diethyl ether gingerol again, and continuous extraction 2～3 times gets thick gingerol behind the recovery ether.
8, synthetic method according to claim 7 is characterized in that: in the described step 2, adopt percolation to obtain the gingerol extraction liquid, be about to ginger powder dress post, make 40%～100% organic solvent slowly by Jiang Fenzhu, obtain the gingerol extraction liquid; Described organic solvent is selected from a kind of of ethanol, methyl alcohol, acetone, ethyl acetate, normal hexane.
9, synthetic method according to claim 2, it is characterized in that described gingerol crude extract is various types of oleoresin gingers, prepares by following steps: the water-containing organic solvent with 20～80% extracts oleoresin ginger, fractionate out the ginger essential oil, get the gingerol crude extract; Described organic solvent is selected from ethanol, methyl alcohol, acetone, ether.
10, gingerol oxime as claimed in claim 1 is used to prepare the purposes that preventing respiratory closes the kitchen virus drugs.
11, gingerol oxime as claimed in claim 1 is used to prepare the purposes of antitumor drug.
12, gingerol oxime as claimed in claim 1 is used to prepare the purposes of sterilant.
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| 山东农业大学学报. 黄雪松等人，511-514，姜酚的提取、分离和鉴定. 1998 食品科技. 韩菊等人，63-66，生姜中姜酚的性能研究. 2004 食品科学. 柳乃奎等人，169-172，姜酮、脱氢姜酮、姜酚肟对两种自由基的清除作用. 2004 |
| 山东农业大学学报. 黄雪松等人，511-514，姜酚的提取、分离和鉴定. 1998 * |
| 食品科学. 柳乃奎等人，169-172，姜酮、脱氢姜酮、姜酚肟对两种自由基的清除作用. 2004 * |
| 食品科技. 韩菊等人，63-66，生姜中姜酚的性能研究. 2004 * |
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